Chronic Myelomonocytic Leukemia Cases Research Articles

The detection of CD14 and CD16 in paraffin-embedded bone marrow biopsies is useful for the diagnosis of chronic myelomonocytic leukemia

Histopathological study of bone marrow biopsy (BMB) in chronic myelomonocytic leukemia (CMML) is often difficult and might benefit from an immunohistochemical approach. We immunostained 15 cases of CMML, focusing at two new antibodies staining for CD14 and CD16 on paraffin-embedded tissues. CD68 (KP1), CD68 (PG-M1), and CD163 were not differentially expressed between CMML and chronic myelogenous leukemia (CML). In CMML BMB, we found a significant increase in the number of CD14+ monocytes. This increase was made of dispersed cells in the interstitium, often exhibiting bilobated nuclei, and being difficult to differentiate from neutrophils. There was no expansion of CD16+ monocyte-like cells. However, we found a significant decrease in the number of granulocytes expressing CD16, MPO, and CD15 in CMML compared to CML and control BMB, probably related to dysgranulopoiesis. Indeed, BMB immunohistochemistry can be helpful in CMML by identifying both the monocyte expansion with CD14 and the dysgranulopoiesis with CD16.

SNP-a Karyotyping Provides a Clonal Molecular Marker and Is Associated with a High Incidence of Segmental Uniparental Disomy in Patients with CMML

Chronic myelomonocytic leukemia (CMML) patients display poor prognosis with median survival of 20 months (range 10–60). The WHO classification recognizes CMML as an overlap syndrome with mixed clinical and histomorphological features of both myelodysplastic syndrome (MDS) and myeloproliferative disorders (MPD). The underlying molecular basis for the poor prognosis and clinical and pathological features including the dysplasia and monocytosis are currently unknown. A lack of clonal markers in the majority of patients impedes diagnosis and prognosis assignment, as well as treatment response monitoring. Individual cases are associated with rare (5;12) translocations, RAS mutations, and unbalanced chromosomal changes. Since chromosomal defects have a major impact on the diagnosis and prognosis of myeloid malignancies, it is likely that cytogenetic methods with higher resolution and an ability to detect uniparental disomy (UPD) could explain clinical heterogeneity and point to potential therapeutic targets in CMML. Our study included 101 cases including 70 cases of both CMML1 and 2 and CMML-derived AML. We also studied 31 M4/M5/monocytic subtype AML cases (8 FLT3+) to allow interpretation of the 17 CMML cases that were analyzed at the time of AML transformation. Both 250K and a more sensitive 6.0 SNP-A array were utilized. Copy number variants (CNV) were identified and excluded from analysis by comparison with simultaneous arrays with CD3+ normal T cells from the test individual, or when not available, by comparison of results with published and our extensive internal CNV databases, and experimentally determined size exclusion parameters from over 500 MDS and AML cases run on the same platform. In total, 67/70 patients had samples sent for metaphase cytogenetics (MC) and 45% of these cases had an abnormal karyotype. Expected lesions commonly associated with MDS/CMML, such as +8 (N=4) and −7/del(7q) (N=5) were observed. When SNP-A was applied to the entire cohort, defects found by MC were confirmed. Also, new chromosomal abnormalities were identified for a total detection rate of 55/70 (79%). When patients with CMML-1, CMML-2 vs CMML-derived AML were analyzed for gains, losses and UPD, several results are apparent. First, subcytogenetic loci on the chromosomes affected by large gains (e.g., 8 and 21) and losses (e.g., del7 or del5) were identified in additional patients. Second, cytogenetic amplification (amp) and deletion (del) regions seen by MC were confirmed by SNP-A. Examples of recurrent amplifications and deletions included amp21q21 (N=8), amp8p23.2–q24.3 (N=5), amp13q31.1(N=4), amp20p(N=3), del7q21.1(N=4), del12p12.2(N=4), del7q34(N=3), and del5q(N=3). We found a high prevalence of segmental UPD, not otherwise detectable by MC, occurring in 54 % of primary CMML/CMML-derived AML. Recurrent lesions were identified on chrs. 1(N=3), 2(N=3), 4(N=5), 6(N=3), 7(N=5), 11(N=7), 13(N=3), 14(N=3), and 21(N=4). Fifteen of seventy (21%) had UPD as a sole or isolated abnormality. In 6/7 of the UPD11 cases, this was present as a sole abnormality. In a separate abstract we present 12 UPD11q cases with MDS/MPD-U and sAML; in 7 of these cases inactivating c-Cbl mutations were found. All were associated with CMML cases associated with AML transformation. Overall survival was compared for patients with CMML (WHO 1 and 2), CMML into AML, and M4/M5 with either normal karyotype and normal SNP-A results (no amplifications, deletions, UPD), relative to those with detectable genomic lesions. Outcome was poor for all CMML groups however. In summary, SNP-A-based karyotyping complements MC and allows for precise definition of chromosomal aberrations in patients with CMML, including copy-neutral LOH/UPD. UPD is common in CMML and overlapping regions may point to potential causative genes. The identification of c-Cbl demonstrates the utility of this approach, identifying pathways to explore for therapeutic intervention. Overall, SNP-A reveals evidence for significant cytogenetic and subcytogenetic genomic instability in CMML, perhaps not surprisingly with the known adverse prognosis of this disorder. UPD 7 and UPD 21 abnormalities are particularly interesting based on the frequency of these lesions, and the known association of monosomy 7 with CMML. They may provide markers for diagnosis and response monitoring, as more novel therapeutics are being developed for this poor prognosis disorder.

