Lymphoma

High expression of several tyrosine kinases and activation of the PI3K/AKT pathway in mediastinal large B cell lymphoma reveals further similarities to Hodgkin lymphoma

Abstract

Mediastinal large B-cell (MBL) and classical Hodgkin lymphoma (HL) have several pathogenic mechanisms in common. As we recently observed aberrant tyrosine kinase (TK) activities in HL, we now analysed also MBL for such activities. Indeed, MBL and HL were the only B-cell lymphomas where elevated cellular phospho-tyrosine contents were typical features. Three TKs, JAK2, RON and TIE1, not expressed in normal B cells, were each expressed in about 30% of MBL cases, and 75% of cases expressed at least one of the TKs. Among the intracellular pathways frequently triggered by TKs, the PI3K/AKT pathway was activated in about 40% of MBLs and essential for survival of MBL cell lines, whereas the RAF/mitogen-activated protein kinase pathway seemed to be inhibited. No activating mutations were detected in the three TKs in MBL cell lines and primary cases. RON and TIE1 were each also expressed in about 35% and JAK2 in about 53% of HL cases. JAK2 genomic gains are frequent in MBL and HL but we observed no strict correlation of JAK2 genomic status with JAK2 protein expression. In conclusion, aberrant TK activities are a further shared pathogenic mechanism of MBL and HL and may be interesting targets for therapeutic intervention.

Introduction

Mediastinal large B-cell lymphoma (MBL) is a distinctive subtype of diffuse large B-cell lymphoma (DLBCL). Patients are usually young, often female and present with localized bulky mediastinal masses, which may contain thymic remnants. The tumor cells express CD19, CD20, CD22, CD79A and PAX5, thus resembling mature B cells.1, 2 However, despite the presence of functionally rearranged immunoglobulin V-genes they do not express any immunoglobulin detectable by immunohistochemistry (IHC),3, 4 and downregulation of the internal IgH enhancer activity likely contributes to this phenomenon.5 Because of the expression of MAL and CD23 in most cases, a derivation from medullary thymic B cells, which express both proteins, is assumed.6, 7

Over the last years, several studies revealed similarities of MBL to the nodular sclerosis (ns) subtype of classical Hodgkin lymphoma (cHL).8 Both entities frequently affect young individuals and nsHL is also often localized in the mediastinum. Morphologically, a more or less extensive sclerosis is a common feature of both entities and, owing to the presence of interspersed small lymphocytes and eosinophils in MBL, some MBL infiltrates may resemble cHL.1

Furthermore, molecular analyses indicated common pathogenetic mechanisms for MBL and nsHL. Comparative genomic hybridization of primary tumor cases revealed that gains of chromosomes 2p13–16 and 9p24 were the most frequent genomic imbalances in both entities.8, 9, 10, 11, 12, 13, 14 Gains in 2p encompass the nuclear factor-κB (NF-κB) family member REL, and constitutive activation of the NF-κB pathway is usually observed in both lymphoma types.15, 16, 17 The 9p24 gains encompass the JAK2 tyrosine kinase (TK), which activates STAT6, and constitutive STAT6 activation is another common feature of both lymphoma types.18, 19 In two global gene expression analyses sets of genes discriminating MBL from other DLBCL were identified, and a substantial fraction of these genes was regulated in a similar manner in MBLs and cHL cell lines as compared with DLBCLs.20, 21

A pathogenetic mechanism frequently observed in tumorigenesis is the aberrant activation of TKs. We recently described the aberrant expression and activation of six different receptor tyrosine kinases (RTK) in cHL as compared with the non-neoplastic counterparts of the tumor cells, which was most pronounced in nsHL and indicated that aberrant TK activity may play an important role in the pathogenesis of nsHL.22 Given the similarity of nsHL and MBL regarding pathogenetic mechanisms, we investigated in the present study if aberrant TK activity and activation of intracellular signalling pathways triggered by TKs could also contribute to MBL pathogenesis.

