Myeloid, B and T lymphoid and mixed lineage thymic lymphomas in the irradiated mouse

Emma Boulton, Helen Cleary and Mark Plumb,1

MRC Radiation and Genome Stability Unit, Chilton Didcot, Oxfordshire OX11 ORD, UK


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Thymic lymphoma is a very common spontaneous and/or induced malignancy in both inbred mice and in transgenic mouse models of human cancer. Although a thymic lymphoma is defined as thymus-dependent T-cell malignancy, diagnostic criteria vary between studies and considerable heterogeneity has been reported. To define and classify the thymic lymphomas that arose in our study of X-irradiated (CBA/HxC57BL/6)F1, F1 backcross and F1 intercross mice, 66 thymic lymphomas were immunogenotyped for immunoglobulin heavy chain (IgH) and T-cell receptor ß (TCRß) gene rearrangements, and/or analysed for expression of lineage-specific markers and allelic loss on chromosome 4. The data indicate that 33% of the thymic lymphomas are very similar to mouse radiation-induced acute myeloid (AML) and mixed lineage (IgHR, TCRßG) pre-B lympho-myeloid (L-MLs) leukaemias, 33% are mixed lineage (IgHR, TCRßR) B/T lymphoid and <33% can be described as single lineage (IgHG, TCRßR) T-cell malignancies. As the myeloid and L-ML leukaemias are not thymus-dependent this suggests that a malignant myeloid or pre-B lympho-myeloid cell can colonize the spleen to give an AML or L-ML leukaemia, or can colonize the thymus where TCRß gene rearrangement(s) may be induced to give the mixed lineage thymic lymphomas. Thus, assuming the single lineage T-cell thymic lymphomas fulfil the criteria of a thymus-dependent T-cell malignancy, thymic lymphomas are comprised of at least three distinct malignancies.

Abbreviations: AML, acute myeloid leukaemia; L-ML, pre-B lympho-myeloid leukaemias; LysM, lysozyme M; MPO, myeloperoxidase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Gene targeting and the generation of transgenic mice which are either deficient for a specific gene, or which over-express a gene, have been extensively used to test the function of oncogenes or tumour suppressor genes implicated in human cancers. Both the inactivation of genes involved in the maintenance of genomic stability or the enforced expression of (proto)-oncogenes result in an increased susceptibility to spontaneous or induced cancer in transgenic mice (1). Although the (proto-)oncogenes and tumour suppressor genes tend to be associated with specific malignancies in humans, the most common malignancy that arises in transgenic mice is thymic lymphoma (1). Many inbred mouse strains are also highly susceptible to radiation- and/or chemical carcinogen-induced thymic lymphoma (2–11), suggesting that thymic lymphoma is the predominant malignancy that arises in mice as a consequence of genetic instability and/or carcinogen exposure.

As a mouse model of an induced haemopoietic malignancy, thymic lymphomas have been extensively analysed to identify tumour suppressor and susceptibility loci. Loss of heterozygosity (LOH) studies of induced mouse thymic lymphomas have identified five tumour suppressor loci on chromosome 4, in addition to loci on chromosomes 2, 5, 6, 11, 12, 16 and 19 (11–21), and the inactivation of the p15INK4B (chromosome 4) and Ikaros (chromosome 11) tumour suppressor genes has been reported (13,20,22–24). Together with susceptibility/resistance loci on chromosomes 2, 4, 5 and 7 identified in genetic studies (25,26), at least 13 loci on nine chromosomes have been implicated in mouse spontaneous and/or induced thymic lymphomagenesis.

The diagnosis of thymic lymphomas in the mouse does not generally follow the strict criteria used to diagnose human haemopoietic malignancies, with diagnosis relying on an enlarged thymus (with laboured breathing) and/or limited serology and immunogenotype analyses, but considerable heterogeneity has been observed (2,3,5–10,12,13,27–30). Although in vivo transplantation experiments following whole body X-irradiation have shown that exposure induces thymus-dependent pre-leukaemic cells which require the thymus microenvironment for progression to full malignancy (32–35), non-thymic mixed phenotype B-myeloid lymphomas arise in thymectomized mice (36,37), evidence that while the thymus may not be essential for malignant transformation, it may determine the type of malignancy.

A number of other mouse strain-specific spontaneous or induced haemopoietic malignancies have been described in inbred mice, and include pre-B lymphomas (38), lymphocytic leukaemia (39), plasmacytoma (40), acute myeloid leukaemia (AML) and a mixed lineage early pre-B lympho-myeloid leukaemia (L-ML) (41,42). Although it is generally accepted that there is a strong genetic component, which determines the type and/or incidence of the spontaneous or induced haemopoietic malignancy in the mouse, like thymic lymphomas, the malignancies tend to be poorly defined. The absence of consistent and stringent diagnostic criteria in the classification of mouse leukaemias and lymphomas raises the possibility of misdiagnosis and makes the comparison of data from different studies and/or in different mouse strains difficult.

There is indirect evidence that mouse thymic lymphomas may represent more than one distinct malignancy: (i) the immunogenotype and phenotype analyses of thymic lymphomas have revealed considerable heterogeneity (2,3,5–10,12,13,27–31); (ii) LOH studies have revealed widespread allelic loss which is in part mouse strain-specific (11–21); (iii) the incidence of p15INK4b tumour suppressor gene inactivation by allelic loss and promoter methylation in radiation-induced thymic lymphomas varies from 20 to 80% depending on the genetic background and carcinogen treatment (22–24); and (iv) mixed lineage L-ML/lymphomas arise in thymectomized mice (36,37).