A novel variant form of MLL-ELL fusion transcript with t(11;19)(q23;p13.1) in chronic myelomonocytic leukemia transforming to acute myeloid leukemia

MLL located at 11q23 is fused with a variety of partner genes by recurrent chromosomal translocations in acute leukemias. ELL, the MLL partner gene located on chromosome 19p13.1, encodes an RNA polymerase II transcriptional elongation factor, which also possesses the N-terminal region involved in the inhibition of transcription initiation. Here we report a case of chronic myelomonocytic leukemia (CMML) with a 46,XY,t(11;19)(q23;p13.1) karyotype that transformed to acute myeloid leukemia (AML) without showing any karyotypic evolution. Interphase fluorescent in situ hybridization analysis showed the split MLL signals in 95% of bone marrow cells when the diagnosis of CMML was made and the percentage of blasts was 1.2%. Sequence analysis of reverse-transcriptional polymerase chain reaction product revealed a novel variant form of MLL-ELL transcript in which MLL exon 10 was fused to ELL exon 3. MLL has been fused to ELL exon 2 in all the previously reported MLL-ELL transcripts, which have always been associated with AML. It is deduced that the variant form of MLL-ELL may be defective not only in inhibition of transcription initiation, but also in transcriptional elongation. Thus, a possibility is raised that the unique clinical presentation of the present case with t(11;19)(q23;p13.1) might be related to the variant form of MLL-ELL.

Isodicentric 20q− in two cases of B-cell acute lymphocytic leukemia with the respective t(9;20)(p11;q11.2) and t(9;22)(q34;q11.2)

The cytogenetic anomaly der(20)del(20)(q11.2q13.3)idic(20)(p11), or idic(20q−) in short form, has been reported in 13 cases of myelodysplastic syndrome, one case of chronic myelomonocytic leukemia, and one case of acute myeloid leukemia since 2004. To our knowledge, it has not previously been described in lymphoid diseases. Here we report the cases of two patients with B-cell acute lymphocytic leukemia (ALL) having a novel idic(20q−). One was a 34-year-old man with B-cell ALL whose leukemic cells at presentation had a karyotype of 45,XY,dic(9;20)(p11;q11.2); at relapse, a small marker chromosome was found coexisting with the dic(9;20). The other was a 39-year-old woman with Ph-positive B-cell-ALL whose leukemic cells contained both t(9;22)(q34;q11.2) and a small marker chromosome. A series of FISH analyses using the appropriate probes revealed the small marker chromosome in both patients to be an idic(20q−), confirming the dic(9;20)(p11;q11.2) in one case and revealing a BCR/ABL fusion gene in the other. One patient achieved complete remission but relapsed; the other did not achieve complete remission. Both patients died with a short survival time, despite receiving intensive chemotherapy. These two cases show that idic(20q−) can appear not only in myeloid diseases but also in lymphoid diseases.

Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of JAK2V617F

The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

Der(20)idic(20)(p11)del(20)(q11q13) Is Seen Not Only in Myeloid Disorders but Also in Lymphoid Disorders: A Report of Further Seven Cases.