Materials and methods

Tissue samples and cell culture

Tissue samples were retrieved from the files of the Department of Pathology of the University of Frankfurt and were originally submitted for diagnostic purposes. Paraffin sections of six cHL with known JAK2 genomic status as detected by fluorescence in situ hybridization (FISH) were retrieved from the Institute of Pathology, Lymph Node Registry Kiel, University Hospital Schleswig-Holstein Campus Kiel. Altogether, 38 MBL samples and, in addition to the HL cases analysed in our previous study,22 46 cHL samples were used for TIE1 and JAK2 stainings and 25 further nsHL and 65 further mixed cellularity (mc) HL cases for the pan-phospho-tyrosine-specific stainings. All tissue samples were studied in accordance with national ethical principles. The MBL derived cell lines Karpas1106P and MedB-1 were grown in RPMI1640 supplemented with 20% fetal-calf serum (FCS) for Karpas1106P and 10% FCS and 50 mM Hepes for MedB-1, and the HL cell lines L1236, L428 and KMH2 were grown in Roswell Park Memorial Institute media (RPMI)-1640 with 10% FCS and HDLM2 in RPMI-1640 with 20% FCS.23, 24

Analysis of global gene expression data

Global gene expression data sets of 76 MBL and 34 DLBCL obtained using U133A and B microarrays of Affymetrix were retrieved from the Supplementary Data of Savage et al. (www.genome.wi.mit.edu/cancer/mediastinal.html).21 The dataset was loaded in the GeneSpring software (Version 7.2, Agilent Technologies, Böblingen, Germany) using per chip and per gene normalisation as recommended. A supervised comparison of MBL and DLBCL samples was performed to identify TKs (all TKs from the human kinome (www.stke.org) for which probe sets were identified on the Affymetrix homepage (www.affymetrix.com/analysis/index.affx), i.e., 90 TKs) with a more than twofold differential expression between MBL and DLBCL groups. For generation of a heat map of all TKs the same dataset was analysed using the Cluster and Tree View software,25 performing median normalization and centering of genes.

Immunohistochemistry

For Immunohistochemistry (IHC) 5 μm sections of the formalin-fixed paraffin-embedded tissues were prepared. After antigen retrieval, sections were blocked with normal goat serum (4 μg/ml; Santa Cruz Biotechnology, Heidelberg, Germany) for 15 min and incubated overnight with the primary antibodies in Tris-buffered saline (TBS) at room temperature. For detection of primary antibodies the Envision system (Dako, Hamburg, Germany) with either alkaline phosphatase and Fast Red or horseradish peroxidase with 3.3-diaminobenzidine (both from Dako) as substrates were used. Antigen retrieval procedures, suppliers and concentrations at which antibodies against PDGFRA, DDR2, EPHB1, RON, TRKB, TRKA and phospho-tyrosine (p-Y)(4G10) were used as described previously.22 For anti-TIE1 and anti-JAK2 antibodies (Santa Cruz Biotechnology, sc-342 and sc-278, respectively), sections were cooked for 15 min in 600 ml citrate buffer (10 mM sodium (Na) citrate pH 6.0) in a microwave oven at maximum power for antigen retrieval. The anti-TIE1-antibody was diluted 1:100 and the anti-JAK2-antibody 1:50 in Tris-buffered saline. Specificity of stainings was tested by pre-incubation of the antibodies with 20-fold amounts of the peptides used for immunization. For both, TIE1 and JAK2, this pre-incubation strongly reduced or completely abolished staining. Specificity of the TIE1 staining was further confirmed using another TIE1-specific antibody (AF619, R&D, Wiesbaden, Germany) at a 1:50 dilution with 15 min cooking in citrate buffer for antigen retrieval. From the cases analysed, the same staining patterns were observed with both anti-TIE1 antibodies.

Intracellular signalling was analysed with antibodies specific for phospho-Ser473-AKT, phospho-Thr202/Tyr204-p44/42-MAPK and phospho-Ser259-RAF (all from Cell Signalling, Beverly, MA, USA no. 4051, no. 4376 and no. 9421). For all three antibodies antigen retrieval was performed by cooking the section for 15 min at maximum power in a microwave oven in 600 ml 10 mM Na citrate pH 6.0. Incubation with primary antibody was performed overnight in TBS with 1:200, 1:100 and 1:50 dilutions, respectively. Specificity of the antibodies was exemplarily verified for each antibody with at least two MBL cases by preincubation with the peptides used for immunization or pretreatment of sections with Lambda protein phosphatase (Upstate, Lake Placid, NY, USA; 1250 U per section in 200 μl buffer (50 mM Hepes pH 7.5, 1 mM ethylenediaminetetraacetic acid, 20 mM manganese chloride and 5 mM Dithiothreitol) for 3 h at 37°C), which in all instances abolished or significantly reduced staining. For detection of bound primary antibody the ABC system with horseradish peroxidase and 3.3′-diaminobenzidine as substrate was used (Dako).

Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) analyses for the detection of chromosomal rearangements of the JAK2 locus in 9p24 were performed using a previously published probe12 and protocol.26 Slides were analyzed using a Zeiss Axioskop-2 fluorescence microscope (Zeiss, Göttingen, Germany) equipped with appropriate filter sets (AHF, Tübingen, Germany) and documented using an ISIS imaging system (MetaSystems, Altlussheim, Germany). Nuclei from Hodgkin-Reed/Sternberg (HRS) cells were identified by their large size and frequent hyperploid genomic status. The ratio between the median number of JAK2 hybridization signals per case and its estimated ploidy level determined as median of number of signals for 12 different loci investigated by FISH, (data not shown) was used to determine the JAK2 genomic status. Ratios between 1.3 and 1.99 were considered as gains whereas ratios equal or higher than two were considered as amplifications.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Cells were grown in 96 well tissue culture plates (starting with 1 × 105 cells/ml) with various concentrations of LY294002 (3–50 μ M). After an incubation time of 24 h 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Calbiochem, Darmstadt, Germany) was added to a final concentration of 0.5 mg/ml. Cells were incubated for further 4 h and then lysed in 5% sodium dodecyl sulfate (SDS) for 12 h at 37°C. Absorbance was measured with a EL311SX microplate reader (Biotek Instruments,Winooski, VT, USA) at 550 nm with the reference wavelength set to 690 nm. Each value was measured as six replicates in three independent experiments. Data were analysed using the GraphPad Prism 4.0 software (GraphPad, San Diego, CA, USA).

Real-time reverse transcriptase–polymerase chain reaction analysis

RNA was isolated from Karpas1106P, MedB-1, L1236, L428, KMH2 and HDLM2 cultures with the RNAeasy kit of Qiagen (Qiagen, Hilden, Germany). One microgram of each RNA was used with random hexamers for first strand complementary DNA (cDNA) synthesis using the Roche first strand cDNA synthesis kit (Roche, Mannheim, Germany) and analysed for RON and TIE1 expression with premade real-time reverse transcriptase–polymerase chain reaction (RT–PCR) Assays-on-Demand of Applied Biosystems (Hs00234013_m1, Hs00892696_m1, Applied Biosystems, Weiterstadt, Germany). B2M was used as reference gene (4326319E of Applied Biosystems).

Western blot analysis and immunoprecipitation

Karpas1106P and MedB-1 cells were lysed by boiling for 10 min in Laemmli buffer. Lysates corresponding to approximately 1 × 105 cells per lane were separated by SDS–polyacrylamide gel electrophoresis (7.5%), blotted onto Polyvinylidene fluoride membranes (BioRad, Munich, Germany), incubated overnight at 4°C with 1:1000 dilutions of anti-TIE1 and anti-RON (Santa Cruz Biotechnology, sc-342 and sc-322) and visualized using the ELC plus system (Amersham Biosciences, Freiburg, Germany). For immunoprecipitation cells were incubated for 2 h with 2 mM Na3VO4 to enrich p-Y-proteins and 1 × 107 cells were lysed in mammalian protein extraction (M-PER) buffer (Pierce, Bonn, Germany). Lysates were precleared with Pansorbin (Calbiochem, Darmstadt, Germany) and incubated with 20 μg 4G10 (or normal mouse Ig as control) antibody bound to Pansorbin at 4°C overnight. Precipitates were washed four times in M-PER buffer and cooked for 5 min in 100 μl Laemmli buffer. For Western blot analysis, 10 μl aliquots were used.

Analysis for mutations in JAK2, RON and TIE1

To analyse for the presence of mutations in the TKs in the cell lines, RNA from Karpas1106P and MedB-1 was extracted using the Qiagen RNAeasy kit (Qiagen) and used for cDNA synthesis with the Roche first strand cDNA synthesis kit and oligo-dT primers (Roche, Mannheim, Germany). For the amplification of the complete open reading frames, 1 μl aliquots of the reactions were used as templates in PCRs with each primer (sequences in the Supplementary Data) at 0.125, 2 mM dNTPs, 2 mM magnesium chloride (MgCl2) and 3.5 U of Expand High Fidelity in 1 × Expand PCR buffer (Roche). Each PCR was run for 45 cycles of 30 s 95°C, 30 s 61°C and 6 min 68°C after an initial denaturation of 2 min at 95°C.