Our recent molecular studies of mouse radiation-induced AML which had been diagnosed by leukaemic blood cell morphology revealed two distinct malignancies: AML and a mixed lineage early B L-ML (41). We have therefore used the same approach to classify thymic lymphomas which arose in 3 Gy X-irradiated (CBA/HxC57BL/6)F1 backcross and intercross mice. Thymic lymphoma DNA was screened for immunoglobulin heavy chain (IgH) and T-cell receptor ß (TCRß) gene rearrangements, and revealed all four possible immunogenotype combinations. Thymic lymphoma RNA was also screened for expression of lineage specific/restricted markers and a significant proportion found to express both lymphoid (VpreB1) and myeloid [myeloperoxidase (MPO), lysozyme M (LysM)] markers. Together with LOH studies of chromosome 4, our data suggest that many of the thymic lymphomas are mixed lineage B and/or T lympho-myeloid malignancies, and in many respects are similar to AML and L-ML leukaemias that arose in the same mice (41). Thus, whilst the thymus does not appear to be essential for malignant progression, the thymus microenvironment may induce TCRß gene rearrangements in myeloid and B L-ML cells which colonize the thymus to yield the observed mixed lineage thymic lymphomas.


    Materials and methods
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 Materials and methods
 Results
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Mouse irradiations
CBA/H and C57BL/6 mice were from the Harwell colony. Mice, 8–12-week-old (CBA/HxC57BL/6)F1 (n = 89), F1xCBA/H (n = 1087), F1xC57BL/6 (n = 314) and F1xF1 (n = 142), were exposed to a single acute dose of 3.0 Gy X-rays at 0.5 Gy/min (250 kV constant potential, HVL 1.2 mm Cu). The animal studies were carried out under guidance issued by the MRC in `Responsibility in the use of animals for medical research' (July 1993) and Home Office Project Licence No. PPL 30/689 and 30/1272.

Leukaemias and lymphomas were diagnosed by micropscopic examination of blood, bone marrow, spleen and thymus. The average weight of an adult mouse thymus is ~0.01 g, and a thymic lymphoma defined in the first instance as a malignancy resulting in an enlarged thymus weighing >0.1 g (42). Thymic lymphomas were snap frozen for subsequent molecular analyses.

Immunogenotype
DNA was prepared from thymic lymphomas and ~15 µg of restriction enzyme-digested DNA was resolved by 1% (w/v) agarose gel electrophoresis and transferred to Genescreen (NEN Life Science Products, Boston, MA) nylon membranes. HindIII DNA digests were probed with the pTcrb-J2 probe to screen for TCRß gene rearrangements, and EcoRI and BamHI DNA double digests probed with p5'(JH) to screen for immunoglobulin heavy chain (IgH) gene rearrangements (41,42).

Lymphoma phenotype
Where possible total cellular RNA was also prepared from ~50% of the thymic lymphoma, and from control adult spleen and thymus, for northern blot analyses. RNA (~10–20 µg) was resolved by 1.0% (w/v) denaturing gel electrophoresis, transferred to Genescreen membranes, and probed with MPO, LysM, CD19, PU1, VpreB1 and Rag1 cDNA probes (41,42). Normal bone marrow and thymus were used as positive controls for VpreB1 and Rag1, respectively. Equivalent RNA loading was confirmed by ethidium bromide staining and/or hybridizations with glutathione peroxidase (GPX) and transforming factor ß (TGFß) cDNA probes. Probes were labelled with [{alpha}-32P]dATP (3000 Ci/mmol; Amersham BioSciences, Amersham, UK) using the Random Prime labelling kit (Invitrogen, Paisley, UK).

Loss of heterozygosity
The polymorphic chromosome 4 microsatellite markers used in LOH anlyses of tail and lymphoma DNA from the same mouse have already been described (41,43). Genetic map positions are from the Mouse Genome Database, and microsatellite primer sequences were from the Jackson Laboratory, except for MP-15-3' on chromosome 4 (44).


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 Abstract
 Introduction
 Materials and methods
 Results
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Thymic lymphoma immunogenotype
In a lifetime study involving 1632 X-irradiated (CBA/HxC57BL/6)F1 and F1 backcross and F1 intercross mice, 66 thymic lymphomas were diagnosed as defined by an enlarged (>10-fold) thymus (42). Thymic lymphomas have been described as T-cell malignancies (2,3,5–10,12,13,27–30), so in a retrospective study we sought to confirm this by immunogenotyping thymic lymphoma DNA for IgH and TCRß gene rearrangements by Southern blot analyses. Unexpectedly, and as illustrated in Figure 1Go and summarized in Table IGo, germline (IgHG and TCRßG) and rearranged (IgHR and TCRßR) alleles were readily detectable in the thymic lymphomas, and all four immunogenotype combinations represented.



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Fig. 1. IgH and TCRß gene rearrangements. Representative Southern blot analyses of thymic lymphoma DNA (~15 µg). (A) EcoRI- and BamHI-digested genomic thymic lymphoma DNA was resolved by 1% (w/v) agarose gel electrophoresis, blotted and probed with p5'(JH) to detect IgH gene rearrangements (41). (B) HindIII-digested genomic thymic lymphoma DNA was resolved by 1% (w/v) agarose gel electrophoresis, blotted and probed with pTcrb-J2 probe to detect TCRß gene rearrangements (42). G: germline configuration.

 

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Table I. Thymic lymphoma immunogenotype and genetic background
 
The detection of mono- and bi-allelic gene rearrangements by Southern blot analysis, together with the relative intensities of the germline and rearranged bands on the Southern blots (Figure 1Go and data not shown), is clear evidence of the clonal expansion of a malignant cell carrying a rearranged gene(s) in the thymus, and we can infer (Figure 1Go) that contamination of the lymphoma by normal untransformed cells in most cases is low. Further evidence that the malignancies are clonal in origin and contain <20% contaminating normal cells can be inferred from LOH studies (see below).

