Variant Of Acute Promyelocytic Leukemia Research Articles

A Novel Fusion of RARA to the PRKAR1A Gene, Encoding the Regulatory Subunit Type-I α of Cyclic AMP Dependent Protein Kinase A, in a Variant Acute Promyelocytic Leukaemia.

The vast majority of acute promyelocytic leukemia (APL) cases are characterized by the formation of a PML/RARA fusion gene. Disruptions of retinoic acid receptor α (RARα) function have also been described in four types of variant APL in which an alternative partner gene (PLZF, NPM, NUMA, or STAT5B) is fused to RARA. We describe a novel variant APL with a RARA fusion formed by a complex gene rearrangement which is undetectable by conventional cytogenetics. A 66 yr old male with a history of mild thrombocytopenia was diagnosed with APL based on the blood and marrow morphology, the coagulopathy, and a microspeckled PML immunofluorescence pattern. The bone marrow immunophenotype was negative for CD2, CD19, CD34, CD56, CD117 and HLA-DR, and with weak expression of CD13, CD33 and CD11b, a pattern atypical for APL. The diagnostic bone marrow karyotype was 47,XY,+22[5]/46,XY[30] with no t(15;17)(q22;q21). FISH with the Vysis LSI PML/RARA dual fusion translocation probe did not show any fusion signals but there was splitting of an RARA signal on one 17q. A second probe, the Vysis LSI RARA break apart probe, showed deletion of the 5′ RARA probe and the 3′ RARA probe appeared to localize more distally than normal. The Cytocell PML/RARA ES probe also showed no fusion signals but one RARA signal appeared smaller. The diagnostic marrow was negative for PML/RARA transcripts by RT-PCR using PML and RARA specific primers, but an atypical product was observed. Sequencing of this product showed partial homology to the PRKAR1A gene that maps to 17q24 and encodes the regulatory subunit type I-alpha (RIα) of cyclic AMP-dependent protein kinase A. RT-PCR using PRKAR1A and RARA specific primers amplified two transcript splice variants of a PRKAR1A/RARA fusion gene. The shorter out-of-frame fusion transcript lacked PRKAR1A exon 3 and encoded a carboxy-truncated RIα protein. The longer in-frame fusion transcript resulted from cryptic splicing of the first 100 bases of PRKAR1A exon 3 to RARA exon 3, and encoded a chimeric RIα-RARα fusion protein that contained the dimerization domain of RIα and the same carboxy terminal domains of RARα that are found in all other known RARA rearrangements in APL. FISH using a BAC probe (RP11–120M18) encompassing the PRKAR1A gene identified signals on both copies of 17q; a strong signal on the normal 17 and a weaker signal on der(17). Before cytogenetic, FISH and molecular results were available, the patient was registered on the Australasian Leukaemia and Lymphoma Group's APML4 treatment protocol which includes ATRA, age-adjusted idarubicin and arsenic trioxide. Arsenic was ceased on day 22 due to toxicity. Morphological and cytogenetic FISH complete remission was documented on day 35. A bone marrow biopsy eleven months from original diagnosis showed no evidence of leukemia and PRKAR1A/RARA RT-PCR was indicative of molecular remission. This novel PRKAR1A/RARA gene rearrangement identified in a variant APL is the fifth variant APL in which the RARA partner gene has been identified and the second known rearrangement of PRKAR1A in a malignant disease.

The NPM-RAR fusion protein associated with the t(5;17) variant of APL does not interact with PML

The PML protein localizes to regions of the nucleus known as nuclear bodies or PODs. However, in t(15;17) Acute Promyelocytic Leukemia (APL) blasts, PML is found in a micro-punctate pattern. In order to test the hypothesis that delocalization of PML from PODs is necessary for APL, we investigated the interaction of the t(5;17) APL fusion protein NPM-RAR with PML. NPM-RAR localizes diffusely throughout the nucleoplasm. NPM-RAR does not alter the localization of PML in transfected HeLa cells, and does not associate with PML in vitro. These studies suggest that NPM-RAR does not interact with PML.

