Overexpression of NPM–ALK induces different types of malignant lymphomas in IL-9 transgenic mice

Abstract

Anaplastic large-cell lymphoma (ALCL) comprises approximately 25% of all non-Hodgkin lymphomas (NHL) in children and young adults, and up to 15% of high-grade NHL in older patients. Over 50% of these tumours carry the translocation t(2;5)(p23;q35). The result of this translocation is the fusion of the nucleophosmin (NPM) gene to the anaplastic lymphoma kinase (ALK) gene. The resulting hybrid protein contains the ALK catalytic domain that consequently confers transforming potential, which contributes to the pathogenesis of ALCL. To further analyse the transforming activity in an animal model, a cDNA encoding the protein product, NPM–ALK, was inserted into the retrovirus vector pLXSN and transduced into mouse bone marrow progenitors. These cells were subsequently used in a bone marrow transplant with the aim of reconstituting the haematopoietic compartments of lethally irradiated recipients. IL-9 transgenic mice were chosen as the animal model system, because dysregulated expression of the IL-9 gene in transgenic mice results in the sporadic development of spontaneous thymic lymphomas. Moreover, IL-9 is known to be expressed in cases of human ALCL. We used 15 IL-9 transgenic mice and eight corresponding wild-type mice (FVB/N) and transplanted them with NPM/ALK infected bone marrow cells. Eight IL-9 transgenic mice, serving as a control group, received pLXSN (vector only)-infected marrow. Reconstituted mice developed NPM–ALK-positive lymphomas, including lymphoblastic lymphomas of T-cell type (T-LB), mature and immature plasmacytoma (PC), and plasmoblastic/anaplastic diffuse large-B-cell lymphoma after about 19–20 weeks. The combined overexpression of NPM–ALK and IL-9 led to the transformation of murine lymphoid cells with accelerated and enhanced development of T-LB in 46% of the mice, which only very rarely occurs in IL-9 transgenic mice only. Of the 15 animals, five (33%) developed plasmacytic/plasmoblastic neoplasms, of which the most aggressive tumours share many features with anaplastic/plasmoblastic diffuse large-B-cell lymphoma on the basis of morphology, a characteristic growth pattern and ALK expression.

Introduction

Chromosomal translocations play an important role in the pathogenesis of both lymphoproliferative disorders and solid tumours by creating hybrid genes that encode fusion proteins with oncogenic properties (Look, 1997; Rabbitts, 2001). Receptor tyrosine kinases (RTKs) are involved in the control of cell proliferation and differentiation, as well as malignant transformation. Constitutive activation of RTKs via somatic mutations or chromosomal translocations can lead to aberrant stimulation of signal transducing pathways, resulting in cellular transformation and neoplasia (Plowman et al., 1999; Robinson et al., 2000).

Anaplastic large-cell lymphoma (ALCL) is frequently associated with t(2;5) translocation (Le Beau et al., 1989; Morris et al., 2001), which results in the fusion of the nucleophosmin (NPM) gene on chromosome 5q35, to the intracytoplasmic region of the anaplastic lymphoma kinase (ALK) gene on chromosome 2p23 (Morris et al., 1994). While NPM is ubiquitously expressed, ALK expression is normally restricted to neural tissues and testis (Morris et al., 1997). In the hybrid gene, ALK is constitutively activated and seems to play a role in the malignant transformation of lymphoid cells in ALCLs (Bischof et al., 1997; Ladanyi, 1997). Additionally, some other fusion partners of ALK have been reported to lead to different ALK-positive lymphomas (Lawrence et al., 2000; Maes et al., 2001; Tort et al., 2001; Touriol et al., 2000). Furthermore, rare cases expressing the full-length ALK receptor protein have been proposed as a new subtype of diffuse large-B-cell lymphomas which lack such translocations (Delsol et al., 1997).

The oncogenic potential of NPM–ALK has already been demonstrated by its ability to transform rat 1a fibroblasts and cell lines from factor-dependent to factor-independent growth (Fujimoto et al; 1996; Wellmann et al., 1997). However, the assessment of the transforming potential of NPM–ALK in an animal model suggests that NPM–ALK expression alone may not be sufficient to induce ALCL, because only large-cell lymphomas of B-cell type were observed (Kuefer et al., 1997).

