A case of acute promyelocytic leukemia with morphologic multilineage dysplastic changes.

Although reports of typical acute promyelocytic leukemia (APL) cases rarely mention dysplastic changes, this report concerns a rare case of APL with tri-lineage dysplastic changes resembling the characteristic features of myelodysplastic syndrome (MDS). The patient, a 77-year-old Japanese male, was diagnosed as having pancytopenia with hematologic morphological abnormalities comprising micro - megakaryocytes, neutrophils with hypo-granulation and negative peroxidase activity, and erythroblasts containing nuclei with abnormalities such as karyorrhexis. Although there is one report of a case of transformation of de novo MDS into APL and several reports of cases of therapy-related MDS transformed into APL, our patient had no history of cytopenia or of either chemo or radiation therapy. Our case can thus be considered to constitute a rare case of APL with dysplastic morphology.

FLT3/ITD associated with an immature immunophenotype in PML-RARα leukemia.

Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARα fusion gene resulting from translocation t(15;17) (q22;q12). Internal tandem duplication (ITD) of the FLT3 gene has been observed in approximately 35% of APLs, and large-scale studies have identified the presence of ITD as an adverse prognostic factor for acute myeloblastic leukemia (AML) patients. Aberrant expressions of surface antigens, such as CD2, CD34, and CD56, have been found in APL, but the implications of this are not well understood. We investigated the incidence of the FLT3/ITD mutation and FLT3/D835 (I836) point mutation in 25 APL patients. Incidence ratios of FLT3/ITD, D835 (I836), and both FLT3/ITD and D835 (I836) were 36%, 36% and 8%, respectively. FLT3/ITD(+) cases showed a predominance of the bcr3 isoform (P=0.008) and M3v morphology (P<0.001). We found that all FLT3/ITD(+) cases expressed CD2 (9 of 9) more frequently than that of FLT3/ITD(-) (1 of 16) (P<0.001), while only one of the CD2(+) cases (1 of 10, 10%) did not harbor FLT3/ITD, and all CD2(+)CD34(+) cases (5 of 5, 100%) harbored FLT3/ITD. In addition, quantitative polymerase chain reaction analysis showed that FLT3 mRNA was more abundantly expressed in FLT3/ITD(+) than that in FLT3/ITD(-) (P=0.025), while there was no difference between D835(I836) (+) and D835(I836)(-) with regards to aberrant surface-antigen expression, expression levels of FLT3 mRNA, M3v morphology, and the bcr3 isoform of PML-RARα mRNA. This study demonstrates that the presence of FLT3/ITD, but not D835 (I836), is closely related to aberrant CD2 expression and high expression levels of FLT3 mRNA. Our findings also suggest that FLT3/ITD as a secondary genetic event may block differentiation at the immature stage of APL.

The HPB-AML-I cell line possesses the properties of mesenchymal stem cells.

BACKGROUND: In spite of its establishment from the peripheral blood of a case with acute myeloid leukemia (AML)-M1, HPB-AML-I shows plastic adherence with spindle-like morphology. In addition, lipid droplets can be induced in HPB-AML-I cells by methylisobutylxanthine, hydrocortisone, and indomethacin. These findings suggest that HPB-AML-I is similar to mesenchymal stem cells (MSCs) or mesenchymal stromal cells rather than to hematopoietic cells. METHODS: To examine this possibility, we characterized HPB-AML-I by performing cytochemical, cytogenetic, and phenotypic analyses, induction of differentiation toward mesenchymal lineage cells, and mixed lymphocyte culture analysis. RESULTS: HPB-AML-I proved to be negative for myeloperoxidase, while surface antigen analysis disclosed that it was positive for MSC-related antigens, such as CD29, CD44, CD55, CD59, and CD73, but not for CD14, CD19, CD34, CD45, CD90, CD105, CD117, and HLA-DR. Karyotypic analysis showed the presence of complicated abnormalities, but no reciprocal translocations typically detected in AML cases. Following the induction of differentiation toward adipocytes, chondrocytes, and osteocytes, HPB-AML-I cells showed, in conjunction with extracellular matrix formation, lipid accumulation, proteoglycan synthesis, and alkaline phosphatase expression. Mixed lymphocyte culture demonstrated that CD3+ T-cell proliferation was suppressed in the presence of HPB-AML-I cells. CONCLUSIONS: We conclude that HPB-AML-I cells appear to be unique neoplastic cells, which may be derived from MSCs, but are not hematopoietic progenitor cells.