For the analysis of presence of JAK2 V617F mutation in primary MBLs, RNA was extracted either from 10 μm sections of frozen (12 cases) or formalin-fixed paraffin-embedded tissues (10 cases) using Trizol reagent (Invitrogen, Karlsruhe, Germany) followed by clean-up with RNAeasy Minikit columns (Qiagen). cDNA was synthesised using 1 μg total RNA, random hexamer primers and the Roche first strand cDNA synthesis kit (Roche). One microlitre of cDNAs was used as template in PCRs with 0.125 mM each primer (sequences in the Supplementary Data), 2 mM dNTPs and 2 mM MgCl2 and 45 cycles of 30 s 95°C 30 s 61°C and 30 s 72°C after an initial denaturation of 2 min at 95°C.

To search for mutations in TIE1 and RON, genomic DNA from formalin-fixed paraffin-embedded primary cases was extracted with the Qiagen DNA mini kit. A portion of 200–800 ng DNA was used as template in PCRs with 0.125 mM each primer (sequences in the Supplementary Data), 2 mM dNTPs and 2 mM MgCl2 and 45 cycles of 30 s 95°C, 30 s 61°C and 45 s 72°C after an initial denaturation of 5 min at 95°C.

All PCR products were visualized on agarose gels, DNA from bands was extracted with the Qiagen gel extraction kit (Qiagen) and sequenced with the corresponding primers on an ABI 3100 automated sequencer with the Big Dye Terminator cycle sequencing kit (Applied Biosystems).

Results

Elevated cellular phospho-tyrosine content is a shared feature of MBL and nsHL among mature B-cell lymphomas

In tumor cells with aberrant TK activity as compared with their normal counterparts cellular phospho-tyrosine (p-Y) content is often elevated and can be detected by IHC with pan-p-Y-specific antibodies (Supplementary Information, Figure 3).27 Our previous analysis of several B-cell lymphomas (mantle cell lymphoma, follicular lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia, DLBCL and lymphocyte-predominance and cHL) with a pan-p-Y-specific antibody revealed that among B-cell non-Hodgkin lymphomas (B-NHL) only minor fractions of cases (0–21%) contained aberrantly high amounts of p-Y (Table 1 and22). Distinct from the B-NHLs, the majority of nsHL cases showed elevated p-Y contents in the HRS cells, and further analysis showed that this was due to the aberrant expression and activation of several RTKs.22

Table 1 Analysis of cellular p-Y content in B-cell lymphomas using a pan-p-Y-specific antibody

In the present study, we analysed 37 cases of MBL for cellular p-Y content by IHC and extended our analysis of nsHL cases (Table 1, Figure 1). Fifteen of the 37 (40%) of the MBL, and 34 of 64 (53%) of the nsHL cases had aberrantly high cellular p-Y contents (Table 1). MBL was thus beside nsHL the only entity among mature B-cell lymphomas with elevated cellular p-Y content in a large fraction of cases. This observation indicated that aberrant TK activity might contribute also to MBL pathogenesis, and we thus tried to identify the aberrantly activated TKs in MBL.

Figure 1
figure1

Immunohistochemical stainings of MBLs. Immunohistochemical stainings of a MBL case with the pan-phospho-tyrosine-specific antibody (4G10, Upstate), (a) and examples of MBL cases with immunohistochemical stainings for RON, TIE1 and JAK2, respectively, are shown (bd). Immunohistochemical stainings of cHL cases for TIE1 and JAK2, respectively, are shown in (e) and (f). Stainings of MBLs for p-AKT and p-RAF-Ser259 are shown in (g) and (h), respectively. Primary antibodies were visualized using horseradish peroxidase with 3.3-diaminobenzidine and in (ac, g, h) and alkaline phosphatase and Fast Red as substrate in (df).