As a newly identified cytogenetic abnormality, idic(20q−) has been reported in thirteen cases of myelodysplastic syndrome (MDS), one case of chronic myelomonocytic leukemia, and one case of acute myeloid leukemia (AML) since 2004. Here we report additional seven cases consisting of 3 MDS, 2 AML and 2 acute lymphocytic leukemia (ALL). A karyotype analysis using R-banding technique revealed an i(20q−) as a sole anomaly in 5 of them and as a secondary change in other 2 cases: one was a B-cell ALL whose leukemic cells at presentation had a karyotype of 45, XY, dic(9;20)(p11;q11). However, an idic(20q−) was found coexistent with the dic(9;20) as relapse; another was a Ph positive ALL whose leukemic cells simultaneously contained t(9;22)(q34;q11) and idic(20q−). A series of FISH using multiple probes such as the centromeric probes specific for chromosomes 9 and 20, the subtelomeric probe for 20q, the locus-specific probe for 20q12, the whole chromosome paint probes for chromosomes 9 and 22 and the translocation probe for t(9;22) demonstrated the idic(20q−) to be actually a der(20)idic(20)(p11)del(20)(q11q13), confirmed the dic(9;20)(p11;q11) in case 6 and revealed a BCR/ABL fusion gene in case 7. Among them, 2 MDS patients survived and anemic except one lost to follow-up, 2 AML and 2 ALL patients did not obtain complete remission and died with short survivals. Our study brings the total number of cases with idic(20q−) to 22 and suggests that the disease spectrum of idic(20q−) is not restricted to myeloid disorders, and it also includes ALL.

巨大肝嚢胞ならびに多臓器への浸潤を来した高齢者慢性骨髄単球性白血病の1剖検例

A 78-year-old man was referred to his local hospital at the beginning of July 2006 with low grade fever and cough, and was admitted on August 22 with pneumonia. Hematology tests on admission revealed leukocytosis, anemia, and thrombocytopenia, so he was subsequently transferred to our hospital on August 24. A diagnosis of chronic myelomonocytic leukemia (CMML) was made from the hematological findings and he was given hydroxycarbamide from September 7. Seventeen days later, abdominal CT revealed hemorrhage into a giant liver cyst, as well as both intraabdominal and intrathoracic hemorrhage. He died of hemorrhagic shock on September 25. Autopsy showed extensive infiltration of myelomonocytic leukemic cells into the walls of the liver cyst, as well as the pleura and multiple other organs. In summary, we report an elderly autopsy case of CMML with infiltration of a giant liver cyst and multiple organs.

Chronic Myelomonocytic Leukemia Evolving From Preexisting Myelodysplasia Shares Many Features With De Novo Disease

The majority of chronic myelomonocytic leukemia (CMML) cases arise de novo; cases evolving from preexisting myelodysplasia (MDS) or myeloproliferative diseases have not been well-studied. We conducted the present study to determine the clinicopathologic features and to study possible underlying molecular and cytogenetic mechanisms involved in this evolution. Between April 1995 and November 2005, we identified 120 CMML cases, of which 20 (16.7%) had a previous diagnosis of MDS. Of the 20 patients with MDS, 6 had relative monocytosis at diagnosis. At the time of MDS to CMML evolution, mutations in JAK2 (V617F), FLT3 (ITD), K-ras-2, or N-ras were not acquired, and only 1 (6%) of 17 evaluable cases showed cytogenetic progression. The median time to evolution from MDS to CMML was 29 months, and the median survival following CMML development was 13 months. Three cases (17%) transformed to acute myeloid leukemia. These findings indicate that in some cases of otherwise typical MDS, the progenitor cells may have some capacity for monocytic proliferation at diagnosis and manifest rapid disease progression once a monocytic proliferation supervenes.