It is now widely accepted that tumorigenesis involves a multistep process in which several genetic lesions have to accumulate in order to produce a fully malignant phenotype. Hence, with this in mind, we used IL-9 transgenic mice as a model in our experiments as: (1) IL-9 expression has been observed in cases of ALCL and HD but not in any other B- or T-cell lymphomas (Merz et al., 1991); (2) IL-9-transfected CD4+ mouse cells have been demonstrated to be tumorigenous in C57B1/6 mice which results in the development of ALCL-like tumours (Bittner et al., 2000); (3) IL-9 transgenic mice develop thymic lymphomas spontaneously only after long latency and with a low incidence rate of 7% (Renauld et al., 1994). Taking these facts into account, we assumed that the overexpression of IL-9 in the presence of NPM–ALK may lead to lymphomagenesis, resulting in the development of ALCL.

Here, we demonstrated that lethally irradiated IL-9 transgenic mice receiving bone marrow cells transduced with an NPM–ALK retrovirus showed constitutive overexpression of NPM–ALK and IL-9 in cells of haematopoietic and/or lymphoid origin. These mice developed malignant lymphomas, but, surprisingly, various lymphoma entities were found. These included T-lymphoblastic lymphoma (T-LB), mature and immature plasmacytoma (PC), as well as anaplastic/plasmoblastic diffuse large-B-cell lymphoma.

Materials and methods

Cell culture

GP+E86 ecotropic packaging cells (Markowitz et al., 1988) and NIH3T3 (ATCC, CCL92) mouse cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 10% foetal calf serum (FCS; Biochrom, Germany).

Retroviral vector and virus-producing cell line

The Moloney murine leukaemia virus-based vector pLXSN (Miller and Rosman, 1989) was used to construct pLNPM–ALKSN. The full-length human NPM–ALK coding sequence was ligated into the pLXSN EcoRI site. Sense orientation of the NPM–ALK fragment was verified by sequencing. Packaging cells GP+E86 were transfected with pLXSN-only and pLNPM–ALKSN DNA by the calcium phosphate method (Graham and van der Eb, 1973). After 12 days of G418 selection (1 mg/ml; Gibco), G418-resistant cell clones were isolated, expanded and titred (Uckert et al., 2000). Clones with litres of 1–3×106 cell colony forming units (CFU)/ml were selected to produce virus vector containing supernatant (virus supernatant) of LXSN and LNPM–ALKSN in tissue culture roller bottles (Costar, Bodenheim, Germany) (Uckert et al., 2000).

Animals

FVB/N (Taketo et al., 1991) mice were used as wild-type controls and were purchased at 4–6 weeks of age from the Jackson Laboratories (Bar Habor, ME). IL-9 transgenic mice were generated by microinjecting the transgene construct into the pronuclei of fertilized eggs of FVB mice as previously described (Renauld et al., 1994). The homozygous transgenic mice strains used in this study were designated as strain Tg54. The Tg54 mice strain has serum IL-9 levels >1 μg/ml, while IL-9 is undetectable in the serum of control FVB/N mice. The transgenic mice were generated at the Ludwig Institute for Cancer Research, Brussels, Belgium, and then bred in the facility at the University of Lübeck, Germany. All experimental groups consisted of a minimum of eight animals, which were transplanted when they were 8–12 weeks old.

Transduction of murine bone marrow cells

Bone marrow was harvested from femurs of 8 to 12-week-old male IL-9 transgenic mice or FVB/N control mice, 6 days after the administration of 150 mg/kg body weight of 5-fluorouracil (Roche, Basel, Switzerland), injected into the lateral tail vein. Cells were prestimulated for 24 h at a concentration of 5×105 cells/ml in DMEM supplemented with 10% FCS, recombinant mouse stem cell factor (100 ng/ml rmSCF), recombinant mouse interleukin-6 (10 ng/ml rmIL-6) and recombinant mouse interleukin-3 (10 ng/ml rmIL-3) (Cytokines from R&Dsystems, Wiesbaden, Germany). Donor bone marrow was transduced with LNPM–ALKSN virus supernatant with a multiplicity of infection (m.o.i.) of >5 for 24 h at 37°C in tissue culture flasks along with DMEM medium supplemented with 10% FCS, rmSCF, rmIL-6, rmIL-3 and 5 μg/ml polybrene (Sigma Chemical Co., St Louis, MO, USA). After 24 h of transduction, the medium was changed for another 24 h. One million cells were injected by lateral tail vein into female recipient mice after they had received a lethal dose of total body irradiation (5 Gy twice within a 3 h interval; 1.75 Gy/min; X-ray). The mice received acidic water (pH 2.8) and sterile food, and were observed for the development of tumours.

