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Purpose: Patients with acute myeloid leukemia (AML) often present with fatigue and severe pancytopenia. We report the case of a 68-year-old woman with no significant medical history who presented with 1 year of progressively worsening bilateral temporomandibular joint (TMJ) pain. She was otherwise asymptomatic. A computed tomography scan revealed degenerative joint disease in both TMJs. Bilateral TMJ replacement was performed., Materials and Methods: The excised TMJ tissue underwent formalin fixation and decalcification, and routine hematoxylin and eosin-stained sections were generated., Results: Immunohistochemical stains showed a population of monotonous cells in the marrow space expressing CD33, CD43, and myeloperoxidase, confirming the diagnosis of myeloid neoplasm. Subsequent bone marrow biopsy with flow cytometry confirmed AML with myelodysplasia-related changes., Conclusions: Adult patients with AML can rarely present with musculoskeletal complaints alone, which could delay the diagnosis. To our knowledge, this is the first report of AML with myelodysplasia-related changes presenting in a patient with TMJ degenerative joint disease that was otherwise asymptomatic., (Copyright © 2014 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

The World Health Organization 2008 Classification emphasizes myeloperoxidase (MPO) detection as sufficient for assigning a blast population to the myeloid lineage. Published MPO positivity thresholds are 10% for flow cytometry (FCM) but 3% for cytochemistry. Here we re-evaluated the FCM-MPO threshold by comparing retrospectively 128 acute lymphoblastic leukaemias and 75 acute myeloid leukaemias without maturation, all assessed by benzidine-based cytochemistry. A 13% threshold was found to be relevant using an isotype control as background-reference (sensitivity 95·1%, specificity 91·7%). Residual normal lymphocytes proved to be an advantageous alternative reference, a threshold of 28% yielding improved 97·4% sensitivity and 96·1% specificity., (© 2013 John Wiley & Sons Ltd.)

Background: According to WHO 2008 guidelines, an important role is designated for cytoplasmic myeloperoxidase (cMPO) as measured by flow cytometry for classifying acute leukemia of myeloid or ambiguous origin (AML or MPAL). However, no threshold with respect to expression level and percentage positive cells is provided. Since the expression of solely cMPO can change the diagnosis from acute lymphoid leukemia into MPAL in the current WHO 2008, a consensus is needed for the cut-off for cMPO., Methods: In this study, we investigated whether or not a cut-off of 10% positivity for cMPO equally defines an acute leukemia as AML or MPAL as compared to a cut-off for cMPO of 20% and compared this with results obtained for Sudan Black B (SBB) staining by cytomorphology., Results: Cell lineage-defining markers and SBB staining were analyzed retrospectively in a cohort of 198 patients who presented with acute leukemia. Eight patients were positive for SBB (>3%), but were considered negative for cMPO (<10%); six patients were negative for SBB (≤ 3%) and positive for cMPO (≥ 10%) staining. In six patients, we found 10-20% cMPO positive leukemic cells. Five of these cases were SBB positive; the sixth patient showed a clear myeloid phenotype without positivity of any lymphoid marker. Using a 10% cut-off instead of 20% would have changed diagnosis from ALL into MPAL in two patients; both cases were SBB positive by morphology., Conclusion: We conclude that a 10% cut-off is a secure lower limit for cMPO expression and can be used independently from SBB expression., (Copyright © 2013 International Clinical Cytometry Society.)

A retrospective analysis of 173 skin biopsy specimens of myeloid leukemia cutis (MLC) was performed to determine histologic and immunophenotypic criteria that could distinguish the varied myeloid disorders from one another. For the study, 11 relevant histologic items were scored and 12 antigens were studied (CD68 [KP1], CD163, CD14, CD4, myeloperoxidase [MPO], CD33, CD117, CD34, CD56, MIB-1, CD303, and CD123). Underlying myeloid disorders were essentially acute myeloid leukemias (65.3%), chronic myelomonocytic leukemias (11.0%), and refractory anemia (10.4%). Skin lesions were de novo in 7.5%, concurrent in 26.6%, and subsequent in 60.7%. Several morphologic characteristics (density, size of tumor cells, inflammatory background) were statistically useful in distinguishing between varied myeloid disorders. De novo MLCs displayed a specific morphologic profile. Association of CD68, CD33, and MPO could diagnose 100% of the cases of MLC. However, the immunohistochemical panel could not distinguish between the varied underlying myeloid disorders, with the exception that CD123 was particularly powerful in recognizing chronic myelomonocytic leukemia and also permitted reclassification of 4 cases as blastic plasmacytoid dendritic cell neoplasm.

Cytochemical detection of myeloperoxidase (MPO) activity, a strong marker for myeloid differentiation, is usually performed by benzidine dihydrochloride staining, with the threshold at 3%. Several reports have demonstrated the potential toxicity of benzidine, and bans have been issued, under French law, prohibiting female technicians from being exposed to the aromatic hydrocarbon group, including benzidine. The aim of this study was to test an alpha-naphthol and pyronine-based substitute using a standardized kit (MYELOPEROXIDASE KIT, RAL [Réactifs RAL, Martillac, France]) to measure MPO activity in blast cells. This prospective, multicenter study made it possible to analyze 101 acute leukemia (AL) cases; it has also demonstrated both the 96% specificity and the 99% sensitivity of the method, with a threshold for positive staining of 3%, as well as good correlation (r = 0.95) between the staining method tested and the benzidine staining method. When using the alpha-naphthol/pyronine-based staining for MPO, the mean number of positive blast cells is statistically lower than that obtained using benzidine, but without incidence on AL classification. These results allow us to conclude that this method makes it possible to classify acute blood diseases by measuring MPO activity using reagents permitted by law, according to a standardized and reproducible protocol.