Although mono- and bi-allelic IgH and TCRß gene rearrangements were detected, two rearranged alleles in addition to the germline configuration allele (Figure 1AGo, lanes 6 and 7; Figure 1BGo, lane 7 and data not shown), and three or more rearranged alleles (Figure 1AGo, lane 10 and data not shown) were also detected. Given that the mean latency of the thymic lymphomas is ~16 months (42), and that the lifetime incidence of the lymphomas is low (Table IGo), the probability that two or more independent lymphomas arose in the same mouse at the same (or very similar) time is negligible. Multiple IgHR and/or TCRßR alleles within individual lymphomas most probably represent subclonal variants of a clonal malignancy as has been described for IgHR alleles in the L-MLs that arose in the same irradiated backcross mice in this study (41) and TCRßR alleles in chemically induced thymic lymphomas (5,30).

As IgH and TCRß gene rearrangements are absolutely specific to B- and T-cell lineages, respectively, at least three distinct malignancies can be classified according to their immunogenotype: B cell (IgHR, TCRßG), T cell (IgHG, TCRßR) and mixed lineage B/T (IgHR, TCRßR). IgHG, TCRßG lymphomas cannot be classified.

Thymic lymphoma phenotype
Mouse haemopoietic malignancies commonly have a leaky differentiation block, so although they are clonal in origin, the proportion of differentiating cells expressing one or more lineage-specific markers can vary considerably. In mouse AML, for example, metamyelocytes can account for 2–40% of the white blood cell count and MPO gene expression in the leukaemic spleen can vary by orders of magnitude (41). Similarly, mixed lineage malignancies are also commonly observed in mice (37,41,46,47). Many mouse haemopoietic malignancies are therefore not composed of a single homogenous cell type, but are classified by the presence of specific immature haemopoietic progenitor cells in haemopoietic tissue(s) which are either not normally present in that tissue, or which are present in much higher numbers than normal.

As the source of malignant thymic lymphoma cells in the mouse is the thymus, and as the thymus is the site of T-cell differentiation in vivo, the use of T-cell-specific cell surface antigens to characterize the lymphomas is potentially complicated by the presence of normal thymic cells, and may explain the considerable heterogeneity observed in thymic lymphoma analyses using CD4, CD8, CD3, TL, IL-2R, MEL-14 and H2-K antigens (3,6,27,31). Although 61% of the thymic lymphomas in this study exhibited an unequivocal T-cell marker (TCRß gene rearrangements, Table IGo), the origin and classification of the IgHR (40%, Table IGo) and IgHG, TCRßG (29%; Table IGo) thymic lymphomas was unclear. To address this issue, thymic lymphomas were analysed for the expression of lineage-specific/restricted genes not normally expressed, or expressed at very low levels, in a normal adult thymus.

To further characterize the thymic lymphomas, total cellular RNA was prepared from 19 thymic lymphomas, which were large enough to permit both DNA and RNA preparations. RNA was analysed for the expression of lineage-specific/restricted markers by northern blot (Figure 2Go).



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Fig. 2. Thymic lymphoma mRNA expression profiles. Representative northern blots containing total cellular RNA (~20 µg) prepared from control spleen (lane 1) or thymus (lane 2) or thymic lymphomas (lanes 3–20). Blots were probed with MPO, LysM, CD19, PU1, VpreB1, Rag1, TGFß and/or GPX cDNA probes (41,42). Thymic lymphoma immunogenotypes are: IgHG, TCRßG: lanes 3, 5–7, 9, 12, 15–19. IgHR, TCRßG: lanes 4, 8, 13. IgHG, TCRßR: lanes 10, 11, 14. IgHR, TCRßR: lane 20.

 
Only a significant difference in the abundance of an mRNA in a thymic lymphoma compared with a normal thymus can be attributed to the lymphoma cells, so enrichment (+) of an mRNA species in an individual lymphoma was initially defined as >2-fold, and depletion (–) as <0.5-fold, compared with its level in control thymus, or as positive (+) when that mRNA is not detectable in thymus (VpreB1). The differences in mRNA levels considered informative are therefore larger than those that might be attributed to gel loading (GPX and TGFß controls, Figure 2Go).

As illustrated in Figure 2Go, and with reference to control adult thymus (lane 2), a significant number of the lymphomas exhibit enrichment of MPO (lanes 7, 9, 16, 18 and 20), LysM (lanes 5, 9, 11, 13, 17, 19 and 20), CD19 (lanes 5, 6, 8–10, 12–14, 18 and 19), VpreB1 (lanes 4, 13 and 16) and PU1 (lanes 3–20) mRNAs. No one thymic lymphoma exhibited significant enrichment of Rag1 mRNA compared with normal thymus, so Rag1 is not informative as a lymphoid marker.

The detection of VpreB1 mRNA by northern blot in three thymic lymphomas is particularly informative as VpreB1 gene expression occurs very early (Pro-B -> Pre-B) in B-cell maturation and is undetectable in normal mouse thymus and spleen, but is detected in bone marrow, which is the site of early B-cell maturation in vivo (41, and data not shown). As it is highly unlikely that normal early B progenitor cells would leave the bone marrow and colonize the thymus in sufficient numbers to give detectable levels of VpreB1 mRNA, the abnormal gene expression observed in the lymphoma, together with the clonal IgH gene rearrangements detected by Southern blot (Figure 1Go and Table IGo), is strong evidence that at least some of the lymphomas are early B-cell malignancies that originated in the bone marrow.

The enrichment of CD19 mRNA compared with normal thymus (Figure 2Go) is observed irrespective of whether IgH gene rearrangements are detected by Southern blot analysis of the lymphoma DNA (Figure 2Go IgHG lanes 5, 6, 9, 12, 14 and 18; IgHR lanes 8 and 13), and is therefore inconsistent with a B lineage lymphoma. We have reported previously that DNA laddering is readily detected in thymic lymphomas (43), and this apoptosis could elicit an immune/inflammatory response. The observed thymic lymphoma mRNA levels of the more mature CD19 and LysM lineage-specific markers, and PU1 which is expressed in normal myeloid and B cells (45) may, therefore, in part, represent the infiltration into the thymic lymphoma of mature untransformed B and macrophage cells in response to apoptosis.