Aberrant expression of the TKs TIE1, JAK2 and RON in MBL

To identify TKs aberrantly expressed in MBL as compared with their proposed normal counterparts, that is, thymic B cells, we performed IHC for candidate TKs on MBLs and normal thymi. Because of the several similarities of MBL and cHL, all six RTKs previously identified as aberrantly expressed in HL were considered as such candidate TKs.22 In addition, we reanalysed published global gene expression data of MBL obtained with Affymetrix U133 gene expression microarrays to identify further candidate TKs.21 Owing to a lack of data for normal B cells in this data set, these analyses were restricted to the identification of TKs more strongly expressed in MBL than in other DLBCLs.21 In a supervised analysis of 34 MBLs and 76 DLBCLs using the GeneSpring software no TK with an at least twofold stronger expression in the MBL group as compared with the DLBCLs was identified. A visual inspection of a heat map of TK expression in both lymphoma groups generated with the Cluster and TreeView software indicated, however, that JAK2 was strongly expressed in 15 of 34 and TIE1 in 12 of 34 MBL cases (Supplementary Information, Figure 1), and both were thus considered as candidate TKs.

IHC for the eight candidate TKs revealed that three were expressed in significant fractions of MBLs but not in normal thymic B cells and, in line with the RNA expression data, in much smaller fractions of DLBCL (Figure 1, Supplementary data Figure 2, Table 2). TIE1 was expressed in 13 of 35 MBLs (37%), JAK2 in 10 of 28 MBLs (36%) and RON in 12 of 38 MBL cases analysed (31%). The other five RTKs (PDGFRA, DDR2, EPHB1, TRKB and TRKA) were only expressed in minor fractions of cases (3–11%) (Table 2). In 75% of all MBL, analysed at least one of the three TKs TIE1, JAK2 or RON was expressed, and in about 30% of cases, two of the three TKs were coexpressed. In addition, in 10 of the 13 cases, which reacted with the pan-p-Y antibody at least one of the three TKs was expressed.

Table 2 Immunohistochemical detection of aberrant TK expression in MBL, nsHL and mcHL

TIE1 and JAK2 are also aberrantly expressed in HRS cells of cHL

Given the similarities between MBL and cHL we analysed whether, beside the already known RON expression in cHL (in 36% of cases), also TIE1 and JAK2 were detectable by IHC in cHL (Figure 1, Table 2). TIE1 expression was observed in 13 of 40 cHL cases (32%), with a preferential expression in the ns subtype, where 10 of 20 (50%) cases were positive, whereas only three of 20 mc cases (15%) showed positivity. JAK2 expression was observed in 35 of 66 cHL cases (53%), and expression was more frequent in ns subtype than in mc subtype (21 of 31 (68%) and 14 of 35 (40%) cases, respectively). In normal lymphatic tissues, (lymph node, tonsil and spleen) neither TIE1 nor JAK2 expression was observed in B cells (Supplementary Information). All three TKs aberrantly expressed in MBL were thus also expressed at comparable frequencies in cHL.

The PI3K/AKT pathway is activated and the RAF/MAPK pathway seems to be inhibited in MBL

Given the aberrant expression of RON and TIE1 in MBL, we analysed whether intracellular signalling pathways frequently triggered by receptor tyrosine kinase (RTK)s, namely the RAF/mitogen-activated protein kinase (MAPK) and the Phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT) pathways, are activated in MBLs.28, 29, 30 Activation of the RAF/MAPK and PI3K/AKT pathways were analysed using antibodies specific for Thr202/Tyr204-phosphorylated MAPK and Ser473-phosphorylated AKT, respectively. Using such antibodies for IHC, we observed that only in two of 27 (7%) MBL cases the MAPK was activated whereas p-AKT was detected in 10 of 24 (42%) MBLs (Table 3, Figure 1). Of the nine p-AKT positive cases where information about TK expression was available, three cases expressed none, two cases expressed one, and four cases expressed two of the three TKs.

Table 3 Intracellular signalling pathways activated in MBL

As AKT can negatively regulate the RAF/MAPK pathway by phosphorylation of RAF in the regulatory domain on Ser259,31, 32 we used an antibody specific for p-Ser259-RAF to analyse whether activated AKT might inhibit the RAF/MAPK pathway in MBL. In line with the low fraction of p-MAPK positive cases, we observed p-Ser259-RAF positivity in 11 of 28 (39%) cases (Table 3, Figure 1). However, in individual cases no correlation between activation of the PI3K/AKT pathway and Ser259 phosphorylation of RAF was observed (of 14 cases for which both, the p-AKT and the p-RAF IHC were informative, eight were p-AKT and nine p-RAF positive, and only three of those were positive for both stainings). Together, these findings indicate that the PI3K/AKT pathway is frequently activated in MBLs whereas the RAF/MAPK pathway seems to be inhibited.