Chronic Myelomonocytic Leukemia Evolving From Preexisting Myelodysplasia Shares Many Features With De Novo Disease

The majority of chronic myelomonocytic leukemia (CMML) cases arise de novo; cases evolving from preexisting myelodysplasia (MDS) or myeloproliferative diseases have not been well-studied. We conducted the present study to determine the clinicopathologic features and to study possible underlying molecular and cytogenetic mechanisms involved in this evolution. Between April 1995 and November 2005, we identified 120 CMML cases, of which 20 (16.7%) had a previous diagnosis of MDS. Of the 20 patients with MDS, 6 had relative monocytosis at diagnosis. At the time of MDS to CMML evolution, mutations in JAK2 (V617F), FLT3 (ITD), K-ras-2, or N-ras were not acquired, and only 1 (6%) of 17 evaluable cases showed cytogenetic progression. The median time to evolution from MDS to CMML was 29 months, and the median survival following CMML development was 13 months. Three cases (17%) transformed to acute myeloid leukemia. These findings indicate that in some cases of otherwise typical MDS, the progenitor cells may have some capacity for monocytic proliferation at diagnosis and manifest rapid disease progression once a monocytic proliferation supervenes.

Chronic myelomonocytic leukemia: the role of bone marrow biopsy immunohistology

The World Health Organization criteria for diagnosing chronic myelomonocytic leukemia (CMML) are largely based on findings observed in the peripheral blood and bone marrow aspirate. A specific diagnostic role for the bone marrow biopsy has not been adequately explored. We examined whether bone marrow biopsy supplemented by immunohistochemistry may be helpful in distinguishing CMML from cases of chronic myelogenous leukemia and atypical chronic myeloid leukemia (aCML). We immunostained 25 cases of CMML with paraffin reactive antibodies which included CD68 (KP1), CD68R (PG-M1), and CD163, and compared the results with those observed in six cases of chronic myelogenous leukemia and in three cases of atypical CML. In addition, we examined whether CD34 immunohistochemistry could be useful in separating cases of CMML with less than 10% blasts (type-1) from cases of CMML with blasts accounting for 10–19% (type-2), and cases of CMML in acute transformation to acute myeloid leukemia (blasts≥20%). The presence of nodules of plasmacytoid monocytes was investigated by CD123 staining. CD42b was used to highlight abnormal megakaryocytes. Our results demonstrate significant differences between the groups. CD34 analysis allowed separating CMML type-1 from type-2 and the former from CMML in acute transformation. CD123-positive plasmacytoid monocyte nodules were found only in CMML and not in the other two disease groups. Overlap between CMML and the other two groups were observed with CD68 immunostaining. CD68R was more restricted to bone marrow macrophages and monocytes than CD68, but the differences between CMML and chronic myelogenous leukemia or atypical CML were still not significant. Although CD42b immunostaining facilitated the detection of dwarf megakaryocytes often present in CMML, the distinction between those and the small forms seen in chronic myelogenous leukemia was still problematic.

A Case of Chronic Myelomonocytic Leukemia Progressing from Dysplastic to Proliferative Phenotype after Acquisition of a RAS Point Mutation.

Chronic myelomonocytic leukemia (CMML) is a myeloid malignancy characterized by high heterogeneity of clinical and hematological features, classified into a new category of myelodysplastic/myeloproliferative diseases by the last World Health Organization. Poor data are available on the biology of CMML and molecular mechanisms related to disease development and progression. Among hematological malignancies, CMML has the highest frequency of point mutations of the RAS gene family (20% to 35% of patients). Patients with proliferative variant of the disease (MP-CMML, WBC > 12 x 109/L) have significantly higher frequency of RAS point mutations than patients with dysplastic variant (MD-CMML, WBC < 12 x 109/L). In fact RAS mutations have been shown to associate with features of cell proliferation and monocytosis rather than dysplasia. However, the exact role of RAS mutations in the pathophysiology of CMML is unknown. Ras family members are small G-proteins that regulate cellular proliferation, differentiation and apoptosis. Switching from the active GTP- to the inactive GDP-bound state is associated with a conformational change of the switch I (residues 30–38) and switch II (60–76) regions. Activating point mutations of codons 12, 13, 59, 61 and 63 have been shown to decrease the weak intrinsic GTPase activity of Ras, leading to its constitutive activation. In April 2003 a patient with diagnosis of MD-CMML (WBC = 6,5 x 109/L) was admitted at our hospital. Genomic DNA extracted from peripheral blood mononuclear cells was amplified with primers for exons 1 and 2 of N- and K-RAS by PCR, and subsequently sequenced. At this stage of the disease, no mutation was detected. In January 2005 the patient experienced a rapid progression to MP-subtype, with WBC counts constantly over 30 x 109/L. Molecular analysis was repeated and a mutation GGA → GAA in codon 60 of N-RAS was found, leading to the substitution of Glycine with Glutamate in the corresponding aminoacidic position. PCR amplification and direct sequencing of both forward and reverse strands from the genomic sample were repeated twice and the presence of mutation confirmed. Gly60 is highly conserved among Ras family members; it is localized in the switch II region which is involved in the binding to GTPase-activating proteins (GAPs) and in the catalytic mechanism. Preliminary data, obtained from a computational model of the Gly60 →Glu mutant protein, show that the negative charge of Glutamate replacing the neutral aminoacid Glycine may exert a highly destabilizing effect by interfering with electrostatic interactions of the “arginine finger” of GAPs, thus altering the conformational changes necessary for Ras activation. Mutations affecting codon 60 of Ras proteins were rarely reported in human cancer and consequently little is known about their transforming potential; however, detection of Gly60 →Glu substitution in a CMML patient after progression from MD- to MP- phenotype suggests a pathogenetic event. To the best of our knowledge this is the first time that a RAS point mutation is identified concomitantly with progression of CMML from dysplastic to proliferative variant. Based on these results, further experiments are on going to characterize the novel mutant protein and thus to define whether N-Ras activation may be effectively involved in the progression of CMML.