The immunogenotype (Table IGo) and mRNA expression profiles (Figure 2Go) of the thymic lymphomas suggest that some may be related to the AML (IgHG, TCRßG, MPO+, CD19-, VpreB1-) or L-ML (IgHR, TCRßG, MPO+/-, LysM+/-, VpreB1+/-, CD19-) leukaemias whose mRNA expression profiles were defined with reference to normal spleen (41). Compared with normal spleen, five of 19 (26%) thymic lymphomas are enriched for LysM mRNA (Figure 2Go, lanes 9, 11, 13, 17 and 19), whereas no significant enrichment of CD19 mRNA was observed. This is remarkably similar to the AMLs and L-MLs which were either negative or not significantly enriched for CD19 mRNA, whereas 6% of the AMLs and 22% of the L-MLs were enriched for LysM mRNA (41).

Excluding CD19, PU1 and Rag1 as not informative, and comparing LysM mRNA levels with control spleen, the immunogenotype and MPO, LysM and VpreB1 mRNA expression profiles (Figure 2Go) permit the further classification of the lymphomas:

  1. IgHG, TCRßG, LysM+/MPO+, VpreB1-: myeloid (lanes 6, 7, 9, 12, 17 and 19).
  2. IgHG, TCRßG, MPO+, LysM-, VpreB1+: proB lympho-myeloid (lane 16).
  3. IgHR, TCRßG, MPO-, LysM+, VpreB1+: preB lympho-myeloid (lane 13).
  4. IgHR, TCRßG, MPO-, LysM-, VpreB1+/-: early B lymphoid (lanes 4 and 8).
  5. IgHG, TCRßR, MPO-, LysM-, VpreB1-: T lymphoid (lanes 10 and 14).
  6. IgHG, TCRßR, MPO-, LysM+, VpreB1-: T lympho-myeloid (lane 11).
  7. IgHR, TCRßR, MPO+, LysM+, VpreB1-: B/T lympho-myeloid (lane 20).
  8. IgHG, TCRßG, MPO-, LysM-, VpreB1-: unknown (lanes 3, 5, 15 and 18).

Thus, the immunogenotype and/or mRNA expression analyses using three lineage-specific markers permits the subclassification of ~80% of the thymic lymphomas and reveals an unexpected complexity.

Loss of heterozygosity
Allelic loss on chromosome 4 is frequently detected in radiation-induced thymic lymphomas, and five thymic lymphoma suppressor regions (TLSR1-5) mapped (17–19,21). As allelic loss at TLSR5 has been preferentially detected in mouse radiation-induced AML and L-ML leukaemias (41), and some of the thymic lymphomas appeared to be related to the leukaemias, the thymic lymphomas in this study were screened for LOH on chromosome 4. As most of the lymphomas arose in F1 backcross or intercross mice (Table IGo), there is a 50% probability of homozygosity at any given microsatellite marker. Similarly, only those lymphoma DNA samples that contained <20% contaminating normal cells will reveal LOH (41,42).

Twenty-five out of 66 thymic lymphomas exhibited LOH on chromosome 4, further evidence that the malignancies are clonal in origin and that there are low levels of contaminating normal cells in the lymphomas. Twenty thymic lymphomas were informative for both proximal and distal breakpoints (Figure 3Go).



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Fig. 3. Chromosome 4 deletion mapping by LOH in informative lymphomas. The polymorphic microsatellite markers employed and their relative genetic positions (cM) are shown, as are the number of thymic lymphomas, which exhibited a particular LOH pattern (AF). Solid boxes show regions of LOH.

 
Ninety-two per cent (23 out of 25) exhibit LOH at D4Mit286, and a 3.4 cM minimally deleted region inferred between D4Mit108 and D4Mit214 irrespective of immunogenotype. This 3.4 cM interval maps to the same TLSR5 tumour suppressor gene locus mapped in both radiation-induced AML (~50% LOH incidence) and L-ML (>95% LOH incidence) leukaemias (41). Furthermore, the maternally transmitted CBA/H allele at D4Mit286 was preferentially lost in 20 out of 23 of the thymic lymphomas as it was in 25 out of 28 of radiation-induced AMLs and L-MLs (41), further evidence that many of the thymic lymphomas are the same or very similar to the radiation-induced leukaemias.

TLSR1 has been associated with the inactivation of the p15INK4B tumour suppressor gene by allelic loss and promoter hypermethylation (22), and the MP15-3' polymorphic microsatellite marker mapped to the INK4 locus (44). Only three thymic lymphomas in our study exhibit LOH at MP15-3', two of which also exhibit LOH at D4Mit286/TLSR5 (Figure 3Go). Two additional thymic lymphomas are not informative at MP15-3', but exhibit LOH at D4Mit286/TLSR5 so LOH at MP15-3' cannot be excluded. Thus, although five out of 25 lymphomas potentially exhibit allelic loss at the TLSR1 locus, only one out of 25 exhibits LOH at TLSR1 (MP15-3') but not TLSR5/D4Mit286, and one lymphoma exhibits allelic loss distal to TLSR1 (Figure 3Go).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Immunogenotype and mRNA expression analyses have revealed that thymic lymphomas, as defined by haemopoietic malignancies that can colonize and undergo clonal expansion in the mouse thymus, include myeloid, B and T lymphoid and mixed lineage malignancies. The IgHG, TCRßG and IgHR, TCRßG thymic lymphomas have the same immunogenotype and similar mRNA expression profiles as radiation-induced AML and mixed lineage early pre-B L-ML leukaemias, respectively (41). Therefore, a malignant myeloid or L-ML cell can undergo clonal expansion in either the spleen (leukaemia) or thymus (thymic lymphoma). Furthermore, as the thymus microenvironment is required for TCRß gene rearrangements, this raises the possibility that on occasion TCRß gene rearrangements can be induced in AML or L-ML cells that colonize the thymus and give rise to the observed B/T and T-myeloid mixed lineage thymic lymphomas. This is consistent with the relative under-representation of IgHR, TCRßG lymphomas in the thymus (0.4%; Table IGo) compared with the lifetime incidence of IgHR, TCRßR lymphomas in the thymus (1.2%; Table IGo) and IgHR, TCRßG L-ML leukaemias in the spleen (~8%) (41).