Summary: Cytochemical myeloperoxidase (MPO) positivity represents the gold standard for discrimination between lymphatic and myeloid blasts. Rarely, cytochemical MPO reaction may be positive in >or=3% of blasts with clear lymphoblastic morphology. We present 5 patients with cytochemically MPO-positive acute leukemia classified as lymphoblastic by cytomorphology and lymphoblastic (n=3) or biphenotypic (n=2) by immunophenotyping, who entered first-line treatment for lymphoblastic leukemia. The former 3 are in first remission and both with biphenotypic leukemia relapsed with acute myeloid leukemia. The study primarily shows that cytochemical MPO expression in childhood acute leukemia revealing typical lymphoblastic morphology and phenotype does rarely exist. Although a small number of patients studied, cytochemical MPO expression in acute leukemia does not seem to require myeloid leukemia treatment in case of otherwise lymphoblastic cytomorphology and phenotype.

Background: Myeloperoxidase (MPO) plays a crucial role in the differentiation of acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL). In this report, we proposed the application of the enhanced chemiluminescent (ECL) technique to the determination of MPO activity in blasts of acute leukemia (AL)., Methods: Bone-marrow samples were obtained from 23 patients with AL (ALL, 5 cases; ANLL, 13 cases; AUL, 1 cases; mixed-lineage AL, 4 cases). Cells were incubated with a standard reaction mixture and chemiluminescence was measured. The mean peak light emission (PLE) was assessed., Results: When the cut-off point of PLE was settled at 2483, which was set for the discrimination between ANLL and ALL (mean + 3 x SD of ALL samples, n=5), all cases of ALL were MPO-negative, and ten of the thirteen ANLL patients were MPO-positive, which was concordant with cytochemical staining. In addition, this technique was able to demonstrate MPO activity in 4 mixed-lineage AL cases which did not stain for MPO in cytochemistry preparations., Conclusions: Our ECL technique is simple, inexpensive, and easier to perform compared to other procedures used to measure MPO activity in AL.

Serglycin is the major cell-associated proteoglycan of hematopoietic cells. Previous work has demonstrated that serglycin may be involved in targeting some proteins to granules of cytotoxic lymphocytes, mast cells and neutrophils. We characterized the expression of serglycin in various hematologic malignancies by immunohistochemistry and ELISA. Serglycin expression was found to distinguish acute myeloid leukemia (AML) from acute lymphoblastic leukemia. In contrast to myeloperoxidase, serglycin was found to be a selective marker for immature myeloid cells, distinguishing AML from Philadelphia chromosome-negative chronic myeloproliferative disorders.

Background: Accurate bone marrow (BM) blast counts (BCs) are essential for diagnosis (dx) of myelodysplasia (MDS), MDS/myeloproliferative (MDS/MPD) disease, or acute myeloid leukemia (AML), and may be difficult in hemodiluted bone marrow aspirates (BMAs). Erythroid precursors (EPs) may be indistinguishable from myeloblasts in BM sections (aspirate clots/cores). We compare the usefulness of immunohistochemistry (IHC) [ie, CD34, CD117, myeloperoxidase (MPO), Hemoglobin A1 (HbA1), and terminal deoxynucleotidyl transferase (TdT)] of BM sections (IHC-BM) with BMA, bone marrow touch preparation (BMTP), and flow cytometry (FC) BCs., Design: The initial BC (48), percentage (%) of Eps (38) (both based on initial 100 to 600-cell counts), and FC expressions of CD34, CD117, Glycophorin A(GLY A), and TdT (44) were tabulated from 50 BMs (MDS, MDS/MPD, or AML). BMAs (48) and BMTPs (25) subsequently received 500-cell counts. IHC-BM was performed (45:formalin, 5:B5-fixed) [CD34 (46), CD117 (45), HbA1 (45), TdT (42), and MPO (45)]., Results: Retrospective BMA BCs revealed a 31% (15/48) discrepant rate between the original/retrospective BMA BCs; 80% revealed an underestimated initial BC. There was a 28% discordance rate between the retrospective BMA and BMTP reviews; 77% showed a higher BMTP BC. IHC showed significantly higher BCs in 19% (9/47), resulting in a different dx (5). However, CD34 and CD117 IHCS revealed lower BCs in 38% and 48%, respectively. The CD34 IHC results were primarily due to CD34-negative blasts by FC. The CD117 IHC results were largely unexplained. EPs were CD34 and CD117-negative., Conclusions: (1) Evaluation for MDS/AML requires 500-cell counts of BMAs and/or BMTPs. (2) CD34 and/or CD117 blasts by FC indicate IHC-BM may increase BC accuracy. (3) CD34 is more reliable than CD117 by IHC; however, in combination, they are most reliable and should be performed on BM clots/cores due to variable reactivity.