PI3K is inhibited by LY294002. We used this compound to analyse the importance of PI3K activation for survival of the two cell lines so far established from MBLs, Karpas1106P and MedB-1 (Figure 2). Both cell lines showed sensitivity towards LY294002 at concentrations causing specific inhibition of AKT in other cell lines (e.g., Jurkat and HRS cell lines) and a LY294002-dependent decrease in AKT phosphorylation,33, 34 suggesting that constitutive PI3K signalling is, as for most cell lines, indeed also an important survival factor for these MBL-derived cell lines.

Figure 2
figure2

Sensitivity of Karpas1106P and MedB-1 towards the PI3K inhibitor LY294002. Karpas1106P, MedB-1 and for comparison also the LY294002 sensitive T-cell leukemia-derived cell line Jurkat were incubated at 1 × 105 cells/ml with various concentrations of LY294002 for 24 h. Survival was monitored with a MTT assay (based on the activity of the mitochondrial respiratory chain) (a). Western blot analysis with a phospho-AKT-specific antibody (b) showed that treatment of the three cell lines with LY294002 for 24 h before lysis significantly reduced the amount of phosphorylated AKT, indicating that the effect of LY294002 on the three cell lines is largely owing to inhibition of PI3K.

No activating mutations are present in JAK2, RON and TIE1

TKs are frequently activated by mutations and we analysed the transcripts encoding JAK2, RON and TIE1 in the two MBL cell lines for potentially activating mutations. JAK2 protein expression in Karpas1106P and MedB-1 has been shown previously,35, 36 and our real-time RT–PCR analysis indicated that RON was also expressed in both cell lines and TIE1 in Karpas1106P (data not shown). In addition, TIE1 transcripts were also detected in the HL cell line KMH2 but not in L1236, L428 and HDLM2 HL cell lines (data not shown). In line with the real-time PCR, Western blot analysis demonstrated TIE1 protein expression in Karpas1106P, whereas significant RON protein expression was only observed in MedB-1, although the real-time PCR analysis indicated only small differences of RON RNA expression levels between Karpas1106P and MedB-1 (Figure 3). We then used RT–PCRs to amplify the complete open-reading frames of JAK2 and RON from both cell lines and TIE1 from Karpas1106P. The sequence analysis of these RT–PCR products revealed neither presence of potentially activating mutations nor could the reason for the lack of RON protein expression in Karpas1106P be clarified. However, despite the lack of mutations the three TKs were activated in the MBL cell lines. Activation of JAK2 in both cell lines as determined by p-JAK2 specific antibodies has been shown previously,35, 36 and immunoprecipitation with a pan-p-Y-specific antibody and subsequent Western blotting with TIE1 and RON-specific antibodies revealed that both RTKs are also phosphorylated (Figure 3).

Figure 3
figure3

Western blot analysis of RON and TIE1 expression and activation in MBL and HL cell lines. Lysates corresponding to approximately 1 × 105 cells were loaded per lane and after blotting to membranes probed with the anti-RON (a) and anti-TIE1 (b) antibodies used for IHC. For RON the HL cell lines L428 and L1236 were included as positive and KMH2 as negative control. Although nearly similar amounts of RON transcripts were observed in Karpas1106P and MedB-1 with real-time RT–PCR (data not shown) only for MedB-1 strong RON protein expression was observed. TIE1 expression was only observed in Karpas1106P and the HL cell line KMH2, in line with real-time PCR analysis, where among the HL cell lines L1236, L428, KMH2 and HDLM2 only KMH2 showed TIE1 expression (data not shown). Loading of equal amounts of proteins was controlled with an anti-actin antibody. For analysis of the activation status of the RTKs, cell lines were incubated for 2 h with 2 mM Na3VO4 before lysis to enrich tyrosine-phosphorylated proteins. P-Y-proteins were immunoprecipitated from lysates of 1 × 107 cells with a pan-p-Y antibody (4G10)(lanes B in (c) and (d) and 1/10 of the lysates were analysed with RON (c) and TIE1 (d) specific antibodies. Normal mouse Ig was used as negative control (lanes A in (c) and (d)).