The thymic lymphomas analysed in this study arose in X-irradiated (CBA/HxC57BL/6)F1, F1 backcross and F1intercross mice. As CBA/H mice are susceptible to radiation-induced AML and L-ML (41), and C57BL/6 are susceptible to radiation-induced thymic lymphoma (4), the genetic background of the hybrid mice analysed must contribute to the complexity of the lymphomas observed. Differences in the thymic lymphoma incidence and/or immunogenotype would be expected in the (CBA/HxC57BL/6)F1, (CBA/HxC57BL/6)F1xCBA/H and (CBA/HxC57BL/6)F1xC57BL/6 mice if susceptibility to radiation-induced T-cell thymic lymphomas was a simple recessive or dominant C57BL/6 genetic trait. However, as shown in Table IGo, a comparison of the lifetime incidence of thymic lymphomas as a whole or subclassified according to their immunogenotype, reveals no obvious or genetically interpretable difference in the five genetic backgrounds studied.

Early dose–response studies of thymic lymphoma induction by ionizing radiation in inbred C57BL mice clearly demonstrated that exposure to fractionated doses of radiation is considerably more efficient in inducing thymic lymphomas than single acute exposure to the same total dose (4), and the generally accepted irradiation regime involves four weekly doses of 1.6–2.5 Gy starting at 4–5 weeks of age (1,2,4,9,12,13,16,17,20,27). In contrast, the thymic lymphomas in our study arose in mice exposed to a single acute dose of 3 Gy X-rays at 8–12 weeks of age, so a more mature haemopoietic system at the time of exposure to a single dose of X-rays may account for the complexity of the thymic lymphomas observed. However, it should be noted that: (i) the last 1.6–2.5 Gy exposure in the thymic lymphoma fractionated irradiation protocol occurs at 7–8 weeks of age; (ii) 40 out of 66 (61%; Table IGo) of the thymic lymphomas analysed in this report can be defined as T-cell malignancies as they have TCRß gene rearrangement(s); (iii) myeloid, plasmacytomas and mixed lineage pre-B L-ML and lymphomas arise in thymectomized mice (36,37) and are very similar to the mixed lineage L-ML and thymic lymphomas in our study; and (iv) spontaneous thymic lymphomas in transgenic mouse models can arise at an age of >18 months (1).

A number of T-cell-specific cell-surface antigens, including CD4, CD8, CD3, TL, IL-2R, MEL-14 and H-2K, have been employed to classify mouse thymic lymphomas, and heterogeneity attributed to either a continuous spectrum of mature and immature phenotypes (3,6,27,31), or to the cortical or medullary origin of the lymphoma cell within the thymus (9). As B or myeloid cell markers are not commonly used, the classification of a T-cell thymic lymphoma based on T-cell-specific markers alone is unsatisfactory, particularly as plasmacytomas, myelomonocytic leukaemia or mixed lineage IgHR, Cd5+, NK1+, Mac1+, LysM+ B lympho-myeloid non-thymic lymphomas arise in thymectomized mice (36,37). Significantly, many studies of mouse haemopoietic malignancies that do use multi-lineage-specific markers have revealed an unexpected complexity (29,36,37,41,46–49), which is comparable with that described here, supportive evidence that at least some of the thymic lymphomas represent malignancies that arose in the bone marrow but fortuitously colonized the thymus where variable T-cell differentiation was induced.

LOH studies of induced mouse thymic lymphomas have mapped 12 tumour suppressor loci on eight chromosomes, including five loci (TLSR1-5) on chromosome 4 (11–21). TLSR1-5 map to positions 42, 71, 64, 39 and 16 cM on chromosome 4, respectively, and the incidence of LOH detected at each locus in (C57BL/6xBALB/c)F1 and/or (C57BL/6x RF/J)F1 radiation-induced thymic lymphomas varies between 20.4 (TLSR5) and 32.6–40% (TLSR2 and TLSR1) (17,18,21,22). In contrast, allelic loss in mouse radiation-induced AML and L-ML leukaemias is considerably more specific when the malignancies are classified according to the criteria used in this study. Allelic loss on chromosome 2 is detected in >95% of AMLs and <15% L-MLs, whereas allelic loss at TLSR5 is detected in >95% of L-MLs and ~50% of AMLs (41). Our observation that the preferential loss of the maternally transmitted CBA/H allele at TLSR5 is observed in 92% of thymic lymphomas (Figure 3Go), and that at most 25% thymic lymphomas exhibit allelic loss at TLSR1, is consistent with the proposal that at least 70% of the thymic lymphomas analysed in this study are related to AML and L-ML leukaemias.

Allelic loss at TLSR1 is associated with promoter methylation of the p15INK4b tumour suppressor gene promoter, but the incidence of p15INK4b gene promoter methylation in radiation-induced thymic lymphomas varies from 20 to 88% depending on genetic background and/or radiation quality (22,24). Prior to the immunogenotype analyses of the thymic lymphomas described in this report, p15INK4b gene promoter methylation was detected in 21% of the thymic lymphomas we analysed (24). A re-evaluation of our data indicates that p15INK4b gene promoter methylation in the thymic lymphomas is immunogenotype-dependent as it is detected in five of 11 (45%) IgHG, TCRßR lymphomas and one of 11 (9%) IgHR, TCRßR lymphomas, but was not detected in IgHG, TCRßG and in IgHR, TCRßG lymphomas (data not shown), further supportive evidence for distinct malignancies.