Variant acute promyelocytic leukemia translocation (15;17) originating from two subsequent balanced translocations involving the same chromosomes 15 and 17 while preserving the PML/RARA fusion

Fluorescence in situ hybridization (FISH) analysis of the bone marrow of a 24-year-old man diagnosed with acute promyelocytic leukemia (APL) revealed a variant pattern with one fusion signal instead of the typical two fusions expected with the probe set used. The combined FISH and conventional chromosome analyses suggested that two subsequent translocations had occurred in this patient involving the same chromosomes 15 and 17. As the prognostic outcome in APL is strictly associated with the presence of a PML/RARA fusion, it is useful and necessary to perform both cytogenetic and FISH analyses of a variant t(15;17) to determine the status of the PML/RARA fusion.

Morphologic heterogeneity of acute promyelocytic leukemia (APL)

6741 Background: Three morphological forms of APL recognized; hypergranular, hypogranular and hyperbasophilic forms. Other subtypes were rare, however, have been described. Methods: We describe a case of AML with PML-RARα fusion gene without above morphology. A 67-year-old caucasian male was hospitalized with diverticulits. He received radioactive seed-implants for prostate cancer 1.5 years ago. CBC showed WBC 9,000/cmm, Hemoglobin 12.4g/dl, platelet 25,000/cmm, neutrophil 5%, band 3%, lymphocyte 30%, monocyte 20%, metamyelocyte 2%, myelocyte 15%, promyelocyte 15%, myeloblast 10%. Clotting profile was normal except INR 1.6 and elevated D-dimers. Bone marrow biopsy was consistent with AML-M2. Induction chemotherapy was initiated resulting in worsening DIC. This raised the suspicion for morphologic variant of APL. The cytogenetic study revealed no diving cells. FISH and PCR studies were positive for PML-RARα fusion gene. He was then started on all-trans retinoic acid. Currently he has received 2 cycles of consolidation chemotherapy. The literature was reviewed for atypical morphologic presentations of APL on MEDLINE. Results: In a review of the literature of t(15:17) in AML subtypes other than M3, there have been 16 reported cases including our case. Male: Female:Unknown- 5:8:3; Subtypes: M1- 6, M2- 5, M4- 1, acute esosinophilic leukemia- 1, M7- 2, undifferentiated L2/M1- 1; DIC: Yes- 4, No-4, unknown- 8; Treatment with ATRA: Yes- 7, No- 6, unknown- 3; Outcomes: Complete hematologic remission - Yes- 7, Died - 3, unknown- 6. Molecular responses: Yes- 1, No- 6, unknown- 9. Conclusion: Patients with t(15;17) are amenable to treatment with ATRA making this most curable AML. The diagnosis of APL can be made from morphology and flow cytometry, but our case and the literature illustrate there are occasions where APL may present with atypical morphology. All efforts must be made rule out PML-RARα gene rearrangement in AML patients, especially with DIC. Summary of APL cases without typical M3 morphology Reference Age/Sex AML subtypes Disseminated Intravascular Coagulation (DIC) Use of All-trans Retinoic Acid (ATRA) Outcome Neame et al 60/F M1 No No Unknown 73F M1 Yes Yes Died 45M M2 Unknown No Unknown 53F M2 Unknown Yes Remission 54M M2 Unknown No Unknown Foley et al 45M L2/M1 Undifferentiated Yes Yes Remission Allford et al 15/F M1 No Yes Died 36/M M2 unknown No Died Head et al unknown M1 unknown unknown unknown unknown M1 unknown unknown unknown unknown M1 unknown unknown unknown Hast et al 22/F M4 Yes No Remission Aventin et al 22/F M7 No Yes Remission Morgan et al 18/F M7 No No Remission Yu et al 66/F Acute eosinophilic leukemia unknown Yes Remission This case 67/M M2 Yes Yes Remission No significant financial relationships to disclose.