Acute myeloid leukemia (AML)'s diagnosis is clinical and biological. We report here 80 AML with cytology and immunophenotype features to establish correlations. 21 AML1, 23 AML2, 12 AML3, 2 AML4, 18 AML5 and 3 AML6 were diagnosed by cytology. Only one case of AML0 was diagnosed by immunophenotype. Myelogysplasia is present in 29.8% cases. CD19 and CD56 expression was significantly associated to AML +t(8;21). Additionally, concomitant negativity of CD34 and HLA-DR was discrimininatif to AML3 diagnosis. Prognostic value to expression some CD needs time backwards.

Background: Flow cytometry of lineage markers is useful in the classification of leukemias. Our aim was to assess whether the study of lineage genes at the RNA level would enable differentiation of acute myeloid leukemias (AMLs) from B-and T-lineage acute lymphoid leukemias (ALLs)., Methods: We measured mRNA of four lineage markers [CD19, CD79a, CD3e, and myeloperoxidase (MPO)] by reverse transcription followed by real-time quantitative (RTQ)-PCR. We investigated 72 acute leukemias (40 AMLs with 23-93% blast cells plus 27 B-lineage ALLs and 5 T-lineage ALLs) defined by morphologic criteria at diagnosis. RTQ-PCR analysis was performed on bone marrow without cell sorting. The expression of each gene was calculated as the difference in the threshold cycle [DeltaCT; CT value of target gene minus CT value of housekeeping gene (Abelson)]., Results: Three patterns of expression were detected. In the first, CD19, CD79a, and MPO mRNAs were less abundant than CD3e. In the second pattern, MPO mRNA was more abundant than the other three mRNAs. In the third, CD19 or CD79a was more highly expressed than CD3e and MPO. The three patterns corresponded to T-ALL, AML, and B-ALL, respectively. The use of cutoffs to establish qualitatively the pattern of coexpression of the four lineage markers provided the same information as the comparison among the four DeltaCT values. Prospective use of the scoring system correctly classified each of 13 additional cases (8 AML, 4 B-lineage ALL, and 1 T-lineage ALL)., Conclusion: Study of lineage markers at diagnosis by RTQ-PCR allows differentiation of AML from B-ALL or T-ALL without cell sorting, even when the bone marrow contains few blast cells.

Lysosomal proteins including myeloperoxidase (MPO), lysozyme (LZ), CD68 and lactoferrin (LF), represent classical immunohistology marker molecules. Additionally, flow cytometry can be used to detect and quantify their expression at the single cell level in phenotypically defined leukocyte subsets. Recent results demonstrated that expression densities of these intracellular proteins vary among myeloid cell subsets, thus enabling insights into novel subset biology and development. Additionally, whole blood staining protocols allow detection of lysosomal proteins in infrequent leukocyte subsets such as circulating CD34+ hematopoietic progenitors and dendritic cells (DC). Thus, information on leukocyte subset distribution and aberrant phenotypes might be gained for diagnositic purposes. Finally, FACS detection of MPO and LZ proved to be of high value for the lineage diagnosis of acute leukemias.

Introduction: In this report, we propose the application of the p-iodophenol-enhanced luminol chemiluminescent technique to the determination of peroxidase (myeloperoxidase and/or platelet peroxidase) activity in blasts of minimally differentiated acute myeloblastic leukemia (AML-M0) and acute megakaryoblastic leukemia (AML-M7)., Methods: The frozen blast cells from 29 patients were thawed and submitted to the optimized protocol., Results: All cases of AML-M7 and AML-M I exhibited integrated light emission greater than 73(10(2) mV x s), which was the arbitrary cutoff point set for the discrimination between AML and acute lymphoblastic leukemia (ALL) (mean + 3 x s.d. of ALL samples, n = 10). In addition, five out of seven cases of AML-MO showed results above the cutoff point., Conclusion: This highly sensitive enhanced chemiluminescent technique may be applied to discriminate between ALL and AML-M7 or AML-MI cases, and most AML-M0 cases. It is very simple, cheap and easy to perform compared to other procedures used to measure MPO activity in AML-leukemias including AML-M7 and AML-M0.

CD56 is a natural killer (NK) cell marker that has been identified in approximately 15-20% of acute myeloid leukemia (AML) cases, where it has been associated with monocytic morphology and chromosomal abnormalities such as trisomy 8, t(8;21), t(15;17), and 11q23 rearrangements. The clinical presentation, chromosomal abnormalities as detected by fluorescent in-situ hybridization (FISH), and clinical outcomes of 7 patients with AML are presented. These cases were characterized by French-American-British (FAB) M5 morphology, myeloperoxidase (MPO) negativity, and co-expression of myelomonocytic and NK cell-associated antigens (CD11c(+), CD13(+), CD15(+), CD33(+), HLA-DR(+), and CD56(+)). All patients presented lymph node, hepatic, or splenic involvement at diagnosis. Despite the homogeneous morphologic and immunophenotypic characteristics the outcomes varied considerably. Two patients died during induction therapy, but the other five patients attained complete remission (CR). Of these five patients, 4 have received a bone marrow transplantation (autologous or allogeneic) and 3 of them are in CR (median follow-up: 45 months). The three patients with 11q23 rearrangements had a poor outcome and died of their disease within 1 year of diagnosis. Further studies with a larger group of patients would help establish the actual prognostic value of these morphologic, immunophenotypic and cytogenetic features., (Copyright 2002 Wiley-Liss, Inc.)