Pathology

A complete necropsy was performed on each mouse. Moribund mice were euthanized and dissected for evidence of tumours. Thymus, lymph nodes, bone marrow, spleen and solid organs were analysed histologically. Specimens were fixed by immersion for 24 h in 4% paraformaldehyde buffered in PBS before embedding in paraffin. Sections of 3–5 μm thickness were cut and placed on glass slides. Tissue specimens were dehydrated, dewaxed and stained with haematoxylin–eosin (H+E) and Giemsa's solution. Some larger lesions were additionally analysed by FACS or snap frozen in liquid nitrogen for immunohistochemical analysis.

RNA expression and reverse transcription PCR analyses

RNA was isolated from various mouse tissues by the guanidinium thiocyanate method (Chomczynski and Sacchi, 1987). An aliquot of 2.5 μg total RNA was reverse transcribed into cDNA using oligo(dT)16 primer and superscript reverse transcriptase (Gibco BRL, Karlsruhe, Germany) according to the manufacturer's recommendations. Each cDNA sample was PCR amplified (1 cycle of 2 min at 95°C (denaturation), 35 cycles of 40 s at 95°C, 45 s at 60°C and 1 min at 72°C, 5 min at 72°C (extension)) using the following primers for neomycin phosphotransferase: sense-strand neo 5′agg atc tcc tgt cat ctc ace ttg etc ctg-3′, antisense-strand neo 5′-aag aac tcg tca aga agg cga tag aag gcg-3′ sense-strand mouse B-actin 5′-tga cga ggc cca gag caa gag ag-3′, antisense-strand mouse β-actin 5′-cta gga gcc aga gca gta atc tc-3′, sense-strand npm–alk 5′-agt tgc aca ttg ttg aag cag agg c-3′ and antisense-strand npm–alk 5′-ttc cat gag gaa ate cag ttc gtc c-3′. Amplified products were 492, 805 and 515 base pairs (bp) for the neomycin phosphotransferase, mouse β-actin and npm–alk gene, respectively. Each reaction (10 μl) was run on a 1% TAE ethidium-bromide stained agarose gel.

Immunocytochemical staining

All tumours were characterized immunophenotypically using the ABC technique (DAKO, Hamburg, Germany). A wide panel of biotinylated antibodies (all purchased from BD Bioscience, Heidelberg, Germany) were used: B-cell markers included CD45R, CD19, CD38 and CD23, and T-cell markers included CD5, CD25, CD4, CD8 and CD3e. For mouse anti-ALK-1 antibody (DAKO) staining, the DAKO ARK™ (animal research kit) was used (DAKO). The polyclonal antibodies anti-lambda, anti-kappa, anti-IgA, anti-IgG and anti-IgM, and secondary biotinylated goat anti-rabbit antibodies were obtained from DAKO.

FACS analysis

Direct immunofluorescence cell staining was performed using the following phycoerythrin (PE)-conjugated rat monoclonal antibodies (mAb): B-cell (CD45R, CD 19, CD38 and CD23) and T-cell (CD5, CD25, CD4, CD8 and CD3e) as well as myeloid cell (CD11c and CD34) associated antigens (BD Bioscience). Using a fluorescence activated cell sorter (FACScan, BD Bioscience), 10 000 cells were analysed.

Transfer of tumour cells into secondary recipient mice

For serial transplants, 2×105 tumour cells from tumour-bearing mice were injected intraperitonally into syngeneic mice.

Results

Bone marrow infection with the NPM–ALK retrovirus

The retroviral construct used for these experiments contains a cDNA sequence encoding NPM–ALK expressed under the long terminal repeat (LTR) promotor control. The construct was transfected into a retroviral packaging cell line and a high-titre retroviral producer cell line was derived. This retroviral producer cell line, as well as NIH 3T3 cells infected with the virus, expressed the NPM–ALK protein as determined by RT–PCR and immunohistochemistry using an anti-ALK-1 antibody. Cells for retroviral infection were harvested from the marrow of the long bones of young TG54 and FVB/N mice and cultivated with infective supernatants. The infected marrow was transplanted into 8 to 10-week-old lethally irradiated syngeneic recipients. Three different experimental groups with infectious retroviral stocks were analysed in this study.

In group (1), a total of 15 female IL-9 transgenic mice (TG54), designated Ml–Ml5 (Table 1a), were used and transplanted with LNPM–ALKSN-transduced bone marrow cells from 10-week-old male IL-9 transgenic donors, resulting in IL-9/NPM–ALK double positive mice.

Table 1 Characteristics of IL-9 transgenic mice transplanted with NPM–ALK-infected marrow (A), control group with mock infected marrow (B) and wild-type mice transplanted with NPM-ALK-infected marrow (C)

In group (2), a total of eight female IL-9 transgenic mice (TG54), designated M16–M23 (Table 1b), served as a control group. They received LXSN-only infected bone marrow from 10-week-old male donors, resulting in IL-9/control mice.

