SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a subtype of acute myeloid leukemia, affecting mainly the elderly. It is thought to be derived from plasmacytoid dendritic cell precursors, which frequently present as cutaneous lesions. We have made a detailed analysis of an infant with BPDCN, who manifested with hemophagocytic lymphohistiocytosis. The peripheral blood leukocytes revealed the t(2;17;8)(p23;q23;p23) translocation and a CLTC-ALK fusion gene, which have never been reported in BPDCN or in any myeloid malignancies thus far. Neonatal blood spots on the patient's Guthrie card were analyzed for the presence of the CLTC-ALK fusion gene, identifying the in utero origin of the leukemic cell. Although the leukemic cells were positive for CD4, CD56, CD123, and CD303, indicating a plasmacytoid dendritic cell phenotype, detailed analysis of the lineage distribution of CLTC-ALK revealed that part of monocytes, neutrophils, and T cells possessed the fusion gene and were involved in the leukemic clone. These results indicated that leukemic cells with CLTC-ALK originated in a multipotent hematopoietic progenitor in utero. This is the first report of the CLTC-ALK fusion gene being associated with a myeloid malignancy, which may give us an important clue to the origin of this rare neoplasm. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological disorder that is categorized as a myeloid leukemia according to the classification of the World Health Organization (Swerdlow, 2008; Vardiman et al., 2009). It is a clinically aggressive tumor, known to be derived from the precursors of plasmacytoid dendritic cells (Chaperot et al., 2001). BPDCN is characterized by cutaneous involvement at diagnosis, with solitary or multiple isolated skin lesions. According to previous reports, >90% of BPDCNs manifested with cutaneous lesions (Petrella et al., 2005), comprising about 0.7% of cutaneous lymphomas (Ng et al., 2006), and are followed by an aggressive progression with rapid systemic dissemination (Feuillard et al., 2002; Petrella et al., 2005; Julia et al., 2013). In rare cases, manifestations as an acute leukemia with bone marrow infiltration but without any skin involvements were observed (Jacob et al., 2003). Although patients could achieve complete remission by initial systemic chemotherapy, they frequently relapse, resulting in the median overall survival of around 1 year (Feuillard et al., 2002; Reimer et al., 2003; Petrella et al., 2005; Chen et al., 2011). BPDCN generally occurs in the elderly, with the median age of affected patients being in the sixth decade of life (Garnache-Ottou et al., 2007), although a few pediatric cases, including an infant case, have also been reported (Hu et al., 2007; Jegalian et al., 2010).

Neoplastic cells in BPDCN are now considered to be derived from the plasmacytoid dendritic cell and are characterized by the coexpression of CD4 and CD56 without any lineage-specific markers other than dendritic cell antigen-2 (BDCA-2, CD303) and interleukin-3 receptor α chain (CD123), which are markers for plasmacytoid dendritic cells (Garnache-Ottou et al., 2007).

The etiology and pathogenesis of BPDCN are largely unknown, with only a few studies published to date. Some chromosome aberrations were described in 21 patients with BPDCN, disclosing 6 major recurrent targets on chromosomes/chromosome arms 5q, 6q, 9, 12p, 13q, and 15q (Leroux et al., 2002). Dijkman et al., (2007) using array-based comparative genomic hybridization, reported recurrent alterations in chromosomes 4, 9, and 13. Lucioni et al. also identified several commonly deleted chromosomal regions, such as 9p21, 13q13-14, 12p13, 13q11-12, and 7p12, by an array-based technique (Lucioni et al., 2011). Specifically, the 9p12 region was reported as being biallelically deleted in several patients, indicating the existence of tumor suppressor gene(s) associated with BPDCN on this chromosomal region, with CDKN2A and/or CDKN2B being the most suspected candidate(s).

The MLL-ENL fusion gene, which is a recurrent fusion observed in acute leukemia, was identified in an adult case of BPDCN with bone marrow infiltration of the tumor cells (Toya et al., 2012). Chromosomal analysis of the bone marrow cell revealed the t(11;19)(q23;p13.3) translocation, which led to the detection of the MLL-ENL fusion in this patient. There are three more BPDCN cases with 11q23 abnormalities in the literature (Bueno et al., 2004; Leung et al., 2006). Although one of these three cases lacked molecular evidence, 11q23 abnormalities with rearrangement of the MLL gene is the only recurrent chromosomal translocation in BPDCN reported thus far.

The t(2;17)(p23;q23) translocation, resulting in clathrin heavy chain (CLTC)-anaplastic lymphoma kinase (ALK) fusion gene formation, has been described as a recurrent chromosome translocation in anaplastic large cell lymphoma (ALCL) (Touriol et al., 2000; Cools et al., 2002; Swerdlow, 2008), ALK-positive large B-cell lymphoma (LBCL; Chikatsu et al., 2003; De Paepe et al., 2003; Gascoyne et al., 2003; Laurent et al., 2009), inflammatory myofibroblastic tumor (Bridge et al., 2001) and extramedullary plasmacytoma (Wang et al., 2011). The tyrosine kinase domain of ALK is constitutively phosphorylated by the formation of CLTC-ALK, the tumorigenicity of which has been verified in vitro and in vivo (Armstrong et al., 2004).