Context: Immunophenotyping of bone marrow (BM) specimens with acute myelogenous leukemia (AML) may be performed by flow cytometric (FC) or immunohistochemical (IH) techniques. Some markers (CD34, CD15, and CD117) are available for both techniques. Myeloperoxidase (MPO) analysis may be performed by enzyme cytochemical (EC) or IH techniques., Objective: To determine the reliability of these markers and MPO by these techniques, we designed a study to compare the results of analyses of these markers and MPO by FC (CD34, CD15, and CD117), EC (MPO), and IH (CD34, CD15, CD117, and MPO) techniques., Materials and Methods: Twenty-nine AMLs formed the basis of the study. These AMLs all had been immunophenotyped previously by FC analysis; 27 also had had EC analysis performed. Of the AMLs, 29 had BM core biopsies and 26 had BM clots that could be evaluated. The paraffin blocks of the 29 BM core biopsies and 26 BM clots were stained for CD34, CD117, MPO, and CD15. These results were compared with results by FC analysis (CD34, CD15, and CD117) and EC analysis (MPO)., Results: Immunodetection of CD34 expression in AML had a similar sensitivity by FC and IH techniques. Immunodetection of CD15 and CD117 had a higher sensitivity by FC analysis than by IH analysis. Detection of MPO by IH analysis was more sensitive than by EC analysis. There was no correlation of French-American-British (FAB) subtype of AML with CD34 or CD117 expression. Expression of CD15 was associated with AMLs with a monocytic component. Myeloperoxidase reactivity by IH analysis was observed in AMLs originally FAB subtyped as M0., Conclusions: CD34 can be equally detected by FC and IH techniques. CD15 and CD117 are better detected by FC analysis and MPO is better detected by IH analysis.

Cytochemical and ultrastructural studies of 42 patients with acute nonlymphoblastic leukemia (ANLL) lacking myeloperoxidase as detected by light microscopy were performed. These ANLL patients were classified into minimally differentiated acute myeloid leukemia (AML-M0) (26 cases), mixed-lineage leukemia (9 cases), and acute undifferentiated leukemia (AUL) (7 cases). Ultrastructural morphology revealed blasts from AML-M0 as very immature myeloid cells, blasts from mixed-lineage leukemia as having partially lymphoid cell features, and blasts from AUL as undifferentiated blasts with myeloid nature to some extent. Ultrastructurally, myeloperoxidase activity was positive in 81% of AML-M0 patients and less positive in the mixed-lineage leukemia and AUL patients. The DNA in the nuclei of the AML-M0 blasts was dispersed, as was observed in the immature myeloid cells. Some of the blasts from the mixed-lineage leukemia patients showed condensation of DNA, resembling blasts from acute lymphoblastic leukemia (ALL). The distribution of periodate-reactive glycoconjugates revealed that glycogen particles were absent or, if present, very scanty in AML-M0. Most of the blasts from the AUL patients lacked glycogen particles, and the blasts from those with mixed-lineage leukemia contained clustered glycogen particles, as was observed in the blasts from all patients. These findings disclosed that three subtypes of immature ANLL showed different cytochemical and ultrastructural features in accordance with their immunophenotypical classification.

A 29-year-old woman having acute myelogeneous leukemia-M1 subtype with the chromosomal abnormality t(16;21)(p11;q22) is presented. Complete blood count at onset showed a hemoglobin level of 7.2 g/dl, a platelet count of 48 x 10(9)/l, and a white blood cell count of 161.2 x 10(9)/l with 99% blasts and 1% lymphocytes. Bone marrow aspiration revealed massive proliferation of blasts that were positive for CD13, CD33, CD34, CD56 and myeloperoxidase, and negative for other T-cell, B-cell and monocytic markers. After achieving complete remission following conventional chemotherapy, she received an HLA-matched bone marrow transplantation (BMT) from her sibling after conditioning with busulfan, etoposide and cyclophosphamide. However, 9 months later, the leukemia relapsed as a painful extramedullary mass in her left femur. In spite of intensive re-induction chemotherapy, she died of progressive disease and sepsis. Although we could not detect the TLS/FUS-ERG fusion transcripts by reverse transcriptase-polymerase chain reaction in pre-BMT remission phase, they were clearly detectable in bone marrow cells obtained 6 months after transplantation with no translocation detected by conventional cytogenetics. We consider that even high-dose chemotherapy with BMT may not be effective in the eradication of this type of leukemia, and that the detection of minimal residual disease possibly contributes to the better planning of the therapeutic strategy., (Copyright 2001 S. Karger AG, Basel)

A patient with a Philadelphia chromosome (Ph)-positive acute mixed-lineage leukemia (AMLL) expressing both major and minor BCR/ABL mRNA transcripts is described. Phenotypic analysis of the leukemic blasts revealed positivity for both myeloid and B-cell lineages. Southern blot analysis showed a rearrangement of the immunoglobulin heavy chain (IgH) gene. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed the expression of both major and minor BCR/ABL mRNA transcripts. To our knowledge, this is the first report of AMLL expressing both major and minor BCR/ABL mRNA transcripts and rearrangement of the IgH gene., (Copyright 2000 Wiley-Liss, Inc.)