In group (3), a total of eight female wild-type mice (FVB/N), designated M24–M31 (Table 1c), were used and transplanted with LNPM–ALKSN-transduced bone marrow from 10-week-old male wild-type donors, resulting in NPM–ALK mice.

Macroscopy and histological analyses of IL-9/NPM–ALK transgenic mice

IL-9 transgenic mice, which received NPM–ALK-transduced haematopoietic precursor cells, developed malignant lymphomas after a latency period of 13–30 weeks (Table 1a). All animals developed tumours in prominent abdominal lymph nodes. Interestingly, the peripancreatic lymph node was frequently involved. Retroperitoneal lymph nodes, spleen, bone marrow and liver were also often infiltrated. Histologic analysis of the tumours showed three distinct disease processes: (1) 7/15 (46%) lymphoblastic lymphoma T-cell type (T-LB) (Figure 1a–c); (2) 7/15 (46%) mature and immature plasmacytoma (PC) (Figure 2a–d); and (3)5/15 (33%) plasmoblastic/anaplastic diffuse large-B-cell lymphoma (plasmoblastic) (Figure 3a–d).

Figure 1
figure1

Lymphoblastic lymphoma of T-cell type (T-LB) in the thymus of an IL-9/NPM–ALK-positive mouse. (a) Transversal section through the anterior part of the mediastinum. Thymus (th) was infiltrated by monomorphic lymphoid cells and the normal structure was completely disrupted. Tumour cells infiltrate the large pericardial vessels (pv) and the pericardial fatty tissue (v: atrial valve; h: ventricular muscle). H+E staining; ×40. (b) Section of the lung with typical perivascular (↔) and peribronchiolar (↔) invasion pattern of the tumour cells. Giemsa staining; ×40. (c) High-power view of the tumour. Tumour cells were slightly larger than small lymphocytes, showed more open chromatin (arrows) and small rims of basophilic cytoplasm. ‘Starry sky’ macrophages were scattered throughout the tumour (asterisk). Giemsa staining; ×630 (c)

Figure 2
figure2

Plasmacytoma (PC) in a lymph node of an IL-9/NPM–ALK-positive mouse. (a) Tumour cells were found in the extended paratrabecular areas and near sinuses; remnants of primary and secondary follicles are spared (rln: residual lymph node parenchym). Haematoxylin & eosin staining; ×25. (b) High-power view of mature PC (mpc). Medium-sized and large plasma cells with dense, amphophilic cytoplasm and eccentric nuclei with clockface-like chromatin (arrows). (c) High-power view of immature PC (ipc). Large plasma cells and many plasmoblasts (arrows) with open chromatin, prominent nucleoli and vacuolated cytoplasm. (d) Immunostaining of the lymph node with the antibody ALK-1. Immature (ipc) and mature (mpc) PC exist in parallel. Some sinuses and residual lymph node parenchyma (rln) were untouched. In the mpc the tumour cells showed only a weak cytoplasmic ALK-1 staining, while in the ipc the tumor cells showed a strong cytoplasmic and/or nuclear staining. DAKO ARK™ (animal research kit) technique; diaminobenzidine-haematotoxylin counterstain; ×100

Figure 3
figure3

Plasmoblastic lymphoma in a lymph node of an IL-9/NPM–ALK-positive mouse. (a) Intrasinusoidal and interfollicular growth pattern of the plasmoblastic lymphoma (pbl) in a lymph node next to normal pancreas (indicated by arrows). Remnants of a primary follicle and of the parafollicular area were marked (rln: residual lymph node parenchyma). (b) Morphologically, the tumour cells are comprised of monomorphic large immunoblast-like cells, frequently containing large central nucleoli (arrows). (c–d) Immunostaining of the lymph node with the antibody ALK-1. The tumour cells showed a strong cytoplasmic and/or nuclear staining (asterisks). DAKO ARK™ (animal research kit) technique; diaminobenzidine-haematotoxylin counterstain; ×400 (c) and ×1000 (d)

In IL-9/NPM–ALK double positive mice, mature and immature PC were simultaneously present. In four out of 15 mice, either PC or plasmoblastic lymphoma was identified in combination with T-LB, where malignant cells of both tumours could sometimes be found in spleen and abdominal lymph nodes. A summary of these three groups of mice is given in Table 2. A detailed pathologic analysis of each of these lymphoma types is presented below.