A constitutively dimerized and activated RON form encoded by a splice variant lacking exon 11 has been described in different tumor types.37 Using quantitative RT—PCR, this splice variant was also detected in MBLs but also in several lymphatic organs with no signs of neoplasia (data not shown), indicating that presence of the splice variant is not necessarily correlated with neoplasia.

Recently, several groups described a G to T transversion in the pseudokinase domain of JAK2, which causes constitutive JAK2 activation in myeloproliferative diseases.38 Given the aberrant JAK2 expression in about a third of MBLs, we analysed 15 cases of MBL by RT–PCR and sequencing for presence of the described G to T transversion. However, all cases displayed the wild-type sequence, in line with a recent analysis of 20 MBL cases, indicating that in MBL V617F replacement in JAK2 does not occur.39

So far no recurrent activating mutations in RON and TIE1 have been observed in human neoplasias. However, as the juxtamembrane regions of RTKs are often affected by activating mutations we amplified the genomic regions encoding these parts (exons 13 and 14) of TIE and RON from TIE1 and RON expressing MBLs. In addition, we sequenced exon 20 of TIE1, because the arginine corresponding to the one affected by the R849W mutation causing constitutive activation of TIE2 in vascular dysmorphogenesis is located in this exon.40 Furthermore, as two experimentally introduced mutations in exon 18 cause constitutive activation of RON, we also sequenced exon 18 from primary cases.41 However, neither in the seven TIE1 expressing (four of these being p-AKT positive) nor in the six RON expressing cases (one of these being p-AKT positive) analysed mutations were observed.

Genomic JAK2 gains are often but not strictly accompanied by JAK2 expression

Gains of the JAK2 gene localized in chromosome band 9p24 are frequently observed in MBL and cHL by comparative genomic hybridization and FISH (about 60 and 30% of cases, respectively),9, 12 and these gains might cause aberrant JAK2 expression.9, 10, 13, 14 In addition, constitutive activation of JAK2 in the MBL-derived cell line MedB-1 has been shown.35 To analyse whether genomic gains of the JAK2 locus correlate with increased JAK2 protein expression, IHC for JAK2 was performed with 26 cases of cHL with known JAK2 genomic status as determined by FISH. Although 14 of 16 cases with genomic JAK2 gains showed expression of JAK2 protein, also six of 10 cases without genomic JAK2 gains were positive in JAK2 IHC. These findings indicate that in addition to the JAK2 genomic gains, also other mechanisms contribute to the high JAK2 expression, at least in cHL.

Discussion

MBL and nsHL show several similarities, and it is likely that also similar transformation mechanisms contribute to lymphomagenesis in both entities. Our analysis now revealed a further similarity in this regard as both entities were the only ones among several types of B-cell lymphomas where significant fractions of cases contained elevated cellular p-Y contents indicative of aberrant TK activity. Taking into account that p-Ys are very unstable and often not well preserved even in cases with constitutively activated TKs,27, 42 it is likely that in the majority of cases of MBL TKs are aberrantly activated, as has been previously shown for nsHL.22

An IHC analysis for aberrantly expressed TKs in MBL identified three TKs, JAK2, RON and TIE1, each of which was expressed in about 30% of cases. Together, about 75% of MBL cases aberrantly expressed at least one of these TKs and about 30% of cases showed coexpression of two of the TKs. The vast majority of cases (10 of 13) showing positivity in the pan-p-Y stainings expressed at least one of the three TKs. This may indicate that the elevated cellular p-Y content of MBLs is largely owing to the aberrant expression of these three TKs. However, as our IHC analysis was largely based on the inspection of the available global gene expression data and therefore limited to a comparison of MBL to DLBCL and not to normal B cells, we may have missed TKs aberrantly expressed in both entities.

For one of the TKs, JAK2, constitutive activation in the MBL-derived cell line MedB-1 has recently been shown,35 and here we show that also TIE1 and RON are activated in the MBL cell lines. With the currently available reagents, however, we could not analyse the activation status of the three TKs in primary MBLs directly. In a search for activating mutations in the TKs in the MBL cell lines and primary cases so far no mutations were detected. RTKs like TIE1 and RON can also be constitutively activated by their ligands via auto- or paracrine mechanisms, but analysis of the global gene expression data indicated that there is no significant expression of the RON ligand MST1 in MBL, and the ligand for TIE1 is unknown.21 However, the selection of the tumor cells for aberrant expression of each of the three TKs in about 30% of cases, the activation of the TKs in the MBL cell lines and the high cellular p-Y content in primary cases strongly argues for an essential role and activation of the TKs in MBL pathogenesis.