We reviewed the clinicopathologic and immunophenotypic profiles of 7 pediatric and 11 adult minimally differentiated acute myelogenous leukemias (AML-M0). We also compared and evaluated myeloperoxidase in leukemic blasts using standard cytochemical and polyclonal antibody immunohistochemical stains. No distinctive clinical findings were noted in either patient group; however, thrombocytopenia typically was more prominent in adults. Adult AML-M0 also was associated with an immature myeloid profile (CD34+, terminal deoxynucleotidyl transferase positive, CD13+, and CD33+), in contrast with pediatric AML-M0, which usually lacked terminal deoxynucleotidyl transferase or CD34 but expressed bright CD33 with weak or negative CD13. Coexpression of the T-cell-associated antigen CD7 was observed in both groups. Antibody immunohistochemical stains were more sensitive than cytochemical stains for detection of myeloperoxidase activity and a useful adjunct for establishing a diagnosis of myeloid leukemia in paraffin-embedded marrow tissues.

CD38 is a transmembrane molecule whose expression varies during hematopoietic cell differentiation. We used stroma-supported cultures of human myeloid cells to assess the effects of CD38 ligation on myeloid differentiation. In 8 experiments with CD34(+ )cells purified from normal bone marrow or cord blood, flow cytometry used with antibodies to CD34 and myeloperoxidase (MPO) identified 4 cell populations after 7 days of culture. Addition of anti-CD38 (T16) to the cultures induced a profound reduction of the most mature (CD34(-)MPO(++)) cell population, which includes promyelocytes, myelocytes and metamyelocytes; mean (+/- SD) cell recovery was 12.8% +/- 9.8% of that in parallel cultures with an isotype-matched control antibody. The suppressive effect of CD38 ligation on phenotypically more immature normal cells was inconsistent but generally less pronounced. Recovery of CD34(++)MPO(-) cells was 63.3% +/- 24.4%, recovery of CD34([+/-] )MPO(- )cells was 95.3% +/- 35.1%, and recovery of CD34(-)MPO(+) cells was 42.0% +/- 18.7% of that in control cultures. However, anti-CD38 suppressed recovery of cells obtained from 6 patients with CD38(+) acute myeloid leukemia; after 7-day cultures, cell recovery was 25.2% +/- 21.7% of that in control cultures. Cell recovery was also reduced by F(ab')(2) or Fab fragments of anti-CD38. CD38 ligation dramatically suppressed recovery of murine 32D myeloid cells transfected with human CD38 and cocultured with stroma (3.8% +/- 7.3%; n = 7). CD38 ligation of CD38( + )32D cells also induced cell aggregation, tyrosine kinase activity, and Ca(++) influx. We conclude that CD38 mediates signals that culminate in suppression of myeloid cell growth and survival. (Blood. 2000;95:535-542)

This study reports findings from a retrospective, comprehensive review of 80 cases of adult AML in regard to cytomorphology, enzyme cytochemistry (EC), flow cytometric immunophenotyping (FCI), and chromosomal analysis. From this review, we conclude that diagnostically challenging cases can only be subtyped by combining the cytomorphology with EC, FCI, and subsequent cytogenetic results. This is particularly true in recognizing the hypogranular variant of AML,M3 (AML, M3m) and distinguishing it from other subtypes. Nonlineage expression of markers (CD1, CD2, CD4, CD5, CD7, and CD56) was nonspecific as to AML subtype. Of interest, CD2 coexpression in acute myelomonocytic leukemia with eosinophilia (M4-Eo) was exclusively associated with inversion of chromosome 16 (inv 16) and was not observed in the other M4-Eo's without inv16. We also recognized a previously undescribed M3m with CD56 coexpression, heightening awareness of this entity which needs to be distinguished from the unique subtype of CD56+ AML with otherwise similar immunophenotypic and morphologic characteristics. In addition, nonlineage expression of CD19 alone was exclusively associated with the cytogenetic finding of t (8;21) (q22; q22) and thus may represent a favorable prognostic indicator by FCI.

Acute leukemias containing > 3% myeloperoxidase (MPO)-positive blast cells, as detected cytochemically, are considered to be myelogenous in origin, regardless of the immunophenotypic markers expressed. Conversely, acute leukemias that express only myeloid antigens are also considered to be acute myelogenous leukemia (AML), even in the absence of MPO. These MPO-negative AMLs, designated AML-M0 in the FAB classification, currently require either immunophenotypic or electron microscopic studies for identification. To examine the association of MPO and myeloid antigen expression in AML, particularly at the early stages of myeloid cell differentiation, we have used in situ hybridization (ISH) to evaluate MPO gene expression in myeloid leukemia cell lines and a variety of well-characterized acute leukemias, including six cases of AML-M0. Strong positivity for MPO mRNA was detected in the myeloid leukemia cell line HL-60 and in 22 of 27 AMLs (three AML-M0, four AML-M1, eight AML-M2, five AML-M4, two AML-M5a). No MPO gene expression was detected in three AML-M0, one AML-M5a, one AML-M7, 5 acute lymphoblastic leukemia, the lymphoid cell lines Molt-4 and Namalwa, or in the early myeloid cell lines KG-1 and KG-1a. Ultrastructural studies for MPO activity were performed on four AML-M0; one leukemia showed both gene expression and cytochemical activity, whereas two others contained neither MPO transcripts nor enzyme. Weak MPO gene expression was evident in one AML-M0 that was negative for enzymatic activity by electron microscopy. These studies show MPO gene expression can be detected by ISH in about half of AML-M0, supporting their presumed myelocytic derivation.(ABSTRACT TRUNCATED AT 250 WORDS)

We demonstrated by ultrastructural examination that the leukemic blasts of 13 patients with acute myeloblastic leukemia (AML) without maturation (M1 in the French-American-British classification) showed heterogeneous features. In 7 patients, the leukemic blasts had a high level of light microscopic myeloperoxidase positivity (> 50%). Ultrastructurally, the cells were myeloblast-promyelocytes with 100% myeloperoxidase positivity, and these 7 patients appeared to have typical AML. In contrast, the remaining 6 patients had leukemic blasts with a low myeloperoxidase positivity (< 50%) and heterogeneous features. Three had ultrastructural features of myelomonocytic or monocytic lineage, 1 had myelomonocytic cells associated with megakaryoblasts, and 1 had undifferentiated blasts. The former group had a better prognosis than the latter, indicating that ultrastructural analysis of M1 leukemia may help predict the response to therapy.