Here, we report an infant with BPDCN with a t(2;17;8)(p23;q23;p23). This is the first report of a congenital BPDCN with CLTC-ALK fusion and could give a significant insight into the pathogenesis of this poorly understood hematological malignancy.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Patient

A female patient with pancytopenia was diagnosed with hemophagocytic lymphohistiocytosis (HLH) on the 24th day after birth. At 2 months of age, leukemic blasts appeared in the peripheral blood, which carried the karyotype 46,XX,t(2;17;8) (p23;q23;p23) (Fig. 1A). The blast cells were resistant to chemotherapy and had infiltrated the bone marrow by 60% at 8 months of age (Fig. 1B), whereupon cytogenetic analysis of the bone marrow showed 45,XX,t(2;17;8)(p23;q23;p23),-7 karyotype in all 20 cells analyzed. There were no apparent skin lesions observed throughout the clinical course. The details of the clinical course are described in the Supporting Information.

image

Figure 1. Karyotype and morphology of leukemic cells. A: Karyotype at 2 months after birth. Three-way translocation involving 2p23 and 17q23 was observed by karyotyping, indicating the presence of the CLTC-ALK fusion. Acquired monosomy 7 was later also observed at the leukemic transformation (8 months after birth). B: Bone marrow smears at 8 months after birth. Large monocytic cells with fine granules were increased in the bone marrow and in the peripheral blood. C: Metaphase fluorescent in situ hybridization (FISH) analysis with the ALK break-apart rearrangement probe confirmed the translocation of the 3′ ALK signal (red) to the long arm of der(17) and the loss of the 5′ ALK signal (green).

Download figure to PowerPoint

Peripheral blood and bone marrow cells were collected from the patient. In accordance with the Declaration of Helsinki, written informed consent was obtained from the parents, and all research was approved by the institutional review board at Ehime University.

Fluorescence In Situ Hybridization (FISH)

The Vysis LSI ALK Break-Apart Rearrangement Probes kit (Abbot, Tokyo, Japan) was used to detect the rearrangement of the ALK gene by FISH analysis, according to the manufacturer's instructions.

Detection of CLTC-ALK Fusion Transcript by Reverse Transcriptase-mediated Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from the bone marrow and peripheral blood mononuclear cells, using an RNeasy Mini kit (Qiagen, Hilden, Germany). The total RNA (100 ng to 1 μg) was reverse-transcribed with a PrimeScript RT-PCR kit (Takara, Otsu, Japan), using a random hexamer according to the manufacturer's instructions. One tenth of the synthesized cDNA was directed to RT-PCR analysis for the detection of the CLTC-ALK fusion transcript. The primers used are listed in Supporting Information Table S1. The PCR products obtained were electrophoresed on agarose gels, and then purified using a QIAquick Gel Extraction kit (Qiagen) and sequenced directly using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). All sequencing was performed on an ABI310 Genetic Analyzer (Applied Biosystems).

Detection of CLTC-ALK Genomic Fusion in Blood Spots

The genomic breakpoints of the CLTC and ALK genes in leukemic cells were identified by inverse PCR, the details of which are described in the Supporting Information. Primer sets used to detect the CLTC and ALK sequences were mixed in a single tube in the first round of PCR together with the primers for the CLTC-ALK fusion to confirm the validity of the PCR with the blood spots. In the second round of PCR, only primers for the CLTC-ALK fusion were used. Details of the primers are described in Supporting Information Table S1 and details of the method are described in Supporting Information.

Flow Cytometry and Cell Sorting

After erythrocyte lysis with RBC Lysis Buffer (BioLegend, San Diego, CA), the peripheral blood and bone marrow cells were stained with a monoclonal antibody according to the manufacturer's instructions and analyzed using a Gallios flow cytometer (Beckman Coulter, Brea, CA) and FlowJo software (TreeStar, Ashland, OR). The cell sorting was done with a FACSAria cell sorter (BD Biosciences, San Jose, CA). Antibodies used for the analysis are described in the Supporting Information.

Quantification of the CLTC-ALK Fusion Gene in Peripheral-blood-sorted Population

The amount of CLTC-ALK fusion gene present in each cell population in the peripheral blood was assessed by a quantitative PCR. Total RNA was extracted from the fluorescence-activated-sorted cells with the RNeasy Micro kit (Qiagen) and reverse-transcribed into cDNA with the PrimeScript RT reagent kit (Takara) according to the manufacturers' instructions. Genomic DNA was extracted from fluorescence-activated-sorted cells with a NucleoSpin Tissue kit (Macherey-Nagel, Duren, Germany). Quantitative PCR was performed using SYBR Premix Ex Taq II (Takara) on a StepOnePlus Real-Time PCR system (Applied Biosystems), using the PCR conditions recommended by the supplier. All measurements were performed in duplicate, and the difference in the duplicate threshold cycle was <1 cycle in all the samples analyzed. All experiments were repeated at least three times. The primers used for quantitative PCR are listed in Supporting Information Table S1

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

The CLTC-ALK Fusion Gene was Formed as the Result of a t(2;17;8)(p23;q23;p23)

The presence of a t(2;17;8)(p23;q23;p23) suggested the involvement of the ALK gene in chromosome band 2p23 and the CLTC gene in 17q23 (Fig. 1A). FISH analysis of the peripheral blood sample with probes covering the ALK gene showed translocation of the 3′ ALK signal to the der(17) and loss of the 5′ ALK signal in 90% of the cells analyzed (Fig. 1C). Total RNA was extracted from peripheral blood, and the expression of the CLTC-ALK fusion gene, which is known to result from translocations between 2p23 and 17q23, was examined by RT-PCR. As shown in Figure 2, amplification of the CLTC-ALK fusion transcript was observed, and in-frame fusion of exon 31 of CLTC to exon 19 of ALK was confirmed by direct sequencing analysis of the RT-PCR product. The reciprocal fusion transcript, ALK-CLTC, was not detected by nested RT-PCR (data not shown). Although the primary lesion was unknown, the existence of the lymphoma-related CLTC-ALK fusion gene prompted us to make a diagnosis of malignant lymphoma, possibly ALCL or ALK-positive LBCL, and to treat the patient with the modified protocol for high-risk ALCL, which ended in relapse at an early stage of the treatment.

image

Figure 2. Detection of the CLTC-ALK fusion transcript in the peripheral blood of the patient at HLH stage (at 2 months after birth). A: RT-PCR analysis showed a clear positive band (indicated by black arrows) in the patient's peripheral blood sample. B: The product of first-round PCR was purified and subjected to direct sequencing analysis. In-frame fusion of exon 31 of CLTC to exon 19 of ALK was identified. PB, peripheral blood.