Quantitative detection of PML-RARalpha fusion transcript by real-time PCR with a single primer pair.

Quantitative detection of minimal residual disease has prognostic value for some leukemias. Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARalpha fusion gene from t(15;17). Added to three PML-RARalpha isoforms, alternative spliced forms of PML exons give rise to multiple isoforms even within a single patient. To date, multiple primer pairs for the detection of the various PML-RARalpha transcripts have been designed, potentially generating some nonspecific amplification products. Here, we established a real-time quantitative PCR (RQ-PCR) strategy with a single primer pair using LightCycler (sp-RQ-PCR), which could simultaneously detect three isoforms with equal specificity and sensitivity as well as alternative spliced forms. Results obtained with sp-RQ-PCR for 39 samples from 15 APL patients and 31 non-APL samples were compared with those with TaqMan assay with three primer pairs. In two of the APL samples, PML-RARalpha was detected in the TM, but not in the sp-RQ-PCR or nested PCR. Furthermore, the sp-RQ-PCR showed no positive results for the 31 non-APL samples, whereas the TM identified 13% (4/31) as positive. Electrophoresis detected some artifacts in the TM, which do not correspond to PML-RARalpha. We conclude that our sp-RQ-PCR is specific enough to identify various forms of PML-RARalpha and yields no false-positive results.

DR negativity is a distinctive feature of M1/M2 AML cases with NPM1 mutation.

Our previous observation of a higher incidence of FLT3-ITD in DR(-) M1/M2 AML than in DR(+) M1/M2 led to an investigation of NPM1 mutation in the same samples, since DR(-) AML and AML with NPM1 mutation share such characteristics as normal karyotype, the absence of CD34, and FLT3-ITD. NPM1 mutation was found in 18 of 26 (69.2%) of DR(-) cases, but not in any of 28 DR(+) cases. FLT3-ITD was noted in 66.7% of the cases with NPM1 mutation. These findings point to DR negativity as another phenotypic feature of AML with NPM1 mutation.

HLA-DR-negative AML (M1 and M2): FLT3 mutations (ITD and D835) and cell-surface antigen expression.

FLT3 mutations and cell-surface antigen were investigated in 29 DR-negative (DR(-)) M1/M2 AML samples in comparison with 30 DR-positive (DR(+)) M1/M2 AML samples. FLT3-ITD was detected in 59.3% and D835 was detected in 7.4% of the samples. The incidence of FLT3-ITD was higher in the DR(-) group (59.3%) than in the DR(+) group (17.9%; P=0.002). The DR(-) status was associated with the CD34(-) (82.8%), CD7(-) (92.9%) and CD45RO(+) status (76%). Our results indicated that FLT3 mutation is the most common gene alteration found in the DR(-) M1/M2 AML. These results are important for further characterizing this phenotypic AML entity.

The Jab1/COP9 signalosome subcomplex is a downstream mediator of Bcr-Abl kinase activity and facilitates cell-cycle progression.

Jab1 is a multifunctional protein associated with the signaling pathway, cell-cycle regulation, and development, and acts as a key subunit of COP9 signalosome (CSN). Jab1 promotes degradation of the cyclin-dependent kinase inhibitor p27(Kip1) by transportation from the nucleus to the cytoplasm. However, there has been no clear evidence for whether and how Jab1 contributes to malignant transformation in human cancers. Here we show that Bcr-Abl tyrosine kinase facilitates the down-regulation of p27 by modulating complex formation of Jab1/CSN through the mitogen-activated protein (MAP) kinase and phosphatidylinositol 3 (PI3) kinase signaling pathways. Nearly half of the chronic myelogenous leukemia cell lines and the murine hematopoietic precursor cells expressing Bcr-Abl exhibited a marked increase in the small loose Jab1 complex located in the cytoplasm. Inhibition of Bcr-Abl kinase by STI571 induced G1 arrest and caused a recovery of the p27 level with reduction of the small Jab1 complex from the cytoplasm. Either blockade of the MAP kinase and PI3 kinase pathways by specific inhibitors or Jab1 knockdown by small interfering RNA (siRNA) prevented p27 down-regulation as well as formation of the small complex. Thus, regulation of p27 via modulation of the Jab1 subcomplex is a novel mechanism whereby Bcr-Abl oncogenic signals accelerate abnormal cell proliferation.