The Pax5 gene maps to the chromosome 4 TLSR5 minimally deleted region mapped in the LOH studies (Figure 3Go). Appropriately stimulated Pax5 deficient pre-B and pro-B cells can develop into erythroid, myeloid and lymphoid cell lineages, suggesting that Pax5 suppresses alternative lineage choices in vivo, or that Pax5 deficient pre-BI cells dedifferentiate as far back as the pluripotent stem cell (50,51). The identification of mixed lineage B-myeloid, B/T-lymphoid and T-myeloid thymic lymphomas and their apparent relationship to AML and mixed lineage L-ML leukaemias, together with the presence of subclonal IgH and TCRß gene rearrangements, suggests that the differentiation block in these murine haemopoietic malignancies is extremely weak and that limited (de-) differentiation can be induced following malignant transformation. Furthermore, the two Pax5 alleles are independently regulated during B-cell development, and one allele is predominantly expressed in early progenitors and mature B cells (52), indicating that the loss of the active allele in the appropriate cell is sufficient to inactivate the gene. As the maternally transmitted CBA/H allele is preferentially lost in murine thymic lymphomas (this report), AMLs and L-MLs (41), Pax5 is an excellent candidate for the TLSR5 tumour suppressor gene.

The classification of mouse haemopoietic malignancies is inconsistent, and is complicated further by differences in genetic background and carcinogen treatments. The data presented here suggest that at least some of the heterogeneity observed in studies of radiation-induced thymic lymphomas can be attributed to the fact that distinct malignancies can undergo clonal expansion in either the thymus or the spleen, and yet in the case of the thymic lymphomas, they fulfil the generally accepted criteria used to define a thymic T-cell malignancy—clonal expansion in the thymus and TCRß gene rearrangements. The data also suggest that allelic loss at specific tumour suppressor gene loci and/or specific tumour suppressor gene inactivation is considerably more specific to a particular haemopoietic malignancy than implied by the 12 thymic lymphoma tumour suppressor gene loci reported in the literature (11–21).


    Notes
 
1 To whom correspondence should be addressed at: Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK Email: map12{at}le.ac.uk Back