Table 2 Summary of the results of the three groups of mice

Lymphoblastic lymphoma T-cell type (T-LB)

Of 15 mice, seven had mediastinal tumour masses in the thymus (Figure 1a). Infiltration of the mediastinum and the lungs as well as nodal and haematogenous spread was frequently observed in advanced stages (Figure 1b). Additionally, bone marrow, kidney, stomach, liver and spleen were affected in several cases. The neoplasms were often composed of sheets of medium-sized blasts with scant basophilic cytoplasm and moderately condensed nuclear chromatin. In a few cells we found cytoplasmic vacuoles and many mitotic figures, a prominent ‘starry sky’ pattern and paracortical spreading in early lymph node metastasis sparing the B-cell follicles and the medulla. As in human T-lymphoblastic lymphoma, the murine tumour cells exhibited polymorphism. Tumour cells were small to medium size, and sometimes had more open chromatin and often visible nucleoli. In some tumours, a considerable number of the cells showed a broader and more basophilic cytoplasm than in their human counterparts, occasionally resembling Burkitt's lymphoma (Figure 1c). When solid organs were infiltrated, a typical perivascular infiltration was found. When using immunohistochemistry and FACS analyses, the lymphomas always tested positive for T-cell antigens; CD3 was always present and CD5 was identified in a small number of the cases. Moreover, some tumours were composed of CD4 and CD8 double positive T-cells, while others showed a single positive staining pattern in the majority of the cells. Some cases were weakly positive for CD25. Neither B-cell nor myeloid antigens could be detected.

Plasmacytoma (PC)

Of 15 mice, seven showed enlarged abdominal lymph nodes with atypical plasma cells. These cells were oval, had round and eccentric nuclei with so-called ‘spoke wheel’ chromatin without visible nucleoli. Other tumour cells had abundant basophilic cytoplasm with a perinuclear halo. Sometimes plasma cells showed a more dispersed nuclear chromatin, various-sized nucleoli and an increase in the nucleus to cytoplasm ratio (Figure 2b). Although murine plasma cells are, in general, more variable than human plasma cells, atypias can be easily identified in the more immature PCs (Figure 2c). We observed cases with plasmacytosis, where large sheets of plasma cells were found in the paratrabecular areas and near sinuses, sometimes extending from there to the paracortical areas and thus increasingly distorting the regular lymph node architecture (Figure 2a). While in some animals only one or a few lymph nodes were involved, others showed a more generalized disease with involvement of the bone marrow, spleen, liver and even solid organs like lungs and kidneys. Immunophenotypic analysis of the PC showed that the tumour cells expressed the plasma cell marker CD38, but were negative for other B- and T-cell markers. Some cases showed weak CD25 expression. Only the more mature plasmacytomas showed slight CD 19 expression. Tumour cells from the lymph nodes where the overall architecture was completely distorted, displayed a light chain restriction either for kappa or lambda. In contrast, lymph nodes that were not disrupted completely, revealed a polyclonal staining pattern.

Plasmoblastic lymphoma

Of these 15 mice, five developed a more aggressive disease with high-grade lymphoma, which could not be easily classified using only cytological criteria. Using Giemsa staining, we identified lymphomas that were composed of large immunoblast-like cells with deeply basophilic and abundant cytoplasm. These cells showed round to slightly pleomorphic clear nuclei with little chromatin staining and prominent, sometimes centrally located, solitary nucleoli (Figure 3b). These observed nucleoli rarely resembled inclusion-like nucleoli similar to those seen in Hodgkin cells. Additionally, some bi- or multinucleated cells were found, some of which resembled Reed–Sternberg cells. When lymph nodes were infiltrated by the tumour cells, a significant sinusoidal growth pattern could be observed (Figure 3a), after which the T-zones were invaded with a sparing of the follicles and, finally, the complete lymph node architecture was effaced. These cytological and morphological features are typical hallmarks of ALCL. Furthermore, a deeply basophilic cytoplasm and the immunoblastic/plasmoblastic appearance of a varying number of tumour cells was observed in some tumours. These features were not completely fitting with those of the human counterpart of ALCL of T- or 0-cell type. Moreover, in some areas of the tumours, a transition from immature plasma cells to plasmablasts and anaplastic cells was seen. These morphological criteria strongly resemble those of a recently reported subtype of diffuse large-B-cell lymphoma with full-length ALK expression in the human system. Using immunohisto-chemistry and FACS analyses, we found strong staining for CD38 in the plasmablastic/anaplastic lymphomas, while all other used T- and B-cell markers were negative. Monoclonality could be detected by immunoglobulin light chain restriction, where three out of five tumours showed kappa light chain restriction and IgA, and two cases showed lambda light chain restriction in combination with IgG.