We examined leukemic blasts from 5 cases of AML-M0 diagnosed according to The French-American-British (FAB) classification for expression of immunological markers as well as myeloperoxidase (MPO) using flow cytometry (FCM) and immunocytochemistry (ICC). In one patient, the myeloid antigens, CD13 and CD33, were negative on FCM, but apparently positive in the cytoplasm by ICC, leading to a diagnosis of AML-M0. We examined MPO with anti-MPO monoclonal antibody in four patients by ICC, and could detect 3% or more MPO positive rates in all cases. These findings indicate that immunological studies for MPO and myeloid markers using ICC are very useful for the diagnosis of AML-M0. Two of 5 patients achieved CR, but they relapsed soon or after one year, respectively. The treatment outcomes suggest that the AML-M0 is an AML subtype with poor prognosis.

The pseudo-Chédiak-Higashi anomaly is characterized by the presence of giant granules in the cytoplasm of blast cells in acute leukemia. We report here a new case of acute myelogenous leukemia (M2 type) with this alteration. The granules were azurophilic or eosinophilic and reacted strongly to peroxidase stain. Ultrastructural studies showed that the granules contained a dense matrix and occasionally "finger print" structures at the periphery; in some inclusions, fibrillar structures of myelinic figures could be seen. The matrix was reactive to peroxidase and small vesicles were prominent in the cytoplasm near the granules. We conclude that the giant granules could have been formed by the fusion of primary granules and/or by the fusion of these small dense vesicles.

We report the clinical presentation and the morphological, immunophenotypic, cytogenetic and molecular genetic characteristics of a 14 1/2-year-old boy who had French-American-British (FAB) type M1 acute non-lymphocytic (ANLL) leukaemia with a common T-ALL immunological phenotype, with no myeloid associated antigen, either on the membrane or in the cytoplasm. ALL-directed induction therapy induced complete remission.

The main clinical, morphological, cytochemical, immunological features and therapy results of eleven patients diagnosed as acute myeloblastic leukemia M0 (AML-M0) are reported here. There were no clinical characteristics, abnormalities on physical examination or initial laboratory parameters that distinguished these eleven patients. Bone marrow aspirates were hypocellular in four patients. The leukemic cells were undifferentiated by light microscopy and myeloperoxidase (MPO) and/or Sudan Black B (SBB) stains were negative in all cases. Myeloid differentiation antigens were present on the leukemic cells of all eleven patients, whereas B and T cell markers were clearly negative except for CD4 and CD7 antigens. Whatever the treatment employed survival was very short. Eight of the eleven patients were treated and two achieved complete remission (CR) but only one of them is alive in continuous CR. Our results like those previously reported, suggest that AML-M0 patients have a very poor prognosis with standard induction therapies and should perhaps be considered for experimental therapeutic approaches.

With the objective of establishing the optimal therapy for minimally differentiated acute myeloid leukemia (AML-M0), we examined the therapeutic results of five AML-M0 cases and reviewed the literature. In a series of 63 patients with newly diagnosed acute leukemia who were admitted to the Main Hospital of Nippon Medical School, five patients fit the criteria for AML-M0: negative myeloperoxidase (MPO) and Sudan black B reaction by light microscopy, negative for B- and T-lineage markers, and positive for myeloid markers. They were treated by means of AdVP [adriamycin, vincristine, and prednisolone (PSL)] therapy and/or BHAC-DMP [behenoylcytosine arabinoside (BHAC), daunorubicin (DNR), 6-mercaptopurine (6-MP), and PSL] therapy. The AdVP therapy was unsuccessful in the two patients who received it, while a complete remission (CR) was achieved with the BHAC-DMP therapy in three of four patients. Although one patient treated with BHAC-DMP did not achieve CR, his blasts were apparently sensitive to the therapy. In assessable cases in the literature where leukemic blasts were MPO-negative, myeloid marker-positive and B- and T-lineage marker-negative, CR was achieved in 54.5% and 44.4% with anti-acute myeloid leukemia therapy and anti-acute lymphocytic leukemia therapy, respectively. Five cases in the literature were treated with a chemotherapeutic regimen containing BHAC [or cytosine arabinoside (Ara-C)], DNR, and 6-MP, and all achieved CR. The regimen containing BHAC (or Ara-C), DNR, and 6-MP may be useful as induction chemotherapy for AML-M0.

Cytochemical analysis of leukemic blasts from 46 patients with acute myeloblastic M2 leukemia (according to the FAB classification) was performed before and after cytostatic therapy, and compared with findings obtained in 20 age- and sex-matched control subjects. Cytochemical findings for myeloperoxidase (MPO), Sudan black B, acid phosphatase and alpha-naphthyl-acetate esterase (ANAE) were related to the achievement of the first complete remission (CR), i.e. data were compared after the patients had been divided into CR and non-CR groups. The analysis clearly showed that a high proportion of myeloperoxidase- and, to a lesser extent, Sudan black B-positive blasts before treatment may have constituted a significantly unfavourable prognostic factor.