CD2 + Tetraploid Acute Promyelocytic Leukemia Variant with Double (15;17) Translocations

We report a patient with a variant form of CD2+ acute promyelocytic leukemia (APL) who had double translocations (15;17) in a single leukemic cell. The patient presented with severe neutropenia, thrombocytopenia, and disseminated intravascular coagulation. The bone marrow showed marked hyperplasia with large leukemic cells that had bizarre nuclear configuration and basophilic, hypogranular cytoplasm. Leukemic cells were positive for CD2, 13, 33, 34, and 56 and negative for HLA-DR. The karyotype of the abnormal clone was characterized as 92,XXYY, t(15;17)(q22;q21)x2. No other additional abnormal clone was found, and the patient's condition was diagnosed as tetraploid APL variant. Fluorescence in situ hybridization assay revealed 2 promyelocytic leukemia and retinoic acid receptor alpha (PML/RARA) fusion signals, and reverse transcription-polymerase chain reaction assay revealed short-form PML/RARA fusion transcript. Tetraploidy in APL is a very rare abnormality. Double translocations were an additional abnormality in this case, and this patient's karyotype might have had some influence on morphological characteristics, expression of CD2, and poor clinical outcome.

Fusion of NPM to RARA in t(5;17) Disrupts Its Ability To Interact with YY1 Transcription Factor.

The transcription factor Ying-Yang 1 (YY1) can function either as a transcriptional activator or transcriptional repressor, depending on context. It has been reported that YY1 is a myeloid transforming gene interfering with neutrophilic differentiation. Further, it has been shown that the nucleolar phosphoprotein Nucleophosmin (NPM) directly interacts with YY1 in cells. As a consequence of NPM overexpression, YY1 repression is diminished. We previously reported the expression of an NPM fusion protein in the t(5;17)(q35;q21) variant of acute promyelocytic leukemia. The fusion protein encodes the N-terminal 118 amino acids of NPM fused to the retinoic acid receptor alpha (RARA). We previously showed that NPM-RAR inhibits myeloid differentiation in both U937 and transgenic model systems. We hypothesized that NPM-RAR might contribute to abnormal myeloid differentiation by altering YY1-mediated transcription pathways. To test this hypothesis, we sought to determine whether the NPM-RAR protein interacted with YY1 in the same manner as wild-type NPM. We generated a eukaryotic expression vector encoding FLAG-tagged YY1, and used it to transform COS cells. Full-length NPM, or NPM-RAR, were subcloned into a vector encoding the maltose binding protein (MBP), in the appropriate reading frame to generate MBP-fusions. The MBP proteins were expressed in bacteria, and purified by affinity chromatography. MBP, MPB-NPM, or MBP-NPM-RAR were incubated with YY1-COS lysates, and complexes were captured on amylose resin. The resin-complexes were washed, and the presence of YY1 was detected by immunoblotting using an anti-FLAG antibody. As expected, MBP-NPM pulled down FLAG-YY1 under these conditions. However, we were unable to detect association between MBP-NPM-RAR and FLAG-YY1. To determine whether this finding might be attributable to lack of post-translational modification of NPM-RAR in the bacterial system, we co-transfected expression vectors for FLAG-YY1 and NPM-RAR into COS cells. Lysates were subjected to immunoprecipitation using anti-RARA polyclonal antisera, and the presence of co-precipitating YY1 was detected by immunoblotting using an anti-FLAG antibody. Again, we were unable to detect direct interaction between the proteins. We conclude that NPM-RAR does not directly interact with YY1. This data does not exclude the possibility that YY1 activity could be altered in t(5;17) by decreased expression of wild-type NPM.

Expression of T-Lineage Affiliated Transcripts and TCR Rearrangements in Hypogranular Variant Acute Promyelocytic Leukemia (APL): Implications for the Cellular Target of the t(15;17).

