Ultrastructural studies on the acute leukemic lymphoblast

Summary Electron microscopic studies were performed on normal lymphoblasts obtained from lymph nodes from 10 surgical patients without hematologic disease. Bone marrow was obtained from 6 patients with acute lymphoblastic leukemia for comparison studies. The normal lymphoblast was significantly larger in total cell area, total cell length, nuclear length, and cytoplasmic area. These differences were discussed in regard to the origin of the leukemic lymphoblast which could be of thymic derivation. In addition, the leukemic lymphoblasts showed bizarre nuclear pockets, increased nucleoli, nuclear membrane duplication, increased numbers of disrupted mitochondria, virus-like particles within mitochondria, and increased numbers of small granules within mitochondria. The location of centrioles outside nuclear pockets may make leukemic lymphoblasts more sensitive to vincristine than leukemic myeloblasts or monoblasts. The disrupted mitochondria with associated virus-like particles were exciting findings which may offer important clues to leukemogenesis. It would appear that quantitative and qualitative ultra-structural findings are important in lymphoblastic leukemia and that the preliminary findings in this study be pursued with more sophisticated techniques.

---

Zusammenfassung Elektronenmikroskopische Untersuchungen wurden ausgeführt an normalen Lymphoblasten aus Lymphknoten von 10 chirurgischen Patienten onhe hämatologische Erkrankungen. Für die vergleichenden Studien wurde Knochenmark von 6 Patienten mit akuter lymphoblastischer Leukämie erhalten. Die normalen Lymphoblasten wiesen eine bedeutend größere gesamte zelluläre Fläche, gesamte zelluläre Länge, nukleäre Länge sowie zytoplasmische Fläche auf. Diese Unterschiede werden mit Bezug auf den Ursprung leukämischer Lymphoblasten, welche thymischer Abstammung sein mögen, besprochen. Zusätzlich wiesen die leukämischen Lymphoblasten bizarre Zellkernsäcke auf, Duplizierung der Zellkernmembranen sowie eine erhöhte Anzahl von Nukleolen und zerstörten Mitochondrien, Virus-gleiche Strukturen und eine erhöhte Anzahl kleiner Granulen innerhalb der Mitochondrien. Die Lokalisierung von Zentriolen außerhalb der Zellkernsäcke trägt möglicherweise für die erhöhte Sensitivität leukämischer Lymphoblasten fürVincristin bei, in Gegensatz zu leukämischen Myeloblasten oder Monoblasten. Die Anwesenheit von Virusgleichen Strukturen in zerstörten Mitochondrien mag für die Zukunft wichtige Hinweise zur Leukemogenase geben. Es scheint, daß bei lymphoblastischer Leukämie qualitative als auch quantitative ultrastrukturelle Beobachtungen gleichsam wichtig sind und daß die bisherigen Ergebnisse dieser Arbeit Anregung für ein eingehenderes Studium mit mehr fortschrittlichen Techniken bieten.

     

Ultrastructural studies on the acute leukemic monoblast

Summary Comparative electron microscopic examination of leukemic monoblasts from 14 patients with various types of acute monoblastic leukemias was performed, using normal myeloblasts from 14 healthy hospital personnel and leukemic myeloblasts from 6 cases of acute myeloblastic or myelomonoblastic leukemias as controls. No significant quantitative differences were found with the exception of increased numbers of polyribosomes in the monoblasts. On the other hand, qualitative differences, which occurred in both types of leukemic blast cells, were numerous and significant. Such qualitative differences manifested themselves in nuclear indentations and pockets, the latter sometimes containing centrioles or mitochondria, and mitochondria in various stages of morphological breakdown, exhibiting DNA fibers as well as virus like particles. A close apposition of such disrupted mitochondria to disintegrating nuclear membranes may suggest a means of interaction or exchange between mitochondria and nucleus in the interphase leukemic cell and indicates a need for further study of this phenomenon.