Download figure to PowerPoint

Leukemic Cells with CLTC-ALK Expression have a Plasmacytoid Dendritic Cell Phenotype

Surface antigens of the blast cells obtained from the patient's bone marrow at the relapsed stage (8 months of age) were analyzed by flow cytometry. Around 40% of bone marrow CD45-positive cells showed low-grade expression of CD4 without CD3 expression, and 27% were positive for both CD4 and CD56 (Fig. 3A). These CD4-positive cells were also positive for the plasmacytoid dendritic cell lineage marker CD123, and the cells with high CD123 expression were also positive for CD303 (another lineage marker for plasmacytoid dendritic cells), leading to the diagnosis of BPDCN (Fig. 3B). These cells with low-grade CD4 expression without CD3 were also observed in the peripheral blood obtained at 2 months of age (Fig. 3C).

image

Figure 3. CD4+CD3 cells possesses CLTC-ALK fusion. FACS analysis of the bone marrow sample at leukemic stage (at 8 months after birth) (A, B), and of peripheral blood at 2 months after birth (at HLH stage; C). A An apparent increase of CD4+CD3 cells was noted in the CD45+ cell population of the bone marrow (middle panel). The intensity of CD4 expression on the CD4+CD3 cells was weaker than that on CD4+CD3+ T cells in this population. About half of the CD4+CD3 cells were also positive for CD56 (right panel). B: Expressions of CD123 and CD303 on CD4+CD3 cells. CD4+CD3 cells expressed both CD123 and CD303, which were mostly negative in CD4CD3 cells. Some cells showed a higher expression of both antigens, albeit most of the expressions of these antigens were weak. C: CD4+CD3 cells were present in the peripheral blood CD45+ population (middle panel) and these cells were also positive for CD56 (right panel). D: Subpopulations of hematopoietic cells in the peripheral blood were separated by FACS, and the presence of the CLTC-ALK fusion transcript was examined by quantitative RT-PCR. T cells, B cells, NK cells, monocytes, neutrophils, and leukemic CD4+CD3CD56+ and CD4+CD3CD56- cells were collected and analyzed. E: Relative expression of the CLTC-ALK fusion in each subpopulation. In addition to CD4+ leukemic subpopulations, T cells, and monocytes were also positive for the CLTC-ALK fusion transcript. No expression of CLTC-ALK was detectable in B cells, NK cells, and neutrophils. The expression of β-actin (ACTB) was used as an internal reference.

Download figure to PowerPoint

To confirm the expression of CLTC-ALK in CD4-positive leukemic cells, the cell populations were separated by fluorescence-activated cell sorting (FACS; Fig. 3D), and the presence of the CLTC-ALK fusion gene in each cell population was evaluated by quantitative RT-PCR. As shown in Figure 3E, CD4+CD3 cells were positive for CLTC-ALK, irrespective of the expression of CD56. The CLTC-ALK fusion transcript was also amplified in CD14-positive monocytes and in CD3-positive T cells but not in the neutrophil, B cell, or NK cell populations.

The CLTC-ALK Fusion Gene Was Formed in Utero

Genomic breakpoints of the CLTC-ALK fusion gene were determined by inverse PCR, which were located in introns 31 and 18 of the CLTC and ALK genes, respectively (Fig. 4A). Primer pairs spanning the genomic fusion point were designed to amplify the CLTC-ALK fusion gene from the neonatal blood spots, by seminested PCR. A total of 24 pieces containing the neonatal blood, punched out from the Guthrie card, were tested for the presence of CLTC-ALK. Of these 24 pieces, 17 were positive for the CLTC-ALK fusion. The actual CLTC-ALK genomic fusion was confirmed by sequence analysis of the PCR products (data not shown). Representative results of the PCR with the blood spots are shown in Figure 4B. These results indicated that the formation of CLTC-ALK, which is considered an initial step of BPDCN development, had occurred in utero.

image

Figure 4. Detection of genomic CLTC-ALK fusion in neonatal blood spots. A: Identification of the genomic fusion point of CLTC-ALK in the patient's leukemic cells. The genomic breakpoint of the CLTC and ALK genes were located in introns 31 and 18, respectively, which are fused to each other with an overlap of 2 base pairs. Black arrows indicate the location of primers used for PCR amplification with the Guthrie blood spots. Three sets of primers facing each other, representing primers for the CLTC, CLTC-ALK fusion, and ALK gene regions, respectively, are shown. As an internal control, parts of intron 30 of CLTC and intron 18 of ALK were also amplified by PCR to confirm the integrity of the DNA on the blood spots. B: Representative results of the PCR with Guthrie blood spots. By the first-round PCR, both the CLTC and ALK regions, like the internal control, were amplifiable in all blood spots examined. The CLTC-ALK genomic fusion was only detectable after the second round of PCR in some blood spots. In total, 17 out of 24 pieces of Guthrie spots were positive for the CLTC-ALK fusion.