Macroscopy and histological analyses of the IL-9/control mice

Mice in the control group were examined at different time points ranging from 9 to 30 weeks after transplantation. During this time course, no signs of disease or spontaneous deaths occurred. A summary of the results obtained for these mice is given in Table 1b. Histological analyses show no tumour growth in 62.5%, but a slight to moderate polyclonal plasmocytosis was a frequent finding in the abdominal lymph nodes. In three cases, lymph node enlargement was observed; here, plasmacytosis was marked with the onset of effacement of the T-zones of the lymph nodes and little variances of plasma cell nuclei. Complete lymph node destruction or atypical plasma cells were not found in the control mice. Metastasis, taken as the most important sign of malignancy, was never seen in the IL-9 transgenic control mice. We investigated clonality in these cases by immunohistochemistry and found a polyclonal pattern. In three out of eight mice, the thymuses showed an enlargement of the cortical areas with a slight increase in the cortex/medulla ratio. One mouse (M21) developed a thymic lymphoma after 23 weeks, as described for IL-9 transgenic mice.

Macroscopy and histological analyses of FVB/N-NPM–ALK wild-type mice

FVB/N mice (wild-type mice) that were transplanted with NPM–ALK—infected haematopoietic precursor cells showed, in all eight mice, tumours after, on average, 6 months of age. Six of these mice developed PC and the other two suffered from more immature to plasmoblastic tumours ( Table 1c). Tumour cells were positive for the plasma cell marker CD38, but were negative for the other B- and T-cell markers as well as for myeloid antigens. The morphology was the same as described for PC in IL-9/NPM–ALK mice.

ALK expression analysis by RT–PCR andALK-l staining

In all but one tumour, strong NPM–ALK expression was detected by RT–PCR analysis. Infiltrated organs of some animals were also analysed for NPM–ALK expression. Here the NPM–ALK cDNA could also be detected in lymph node, bone marrow, spleen, liver and thymus. The NPM–ALK expression pattern is indicated in Table 1. NPM–ALK was not detected in the marrow, thymus or spleen of the control group, pLXSN reconstituted mouse. Figure 4 depicts typical results exemplary for M3 (IL-9/NPM–ALK) and Ml7 (IL-9/mock). NPM–ALK expression was also detected at the protein level by immunohistochemistry using ALK-1 antibody. As expected, the plasmoblastic lymphomas expressed high levels of NPM–ALK and exhibited strong cytoplasmic staining with some cells showing nuclear staining (Figure 3c,d). PC show weak to moderate expression of NPM–ALK. Even in cases where immature and mature PC exist simultaneously, the immature PC show a stronger staining pattern in contrast to mature PC (Figure 2d). NPM–ALK expression can hardly ever be detected on protein level in tumour cells of T-LB. The ALK-1 staining pattern is indicated in Table 1.

Figure 4
figure4

RT–PCR detection of NPM–ALK transcript of various organs of IL-9/NPM–ALK reconstituted mouse (M3). Lanes 2–5 demonstrate detection of the predicted NPM–ALK 515 bp band in liver, tumour, spleen and bone marrow. Mock transduced IL-9 transgenic mice (Ml 7) were used as a negative control and showed no evidence of NPM–ALK transcripts in spleen (lane 6) and bone marrow (lane 7). Lane 8 negative control: no template cDNA. The bottom part shows the 805 bp mouse β-actin DNA fragment in the same samples. HMS: high molecular weight DNA marker

Serial transplantation of NPM–ALK–positive tumour cells into secondary recipients

Tumour cells derived from lymph nodes with plasmoblastic features were injected intraperitoneally into secondary recipient mice. These mice developed tumours within 4–10 weeks. The lymphomas observed in these secondary recipient animals were histologically and immunophenotypically identical to the primary tumours.

Discussion

Mutations in pathways that control cell proliferation, survival and apoptosis are critically involved in many, if not most, cancers. An imbalance between cellular growth and dying is the most important feature of tumorigenesis. Genes involved in these pathways encode for growth factors, their receptors and signal transduction molecules, or affect cell cycle control or apoptosis. IL-9 is a multifunctional cytokine produced by activated T-cells (Demoulin and Renauld, 1998). IL-9 seems to be involved in both human and murine tumorigenesis. In vitro, IL-9 induced proliferation of T-cell lymphomas and protects them against dexamethasone-induced apoptosis (Vink et al., 1993; Renauld et al., 1995). In vivo, IL-9 transgenic mice spontaneously developed precursor T-lymphoblastic lymphomas (Renauld et al., 1994). Within the mouse system, IL-9-dependent T-cells were transfected with IL-9, which resulted in an autocrine production of IL-9 and independent clonal T-cell growth. The injection of these cells into syngeneic mice resulted in the development of malignant lymphomas in all animals (Uyttenhove et al., 1991). The detailed histological analysis of such tumours revealed a high-grade lymphoma of T-cell type with many features of human ALCL, including cytological, morphological, phenotypical and genetic characteristics (Bittner et al., 2000). Finally, the existence of an IL-9-mediated autocrine loop in ALCL and Hodgkin's disease has been suggested. In cases of human ALCL of T- or 0-cell type, IL-9 expression was detectable; thus, we argued that IL-9 may be an important growth factor for tumour cells of this lymphoma entity (Merz et al., 1991).

