From brain to testis: immune escape and clonal selection in a B cell lymphoma with selective out-growth in two immune sanctuariesy

We describe a patient with a primary diffuse large B-cell lymphomaof the central nervous system who developed a localized testicularrelapse after 8 years. Both tumours lacked HLA-DR expression,the relapse additionally lost HLA class I expression. Immunoglobulinheavy chain gene rearrangements were identical in both lymphomaswith extensive and ongoing somatic hypermutations resultingin extensive idiotype modulation. We hypothesize that theseimmune sanctuaries initially provided a safe haven for the tumourcells. When the environment becomes more permissive for an anti-tumourresponse, the continuous idiotype modulation and progressiveloss of HLA expression on the tumour cells facilitates furtherimmune escape.

Primary central nervous system lymphoma (PCNSL) and lymphomaof the testis are rare forms of diffuse large B cell lymphoma(DLBCL). Both lymphoma types are immune-privileged site-associatedDLBCL (IP-DLBCL), are EBV negative (except for immune-compromisedpatients), and almost exclusively have an activated B cell-like(ABC) phenotype.^1,2 Lymphomas are considered to develop andprogress in a multistep manner due to accumulation of geneticaberrations. Where a lymphoma is subject to selective pressureof the immune system, such aberrations may give rise to an immuneescape phenotype.³ A common aberration leading to immune escapeof PCNSL and testicular DLBCL is loss of HLA expression.^4,5,6Another common feature of both lymphoma types is a high levelof (often ongoing) somatic hypermutation (SHM) in the immunoglobulinheavy chain (IgH) genes.^1,7,8

Although usually presenting with stage IE disease, patientswith PCNSL or testicular DLBCL have a poor prognosis that hasonly recently improved upon introduction of novel chemotherapystrategies.^9,10 Primary testicular DLBCL frequently relapsein the CNS up to 10 years after initial presentation.^10,11 Relapseof PCNSL is almost always (90–95%) confined to the CNS.One patient has been reported with a relapse of PCNSL in thetestis, accompanied by extensive systemic involvement,¹² butno patients have been described with a relapse solely in thetestis. We describe a unique patient having a CNS lymphoma witha relapse confined to the testis 8 years after diagnosis. Theongoing modulation of the idiotype, in addition to progressiveloss of HLA class II and I proteins, might have provided anefficient tumour escape mechanism in these lymphomas.

Materials and methods

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Materials and methods

Histology
Formalin-fixed paraffin-embedded material of both CNS and testicularlymphomas was available. Staining with anti-CD20 (L26, DAKO,Glostrup, Denmark) confirmed the B cell origin. Stainings withanti-CD3 (PS1, Monosan, Uden, The Netherlands), anti-CD8 (C8/144B,DAKO) and anti-CD68 (PG-M1, DAKO) were performed to determinethe presence of T cells and macrophages in the tumour microenvironment.Stainings with anti-Bcl6 (PG-B6p, DAKO), anti-CD10 (56C6, Novocastra,Newcastle upon Tyne, UK) and anti-MUM1/IRF4 (MUM1p, DAKO) wereconsidered positive if more than 30% of neoplastic cells werestained. Stainings with anti-HLA-A/G, anti-HLA-B/C (HCA2 andHC10; Dr. J. Neefjes, NKI, Amsterdam) and anti-HLA-DR (LN3,Biotest AG, Dreieich, Germany) were considered negative whenstaining of tumour cells was absent in the presence of a positiveinternal control. EBER in situ hybridization for EBV was performedaccording to manufacturer’s protocol (DAKO).

Immunoglobulin mutation analysis
DNA was isolated from paraffin tissue and IgH multiplex PCRand GeneScan analysis were performed as described.¹ UnlabeledPCR products were cloned and from both tumours 20 colonies (10for each duplicate PCR) were sequenced. Sequences were analyzedusing IMGT/VQUEST13 and aligned using ClustalW14 and were depositedin GenBank (EF205597 to EF205627).

Results

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Results

In March 1996 a male patient, age 60, was diagnosed with a stageIE, right temporal PCNSL. Complete remission was achieved withchemotherapy (alternating high doses of MTX/Teniposide and highdoses of MTX for 4 weeks, with 4x intermittent intrathecal MTX),followed by radiotherapy (40 Gy in 20 fractions). In June 2004,the patient presented with a tumour of the left testicle anda diagnostic orchidectomy revealed DLBCL. Restaging disclosedstage IE disease. The patient was treated with 4x CHOP withintrathecal MTX and radiotherapy of the left groin area andscrotum (30 Gy in 15 fractions). In August 2005, the patientdeveloped neurological symptoms, most probably due to post-radiationencephalopathy. Until September 2006 no disease activity wasfound. Both PCNSL and testicular DLBCL were CD20 positive andEBV negative. The PCNSL was heterogeneous for CD10, negativefor Bcl6 and positive for MUM1; the testicular DLBCL was negativefor CD10 and positive for Bcl6 and MUM1, compatible with anABC-like immunophenotype.¹ Both localisations showed loss ofHLA-DR expression, the testicular DLBCL showed additional lossof HLA class I expression. Very few T cells (including CD8-positivecytotoxic T cells) and macrophages were present in the microenvironmentof the CNS lymphoma, while the numbers for both cell types werehigher in the microenvironment of the testicular lymphoma. TheT cells that were present were mostly CD8 positive.