Download figure to PowerPoint

The sensitivity of the PCR for the CLTC-ALK genomic fusion was assessed on the serially diluted CLTC-ALK amplified PCR product. By calculation, 1 copy of double-stranded DNA of the PCR product corresponds to 2 × 10−7 pg of DNA. The primer set used for the analysis was able to detect as little as 10−5 pg of fusion DNA, which roughly corresponds to 50 copies of CLTC-ALK (50 leukemic cells; Supporting Information Fig. S1).

Characterization of Leukemic Cells in BPDCN

According to a typical immunophenotypic profile previously reported, BPDCN is characterized by the co-expression of CD4, CD56, and CD123 and a lack of specific myeloid-, T-, B-, or NK-lineage markers (Feuillard et al., 2002; Petrella et al., 2005). However, leukemic cells of BPDCN can express aberrant lineage markers, mostly myeloid or B or T lymphoid markers (Garnache-Ottou et al., 2009). In our case, leukemic cells expressed some T-cell antigens (CD2, CD7), myeloid lineage antigens (CD13, CD33), and the hematopoietic stem cell marker CD34, as determined by routine laboratory tests (Supporting Information Table S2). Multicolor flow cytometry analysis of CD4+CD3 cells of the patient also revealed the leukemic cells to be positive for some myeloid lineage markers. Most of the leukemic CD4+CD3 cells were positive for CD33, and nearly half of the CD33-positive cells were positive for CD14 (Fig. 5A). Although most of the CD4+CD3 cells were weakly positive for CD34, this marker was still highly positive in about 6% of CD4+CD3 cells, and these cells were also positive for CD33 but negative for CD14 (Fig. 5A). As for the plasmacytoid dendritic cell marker, expression of CD123 was weak in the CD56-positive cell fraction, and cells with a high expression of CD123 were virtually negative for CD56 (Fig. 5B). These data indicated that CD4+CD3 cells had differentiated not only into plasmacytoid dendritic cells (which are highly positive for CD123) but also into other myeloid lineages such as CD14-positive monocytes.

image

Figure 5. FACS analysis of leukemic cells at leukemic stage (8 months after birth). A: Expression of myeloid markers in leukemic CD4+CD3 cells and in nonleukemic CD4CD3 cells. Note that most of the leukemic cells are positive for CD33, and some are positive for CD14 and/or CD34. B: CD123 expression in CD4+CD3 cells in the bone marrow. CD123-positive cells are clearly separated from the CD56-positive or CD14-positive cells. C: CD30 expression in CD45-positive and CD4+CD3 cells. CD30-positive cells existed in the CD4+CD3 population (lower panel), but not in any of the populations positive for the lineage markers (upper panel).

Download figure to PowerPoint

CD30 is a frequently expressed marker in ALK-positive ALCL, and its expression in the physiological state is reported to be restricted to subpopulations of activated B and T cells. In the present case, CD30 expression was observed in about 8% of CD45-positive cells, mainly in the CD4+CD3 fraction, and CD30-positive cells were negative for T-, B-, and monocytic-lineage markers (Fig. 5C).

Of note was that some anti-CD56 antibodies (i.e., NCAM16.2 from BD Bioscience, and HCD56 from Biolegend) failed to detect CD56 expression on the leukemic cells, as shown in Supporting Information Fig. S2A and Table S2. The actual expression of CD56 was verified by another anti-CD56 antibody (MEM-188 from AbD Serotec), as shown in Figure 3A.

Possible Hematopoietic Progenitor Cell Origin of the CLTC-ALK Fusion Gene

To explore the origin of leukemic transformation in the patient, the presence of the CLTC-ALK fusion gene was examined using genomic DNA extracted from subpopulations sorted from the patient's peripheral blood at leukemic stage (8 months of age, Fig. 6). FACS was used to separate the T cells, B cells, NK cells, neutrophils, monocytes, and CD4+CD3CD56+ cells (Fig. 3D) as well as CD123+CD303+ cells (Fig. 3B). The separation of CD4+CD34+ and CD4CD34+ cells is shown in Supporting Information Fig. S2B.

image

Figure 6. Proportion of CLTC-ALK-positive cells in each subpopulation of hematopoietic cells. A: Primers used for the quantitative PCR. A pair of primers spanning the genomic fusion point was used to amplify the CLTC-ALK fusion gene (a). The genomic regions upstream to the break point in intron 31 of CLTC (b) and downstream to the breakpoint in intron 18 of ALK (c) were also amplified as the internal control for quantification. B: Quantification of the CLTC-ALK gene dosage in CD4+CD3+ cells and in leukemic CD4+CD3CD56+ cells. The PCR product of the CLTC-ALK genomic fusion containing exon 31 of CLTC to exon 19 of ALK was used to normalize the value and set as 1. The CLTC gene was used as an internal reference. C: Relative gene dosage of CLTC and ALK in each subpopulation, as assessed by quantitative PCR. Amplification of the beta-2-microglobulin (B2M) gene region was used as an internal reference. Values obtained with CD4+CD3CD56+ cells were used to normalize the value and set as 1, because this population is speculated to be entirely composed of leukemic cells. D: Relative gene dosage of the CLTC-ALK genomic fusion in each subpopulation, as assessed by quantitative PCR. The B2M gene was used as an internal reference and the value obtained with the CD4+CD3CD56+ cells was set as 1.