APL is generally considered to be the most differentiated subtype of acute myeloid leukemia (AML) and to derive from late myeloid-restricted progenitors. This is, however, difficult to reconcile with the observation that approximately 25% of APL cases, particularly those with the hypogranular variant (M3v) form, express the T-lineage affiliated marker, CD2. To gain further insight into the extent of T-lineage deregulation in primary APL blasts and its implications for the likely target progenitor for the t(15;17), we quantified the expression of pre-Tα(pTα), T earlyα(TEA) and RAG1 transcripts by RQ-PCR (expressed as ΔCt ABL-target, whereby ΔCt =−2 is approximately 3 log higher than ΔCt =−12) and determined TCRδ and γ status in 36 cases of PML-RARA positive APL, including 21 classical hypergranular (M3) and 15 M3v forms. pTαforms part of the pre-TCR necessary for TCRαβ lineage differentiation and TEA represents a sterile transcript involved in the early stages of TCRαrearrangement.107 T-ALLs15 M3v21 M314 M0/1/212 M4/5Mean ΔCt pTα−2.1−2.38−4.64−5.7−12.23Mean ΔCt TEA−3.21 (34 pt)−3.55−6.58−5.23−6.95TCR rearrangementNA4/15 (27%)1/17 (6%)0/121/8Both classical and M3v APL expressed pTαat higher levels compared to 26 non-APL AML (p<0.001), with the levels in M3v being similar to those found in T-ALL. Amongst control AMLs, pTαlevels were lower (p<0.001) in FAB M4/5 AML compared to FAB M0/1/2 AML, which were not different from pTαlevels in classical APL (p=0.31). Unlike T-ALL, in which pTαand RAG1 expression cosegregate, M3v and classical APL did not express RAG1 transcripts. However, TEA was expressed at significantly higher levels in M3v APL compared to classical APL (p<0.001) and non-APL AML (p=0.002). TCR γ and δ rearrangements tended to be more common in M3v as compared with classical APL (p=0.11); moreover, expression of cytoplasmic CD3 (2/12) and TdT (3/13) were also restricted to these cases (0/3 and 0/5 in classical APL). Only M3v morphology and CD34 expression correlated with higher levels of pTαexpression amongst APL. Notably, CD2 expression, known to correlate with M3v APL, including in this series, did not. These data demonstrate that M3v APL blasts exhibit several features suggestive of early T-lymphoid orientation which may reflect the progenitor subject to leukemic transformation. They suggest that the PML-RARA fusion gene may arise in more primitive hematopoietic stem cells in M3v than previously thought to be the case and that the biological differences between classical and hypogranular variant forms of the disease may in part reflect differing cellular origins. Moreover these data are consistent with the existence of bipotent myeloid/T-lymphoid progenitors recently proposed by Katsura and colleagues and which could be the progenitor population subject to leukemic transformation in the hypogranular variant form of APL.

Immunophenotyping of acute leukemias and myelodysplastic syndromes.