The receptor tyrosine kinases (RTKs) represent a family of cell surface receptors that play an important role in regulating the survival, growth and differentiation of haematopoietic cells. Deregulation of RTKs activation has been shown to result in a variety of neoplastic disorders. One such RTK is ALK, a novel RTK of the insulin receptor subfamily, whose expression is normally restricted to neural tissues and testis (Morris et al., 1997). Karyotypic analysis has identified a t(2;5)(p23;q35) chromosomal translocation in more than half of the cases of ALCL (Le Beau et al., 1989; Morris et al., 2001). The relevance of this is that ALK is deregulated because of the translocation, and in this form it has oncogenic potential. Cloning of the t(2;5) translocation shows that the resulting chimeric protein consists of the amino-terminal portion of NPM linked to the cytoplasmic catalytic domain of ALK. The NPM–ALK fusion gene encodes for a constitutively activated tyrosine kinase, which contributes significantly to malignant transformation. The oncogenic potential of NPM–ALK has already been demonstrated by its ability to transform rodent fibroblasts and transform cell lines from factor-dependent to factor-independent growth (Fujimoto et al., 1996; Wellmann et al., 1997). However, the observations from the experiments from Kuefer et al. suggested that NPM–ALK expression alone may not be sufficient to induce lymphomagenesis, especially ALCL (Kuefer et al., 1997). NPM–ALK overexpression in haematopoetic precursor cells of mice rarely caused lymphoid malignancies after a long latency period, and when it did, this involved the mesenteric lymph nodes, with metastases to the lungs, kidneys, liver and spleen. Immunophenotypic and genotypic analyses demonstrated a B-cell origin for the lymphomas induced by NPM–ALK in this tumour model. The animal model and human ALCL demonstrated that overexpression of ALK may have effects on both T- and B-cells. This conclusion was supported by the finding that, in a subtype of human diffuse large-B-cell lymphomas rare cases exist, which expressed the full-length ALK receptor protein while lacking t(2;5) translocation (Delsol et al., 1997). Taken together, both IL-9 and ALK exert growth regulating and antiapoptotic properties, and thus we argued that IL-9 and NPM–ALK may cooperate in the pathogenesis of human ALCL.

To verify this hypothesis, we used IL-9 transgenic mice reconstituted with NPM–ALK. All of these animals developed lymphoid neoplasias after a short time of latency, but T-type ALCL—as would be expected—was not observed. Instead, we found mature and immature plasmacytoma in one-third of the animals, in another third plasmablastic/anaplastic lymphoma of B-cell type, and in nearly a half T-cell lymphoblastic lymphoma. Hence, NPM–ALK reinforces and accelerates the development of T-LB in IL-9 transgenic mice. IL-9 transgenic mice showed a sporadic development (7%) of spontaneous thymic lymphomas at the age of 6–9 months, without histological changes in secondary lymphoid organs. IL-9/NPM–ALK mice develop T-LB with spreading into multiple organs, only 14 weeks after transplantation. Interestingly, one-fourth of the IL-9/NPM–ALK mice suffer simultaneously from two different lymphoma entities, either lymphoblastic lymphoma and plasmacytoma/plasmoblastic lymphoma or from plasmacytoma and plasmoblastic lymphoma. In cases with growing T-LB, some of the infiltrated abdominal lymph nodes or the spleen also showed significant plasmacytosis and sometimes also atypical plasma cells. Additionally, we found animals with a varying cytological picture, with one lymph node displaying just plasmacytosis and another already showing mature or immature plasma cytoma or even plasmoblastic/anaplastic high-grade lymphoma.