Download figure to PowerPoint

The proportion of leukemic cells in each population was estimated by quantitative PCR, to compare the gene dosage of the CLTC-ALK fusion gene with the CLTC or ALK gene regions. A 2.5 kb genomic region encompassing intron 31 of CLTC fused to intron 18 of ALK was amplified by PCR, using the patient's DNA. This PCR product was used as a reference DNA, containing equal amounts of CLTC, ALK, and CLTC-ALK fusion genomic sequences (Supporting Information Fig. S3). When this PCR product was used as the reference, values obtained for the CLTC-ALK fusion gene with the PCR product were halved, since the amount of CLTC-ALK fusion gene (1 allele) in actual leukemic cells is predicted to be half that of the 5′ CLTC or 3′ ALK genomic sequences (2 alleles; Fig. 6A). Preliminary analysis showed that in the CD4+CD3CD56+ population containing mostly leukemic cells the CLTC-ALK fusion gene were clearly detected, indicating that most of the cells in this population possess the fusion gene (Fig. 6B). However, in the CD4+CD3+ T cells containing mostly non-leukemic cells the percentage of cells carrying CLTC-ALK was suspected to be low. Quantification of the 5′ CLTC and 3′ ALK genomic regions, which were used as internal controls, showed almost equal amounts in every population examined (Fig. 6C). The amount of CLTC-ALK fusion varied among the sorted population. Most of the cells in the CD123+CD303+ subpopulation and the monocytes had the CLTC-ALK fusion (Fig. 6D). In contrast, the percentage of cells with CLTC-ALK was low for T cells, B cells, and neutrophils, and the fusion gene was almost undetectable in NK cells. Some of the CD4CD34+lineage cells containing hematopoietic progenitors were also positive for CLTC-ALK (Fig. 6D). Although contamination by a few CLTC-ALK-harboring leukemic cells at the cell separation by FACS could not be ruled out, these results indicated that in addition to leukemic cells with a plasmacytoid dendritic cell phenotype, some T cells, B cells, and neutrophils were also involved in leukemic clone with CLTC-ALK fusion gene.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

BPDCN is a subtype of myeloid leukemia mainly affecting the elderly and often accompanied by cutaneous lesions. It is thought to be derived from plasmacytoid dendritic cell precursors, but neither the genetic nor the clonal origin of the disease is known. We have made a detailed analysis of an infant, who manifested with HLH and later diagnosed as BPDCN, to understand the origin of the tumor cells and the leukemic process involved.

Surface antigens of the blast cells in the patient showed low-grade expression of CD4 without CD3 expression, and some of the CD4-expressing cells were also positive for plasmacytoid dendritic cell lineage marker CD123 and CD303, leading to the diagnosis of BPDCN (Fig. 3). Flow cytometry analysis of the CD4+CD3 leukemic cells indicated that some of these cells also had a characteristic of monocytic cells with CD14 positivity. CLTC-ALK fusion gene was identified in the CD4-expressing leukemic cells including CD123+CD303+ plasmacytoid dendritic cells and in the CD14-positive monocytes (Figs. 3E and 6D). In hematopoietic malignancy, CLTC-ALK has been reported only in malignant lymphomas, a lymphoid lineage neoplasm. This is the first report showing the presence of the CLTC-ALK fusion gene in a myeloid malignancy, BPDCN.

A certain percentage of HLH is known to be induced by hematological malignancies, including lymphoma and leukemia (Ishii et al., 2007). In these secondary HLHs, the uncontrolled antitumor activity of macrophages, histiocytes, and T lymphocytes, resulting in hypercytokinemia (including INF-γ) and possibly hyperchemokinemia, is considered to be the cause of the hemophagocytic syndrome (Filipovich, 2009). Although the hemophagocytic syndrome itself is not a frequent complication in BPDCN, the patient was first diagnosed as having HLH before the leukemic transformation had become apparent. Only three cases of HLH preceding the overt acute myeloid leukemia (AML) were reported previously (Kumar et al., 2000; Tadmor et al., 2006; Chang et al., 2011) and two cases showed clonal chromosomal abnormalities, which were identified before the leukemia became obvious like our case. In this patient, inappropriately activated hematopoietic cells harboring the CLTC-ALK fusion, such as monocytes, may have been the cause of the HLH symptoms observed. Clonal abnormalities observed in HLH may suggest the presence of an occult neoplastic disease. Cytogenetic and molecular analysis should be mandatory to verify the clonal proliferating hematopoietic cells as being one of the causes of HLH and to make a precise diagnosis in cases of intractable HLH.

Identification of the CLTC-ALK fusion from the neonatal blood on the Guthrie card (Fig. 3A) indicated that the initial step of BPDCN development had occurred in utero. Some fusion genes observed in leukemia such as ETV6-RUNX1 (TEL-AML1) or MLL-AF4 have been proven to be derived in utero. This prenatal generation of leukemogenic gene mutations are thought to be the first hit to leukemic transformation, which is later accompanied by additional genetic changes when manifesting as postnatal overt leukemia (Greaves and Wiemels, 2003).

CD4-positive hematopoietic progenitors are known to be present in human fetal liver, fetal bone marrow, and umbilical cord blood progenitors, which also express CD34, CD13, CD33, CD117, and CD123 (Muench et al., 1997). The similarity in characteristics of the patient's leukemic cells to that of the fetal hematopoietic stem cells may suggest that leukemic cells had originated in hematopoietic progenitor cells in the fetus during the prenatal period. The finding that the CLTC-ALK fusion gene existed not only in the leukemic cells but also in various cell lineages supports this hypothesis.