Presence of somatic hypermutation and activation-induced cytidine deaminase in acute lymphoblastic leukemia L2 with t(14;18)(q32;q21).

AIM: Acute lymphoblastic leukemia (ALL) with L2 (FAB) morphology has rarely been reported to show t(14;18)(q32;q21). We aimed to delineate the stage at which this type of ALL is derived in B-lineage differentiation. METHODS: The somatic hypermutation (SHM) of the variable region of immunoglobulin heavy chain (IgV(H)) gene and the expression of terminal deoxynucleotidyl transferase (TdT), recombination-activating gene 1 and 2 (RAG-1 and -2), and activation-induced cytidine deaminase (AID) were investigated in three cell lines and two fresh samples, including a pair of matched fresh and cell line cells. RESULTS: TdT, RAG-1, and RAG-2 were variably expressed. AID was expressed in four of five samples. SHM of the IgV(H) gene was found in all samples with high average frequency (11.84%) comparable with that in follicular lymphoma. Ongoing mutation was seen in two fresh samples. CONCLUSION: As AID and SHM are generally regarded as properties exhibited by mature B cells, the presence of AID and SHM in this study seems to be incompatible with the general understanding of the early stage derivation of ALL in B-lineage differentiation. The results here give some insight into the relationship between disease type (ALL or lymphoma) and derivation stage, the overlapping of the early stage phenotype and the mature genomic characteristics, and the probable relationship between the mechanism of the occurrence of t(14;18)(q32;q21) and the machinery causing SHM.

Expression of activation-induced cytidine deaminase (AID) in Burkitt lymphoma cells: rare AID-negative cell lines with the unmutated rearranged VH gene.

Activation-induced cytidine deaminase (AID) is an enzyme that catalyzes somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) gene. The expression of AID was reported to be confined to the germinal center (GC). Burkitt lymphoma (BL) cells are regarded as being derived from GC cells. BL cells have been reported to have SHM in the Ig gene with variety in terms of degree, antigen selection and ongoing mutation characteristic. We investigated the expression of AID in 15 BL cell lines by RT-PCR. In only 2 BL cell lines, Tree92 and Black93, AID expression was not detected, and the 1gVH gene of these 2 cell lines was not mutated. Tree92 expresses terminal deoxy-nucleotidyl transferase (TdT) and recombination activating gene (RAG)1, but Black93 does not, as is typical of the BL phenotype. BL cells are generally derived from GC B-cell expressing AID, but are rarely derived from the stage of B-lineage differentiation in which AID is not yet expressed, causing the absence of mutation in the IgVH.

[Two cases of acute lymphoblastic leukemia with cytoplasmic granules].

The presence of cytoplasmic granules in blastoid cells of patients with acute leukemia is generally accepted as a useful morphological marker for differentiation of myeloid leukemia from lymphoblastic leukemia. We diagnosed two cases of acute lymphoblastic leukemia(ALL) with cytoplasmic granulation. Surface marker analysis of leukemic cells revealed they were positive for CD10, 19, 20, 33, 34 and HLA-DR. Immunoglobulin gene rearrangement was detected by means of Southern hybridization with an Ig-JH probe for both patients. On the basis of these findings, the patients were diagnosed as having B-precursor ALL. Electron microscopic observation showed no myeloperoxidase activity, so that the granules were considered to be related to autophagolysosomes. This experience demonstrates that the recognition of the presence of granular ALL is necessary for making an accurate differential diagnosis of acute leukemias.

A mini-review of CD13 antigen in AML: easy induction or enhancement of expression in in vitro culture and necessary consideration for assessment.

CD13 is a pan-(MPO-positive) AML antigen, while it is expressed in some B-lineage neoplasms (ALL and PLL) and in T-linaege neoplasms at pro-thymic stage. Some reports have described the lack of this antigen in MPO-positive AML, and tried to regard such MPO-postive AML cases as an clinical entity. However, considering the easy induction or enhancement of the expression in in vitro culture, it is pertinent to interpret that the expression of CD13 in these "CD13-negative and MPO-positive AML" cases is marginally positive, which is readily induced or enhanced in expression in in vitro culture. It can, however, be pointed out that the CD13 expression in ex vivo-positive cases are significantly stronger than that in ex-vivo-negative in vitro-positive cases. Such consideration is necessary particularly in interpreting the results obtained after overnight transportation in commercial laboratories.