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain

All-Trans-Retinoic Acid andBleeding/Thrombosis

Hematology-Oncology Department Ospedali Riuniti Largo Barozzi, 1 24128 Bergamo (Italy) Tel. 39 035 269492, Fax 39 035 266659 E-Mail annafalanga@yahoo.com All-trans-retinoic acid (ATRA) inhibits cell growth and proliferation by inducing cyto-differentiation and/or apoptosis in several cell types. These effects have become a therapeutic objective in human cancers. In addition to these functions, ATRA has been shown to affect cellular hemostatic properties [1]. Much understanding in this field has come from the clinical and experimental studies of human acute promyelocytic leukemia (APL), a distinct subtype of acute myelogenous leukemia (AML-M3), cytogenetically characterized by the balanced reciprocal translocation between chromosomes 15 and 17. In these cells, the fusion of the nuclear retinoic acid receptor (RAR ) gene on chromosome 17 with part of the PML gene on chromosome 15 results in the expression of a chimeric PML/RAR protein, which is involved in the leukemogenesis and is the target for the myeloid differentiation effect induced by ATRA. The disease typically presents with a life-threatening hemorrhagic diathesis, which is worsened by cytotoxic chemotherapy. The bleeding disorder is particularly severe in the microgranular variant of APL (M3v), which is characterized by marked hyperleukocytosis. Before the introduction of ATRA for the management of APL patients, fatal hemorrhages due to the associated coagulopathy were a major cause of induction remission failure. In a large retrospective study, the overall remission rate was 62% and the prevalence of hemorrhagic deaths in induction 14% [2]. The use of ATRA for the remission induction therapy of APL has raised the complete remission rate to greater than 90%, together with a rapid resolution of the coagulopathy, without causing bone marrow hypoplasia. ATRA promotes the terminal differentiation of leukemic promyelocytes, which is accompanied by prompt improvement of the coagulopathy typical of this disease [3]. Consistent with that, normalization in the plasma levels of hypercoagulation markers and downregulation of the two major blast cellassociated procoagulants (i.e. tissue factor, TF, and cancer procoagulant, CP) were observed [4]. A number of laboratory studies have subsequently confirmed the decrease or normalization of clotting and fibrinolytic variables during the first 1 or 2 weeks of therapy with ATRA. Inspite of that, the impact of ATRA therapy on early hemorrhagic deaths and CR rate in APL remains uncertain compared to chemotherapy alone. In nonrandomized studies, APL patients administered ATRA showed 9% to 20% improvement of CR rate and 5% to 6% reduction of early hemorrhagic deaths, compared to historical controls receiving conventional chemotherapy [1, 5–8]. These preliminary findings have been more recently confirmed by randomized clinical trials. APL patients treated with different combinations of ATRA plus chemotherapy show a prevalence of early hemorrhagic deaths from 2.4% to 6.5% [9–13]. However, both nonrandomized and randomized clinical trials clearly show an improvement of event free and overall survival in patients receiving induction therapy All-Trans-Retinoic Acid and Bleeding/Thrombosis

HLA-DR antigen-negative acute myeloid leukemia.

Human leukocyte antigen (HLA) Class II antigens are variably expressed on acute myeloid leukemia (AML) blasts. The biological and clinical significance of HLA Class II antigen expression by AML cells is not known. Therefore, we sought to characterize cases of AML without detectable HLA-DR expression. Samples from 248 consecutive adult AML patients were immunophenotyped by multiparameter flow cytometry at diagnosis. HLA-DR antigens were not detected on AML cells from 43 patients, including 20 with acute promyelocytic leukemia (APL), and 23 with other subtypes of AML. All APL cases had t(15;17), but there were no characteristic chromosome abnormalities in non-APL cases. No direct expression of other antigens was identified in HLA-DR-negative APL and non-APL cases. Interestingly, cells from three HLA-DR-negative non-APL patients had similar morphology to that of the hypogranular variant of APL. This morphology, however, was not present in any HLA-DR-positive AML cases. Treatment response was similar in the 23 HLA-DR-negative non-APL and the 205 HLA-DR-positive patients. Finally, relapse was infrequently associated with changes in HLA-DR antigen expression, as the HLA-DR antigen was lost at relapse in only 4% of HLA-DR-positive cases, and was gained at relapse in only 17% of HLA-DR-negative cases. We conclude that HLA-DR-negative AML includes approximately equal numbers of APL and non-APL cases, and that the morphology of HLA-DR-negative non-APL cases can mimic the hypogranular variant of APL. The diagnosis of APL cannot be based on morphology and lack of HLA-DR antigen expression; rather, it requires cytogenetic or molecular confirmation.

Leukemias resembling acute promyelocytic leukemia, microgranular variant.