Pretreatment peripheral and/or bone marrow blasts from 14 patients with acute unclassifiable leukemia (AUL) expressing myeloid related cell-surface antigen (CDII) or megakaryocyte-platelet related cell-surface antigen (OKM6), were isolated for further analysis in this study. Among 11 cases of CD11+AUL, despite a lack of myeloperoxidase (MPO) activity, one patient's blasts possessed Auer rod in a basophilic cytoplasm and another one's blasts expressed MPO maintaining the same surface phenotype after 20 months of his clinical course. The blast from 2 cases possessed both myelomonocytic and monocyte-specific antigens on the cell-surface, whereas the remaining nine cases completely lacked monocyte-specific antigen which is detectable by monoclonal antibodies, Mo2, My4 and Leu M3 (CD14). In addition, we revealed the presence of MPO protein in the cytoplasm of 3 cases of AUL patients by cytoplasmic immunofluorescence test utilizing monoclonal antibody (MA1). Following these results, the former was diagnosed as acute myelomonocytic leukemia (AMMoL) and the latter as acute myelogenous leukemia (AML) by immunophenotypic analysis using flow cytometry (FACS IV) and cytoplasmic immunofluorescence test. We have also described three cases of acute megakaryocytic leukemia which were demonstrated by the presence of megakaryocyte-platelet-related cell-surface antigens detected by utilizing flow cytometry and monoclonal antibodies in addition to both the PPO activity which was shown by ultrastructural cytochemistry, and the emergence of differentiation antigens while culturing these leukemic cells. The blast of 1 case possessed both platelet GPIb and GPIIb/IIIa cell-surface antigens detected by 5F1 (CD36), AN51 (CDw42), and J15, P2 and HPL2 (CDw41), respectively, whereas the remaining two cases almont lacked the GPIb cell-surface antigen. Hence, the former was diagnosed as immature (pro) megakaryocytic leukemia and the latter as acute megakaryoblastic leukemia from the viewpoint of immunophenotypic analysis as will be discussed in this article. These leukemic blasts did not express both T-cell lineage antigens which are detectable by monoclonal antibodies, T6 (CD1), T11 (CD2), T3 (CD3), T4 (CD4), T1 (CD5), Tp40, Leu9 (CD7), T8 (CD8), and B-cell lineage antigens which are detectable by monoclonal antibodies, B4 (CD19), B1 (CD20), B2 (CD21) and J5 (CD10).(ABSTRACT TRUNCATED AT 400 WORDS)

The permanent promyelocytic cell line HL-60 was subjected to stimulation with dimethyl sulfoxide (DMSO) and retinoic acid (RA), as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and lymphokine conditioned media for the induction of granulocytic or monocytic differentiation, respectively. Cells were investigated cytochemically using alpha-naphthylacetate esterase (acid esterase; AcE), naphthol AS-D chloroacetate esterase, and peroxidase reactions. In addition, the granulocyte or monocyte specific isoenzyme patterns of AcE as an intracytoplasmic property and the immunoreactivity to monoclonal antibodies recognizing granulocytes and monocytes (Ki-M2, Ki-M5) or monocytes alone (Ki-M1) were considered. The results indicated that HL-60 cell line bear the potency to evolve into granulocytes as well as monocytes. Additional studies performed on normal human bone marrow stained for AcE led to the conclusion that the myeloid cell line remains bipolar until the maturation stage of promyelocytes. Myelocytes being AcE positive only in 11.5 +/- 5.0 are heterogeneous and display the first indications of separated monocytic or granulocytic differentiation.

Multiple monoclonal antibodies and enzyme assays were used to study maturity markers (myelo-peroxidase) and immaturity markers (terminal transferase, HLA-2) in acute myeloid leukemia cells from 35 patients. In 8 of the patients, indications were found of an expression of maturity and immaturity markers on the same cells, here in called maturation asynchrony. It is suggested that the orderly appearance and disappearance of markers during the maturation of normal cells is disordered in malignant cells, and that single markers should be used with caution for the maturation classification of tumors. The simultaneous expression of maturity and immaturity marker by tumor cells could explain also why such cells can be recognized as abnormal even in the absence of tumor specific antigens.

In acute myeloid leukemia the leukemic cells are thought to be blocked in their normal differentiation. The stage of differentiation is the basis for the FAB classification. Induction of cell differentiation is a new and promising development in the treatment of some myeloproliferative diseases. The criteria for cell maturation and differentiation are almost all based on the morphology of the leukemic cells. In this study we tried to specify the maturation stage of the leukemic cells by quantitative enzyme analysis. In the HL60 (promyelocytic) cell line cells we determined the following enzymes: myeloperoxidase; alpha naphthyl acetate esterase; alpha naphthyl butyrate esterase and lactate dehydrogenase. The enzyme profiles obtained after culturing the cells in the presence of the differentiation inducing agents 1:25 dihydroxy vitamin D3 and DMSO were compared with several cytochemical and functional parameters. The results obtained in this study show that quantitative enzyme analysis is a useful tool in the study of myeloid or monocytic differentiation.