Flow Cytometric Analysis of Monocytes as a Tool for Distinguishing Chronic Myelomonocytic Leukemia From Reactive Monocytosis

To determine whether immunophenotypic features of monocytes are useful in differentiating chronic myelomonocytic leukemia (CMML) from reactive monocytosis, multiparameter flow cytometry was used to immunophenotype 20 bone marrow samples from patients with CMML, 10 normal marrow samples, and 20 marrow samples with reactive monocytosis. Monocytes in CMML exhibited aberrant antigen expression in all 20 cases. Abnormal antigen expression also was observed in monocytes in 11 of 20 reactive marrow samples. However, aberrant expression of 2 or more antigens was significantly less frequent in reactive monocytosis than in CMML (P = .002). CD56 expression with underexpression of a myeloid marker was unique to CMML monocytes. Subpopulations of monocytes with moderate levels of CD14 were present in all 3 groups. The proportion of CD14(moderate) monocytes was highest in CMML and was 20% or more in 13 of 20 CMML cases vs 3 of 20 reactive marrow samples (P = .003) and 2 of 10 normal marrow samples (P = .007). A combination of monocytosis with 2 or more immunophenotypic aberrancies with 20% or more of marrow monocytes showing moderate CD14 expression was 67% sensitive and 100% specific for CMML.

Flow Cytometric Analysis of Monocytes as a Tool for Distinguishing Chronic Myelomonocytic Leukemia From Reactive Monocytosis

To determine whether immunophenotypic features of monocytes are useful in differentiating chronic myelomonocytic leukemia (CMML) from reactive monocytosis, multiparameter flow cytometry was used to immunophenotype 20 bone marrow samples from patients with CMML, 10 normal marrow samples, and 20 marrow samples with reactive monocytosis. Monocytes in CMML exhibited aberrant antigen expression in all 20 cases. Abnormal antigen expression also was observed in monocytes in 11 of 20 reactive marrow samples. However, aberrant expression of 2 or more antigens was significantly less frequent in reactive monocytosis than in CMML (P = .002). CD56 expression with underexpression of a myeloid marker was unique to CMML monocytes. Subpopulations of monocytes with moderate levels of CD14 were present in all 3 groups. The proportion of CD14(moderate) monocytes was highest in CMML and was 20% or more in 13 of 20 CMML cases vs 3 of 20 reactive marrow samples (P = .003) and 2 of 10 normal marrow samples (P = .007). A combination of monocytosis with 2 or more immunophenotypic aberrancies with 20% or more of marrow monocytes showing moderate CD14 expression was 67% sensitive and 100% specific for CMML.

Intermittent Low Dose Interleukin 11 Can Lead to Transfusion Independence in Patients with Chronic Myelo-Monocytic Leukemia and Thrombocytopenia.