GeneScan analysis of IgH rearrangements showed identical rearrangementsin both localisations, confirming that the testicular DLBCLwas a relapse from the PCNSL. Sequence analysis revealed 31clones derived from the same rearrangement (C1-C18 and T1-T13).CDR3 V-D-J junctional sequences showed highest homology to IGVH3-30*18,DH2-2 and JH5*02. A hypothetical consensus sequence, sharedCNS, was considered as PCNSL founder clone and carried 52 mutations(23%) compared to the germline V3-30*18 allele, of which 35were located in a mutation hotspot or a directly adjacent codon(Figure 1). Using the multinomial model15, significant negativeselection pressure on the framework regions was observed (p $≤$ 0.005).Considerable intraclonal heterogeneity was found at both lymphomasites (Supplementary Figure 1).

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Figure 1. Alignment of hypothetical IgH consensus ‘shared CNS’ sequence with germline V3-30*18, D2-2 and J5*02 allele sequences. Codon numbering according to IMGT¹³. Mutations in the ‘shared CNS’ consensus are indicated by asterisks under the germline sequence. Mutation hotspots (RGYW, WRCY and TAA motifs) are indicated in bold. Primer sequences are highlighted in grey. The complete alignment of all lymphoma sequences is available as Supplementary Figure 1.

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Supplementary Figure 1. Alignment of lymphoma IgH sequences with germline V3-30*18, D2-2 and J5*02 allele sequences. C: sequences from PCNSL, T:sequences from testicular DLBCL. Codon numbering according to IMGT13. The sequence of the hypothetical consensus ‘shared CNS’ is included and mutations in this consensus are indicated by asterisks under the germline sequence. Additional mutations in lymphoma sequences are indicated relative to this consensus sequence. If an additional mutation in a lymphoma clone leads to an amino acid replacement compared to the consensus sequence, the new amino acid is indicated below the sequence of T11. Mutation hotspots (RGYW, WRCY and TAA motifs) are indicated in bold. Primer sequences are highlighted in grey.

Discussion

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Discussion

We describe a unique patient with primary DLBCL of one immunesanctuary (CNS) and a very late, isolated relapse to anotherimmune sanctuary (testis). Both localisations were clonallyrelated, and showed a high level of ongoing SHM and progressiveloss of HLA expression. The observed SHM frequencies are considerablyhigher than in normal GC and post-GC B-cells¹⁶ and nodal DLBCL.¹⁷but comparable to the high SHM frequencies that are common inDLBCL of the testis and CNS.^1,7,8

We used IgH SHM analysis to deduce a model for the developmentof these lymphomas (Figure 2). Since there was a common tumoursubclone between both localizations (C15 and T2/T5/T7/T12/T13were identical), we considered a subclone selection model asthe most appropriate.¹⁸ According to this model, PCNSL subcloneC15 founded the testicular lymphoma. This model fits with theclinical presentation (the testicular lymphoma developed afterthe CNS lymphoma). None of our sequences contained nonsenseor frameshift mutations. Moreover, all sequences had a significantlylower than expected R/S ratio in the framework region indicatingmaintenance of the overall structure of the functional B cellreceptor and the presence of selection pressure. This is reminiscentof the oligoclonal B cells from the CSF of patients with MSwith a high load of ongoing SHM and strong preservation of theFR regions,¹⁹ and CSF derived B-cells implicated in the generationof autoantibodies against GM1 gangliosides in neuropathy.²⁰A BLAST analysis of the PCNSL CDR3 sequence, as described beforefor MALT lymphomas,²¹ did not reveal any homology to antibodiesdirected against known autoantigens (data not shown). Basedon the results from the current study and previous reports wepropose a hypothesis for the biological behaviour of immune-privilegedsite-associated DLBCL. Both PCNSL and testicular DLBCL haveIgH open reading frames with an extremely high load of ongoingsomatic mutations, many leading to amino acid and idiotype changes.^1,7,8In immune sanctuaries, a delicate balance exists between a tolerant/inhibitoryimmune response and an active cytotoxic immune response. Severalmechanisms act together to provide an environment in which thisbalance is skewed towards tolerant or inhibiting responses.^22,23We hypothesize that the high load of mutations makes the tumourcells highly immunogenic and subject to an anti-idiotype immuneresponse, and that in consequence the tumour cells initiallycan only survive within an immune sanctuary where this responseis absent. When subsequently these lymphomas start to grow,the balance will eventually be disturbed, rendering the environmentmore permissive for an anti-tumour cytotoxic immune response.This is substantiated by high numbers of infiltrating cytotoxicT cells in PCNSL and testicular DLBCL at the moment of clinicaldiagnosis.⁶ In the case presented here, this infiltrate is morepronounced in the testicular relapse than in the CNS localisation.Under pressure of this immune reaction the ongoing remodellingof the idiotype, in addition to progressive loss of HLA classII and HLA class I expression, might thus provide escape mechanismsfor the tumour cells, necessary for their sustained survivaland growth at these sites.

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Figure 2. Hypothetical model of the development of the PCNSL and its subsequent relapse in the testis. At a certain point in PCNSL development (in or before 1996), ongoing hypermutation of a ‘shared CNS’ lymphoma clone resulted in 2 subclones (represented by open and filled cells respectively). While the major sub-clone (open cells) continued to diversify within the CNS environment, the minor subclone (filled cell) migrated through the bloodstream to the testis. Here it possibly stayed clinically dormant for many years to finally result, after 8 years, in a clinically manifest lymphoma (filled cells), which continued to accumulate mutations. In this figure mutations of individual clones relative to the consensus shared CNS sequence are indicated by the mutated nucleotide number.

References

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