Acute promyelocytic leukemia (APL) should be distinguished from other subtypes of acute myeloid leukemia (AML) because of the increased risk of disseminated intravascular coagulation (DIC) and its response to arsenic compounds and retinoids. Some cases of AML seem morphologically similar to the microgranular variant of APL (French-American-British [FAB] AML-M3v) but lack the t(15;17). We evaluated 8 cases of APL-like leukemias for subtle morphologic, cytochemical, immunophenotypic, and cytogenetic differences compared with 5 cases of promyelocytic leukemia/retinoic receptor alpha (PML/RARalpha)-positive APL (FAB AML-M3v). We also evaluated both groups for the presence of DIC. No differences among the groups were noted in blast size, chromatin pattern, nuclear morphologic features, intensity of myeloperoxidase staining, or presence of Auer rods. Immunophenotypes were similar; both types of cases lacked CD34 and HLA-DR and were CD13+ and CD33+. Two cases of APL-like leukemias also were CD56+. DIC was present in 2 patients with M3v. Our study shows that there are no definitive morphologic, cytochemical, or immunophenotypic findings that can distinguish these cases from PML/RARalpha-positive APL.

Translocation (15;17) and trisomy 21 in the microgranular variant of acute promyelocytic leukemia

Cytogenetic abnormalities in acute myelogenous leukemia have been identified as one of the most important prognostic factors. Favorable chromosomal changes such as inv(16), t(8;21), and t(15;17) are associated with higher rates of complete remission and event-free survival. Translocation t(15;17) characterizes acute promyelocytic leukemia (APL) (French-American-British [FAB] class M3) in almost all patients. Secondary chromosomal abnormalities are also present in approximately one-third of patients with newly diagnosed APL. We present a 26-year-old Hispanic man diagnosed with the microgranular variant of APL (FAB class M3v) whose initial cytogenetics included t(15;17) and trisomy 21. The prognostic implications of trisomy 21 and other secondary cytogenetic aberrations in APL are reviewed. To our knowledge, this is the first reported case of trisomy 21 with t(15;17) in the microgranular variant of APL.

The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties

The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RAR). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RAR sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RAR. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RAR in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RAR. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RAR. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RAR to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.

The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties

AbstractThe t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RARα). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RARα sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RARα. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RARα in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RARα. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RARα. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RARα to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.

Target acquisition and acute promyelocytic leukemia.

Target acquisition and acute promyelocytic leukemia.

Light scatter and immunophenotypic characteristics of blast cells in typical acute promyelocytic leukemia and its variant

Acute promyelocytic leukemia (APL), defined by the French-American-British (FAB) classification as the M3 subtype of acute myeloblastic leukemia (AML), is readily diagnosed by direct cytomorphologic examination. The potential difficulty in recognizing the microgranular variant of APL (M3v) stems from the morphological characteristics of APL leukemic cells, which resemble monocytes. From 109 newly diagnosed acute leukemic patients, 48 were classified as AML and 16 of these patients (33%) had APL. Mononuclear cells (MNC) of all APL patients were analyzed by flow cytometry for immunophenotypic features and forward and right angle scatter (FW-SC/RT-SC) light characteristics. Two clearly different FW-SC/RT-SC distribution patterns were recognized and defined as hypergranular (mature cells) and hypogranular (immature cells). Correlation between FW-SC/RT-SC patterns and the FAB system was poor: from eight patients classified as M3, six had the hypergranular pattern and two had the hypogranular pattern; from eight cases with M3, five and three patients had hypogranular and hypergranular patterns, respectively. The most relevant cellular immunophenotypic feature was the high frequency of CD34+ cases (5/7) recorded exclusively in patients with the hypogranular pattern. An interesting finding was that MNCs of all APL patients changed color from beige to green in the course of 3–4 h, whereas none of the 93 specimens of non-M3 acute leukemia cases showed this phenomenon. Summarizing, the FW-SC/RT-SC characteristics of APL blast cells along with the immunophenotype may represent an objective and reproducible measurement system to distinguish microgranular (M3v) from typical (M3) APL. In addition, the present study has identified another unique feature of APL based on the green color of their blast cells imparted by the high content in myeloperoxidase. Cytometry 32:286–290, 1998. © 1998 Wiley-Liss, Inc.