Recombinant Interleukin- 11 (IL-11) is a thrombopoietic cytokine that stimulates megakaryocytopoiesis in vitro and platelet production in vivo. It attenuates post chemotherapy thrombocytopenia at a dose of 50 mcg/ kg/ day subcutaneously (SC). Unfortunately, prolonged administration is associated with significant toxicity including peripheral and pulmonary edema at this dose. Administration of low dose IL-11 at 10 mcg/kg/day SC has shown efficacy in bone marrow failure states without significant toxicity. We report two cases of chronic myelo-monocytic leukemia (CMML) with transfusion dependent thrombocytopenia who received intermittent low dose IL-11 without significant toxicity. Case Reports- Patient 1 is a 79-year-old male with history of CMML with pancytopenia of three years duration. Recently he required transfusion of platelets with his platelet counts falling to less than 15 x 109/L. His cytogenetic study showed normal karyotype, 46 XY. He required platelet transfusion at every 14 days. He initially was started on IL-11 at 10mcg/kg/day, 5 days per week.. His platelet count increased to above 30 x 109/L and he became transfusion independent within two weeks. Unfortunately on this schedule he developed edema and mild CHF. IL-11 was stopped for two weeks and upon resolution of toxicity, restarted at 10 mcg/kg/day on Monday, Wednesday and Friday. He has remained transfusion independent without recurrence of edema at 5 months on this schedule. Patient 2 was a 63-year-old male with previous history of chronic lymphocytic leukemia and diffuse large B cell lymphoma who developed CMML with severe pancytopenia. His karyotype was 46, XY, −7, +21. His platelet count was consistently less than 10 x 109/L. He required platelet transfusion twice a week. He was started on IL-11 at 10mcg/kg/day for 5 days per week, two weeks on and two weeks off. His platelet count increased to as high as 64 x 109/L after 2nd cycle. His platelet transfusion requirement decreased from every 3rd day to every 10th-14th day. He experienced no peripheral or pulmonary edema. Conclusion: Administration of low dose IL-11 in other bone marrow failure states has been reported but its use has not been described in CMML. Our observation in these 2 patients suggests that IL-11 has efficacy in CMML and is very well tolerated at low doses on an intermittent administration schedule. IL-11 may decrease the transfusion requirement in transfusion dependent patients. Further studies are needed to evaluate overall impact on larger number of patients who require regular platelet transfusion.

CD163

Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD163 to evaluate the antibody’s usefulness in identifying cells of monocyte/macrophage lineage in normal and neoplastic conditions. Normal human tissue samples and samples from 211 hematopoietic disorders and 115 nonhematopoietic neoplasms were examined. The distribution of KP1 and PG-M1, monoclonal antibodies to the macrophage-associated CD68 antigen, also were evaluated for comparison. CD163 immunoreactivity was observed in resident macrophages of all normal tissue samples except splenic white pulp macrophages and germinal center tingible body macrophages. Among hematopoietic disorders and nonhematopoietic neoplasms, CD163 expression was restricted largely to cases of chronic myelomonocytic leukemia, histiocytic sarcoma, sinus histiocytosis with massive lymphadenopathy, and littoral cell angioma. Acute myeloid leukemias (AMLs) with monocytic differentiation were CD163– with the exception of 1 case of acute monoblastic leukemia. Most myeloid sarcomas also were CD163–. Compared with the CD68 antibodies, CD163 demonstrated greater specificity as a marker of disorders of monocyte/macrophage origin. However, immunohistochemical evaluation of CD163 expression does not seem to be a sensitive means of determining monocytic differentiation in AMLs in paraffin sections or establishing a diagnosis of myeloid sarcoma.

CD163: A Specific Marker of Macrophages in Paraffin-Embedded Tissue Samples

Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD163 to evaluate the antibody's usefulness in identifying cells of monocyte/macrophage lineage in normal and neoplastic conditions. Normal human tissue samples and samples from 211 hematopoietic disorders and 115 nonhematopoietic neoplasms were examined. The distribution of KP1 and PG-M1, monoclonal antibodies to the macrophage-associated CD68 antigen, also were evaluated for comparison. CD163 immunoreactivity was observed in resident macrophages of all normal tissue samples except splenic white pulp macrophages and germinal center tingible body macrophages. Among hematopoietic disorders and nonhematopoietic neoplasms, CD163 expression was restricted largely to cases of chronic myelomonocytic leukemia, histiocytic sarcoma, sinus histiocytosis with massive lymphadenopathy, and littoral cell angioma. Acute myeloid leukemias (AMLs) with monocytic differentiation were CD163- with the exception of 1 case of acute monoblastic leukemia. Most myeloid sarcomas also were CD163-. Compared with the CD68 antibodies, CD163 demonstrated greater specificity as a marker of disorders of monocyte/macrophage origin. However, immunohistochemical evaluation of CD163 expression does not seem to be a sensitive means of determining monocytic differentiation in AMLs in paraffin sections or establishing a diagnosis of myeloid sarcoma.