The most interesting feature of this case was the presence of the CLTC-ALK fusion gene in a myeloid neoplasm, BPDCN. In this case, the CD123+CD303+ plasmacytoid dendritic cell was not the only cell lineage possessing the CLTC-ALK fusion. In addition to CD123lowCD303 cells which in part possessed the monocytic lineage marker CD14, some T cells, B cells and neutrophils were also involved in the leukemic clone with the CLTC-ALK fusion gene (Fig. 6D), indicating that the transformation occurred at the hematopoietic progenitor level. In accord with this supposition, some of the CD4CD34+lineage- cells containing hematopoietic progenitors were also positive for CLTC-ALK fusion gene (Fig. 6D). However, T cells and B cells could not expand to form overt leukemia indicating presence of CLTC-ALK alone is not enough for the leukemic transformation of these lymphoid cells.

In acute leukemia, at least two types of gene mutation are considered necessary for leukemogenesis. One is the mutation, which provides a proliferative and/or survival signal such as tyrosine kinase activation (Class I mutation) and the other serves to impair differentiation such as transcriptional factor mutation (Class II mutation) (Gilliland, 2002). In this regard, it is appropriate to guess that the second hit accompanying the CLTC-ALK fusion gene may be important in determining the cell lineage of the malignant cell rather than CLTC-ALK itself, which is an activated tyrosine kinase and can exist both in lymphoid and myeloid malignancy.

In the case reported here, although CLTC-ALK was obtained at the hematopoietic progenitor level, the acquisition of monosomy 7, which is one of the progressive factors in myeloid malignancies, might be responsible for the disease's progression to an overt myeloid leukemia, rather than lymphoid malignancies, with the CLTC-ALK fusion (Fig. 7). However, it is also possible that there are other mutations or copy number alterations other than monosomy 7, which are not visible by standard cytogenetic analysis that could constitute second hits leading to myeloid malignancy.

image

Figure 7. A hypothetical model of leukemogenesis as BPDCN by the CLTC-ALK fusion. It is speculated that the CLTC-ALK fusion gene is formed in one of the CD34+lineage- hematopoietic stem cells in utero. This CLTC-ALK-harboring hematopoietic stem cell can differentiate into monocytes, neutrophils, and plasmacytoid dendritic cells as well as T cells and B cells, because CLTC-ALK is essentially an activated kinase that does not have any inhibitory activity on the hematopoietic differentiation pathway. One of the CLTC-ALK-positive cells that acquire the second genetic hit, possibly monosomy 7, differentiates into CD4+CD34+lineage cells and then into various lineages such as plasmacytoid dendritic cells, CD4+CD3CD56+ cells, monocytes, neutrophils, and CD30+CD56 cells. Within these cells, the population that obtains growth advantage by the CLTC-ALK and the second hit becomes dominant in the bone marrow and the peripheral blood. CD30+ cells may expand and become ALCL if the second hit has preference toward the lymphoid lineage, wheaeas in this case acquisition of monosomy 7 may have caused expansion of myeloid lineage cells such as plasmacytoid dendritic cells and monocytes. Aberrantly activated monocytes may cause the HLH symptom.

Download figure to PowerPoint

There was also a small population of CD30-positive cells that showed the CD4+CD56 phenotype. Considering that the CLTC-ALK fusion gene is usually observed in ALCL, which is also accompanied by CD4 positivity (Juco et al., 2003; Kesler et al., 2007), these CD30+CD4+ cells might correspond to the malignant cells in ALCL. ALCL occurs more frequently in children than in adults, comprising 10–15% of pediatric non-Hodgkin's lymphoma (Wright et al., 1997). Although definitive conclusions cannot be drawn because of the lack of detailed analysis of the lineage involvement and clonal evolution of ALCL, it is an attractive hypothesis to recognize CD30+CD4+ cells as the origin of ALCL. Single CLTC-ALK-harboring hematopoietic stem cells, when acquired with yet-unspecified second genetic change(s) that can drive tumor cells to the lymphoid lineage, may progress to form ALCL.

In conclusion, the CLTC-ALK gene was identified for the first time as the leukemia-promoting abnormality in an infant case of myeloid neoplasm, BPDCN, indicating the tumorigenic potential of CLTC-ALK in myeloid progenitor cells. Formation of the CLTC-ALK fusion was shown to have occurred in utero, and the subsequent acquisition of monosomy 7, one of the myeloid lineage-oriented abnormalities, might have determined the cell fate to a myeloid neoplasm in this patient.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