    Acknowledgments
 
Supported by the Medical Research Council and by the Leukaemia Research Fund. H.C. was supported by an MRC Research Studentship.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. TBASE 2001. Transgenic/targeted mutation database; The Jackson Laboratory. http://tbase.jax.org/
  2. Diamond,L.E., Berman,J.W. and Pellicer,A. (1987) Differential expression of surface markers on thymic lymphomas induced by two carcinogenic agents in different mouse strains. Cell. Immunol., 107, 115–120.[ISI][Medline]
  3. Frank,A.A., Collier,J.M., Forsyth,C.A., Oughton,J.A. and Kerkvliet,N.I. (1992) Flow cytometric analysis of thymic lymphosarcoma induced by N-methyl-N-nitosourea in C57BL/6J mice. Carcinogenesis, 13, 509–512.[Abstract]
  4. Kaplan,H.S. and Brown,M.B. (1952) A quantitative dose–response study of lymphoid-tumour development in irradiated C57 Black mice. J. Natl Cancer Inst., 13, 185–192.[ISI]
  5. Dasgupta,U.B. and Lilly,F. (1988) Chemically induced murine T lymphomas: continued rearrangement within the T-cell receptor beta-chain gene during serial passage. Proc. Natl Acad. Sci. USA, 85, 3193–3197.[Abstract]
  6. Gomez,G., Kraig,E., Infante,A.J., Holloway,M. and Richie,E.R. (1997) Characterization of spontaneous and carcinogen-induced thymic lymphomas. J. All. Clin. Immunol., 99, 197–202.[Medline]
  7. Kubo,E., Muto,M., Sado,T., Takeshita,S., Shimizu,T. and Yamagishi,H. (1992) Novel TCR gene rearrangements and expression in radiation-induced thymic lymphomas. J. Radiat. Res., 33, 227–242.[ISI][Medline]
  8. Newcomb,E.W. (1997) Clonal evolution of N-methylnitosourea-induced C57BL/6J thymic lymphomas by analysis of multiple genetic alterations. Leuk. Res., 21, 199–200.[ISI][Medline]
  9. Newcomb,E.W., Steinberg,J.J. and Pellicer,A. (1998) ras oncogenes and phenotypic staging in N-methylnitrosourea- and {gamma}-irradiation-induced thymic lymphomas in C57BL/6J mice. Cancer Res., 48, 5514–5521.[Abstract]
  10. Shimizu,T., Muto,M., Kubo,E., Sado,T. and Yamagishi,H. (1993) Multiple pre-neoplastic events and clonal selection of radiation induced thymic lymphomas shown by TCR gene rearrangements. Leuk. Res., 17, 959–965.[ISI][Medline]
  11. Zhuang,S.-M., Eklund,L.K., Cochran,C., Rao,G.N., Wiseman,.R.W. and Soderkvist,P. (1996) Allelotype analysis of 2',3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice. Cancer Res., 56, 3338–3343.[Abstract]
  12. Matsumoto,Y., Kosugi,S., Shinbo,T. et al. (1998) Allelic loss analysis of {gamma}-ray-induced mouse thymic lymphomas: two candidate tumor suppressor gene loci on chromosomes 12 and 16. Oncogene, 16, 2747–2754.[ISI][Medline]
  13. Okano,H., Saito,Y., Miyazawa,T., Shinbo,T., Chou,D., Kosugi,S., Takahashi,Y., Odani,S., Niwa,O. and Kominami,R. (1999) Homozygous deletions and point mutations of the Ikaros gene in {gamma}-ray-induced mouse thymic lymphomas. Oncogene, 18, 6677–6683.[ISI][Medline]
  14. Okumoto,M., Park,Y.G., Song,C.W. and Mori,N. (1999) Frequent loss of heterozygosity on chromosomes 4, 12 and 19 in radiation-induced lymphomas in mice. Cancer Lett., 135, 223–228.[ISI][Medline]
  15. Sitarz,M., Wirth-Dzieciolowaska,E. and Demant,P. (2000) Loss of heterozygosity on chromosome 5 in vicinity of the telomere in gamma-radiation-induced thymic lymphomas in mice. Neoplasm, 47, 148–150.[ISI]
  16. Shinbo,T., Matsuki,A., Matsumoto,Y., Kosugi,S., Takahashi,Y., Niwa,O. and Kominami,R. (1999) Allelic loss mapping and physical delineation of a region harbouring a putative thymic lymphoma suppressor gene on mouse chromosome 12. Oncogene, 18, 4131–4136.[ISI][Medline]
  17. Santos,J., Perez de Castro,I., Herranz,M., Pellicer,A. and Fernandez-Piqueras,J. (1996) Allelic loss on chromosome 4 suggest the existence of a candidate tumor suppressor gene region of about 0.6 cM in {gamma}-radiation-induced mouse primary thymic lymphomas. Oncogene, 12, 669–676.[ISI][Medline]
  18. Santos,J., Herranz,M., Perez de Castro,I., Pellicer,A. and Fernandez-Piqueras,J. (1998) A new candidate site for a tumour suppressor gene involved in mouse thymic lymphomagenesis is located on the distal part of chromosome 4. Oncogene, 17, 925–929.[ISI][Medline]
  19. Santos,J., Herranz,M., Fernandez,M., Vaquero,C., Lopez,P. and Fernandez-Piqueras,J. (2001). Evidence of a possible epigenetic inactivation mechanism operating on a region of mouse chromosome 19 in {gamma}-radiation-induced thymic lymphomas. Oncogene, 20, 2186–2189.[ISI][Medline]
  20. Shimada,Y., Nishimura,M., Kakinuma,S., Okumoto,M., Shiroishi, T., Clifton,K.H. and Wakana,S. (2000) Radiation-associated loss of heterozygosity at the Znfn1a1 (Ikaros) locus on chromosome 11 in murine thymic lymphomas. Radiat. Res., 154, 293–300.[ISI][Medline]
  21. Melendez,B., Santos,J. and Fernandez-Piqueras,J. (1999) Loss of heterozygosity at the proximal-mid part of mouse chromosome 4 defines two novel tumour suppressor gene loci in T-cell lymphomas. Oncogene, 18, 4166–4169.[ISI][Medline]
  22. Malumbres,M., Perez de Castro,I., Santos,J., Melendez,B., Mangues,R., Serrano,M., Pellicer,A. and Fernandez-Piqueras,J. (1997) Inactivation of the cyclin-dependent kinase inhibitor p15INK4b by deletion and de novo methylation with independence of p16INK4a alterations in murine primary T-cell lymphomas. Oncogene, 14, 1361–1370[ISI][Medline]
  23. Zhuang, S-M., Schippert,A., Haugen-Strano,A., Wiseman,R.W. and Soderkvist P. (1998) Inactivations of p16INK4a-{alpha}, p16INK4a-ß and p15INK4b genes in 2',3'-dideoxycytidine- and 1,3-butadiene-induced murine lymphomas. Oncogene, 16, 803–808.[ISI][Medline]
  24. Cleary,H.J., Boulton,E. and Plumb,M. (1999) Allelic loss and promoter hypermethylation of the p15INK4b gene features in mouse radiationinduced lymphoid—but not myeloid—leukaemias. Leukemia, 13, 2049–2052.[ISI][Medline]
  25. Saito,Y., Ochiai,Y., Kodama,Y. et al. (2001) Genetic loci controlling susceptibility to {gamma}-ray-induced thymic lymphoma. Oncogene, 20, 5243–5247.[ISI][Medline]