Expression of constitutively active Nuclear-κB RelA transcription factor in blasts of acute myeloid leukemia

The nuclear transcription factor NF-κB regulates cell survival, proliferation, and differentiation. Little is known about NF-κB in myeloid malignancies. In this report, we assessed NF-κB in a group of myeloid neoplasms by using an electrophoretic mobility shift assay (EMSA) and immunofluorescence methods in freshly isolated leukemia cells. We analyzed 30 cases of acute myeloid leukemia (AML), 5 cases of myelodysplastic syndrome (MDS), 3 cases of chronic myelomonocytic leukemia (CMML), 15 cases of chronic myeloid leukemia in chronic phase (CML-CP), and 2 cases of chronic myeloid leukemia in blast crisis (CML-BC). Unstimulated cells (bone marrow and peripheral blood) from 17 normal donors and apheresis samples from 6 peripheral blood stem cell donors treated with granulocyte colony-stimulating factor (G-CSF) were used as controls. When EMSA was used, NF-κB was elevated in 14 of 30 (47%) cases of AML, in both cases of CML-BC, and in all reference donors treated with G-CSF, but it was at basal levels in all cases of MDS and CML-CP and in normal donors ( P = < .01). Immunofluorescence analysis confirmed strong nuclear RelA/NF-κB immunoreactivity in AML blasts but not in normal bone marrow. Bcl-2, a downstream molecule, was expressed in cases with elevated NF-κB, but not in cases with basal levels of NF-κB, suggesting that NF-κB is active and provides the cells with survival advantages in vivo. These results suggest that suppression of NF-κB may be a useful therapeutic strategy for a subset of patients with AML.

Aberrant methylation and impaired expression of the p15(INK4b) cell cycle regulatory gene in chronic myelomonocytic leukemia (CMML).

The important cell cycle regulatory gene p15(INK4b) has been shown to be inactivated in acute myeloid leukemia and myelodysplastic syndrome. Little is known about the expression and epigenetic modification of this gene in chronic myelomonocytic leukemia (CMML) that belongs to the myelodysplastic/myeloproliferative disorders (MDS/MPD) with a high proportion of blastic transformation. Analysis of bone marrow trephines in a series of 33 CMML cases showed an aberrant p15(INK4b) gene methylation in up to 58% of cases. Methylation was analyzed employing different methylation-specific PCR and genomic sequencing protocols. It turned out to be spread over a broad area of the 5' region and exhibited substantial heterogeneity between cases and even in individual patients. The degree of aberrant methylation was correlated with a reduced mRNA as well as reduced protein expression, and was associated with a higher expression of DNA methyltransferase DNMT 3A. We conclude that aberrant gene methylation is a frequent event in CMML that might contribute to the pathogenesis of this MDS/MPD.

Two balanced and novel chromosomal translocations in myeloid malignancies: characterization by multiplex fluorescence in situ hybridization

We describe two novel chromosomal translocations in two cases of leukemia in which these translocations were further characterized as the sole acquired karyotypic abnormality by mutliplex fluorescence in situ hybridization (M-FISH). They comprised a case of acute myeloid leukemia with t(6;10)(q21;p12) and a case of chronic myelomonocytic leukemia with t(5;12)(q34;q24). To the best of our knowledge, these two balanced translocations are novel and are hitherto unrecognized in hematologic malignancies. While the clinical and pathogenic significance of these translocations remains to be defined, the present report illustrates that M-FISH technology contributes to the exclusion of subtle or cryptic translocations in sole karyotypic aberrations and the confirmation of novel chromosomal arrangements in neoplastic disorders.

Receptor tyrosine kinase mutations in myeloid neoplasms.

Receptor tyrosine kinase mutations in myeloid neoplasms.