In contrast, IL-9 transgenic mice that received mock infected bone marrow cells showed inconspicuous macroscopy and histology of lymph nodes and haematopoietic organs. Half of the animals showed slight to moderate plasmacytosis of lymph nodes and occasionally of the spleen. However, neither severe atypical plasma cells nor metastasis or organ destructive infiltrates could be found. This plasmacytosis is probably IL-9 triggered and was most prominent in abdominal lymph nodes. IL-9 is known to act as a differentiation factor on late-stage B-cells (Ig switch) (Dugas et al., 1993; Petit-Frere et al., 1993). True signs of plasma cell malignancy were not found in IL-9 transgenic mice. We investigated clonality in these cases by immunohistochemistry and found a polyclonal pattern. However, it has to be pointed out that murine plasma cells are mostly of kappa type (kappa : lambda =10 : 1), and therefore the detection of kappa light chain restriction using immunohistochemistry in the mouse system is sometimes difficult. In a larger series of transgenic animals, including 2 mouse lines with different IL-9 insertions, PCs were not observed (Renauld et al., 1994). Advanced age and irradiation greatly increase the spontaneous incidence of lymphoma in this mice strain, which might also explain the detection of prominent plasmacytosis in the control a mice. Thus, it appears that ALK expression may be more important for the development of plasmacytoma and plasmacytic/plasmoblastic tumours than IL-9. Indeed, when we examined wild-type FVB/N mice reconstituted with NPM–ALK, we observed plasmacytomas and plasmoblastic lymphomas in all animals, yet lymphoblastic lymphomas were not detected. The mice developed tumours later compared to the double manipulated mice (28 weeks versus 17 weeks) and the frequency of the high-grade plasmoblastic/anaplastic lymphomas seemed to be lower (20 versus 33%). Although a detailed morphological analysis was given in the Results section, a few major points deserve to be highlighted again here.

First, the T-LB were slightly more heterogeneous than their human counterparts, e.g. they had more open chromatin, larger nuclei and detectable nucleoli, and appeared more blastic than human lymphoblastic cells such as Burkitt's lymphoma. Nevertheless, they always developed in the thymus and showed other characteristic features of T-LB (starry sky pattern and immunophenotype) and metastasized like human T-LB.

Second, the observed PCs developed mostly in the peripancreatic or abdominal lymph nodes, although spleen and bone marrow were frequently involved. In humans, primary nodal PC is a rare finding. We speculate that the preferential involvement of abdominal lymph nodes may be related to antigen loading and stimulation by the gastrointestinal tract.

Third, the plasmablastic/anaplastic lymphomas exhibit morphological features of a recently described rare variant of human diffuse large-B-cell lymphoma, with expression of full-length ALK. Morphologically, this lymphoma entity is comprised of monomorphic large immunoblast-like cells, containing large central nucleoli, and the tumour cells tend to invade lymphatic sinuses. These lymphomas express epithelial membrane antigens; also contain intracytoplasmic IgA of a single light chain type and the plasma cell marker CD38, but they lack CD30 (Delsol et al., 1997).

In anaplastic/plasmoblastic lymphomas of the IL-9/NPM–ALK mice, we found immunoblastic, plasmoblastic and anaplastic differentiation as well as prominent intrasinusoidal and interfollicular (T-zone-) growth patterns. These lymphomas were also shown to be, at least in part, positive for IgA and CD38. The intrasinusoidal growth pattern is also characteristic for human ALCL of T- and null-cell type, carcinomas and melanomas, and was not observed in any other human lymphoid neoplasia. Moreover, it seems that there is a transition between mature/immature plasmacytoma and plasmoblastic/anaplastic high-grade lymphoma. We suggest the progressive development of plasmacytic/plasmablastic lymphomas shift in our model as follows:

It will be interesting to further analyse human immature plasmacytoma and plasmoblastic/anaplastic lymphomas for ALK expression. So far, ALK expression has not been reported in human plasmacytoma or multiple myeloma.

In summary, we have shown that NPM–ALK expression in haematopoietic cells and in the settings of IL-9 transgene expression leads to the development of murine plasmacytoma, plasmoblastic/anaplastic lymphoma and precursor T-lymphoblastic lymphoma. This suggests that there are significant additive effects on lymphomagenesis by each of the two ‘genetic’ hits. However, the precise mechanism through which the transgene IL-9 and NPM/ALK operates in each individual tumour is not yet clear, and remains to be determined.

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Correspondence to Karin Lange.

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Lange, K., Uckert, W., Blankenstein, T. et al. Overexpression of NPM–ALK induces different types of malignant lymphomas in IL-9 transgenic mice. Oncogene 22, 517–527 (2003). https://doi.org/10.1038/sj.onc.1206076

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Keywords

  • t(2;5) translocation
  • anaplastic lymphoma kinase (ALK)
  • IL-9 transgenic mice
  • retroviral-mediated gene transfer

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