Light scatter and immunophenotypic characteristics of blast cells in typical acute promyelocytic leukemia and its variant

Acute promyelocytic leukemia (APL), defined by the French-American-British (FAB) classification as the M3 subtype of acute myeloblastic leukemia (AML), is readily diagnosed by direct cytomorphologic examination. The potential difficulty in recognizing the microgranular variant of APL (M3v) stems from the morphological characteristics of APL leukemic cells, which resemble monocytes. From 109 newly diagnosed acute leukemic patients, 48 were classified as AML and 16 of these patients (33%) had APL Mononuclear cells (MNC) of all APL patients were analyzed by flow cytometry for immunophenotypic features and forward and right angle scatter (FW-SC/RT-SC) light characteristics. Two clearly different FW-SC/RT-SC distribution patterns were recognized and defined as hypergranular (mature cells) and hypogranular (immature cells). Correlation between FW-SC/RT-SC patterns and the FAB system was poor: from eight patients classified as M3, six had the hypergranular pattern and two had the hypogranular pattern; from eight cases with M3, five and three patients had hypogranular and hypergranular patterns, respectively. The most relevant cellular immunophenotypic feature was the high frequency of CD34+ cases (5/7) recorded exclusively in patients with the hypogranular pattern. An interesting finding was that MNCs of all APL patients changed color from beige to green in the course of 3-4 h, whereas none of the 93 specimens of non-M3 acute leukemia cases showed this phenomenon. Summarizing, the FW-SC/RT-SC characteristics of APL blast cells along with the immunophenotype may represent an objective and reproducible measurement system to distinguish microgranular (M3v) from typical (M3) APL In addition, the present study has identified another unique feature of APL based on the green color of their blast cells imparted by the high content in myeloperoxidase.

An immunofluorescent assay for acute promyelocytic leukemia cells.

Sequential treatment with all-trans retinoic acid followed by chemotherapy significantly improves the long-term survival of patients who have acute promyelocytic leukemia (APL). Consequently, a simple and accurate test is needed to establish the diagnosis of APL and to identify those patients having a relapse of the disease. We describe an accurate, 2-hour indirect immunofluorescent assay for identifying APL cells in bone marrow specimens. The assay uses the PML (PG-M3) murine monoclonal antibody that is directed against the amino-terminal portion of the PML gene product. We observed a distinctive, finely speckled pattern of fluorescence in the NB4 cell line (a positive control), as well as in 15 clinical specimens that were confirmed to have APL by cytogenetic, cytochemical, and immunophenotypic studies, including four cases of microgranular variant of APL. By contrast, a coarse globular pattern of fluorescence was observed in 53 other clinical specimens that did not contain APL. When we performed dilution studies using artificial mixtures of APL cells with normal bone marrow cells, we detected as few as 5% APL cells in the mixture. Finally, there was complete concordance between the immunofluorescent assay and a polymerase chain reaction-based assay for the PML-retinoic acid receptor alpha chimeric gene in 12 other clinical specimens. We conclude that the immunofluorescent assay for PML protein is a rapid, sensitive, and accurate method for determining the presence of APL cells in clinical specimens. This assay therefore should be considered as a cost-effective alternative to other diagnostic tests, such as karyotyping or polymerase chain reaction, for the diagnostic evaluation of APL.

A microgranular variant of acute promyelocytic leukemia with atypical morpho-cytochemical features and an early myeloid immunophenotype

This report describes a unique case of acute promyelocytic leukemia (APL) showing elusive morphologic features, an atypical pattern of cytochemical reactions, and a previously unreported immunophenotype consistent with a very early myeloid form: CD13 (+), CD33 (+), CD9 (+), CD2 (+), HLA-DR (−), CD34 (+), CD117 (+), and TdT (+). The diagnosis of AML M3 variant was made only after genotypic analyses revealed the PML/RARα rearrangement associated with the typical (15;17) (q22;q21) translocation. This example of ‘asynchronous differentiation’ emphasizes the need for a multiparameter approach to the diagnosis of acute leukemia.