The authors thank Ms. Chihiro Tanaka and Ms. Tokiko Mizushiro for their technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information
  • Armstrong F, Duplantier MM, Trempat P, Hieblot C, Lamant L, Espinos E, Racaud-Sultan C, Allouche M, Campo E, Delsol G, Touriol C. 2004. Differential effects of X-ALK fusion proteins on proliferation, transformation, and invasion properties of NIH3T3 cells. Oncogene 23:60716082.
  • Bridge JA, Kanamori M, Ma Z, Pickering D, Hill DA, Lydiatt W, Lui MY, Colleoni GW, Antonescu CR, Ladanyi M, Morris SW. 2001. Fusion of the ALK gene to the clathrin heavy chain gene, CLTC, in inflammatory myofibroblastic tumor. Am J Pathol 159:411415.
  • Bueno C, Almeida J, Lucio P, Marco J, Garcia R, de Pablos JM, Parreira A, Ramos F, Ruiz-Cabello F, Suarez-Vilela D, San Miguel JF, Orfao A. 2004. Incidence and characteristics of CD4(+)/HLA DRhi dendritic cell malignancies. Haematologica 89:5869.
  • Chang TY, Jaffray J, Woda B, Newburger PE, Usmani GN. 2011. Hemophagocytic lymphohistiocytosis with MUNC13-4 gene mutation or reduced natural killer cell function prior to onset of childhood leukemia. Pediatr Blood Cancer 56:856858.
  • Chaperot L, Bendriss N, Manches O, Gressin R, Maynadie M, Trimoreau F, Orfeuvre H, Corront B, Feuillard J, Sotto J-J, Bensa J-C, Brière F, Plumas J, Jacob M-C. 2001. Identification of a leukemic counterpart of the plasmacytoid dendritic cells. Blood 97:32103217.
  • Chen J, Zhou J, Qin D, Xu S, Yan X. 2011. Blastic Plasmacytoid Dendritic Cell Neoplasm. J Clin Oncol 29:e27e29.
  • Chikatsu N, Kojima H, Suzukawa K, Shinagawa A, Nagasawa T, Ozawa H, Yamashita Y, Mori N. 2003. ALK+, CD30-, CD20- large B-cell lymphoma containing anaplastic lymphoma kinase (ALK) fused to clathrin heavy chain gene (CLTC). Mod Pathol 16:828832.
  • Cools J, Wlodarska I, Somers R, Mentens N, Pedeutour F, Maes B, De Wolf-Peeters C, Pauwels P, Hagemeijer A, Marynen P. 2002. Identification of novel fusion partners of ALK, the anaplastic lymphoma kinase, in anaplastic large-cell lymphoma and inflammatory myofibroblastic tumor. Genes Chromosomes Cancer 34:354362.
  • De Paepe P, Baens M, van Krieken H, Verhasselt B, Stul M, Simons A, Poppe B, Laureys G, Brons P, Vandenberghe P, Speleman F, Praet M, De Wolf-Peeters C, Marynen P, Wlodarska I. 2003. ALK activation by the CLTC-ALK fusion is a recurrent event in large B-cell lymphoma. Blood 102:26382641.
  • Dijkman R, van Doorn R, Szuhai K, Willemze R, Vermeer MH, Tensen CP. 2007. Gene-expression profiling and array-based CGH classify CD4+CD56+ hematodermic neoplasm and cutaneous myelomonocytic leukemia as distinct disease entities. Blood 109:17201727.
  • Feuillard J, Jacob MC, Valensi F, Maynadie M, Gressin R, Chaperot L, Arnoulet C, Brignole-Baudouin F, Drenou B, Duchayne E, Falkenrodt A, Garand R, Homolle E, Husson B, Kuhlein E, Le Calvez G, Sainty D, Sotto MF, Trimoreau F, Bene MC. 2002. Clinical and biologic features of CD4(+)CD56(+) malignancies. Blood 99:15561563.
  • Filipovich AH. 2009. Hemophagocytic lymphohistiocytosis (HLH) and related disorders. Hematology Am Soc Hematol Educ Program 127131.
  • Garnache-Ottou F, Feuillard J, Saas P. 2007. Plasmacytoid dendritic cell leukaemia/lymphoma: Towards a well defined entity? Br J Haematol 136:539548.
  • Garnache-Ottou F, Feuillard J, Ferrand C, Biichle S, Trimoreau F, Seilles E, Salaun V, Garand R, Lepelley P, Maynadie M, Kuhlein E, Deconinck E, Daliphard S, Chaperot L, Beseggio L, Foisseaud V, Macintyre E, Bene MC, Saas P, Jacob MC. 2009. Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia. Br J Haematol 145:624636.
  • Gascoyne RD, Lamant L, Martin-Subero JI, Lestou VS, Harris NL, Muller-Hermelink HK, Seymour JF, Campbell LJ, Horsman DE, Auvigne I, Espinos E, Siebert R, Delsol G. 2003. ALK-positive diffuse large B-cell lymphoma is associated with Clathrin-ALK rearrangements: Report of 6 cases. Blood 102:25682573.
  • Gilliland DG. 2002. Molecular genetics of human leukemias: New insights into therapy. Semin Hematol 39:611.
  • Greaves MF, Wiemels J. 2003. Origins of chromosome translocations in childhood leukaemia. Nat Rev Cancer 3:639649.
  • Hu SC-S, Tsai K-B, Chen G-S, Chen P-H. 2007. Infantile CD4+/CD56+ hematodermic neoplasm. Haematologica 92:e91e93.
  • Ishii E, Ohga S, Imashuku S, Yasukawa M, Tsuda H, Miura I, Yamamoto K, Horiuchi H, Takada K, Ohshima K, Nakamura S, Kinukawa N, Oshimi K, Kawa K. 2007. Nationwide survey of hemophagocytic lymphohistiocytosis in Japan. Int J Hematol 86:5865.
  • Jacob MC, Chaperot L, Mossuz P, Feuillard J, Valensi F, Leroux D, Bene MC, Bensa JC, Briere F, Plumas J. 2003. CD4+ CD56+ lineage negative malignancies: A new entity developed from malignant early plasmacytoid dendritic cells. Haematologica 88:941955.