---

Zusammenfassung Es wird über vergleichende elektronenmikroskopische Untersuchungen leukämischer Monoblasten von 14 Patienten mit verschiedenen Typen akuter monoblastischer Leukämie berichtet, wobei normale Myeloblasten von 14 gesunden Krankenhausangestellten und leukämische Myeloblasten von 6 Kranken mit akuter myeloblastischer oder myelomonoblastischer Leukämie als Kontrollen dienten. Bedeutsame quantitative Differenzen wurden nicht gefunden mit Ausnahme einer erhöhten Anzahl von Polyribosomen in Monoblasten. Andererseits waren die qualitativen Unterschiede, welche in beiden Arten von leukämischen Blastzellen beobachtet wurden, zahlreich und bedeutend. Diese qualitativen Unterschiede bezogen sich auf Einbuchtungen und Säcke im Zellkern, wobei solche Säcke oft Zentriolen oder Mitochondrien enthielten, sowie auf Mitochondrien in verschiedenen Stufen morphologischer Zerstörung, die dabei DNS-Fasern und Virus-gleiche Strukturen aufwiesen. Die Apposition solcher zerstörten Mitochondrien an ebenfalls zerstörten nukleären Membranen mag eine Art Interaktion oder Austausch zwischen Mitochondrien und Zellkern während der Interphase andeuten; dieses Phänomen bedarf jedoch weiteren Studiums.

     

急性髓细胞白血病细胞诱导分化成为树突状细胞的实验研究

目的 :探讨急性髓细胞白血病 (AML)患者的白血病细胞体外是否可诱导分化成为树突状细胞(DC)。方法 :从 11例AML患者的骨髓或外周血中获取非贴壁细胞 ,利用细胞因子 (rhGM CSF、rhIL 4、rhTNF α)联合培养 ,每 12d收获细胞。培养前后分别用倒置显微镜、电镜观察细胞形态 ,用流式细胞术测定细胞表面标志 ,用MTT法检测收获细胞激发混合淋巴细胞反应的能力。结果 :8例AML标本分化成为具有典型树突状形态的细胞 ,培养后的白血病细胞的HLA DR、CD86、CD1a表达较培养前明显增高 ,差异有统计学意义 (P <0 .0 1)。在混合淋巴细胞反应中 ,DC具有强烈激发同种T淋巴细胞增殖的能力 ,且随数量增加而作用增强。M4/M5型AML DC的表面标志的表达率明显高于非M4/M5型AML DC(P <0 .0 1)。结论 :急性髓细胞白血病细胞可以诱导分化成为具有DC形态、表型、功能的细胞。     

Molecular diagnosis of leukemic cerebrospinal fluid cells in children with newly diagnosed acute lymphoblastic leukemia

Cytomorphology and IgH/T-cell receptor g clonal gene rearrangements detected by polymerase chain reaction (PCR) homo/heteroduplex analysis and direct sequencing were evaluated in cerebrospinal fluid (CSF) free of red-blood cells at diagnosis of 37 children with acute lymphoblastic leukemia. Molecular CSF involvement was greater as detected by molecular analysis than observed by morphologic criteria (45.9% vs 5.4%). The 4-year event-free survival was lower in the group with molecularly detected CSF involvement (p = 0.01).     

Methylthioadenosine phosphorylase as target for chemoselective treatment of T-cell acute lymphoblastic leukemic cells

We analyzed the role of methylthioadenosine phosphorylase (MTAP) for chemoselective treatment of T-cell acute lymphoblastic leukemia (T-ALL). MTAP converts methylthioadenosine into adenine which serves as an alternative purine source, if de novo purine biosynthesis is inhibited by antimetabolites (i.e., methotrexate). The idea of the chemoselectivity concept is that tumors with MTAP deletion at chromosome 9p21 are more susceptible to antimetabolites than normal cells without such a deletion. First, we screened 13 T-ALL lines for 9p21 deletions by comparative genomic hybridization. Five cell lines revealed deletions at the short arm of chromosome 9, dim(9p21pter). Further analyses were performed with CEM cells in which the 9p21 deletion was corroborated by fluorescence in situ hybridization. CEM cells were transfected with an MTAP expression vector. A green fluorescent protein (GFP) plasmid was cotransfected, to monitor the transfection efficacy by flow cytometry. The response of MTAP-transfected cells to the antimetabolites methotrexate (MTX), trimetrexate (TMX), and L-alanosine (ALA) was decreased compared to mock control transfectants using growth inhibition assays. The activity of doxorubicin (DOX) which is not involved in DNA biosynthesis was not changed in MTAP transfectants. As the p16(INK4a) tumor suppressor gene resides also at 9p21, we transfected CEM cells with a p16(INK4a) expression vector. These transfectant cells were more resistant to all four drugs indicating that p16(INK4a) did not specifically affect antimetabolites. The chemoselective effect of antimetabolites in MTAP-deleted tumor cells may, however, be compensated by the development of drug resistance. To prove this possibility, we analyzed an MTX-resistant subline, CEM/MTX1500LV, in which the MTX-resistance conferring dihydrofolate reductase (DHFR) gene was amplified. While TMX exhibited considerable cross-resistance in CEM/MTX1500LV cells, ALA did not. Thus, ALA could exhibit chemoselectivity in 9p21/MTAP-deleted cells, even if DHFR amplification occurs. We conclude that ALA may be more suitable than MTX or TMX for MTAP-mediated chemoselective treatment of T-ALL. Pretherapeutical detection of 9p21 and MTAP deletion may be helpful in developing a predictive molecular chemosensitivity test for T-ALL.     