  26. Yamada,Y., Shisa,H., Matsushiro,H., Kamoto,T., Kobayashi,Y., Kawari,A. and Hiai,H. (1994) T lymphomagenesis is determined by a dominant host gene thymic lymphoma susceptible mouse-1 (TLSM-1) in mouse models. J. Exp. Med., 180, 2155–2162.[Abstract]
  27. Shimada,Y., Nishimura,M., Kakinuma,S., Takeuchi,T, Ogiu,T., Suzuki,G., Nakata,Y., Sasanuma,S. and Sado,T. (2001) Characteristic association between K-ras gene mutation with loss of heterozygosity in X-ray-induced thymic lymphomas of the B6C3F1 mouse. Int. J. Radiat. Biol., 77, 465–473.[ISI][Medline]
  28. Sen-Majumdar,A., Guidos,C., Kina,T., Lieberman,M. and Weissman,I.L. (1994) Characterization of preneoplastic thymocytes and of their neoplastic progression in irradiated C57BL/Ka mice. J. Immunol., 153, 1581–1592.[Abstract/Free Full Text]
  29. Thomas,C.Y., Buxton,V.K., Roberts,J.S., Boykin,B.J. and Innes,D. (1989) Phenotype heterogeneity of spontaneous lymphomas of CWD mice. Blood, 73, 240–247.[Abstract]
  30. Sloan,S.R. and Pellicer,A. (1991) Staging of T-cell receptor beta chain gene rearrangements and ras oncogene mutations in the development of murine thymic lymphomas. Cancer Res., 51, 1627–1631.[Abstract]
  31. Boudreau,R., St-Pierre,Y., Beauchemin,C. and Potworowski,E.F. (1998) TL antigen is not linked to radioinduced thymic lymphoma. Cell Immunol., 184, 161–167.[ISI][Medline]
  32. Boniver,J., Decleve,A., Lieberman,M., Honsik,C., Travis,M. and Kaplan,H.S. (1981) Marrow-thymus interactions during radiation leukaemogenesis in C57BL/Ka mice. Cancer Res., 41, 390–392.[Abstract]
  33. Muto,M., Kubo,E. and Sado,T. (1987) Development of prelymphoma cells committed to thymic lymphomas during radiation-induced thymic lymphomagenesis in B10 mice. Cancer Res., 47, 3469–3472.[Abstract]
  34. Rongy,A.M., Humblet,C., Lelevre,P., Greimers,R., Defresne,M.P and Boniver,J. (1990) Abnormal thymocyte subpopulations in split dose irradiated C57BL/Ka mice before the onset of lymphomas. Effects of bone marrow grafting. Thymus, 16, 7–27.[ISI][Medline]
  35. Upton,A.C., Wolff,F.F., Furth,J. and Kimball,A.W. (1958) A comparison of the induction of myeloid and lymphoid leukaemias in X-irradiated RF mice. Cancer Res., 18, 842–848.[ISI]
  36. Mori,N. and Takamori,Y. (1990) Development of nonthymic lymphomas in thymectomized NFS mice exposed to split-dose X-irradiation. J. Radiat. Res., 31, 389–395.[ISI][Medline]
  37. Lu,L.M. and Hiai,H. (1999) Mixed phenotype lymphomas in thymectomized (SL/KHxAKR/Ms)F1 mice. Jpn. J. Cancer Res., 90, 1218–1223.[ISI][Medline]
  38. Yamada,Y., Matsushiro,H., Ogawa,M.S., Okamoto,K., Nakakuki,Y., Toyokuni,S., Fukumoto,M. and Hiai,H. (1994) Genetic predisposition to pre-B lymphomas in SL/Kh strain mice. Cancer Res., 54, 403–407.[Abstract]
  39. Duran-Reynals,M.L., Kadish,A.S. and Lilly,F. (1986) Genetic and epigenetic factors that influence the occurrence of spontaneous lymphoid tumors in crosses of mice of high- and low-incidence strains. Int. J. Cancer, 37, 155–160.[ISI][Medline]
  40. Mock,B.A., Krall,M.M. and Dosik,J.K. (1993) Genetic mapping of tumour susceptibility genes involved in mouse plasmacytomagenesis. Proc. Natl Acad. Sci. USA, 90, 9499–9503.[Abstract]
  41. Cleary,H., Boulton,E. and Plumb,M. (2001) Allelic loss on chromosome 4 (Lyr2/TLSR5) is associated with myeloid, B-lympho-myeloid and lymphoid (B & T) mouse radiation-induced leukaemias. Blood, 98, 1549–1554.[Abstract/Free Full Text]
  42. Boulton,E., Cleary,H., Papworth,D. and Plumb,M. (2001) Susceptibility to radiation-induced leukaemia/lymphoma is genetically separable from sensitivity to radiation-induced genomic instability. Int. J. Radiat. Biol., 77, 21–29.[ISI][Medline]
  43. Cleary,H.J., Wright,E. and Plumb,M. (1999) Specificity of loss of heterozygosity in radiation-induced mouse myeloid and lymphoid leukaemias. Int. J. Radiat. Biol., 75, 1223–1230.[ISI][Medline]
  44. Malumbres,M., Perez de Castro,I., Santos,J., Perez-Olle,R., Fernandez-Piqueras,J. and Pellicer,A. (1998) An AC-repeat adjacent to mouse Cdkn2b allows the detection of specific allelic losses in the p15INK4b and p16INK4a tumor suppressor genes. Mamm. Genome, 9, 183–185.[ISI][Medline]
  45. Klemsz,M.J., McKercher,S.R., Celada,A., Van Beveren,C. and Maki,R.A. (1990) The macrophage and B cell-specific transcription factor PU. 1 is related to the ets oncogene. Cell, 61, 113–124.[ISI][Medline]
  46. Gibbons,D.L., Cleary,H., Macdonald,D., Plumb,M., Wright,E.W. and Greaves,M.F. (1999) An Eµ-BCL-2 transgene facilitates leukaemogenesis by ionising radiation. Oncogene, 18, 3870–3877.[ISI][Medline]
  47. Defresne,M.P., Borremans,B., Verhofsted,C., Peled,A., Thiry,A., Greimers,R., Robberecht,P., Nabarra,B., Verschaeve,L. and Hooghe,R. (1993) Mixed phenotype murine leukemias. Leukemia, 7, 1253–1260.[ISI][Medline]
  48. Herr,W., Perlmutter,A.P. and Gilbert,W. (1983) Monoclonal AKR/J thymic leukemias contain multiple Jh immunoglobulin gene rearrangements. Proc. Natl Acad. Sci. USA, 80, 7433–7436.[Abstract]
  49. Vasmel,W.L.E., Radaszkiewicz,T., Miltenburg,A.M., Zjilstra,M. and Melief,C.J.M. (1987) Refinement and precision in the classification of murine lymphomas by genotyping with immunoglobulin and T cell receptor probes. Leukemia, 1, 155–162.[ISI][Medline]
  50. Nutt,S.L,, Heavey,B., Rolink,A.G. and Busslinger,M. (1999) Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature, 401, 556–562.[ISI][Medline]
  51. Schaniel,C., Bruno,L., Melchers,F. and Rolink,A.G. (2002) Multiple haemopoietic cell lineages develop in vivo from transplanted Pax5-deficient preB I-cell clones. Blood, 99, 472–478.[Abstract/Free Full Text]
  52. Nutt,S.L., Vambrie,S., Steinlein,P., Kozmik,Z., Rolink,A., Weith,A. and Busslinger,M. (1999) Independent regulation of the two Pax5 alleles during B-cell development. Nat. Genet., 21, 390–395.[ISI][Medline]
Received February 5, 2002; revised March 26, 2002; accepted April 2, 2002.