  • Jegalian AG, Buxbaum NP, Facchetti F, Raffeld M, Pittaluga S, Wayne AS, Jaffe ES. 2010. Blastic plasmacytoid dendritic cell neoplasm in children: Diagnostic features and clinical implications. Haematologica 95:18731879.
  • Juco J, Holden JT, Mann KP, Kelley LG, Li S. 2003. Immunophenotypic analysis of anaplastic large cell lymphoma by flow cytometry. Am J Clin Pathol 119:205212.
  • Julia F, Petrella T, Beylot-Barry M, Bagot M, Lipsker D, Machet L, Joly P, Dereure O, Wetterwald M, d'Incan M, Grange F, Cornillon J, Tertian G, Maubec E, Saiag P, Barete S, Templier I, Dalle S. 2013. Blastic plasmacytoid dentritic cell neoplasm:Clinical features in 90 patients. Br J Dermatol 169:579586.
  • Kesler MV, Paranjape GS, Asplund SL, McKenna RW, Jamal S, Kroft SH. 2007. Anaplastic large cell lymphoma: A flow cytometric analysis of 29 cases. Am J Clin Pathol 128:314322.
  • Kumar M, Boggino H, Hudnall SD, Velagaleti GV. 2000. Acute myeloid leukemia associated with hemophagocytic syndrome and t(4;7)(q21;q36). Cancer Genet Cytogenet 122:2629.
  • Laurent C, Do C, Gascoyne RD, Lamant L, Ysebaert L, Laurent G, Delsol G, Brousset P. 2009. Anaplastic lymphoma kinase–positive diffuse large b-cell lymphoma: A rare clinicopathologic entity with poor prognosis. J Clin Oncol 27:42114216.
  • Leroux D, Mugneret F, Callanan M, Radford-Weiss I, Dastugue N, Feuillard J, Le Mée F, Plessis G, Talmant P, Gachard N, Uettwiller F, Pages M-P, Mozziconacci M-J, Eclache V, Sibille C, Avet-Loiseau H, Lafage-Pochitaloff M. 2002. CD4+, CD56+ DC2 acute leukemia is characterized by recurrent clonal chromosomal changes affecting 6 major targets: A study of 21 cases by the Groupe Français de Cytogénétique Hématologique. Blood 99:41544159.
  • Leung R, Chow EE, Au W-Y, Chow C, Kwong Y-L, Lin S-Y, Ma ES, Wan TS, Wong K-F. 2006. CD4+/CD56+ hematologic malignancy with rearranged MLL gene. Hum Pathol 37:247249.
  • Lucioni M, Novara F, Fiandrino G, Riboni R, Fanoni D, Arra M, Venegoni L, Nicola M, Dallera E, Arcaini L, Onida F, Vezzoli P, Travaglino E, Boveri E, Zuffardi O, Paulli M, Berti E. 2011. Twenty-one cases of blastic plasmacytoid dendritic cell neoplasm: focus on biallelic locus 9p21.3 deletion. Blood 118:45914594.
  • Muench MO, Roncarolo MG, Namikawa R. 1997. Phenotypic and functional evidence for the expression of CD4 by hematopoietic stem cells isolated from human fetal liver. Blood 89:13641375.
  • Ng A, Lade S, Rutherford T, McCormack C, Prince H, Westerman D. 2006. Primary cutaneous CD4+/CD56+ hematodermic neoplasm (blastic NK-cell lymphoma): A report of five cases. Haematologica 91:143144.
  • Petrella T, Bagot M, Willemze R, Beylot-Barry M, Vergier B, Delaunay M, Meijer CJ, Courville P, Joly P, Grange F, De Muret A, Machet L, Dompmartin A, Bosq J, Durlach A, Bernard P, Dalac S, Dechelotte P, D'Incan M, Wechsler J, Teitell MA. 2005. Blastic NK-cell lymphomas (agranular CD4+CD56+ hematodermic neoplasms): A review. Am J Clin Pathol 123:662675.
  • Reimer P, Rudiger T, Kraemer D, Kunzmann V, Weissinger F, Zettl A, Konrad Muller-Hermelink H, Wilhelm M. 2003. What is CD4+CD56+ malignancy and how should it be treated? Bone Marrow Transplant 32:637646.
  • Swerdlow SH. 2008. WHO Classification of Tumors Of Hematopoietic And Lymphoid Tissues, 4 ed. Lyon: International Agency for Research on Cancer.
  • Tadmor T, Vadazs Z, Dar H, Laor R, Attias D. 2006. Hemophagocytic syndrome preceding acute myeloid leukemia with der t[7:17][q12;q11], monosomy, 17 and 5p. J Pediatr Hematol Oncol 28:544546.
  • Touriol C, Greenland C, Lamant L, Pulford K, Bernard F, Rousset T, Mason DY, Delsol G. 2000. Further demonstration of the diversity of chromosomal changes involving 2p23 in ALK-positive lymphoma: 2 Cases expressing ALK kinase fused to CLTCL (clathrin chain polypeptide-like). Blood 95:32043207.
  • Toya T, Nishimoto N, Koya J, Nakagawa M, Nakamura F, Kandabashi K, Yamamoto G, Nannya Y, Ichikawa M, Kurokawa M. 2012. The first case of blastic plasmacytoid dendritic cell neoplasm with MLL-ENL rearrangement. Leuk Res 36:117118.
  • Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hellstrom-Lindberg E, Tefferi A, Bloomfield CD. 2009. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 114:937951.
  • Wang WY, Gu L, Liu WP, Li GD, Liu HJ, Ma ZG. 2011. ALK-positive extramedullary plasmacytoma with expression of the CLTC-ALK fusion transcript. Pathol Res Pract 207:587591.
  • Wright D, McKeever P, Carter R. 1997. Childhood non-Hodgkin lymphomas in the United Kingdom: Findings from the UK Children's Cancer Study Group. J Clin Pathol 50:128134.

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
gcc22119-sup-0001-suppInfo.doc106KSupporting Information
gcc22119-sup-0002-suppFig1.jpg1120KSupporting Information Figure 1
gcc22119-sup-0003-suppFig2.jpg5126KSupporting Information Figure 2
gcc22119-sup-0003-suppFig3.jpg2689KSupporting Information Figure 3

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.