Ten cases of acute granulocytic leukemia with blast cells containing Auer bodies (ABs) have been studied by electron microscopy after cytochemical demonstration of myeloperoxidase. The cytochemical dense reaction product has been used as a dark field to visualize unreactive protein of ABs which may then be easily identified by its negative contrast. This method has allowed us to identify three types of ABs which differ in their substructure. In type I, (five patients with promyelocytic leukemia), our study confirmed that all of the ABs consisted of a hexagonal arrangement of hollow tubes. Cells from four cases of acute myeloblastic leukemia displayed type II ABs, in which a unique pattern of protein associated in a regular linear arrangement with well defined periodicity was identified. Type III appeared characteristic of a subclass of acute myeloblastic leukemia in which large inclusions with Chediak-Higashi-like granules containing numerous micro-ABs were seen. The configuration, size, and organization of the protein in the crystal were distinct from those seen in the two other types of ABs. These features suggest that the nature of the protein in ABs may be heterogeneous.

The classification of acute leukemia is important for the selection of optimal therapy. Classification often rests on morphologic, cytochemical, and immunologic criteria, and the marker enzyme terminal deoxynucleotidyl transferase (TdT) has been considered to be a reliable indicator of lymphoblastic leukemias. Because TdT-positive cells sometimes are seen in leukemias otherwise identified as myeloblastic, the authors evaluated blasts identified as myeloid by the presence of myeloperoxidase (MPO) for the simultaneous expression of TdT. The blasts in the bone marrow aspirate or peripheral blood of unselected patients with hematologic malignancies were evaluated and 60 cases are shown. The French-American-British system and, in some patients, cytochemical and immunologic studies were used to classify the leukemias. The authors demonstrated that blasts simultaneously contained MPO and TdT in 29% of patients with acute myeloblastic leukemia and 3% of patients with acute lymphocytic leukemia (ALL). This finding supports the hypothesis that TdT is an expression of cell primitivity rather than a marker for lymphoblastic cells.

Terminal deoxynucleotidyl transferase (TdT) is a biochemical marker for acute lymphoblastic leukemia (ALL). In studies of ALL at diagnosis, there are usually greater than 40% TdT-positive cells by indirect immunofluorescence, whereas acute myeloblastic leukemia (AML) shows less than 1% TdT-positive cells. Rare cases of TdT-positive AML have been reported. We present here three AML patients with TdT in 15%, 45%, and 90% of the leukemic blasts. The diagnosis of AML was established on the basis of morphology and cytochemistry, and the cases included one patient with Auer rods. Myeloperoxidase was present respectively in 20%, 90%, and 5% of the blasts. There was no Philadelphia chromosome present in the three cases. These results may indicate the simultaneous presence of lymphoid and myeloid populations, or the presence of a blast cell with both lymphoid and myeloid markers.

The ultrastructural localization of peroxidase activity has been studied in the circulating neutrophils and in a neutrophilic series of bone marrow cells from a patient with preleukaemia. Light microscopic examination showed 36% of the polymorphonuclear leucocytes to be totally devoid of myeloperoxidase, while 50% were normally stained and 14% were slightly positive for this enzyme. Electron microscopic studies revealed considerable heterogeneity in the promyelocyte population, since the number of peroxidase-deficient azurophil granules was seen to vary from 0 to 100% in these cells. Thus, several types of promyelocytes were identified. One cell type, which resembled that seen in normal subjects, contained myeloperoxidase within its azurophil granules and also within the cisternae of the rough endoplasmic reticulum and Golgi complex. A second type of promyelocyte, which was at an early stage of development, lacked myeloperoxidase in its secretory apparatus. These cells contained two species of azurophil granules, the first of which was devoid of peroxidase activity whereas the other reacted positively. These observations suggest that the premature arrest of myeloperoxidase synthesis in the promyelocytes from a preleukaemic patient may give rise to several populations of circulating neutrophils which can exhibit varying contents of myeloperoxidase.

Aminopeptidase (AMP) activity of leukemic cells in acute non-lymphocytic leukemia (36 cases) was examined by comparing peroxidase (PO) activity. The examination of AMP activity was useful for the diagnosis of non-lymphocytic leukemia in 19 of 36 cases rather than PO activity. Especially in acute monocytic leukemia its tendency was conspicuous. Furthermore, in 5 cases AMP was positive whereas there was no evidence of PO activity. On the contrary, PO was evident in 11 cases. The cases which revealed the same degree of both activities were only 4 and in 2 cases they were completely negative. Although the total number of PO negative cases was 7, ultrastructural endogenous myeloperoxidase reaction was clearly observed in granules of leukemic cells in all cases examined (6 cases). The diagnosis and classification of leukemia must be made using several techniques, and especially in non-lymphocytic leukemia cases, not only PO activity but also the examination of AMP activity is important. Therefore, one must be prudent in applying the FAB classification in acute leukemia.

Blast cells from untreated cases of acute leukemia were examined for expression of terminal transferase enzyme (TdT) by immunofluorescence and for myeloid lineage commitment as demonstrated by the presence of myeloperoxidase enzyme at the ultrastructural level. In three cases, an overlapping expression of the "lymphoid-specific" marker TdT was found on peroxidase-positive myeloblasts. These results indicate either the clonal expansion of a rare TdT-positive myeloid precursor or inappropriate expression of TdT by malignant myeloblasts, and further illustrate that TdT expression must be interpreted with caution when distinguishing between lymphoid and myeloid leukemias.