T-cell acute lymphoblastic leukemia with severe leukopenia: evidence for suppression of myeloid progenitor cells by leukemic blasts

A patient with T-cell acute lymphoblastic leukemia presented with leukopenia due to neutropenia, no circulating blasts and normal hemoglobin level. Marrow leukemic T lymphoblasts inhibited in vitro normal CFU-GM colony growth and released an activity that stimulated normal BFU-E growth. This patient demonstrates that immature T cells may modify hematopoietic stem cell growth both in vitro and in vivo.     

Fucose binding lectin for characterizing acute myeloid leukemia progenitor cells

The reactivity of acute myeloid leukemia cells (AML) was determined in 29 patients using the fucose binding lectin Ulex europaeus agglutinin (UEA) as surface marker. We show a marked heterogeneity in the UEA- binding abilities of the cells in these patients as determined by fluorescence analysis of the blasts labeled with the UEA coupled to the fluorescent molecule FITC. The results suggest a correlation between the capability of AML blast cells to bind UEA and cytologic maturation, because in 1 of 10 M1, 3 of 8 M2, 6 of 8 M4, and 1 of 3 M5 cytology types UEA binding to the leukemic cells was apparent. In 13 cases, the cells gave rise to colonies in vitro. The amount of UEA binding to AML colony-forming cells (AML-CFU) was determined by cell sorting and subsequent colony culture of UEA-negative, intermediately positive, and highly fluorescent cells. AML-CFU from none of the four patients with M1 cytology were UEA positive, whereas they showed intense reactivity with the lectin in 1 of 4 cases with M2 cytology and in all 4 cases of M4. In these five cases with strongly UEA positive AML-CFU, the fluorescence distribution of the colony formers differed from that of the total leukemia population, indicating that AML-CFU represent a subpopulation of AML cells with specific UEA-binding properties. Normal bone marrow myeloid and multipotential colony-forming cells (CFU-GM, CFU-GEMM) showed low or no binding of UEA. UEA-FITC appears a useful reagent for membrane analysis of AML-CFU. In certain cases, UEA-FITC labeling may be applied to discriminate AML-CFU from normal hematopoietic progenitors.     

Bullous exudative retinal detachment due to infiltration of leukemic cells in a child with acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is known to cause several ocular involvements, but exudative retinal detachment is a rare complication. We describe a case report of a 4-year-old boy with T cell ALL who developed bilateral exudative retinal detachment caused by leukemic infiltration in the retinas after achieving hematological remission. Intravenous steroid pulse therapy and local irradiation reversed the condition, but it recurred concurrently with disease progression after a second relapse in the bone marrow. It is suggested that ophthalmic examination is crucial for ALL patients, especially for those whose white blood cell count is very high at onset.     