AIDA (All-Trans Retinoic Acid + Idarubicin) in Newly Diagnosed Acute Promyelocytic Leukemia: A Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (GIMEMA) Pilot Study

From March 1993 to October 1993, 20 consecutive, newly diagnosed acute promyelocytic leukemia (APL) patients from 13 Italian institutions entered in a pilot study named AIDA, combining all-trans retinoic acid (ATRA) with idarubicin (IDA). ATRA was administered orally beginning on the first day of induction at the dosage of 45 mg/m2/d until complete remission (CR), whereas IDA was administered intravenously at the dosage of 12 mg/m2/d on days 2, 4, 6, and 8 of the induction. Patients who achieved CR were consolidated with 3 courses of chemotherapy without ATRA; thereafter, they were followed up for molecular and hematologic CR. The median age was 35.3 years (range, 6.5 to 67.6 years); 8 patients were males and 12 females; 4 had the hypogranular variant of APL (M3v), and 4 (2 with M3v) presented with leukocyte counts > or = 10,000/microL. Molecular analysis for the promyelocytic leukemia-retinoic acid receptor alpha (PML-RAR alpha) hybrid gene at diagnosis was performed in 16 patients by means of reverse transcription-polymerase chain reaction (RT-PCR) analysis, and all were RT-PCR+ for the hybrid gene. In the remaining 4 patients, the cytogenetic study showed the presence of the t(15;17). After a median time of 36 days (range, 28 to 52 days) 18 (90%) patients achieved CR; the remaining 2 patients died 12 and 34 days after diagnosis from myocardial infarction caused by fungal myocarditis and from massive hemoptysis, respectively. ATRA syndrome was observed in only 2 patients, and, after the prompt discontinuation of ATRA and initiation of dexamethasone, both recovered from the syndrome. However, after recovering, 1 patient achieved CR, whereas the other died at day 34 because of massive hemoptysis; other side effects were very limited. At recovery from the third consolidation course, only 3 of 14 (21.4%) tested patients were RT-PCR+ for the PML-RAR alpha hybrid gene. Of these, 2 relapsed shortly afterwards; however, in the last patient, the PML-RAR alpha disappeared at successive testing performed 2 months later. As of September 30, 1995, after a median follow-up period from diagnosis of 27 months (range, 24 to 31 months), the overall survival and event-free survival durations are 85% and 69%, respectively; moreover, 14 of 18 (78%) patients who achieved CR are still alive and in first molecular and hematologic CR. Of the 4 relapsed patients, 3 achieved a second CR with ATRA and, after further treatment, are now in molecular and hematologic CR after 4+, 16+, and 17+ months from the second CR. These results indicate that (1) the AIDA protocol is highly effective in treating APL; (2) after 3 consolidation courses, the majority of patients who achieved CR are RT-PCR- for the hybrid gene PML-RAR alpha; (3) the persistence of an RT-PCR positivity for the PML-RAR alpha hybrid gene after 3 consolidation courses is indicative of early relapse, thus these patients still require additional treatment. These results have prompted the Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (GIMEMA) to initiate, in cooperation with the Associazione Italiana di Ematologia ed Oncologia Pediatrica and some European Organization for Research and Treatment of Cancer (EORTC) centers, a new multicentric clinical trial named AIDA LAP 0493 for the treatment of adult and pediatric APL patients. All patients are considered eligible if APL diagnosis is confirmed with molecular or cytogenetic studies for PML-RAR alpha hybrid gene or t(15;17) and are enrolled to receive the same induction and consolidation therapy of this pilot study. After consolidation, patients who are RT-PCR- for PML-RAR alpha hybrid gene are randomized to four arms, whereas patients who are RT-PCR+ after consolidation undergo, if eligible, an allogenic transplantation procedure.