The aberrant JAK2 activity in MBL seems to be due to two different genetic aberrations. On the one hand, inactivation of the SOCS1 protein owing to deletions or mutations prevents degradation of the JAK2 protein,35, 43 and on the other hand JAK2 gene copy number gains may cause a gene dosage effect.12 For the latter mechanism, however, our data indicate that JAK2 gene copy gains in MBL are only in a fraction of cases accompanied by JAK2 protein expression detectable by IHC (60% of cases show JAK2 gene gains in previously published series,8 but we observed expression only in 36% of cases). A direct correlation of JAK2 expression with JAK2 gene dosage in cHL revealed that most cases with genomic gains showed also JAK2 protein expression, but that also the majority of cases without genomic JAK2 gains expressed the protein. JAK2 expression is thus not strictly correlated to the JAK2 genomic status.

In most tumors where aberrant TK activity has been observed, activation of a single TK (e.g., ALK in anaplastic large cell lymphoma or BCR-ABL in chronic myeloid leukemia)44 or two TKs from the same family of TKs (e.g. KIT or PDGFRA in gastrointestinal stroma tumors)45 were found in the majority of cases. It is thus an interesting question whether these three TKs, which belong to different families of TKs, contribute in the same way to MBL pathogenesis or whether each is relevant only for a specific aspect of the MBL phenotype. For JAK2 it seems clear that activation of STAT5 and STAT6 is a major consequence.19, 35 It should, however, be noted that STATs can also be activated by several RTKs,28 and that there is thus the possibility that also TIE1 and RON could contribute to the STAT5/6 activation in MBL. For TIE1 and RON activation of the PI3K/AKT pathway has been described,29, 30 and we indeed were able to demonstrate the activation of this pathway in MBL. Furthermore, JAKs can also activate the PI3K/AKT signalling cascade,46 and it is thus conceivable that all three TKs contribute to MBL pathogenesis by constitutive activation of the same signalling molecules, namely PI3K/AKT and STATs. The PI3K/AKT pathway is constitutively activated in several different types of tumors, among them also HL,33, 47, 48 and can, beside the inhibition of apoptosis, also enhance cell proliferation and growth.49 Constitutive STAT6 activation has so far only been observed in HL and prostate cancer.18, 50 The consequences of the activation of these signalling molecules in MBL await further investigation. In this regard, our experiments with the PI3K inhibitor LY294002 and the MBL cell lines gave first hints for a role of PI3K activation in promoting survival of MBL cells.

Taken together, aberrant TK activities in significant fractions of cases are a common feature of MBL and nsHL and distinguish both entities from other mature B cell lymphomas. Both entities have the aberrant expression of JAK2, RON and TIE1 in common. Thus, our analysis adds MBL to the list of tumors where aberrant TK activity likely plays an important role in pathogenesis. Together with the identification of the constitutively activated PI3K/AKT pathway these findings may form the basis for novel therapeutic strategies.

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Acknowledgements

We thank Yvonne Blum, Sabine Albrecht, Ekaterini Hadzoglou, Claudia Becher and Dorit Schuster for excellent technical assistance. Supported by grants from the DFG (RK 1315/2-1 and 2-2 to RK and BR 1238/6-1 and 6-2 to AB), the Deutsche Krebshilfe Network (70-3173-Tr3 to MLH, PM and RS) and the Deutsche José Carreras Leukämie Stiftung (to ET).

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Correspondence to A Bräuninger.

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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Renné, C., Willenbrock, K., Martin-Subero, J. et al. High expression of several tyrosine kinases and activation of the PI3K/AKT pathway in mediastinal large B cell lymphoma reveals further similarities to Hodgkin lymphoma. Leukemia 21, 780–787 (2007). https://doi.org/10.1038/sj.leu.2404594

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Keywords

  • mediastinal large B cell lymphoma
  • Hodgkin lymphoma
  • tyrosine kinase
  • PI3K/AKT pathway
  • RAF/MAPK pathway

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