Increased frequency of mannose-binding lectin insufficiency among children with acute lymphoblastic leukemia

Epidemiological data indicate that acute lymphoblastic leukemia (ALL) could be induced by interactions between the immune system and early childhood infections. Mannose-binding lectin (MBL) plays a critical role in the immune response in early childhood before specific immune protection develops. We investigated whether there may be an association between childhood ALL and low-producing MBL genotypes. Serum MBL levels depend on normal (A) or defective (O) alleles, and on normal (Y) or reduced (X) promoter activities. For this study, 137 noninfants with ALL and 250 controls were classified into 3 MBL genotype groups according to their influence on the serum level of functional MBL: group I, YA/YA and YA/XA (higher levels); group II, XA/XA and YA/O (intermediate levels); and group III, MBL insufficiency with XA/O or O/O (MBL-deficient) genotypes. Compared with controls, cases more often had low-level genotypes (I/II/III: 63 [46%]/44 [32%]/30 [22%] vs 145 [58%]/65 [26%]/40 [16%]; P =.02) and MBL deficiency (8.8% vs 2.8%; P =.009). Thus, the ALL odds ratio for MBL-deficient versus nondeficient individuals was 3.3 (95% CI, 1.3-8.7), whereas the ALL odds ratio for group I versus group II/III genotypes was 0.62 (95% CI, 0.41-0.94). MBL group III patients were significantly younger at diagnosis than patients in group I/II (median, 3.9 vs 5.2 years; P =.04). The study shows that the presence of low-level MBL genotypes is associated with an increased risk of childhood ALL, particularly with early age at onset.     

Interaction of peanut agglutinin with normal human lymphocytes and with leukemic cells.

The interaction of peanut agglutinin (PNA) with human thymocytes, peripheral blood lymphocytes, and peripheral blood cells of various types of leukemia was investigated by using fluorescein isothiocyanate-conjugated PNA. The majority of human thymocytes (60-80%) bind the lectin. The major subpopulation of thymocytes that is PNA-positive was separated from the PNA-negative cells by differential agglutination with the lectin. The two thymocyte subpopulations were tested in the mixed lymphocyte reaction and with the phytohemagglutinin of Phaseolus vulgaris. The poor response of the PNA-positive thymocytes to these stimuli indicates that these thymocytes are functionally immature. The fluorescein isothiocyanate-PNA-binding test with peripheral blood lymphocytes of leukemic patients revealed that in most acute leukemias the PNA receptor is exposed on the blastic cells, whereas in most cases of chronic leukemia the peripheral blood lymphocytes are PNA-negative. The validity of PNA as a marker of immature blood cells and its potential clinical application are discussed.     

Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL).

An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.     

Overexpression of CD123 correlates with the hyperdiploid genotype in acute lymphoblastic leukemia

We evaluated CD123 expression in 95 pediatric and 24 adult ALL patients and compared the results with the CD123 expression in normal B-cell precursors. Early B-cell precursors were negative while intermediate precursors and mature B cells showed weak CD123 expression. Leukemic blasts in 31% of precursor-B ALL samples exhibited strong expression of CD123, 61% had moderate CD123 expression and 8% were negative; 81.5% of ALL with hyperdiploid karyotype (≥ 52 chromosomes) showed strong CD123 overexpression. In contrast, cases with ETV6/RUNX1 rearrangement had weak CD123 expression. Our study suggests that overexpression of CD123 is an aberrant phenotype present in a subset of precursor-B ALL with hyperdiploid genotype, and represents an additional marker of good prognosis in pediatric precursor-B ALL. Moreover, aberrant CD123 expression in ALL is a good marker for monitoring of minimal residual disease.     

Motility of leukemic cells in collagen gel related to immunological phenotype in childhood acute lymphoblastic leukemia

Motility of leukemic cells was measured in a three-dimensional collagen matrix assay. Leukemic cells from 16 children with acute lymphoblastic leukemia (ALL) and normal peripheral blood lymphocytes (NPBL) from 6 healthy volunteers, were allowed to migrate into this collagen matrix for 48 h at 37 degrees C. NPBL migrated much further (300-600 micron) than leukemic cells (0-200 micron). Among the leukemic cases, only common ALL and one case of null ALL showed some migration (0-200 micron). T-All cells did not migrate at all under the circumstances of this experiment.     

Capping of leukemic cells with monoclonal anti-HLA-A,B,C related to prognosis in childhood acute lymphoblastic leukemia

Capping of leukemic cells with a monoclonal antibody against HLA A,B,C determinants was studied in 53 cases of childhood acute lymphoblastic leukemia (ALL). Determination of the percentages of capped cells after different times of incubation with anti-HLA A,B,C show that T ALL and common ALL do have quite different kinetics of HLA capping. In T ALL all cases reach levels of percentage of capped cells above 30%, in common ALL only 11 of 31 cases cap well. Dilution of the antiserum in 6 common ALL cases results in an increase of capped cells, but the original kinetics of the common ALL capping remain. ALL cases with capping curves above 30% have a worse prognosis (shorter continuous complete remission) than cases with capping curves below 30% in the total group as well as in the non-high-risk group.     

Cytogenetically different leukemic clones at relapse of childhood acute lymphoblastic leukemia

Sequential analysis of blast cell chromosomes in 98 cases of acute lymphoblastic leukemia (ALL) disclosed entirely different karyotypes for nine patients at the time of relapse. The presenting clinical, immunophenotypic, and cytogenetic features of this subgroup were similar to those of the 89 patients without major karyotypic shifts. The median length of initial remissions in these nine patients, all of whom received intensive multiagent therapy, was 24 months (range, 6 to 35); responses to subsequent treatment have been uniformly poor. Prominent cytogenetic changes included a gain of modal chromosome numbers in five cases, a loss of chromosomes in two, and the acquisition of an 11q23 rearrangement in three. We propose several different mechanisms to account for these findings. In one, the presence of an entirely different ALL karyotype at relapse may represent induction of secondary leukemia analogous to the well- described entity of epipodophyllotoxin-related secondary acute myeloid leukemia (AML).     

Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia

Early clearance of leukemic cells is a favorable prognostic indicator in childhood acute lymphoblastic leukemia (ALL). However, identification of residual leukemic cells by their morphologic features is subjective and lacks sensitivity. To improve estimates of leukemia clearance, we applied flow cytometric techniques capable of detecting 1 leukemic cell in 10,000 or more normal cells and prospectively measured residual leukemia in bone marrow samples collected on day 19 of remission-induction chemotherapy from 248 children with newly diagnosed ALL. In 134 samples (54.0%), we identified at least 0.01% leukemic cells (0.01%-< 0.1% in 51 samples [20.6%], 0.1%-< 1% in 36 [14.5%], and > or = 1% in 47 [19.0%]). Among 110 children treated within a single chemotherapy program, the 5-year mean +/- SE cumulative incidence of relapse or failure to achieve remission was 32.2% +/- 6.5% for the 59 patients with 0.01% residual leukemic cells or greater on day 19 and 6.0% +/- 3.4% for the 51 patients with less than 0.01% leukemic cells (P <.001). The prognostic value of day-19 bone marrow status defined by flow cytometry was superior to that defined by morphologic studies and remained significant after adjustment for other clinical and biologic variables. Lack of detectable leukemic cells on day 19 was more closely associated with relapse-free survival than was lack of detectable residual disease at the end of remission induction (day 46). Thus, approximately half of the children with ALL achieve profound clearance of leukemic cells after 2 to 3 weeks of remission-induction chemotherapy, and these patients have an excellent treatment outcome.     

Basophilic differentiation of leukemic cells in a patient with acute leukemia carrying minor bcr/abl chimeric mRNA

A 77-year-old woman was referred to our hospital because of leukocytosis and leukoblastosis in September 1999. She was healthy except for hypertension, and no abnormal findings in the peripheral blood had been observed up to December 1998. Physical examination revealed neither hepatosplenomegaly nor superficial lymphadenopathy. A bone marrow film showed massive proliferation of blast cells (87.8%), some of which contained coarse basophilic granules (38.6%). The cells were negative for peroxidase and esterase (alpha-naphtyl butyrate and ASD-chloroacetate) staining, but the granules showed metachromasia upon toluidine blue staining. As immunophenotypic analysis of the cells showed double positive for CD13/CD19 but negativity for CD33, this case did not meet the diagnostic criteria for biphenotypic acute leukemia. Chromosome and gene analysis showed positivity for the Ph1 chromosome with minor bcr/abl chimeric mRNA. A homogenate of the peripheral mononuclear cells demonstrated a high concentration of histamine. Electron microscopy analysis confirmed that some of the blast cells contained dense granules, which closely resembled "immature basophil granules" morphologically. These results suggested that the blast cells showed basophilic differentiation. As the clinical course and peripheral blood findings were different from blastic crisis of chronic myelogenous leukemia (CML) and CML with minor bcr/abl chimeric mRNA, the present case was diagnosed as "multiphenotypic acute leukemia", a type of acute basophilic leukemia classified by Duchayne.