Modern Pathology (2009) 22, 416–425 & 2009 USCAP, Inc All rights reserved 0893-3952/09 $32.00 www.modernpathology.org Hypermutation in mantle cell lymphoma does not indicate a clinical or biological subentity Margit Schraders1,5, Sabine Oeschger2,5, Philip M Kluin3, Konnie Hebeda1, Ed Schuuring3, Patricia JTA Groenen1, Martin-Leo Hansmann4 and Johan HJM van Krieken1 1Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2Department of Pathology, University Hospital Giessen, Giessen, Germany; 3Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands and 4Department of Pathology, University Hospital Frankfurt, Frankfurt, Germany Mantle cell lymphoma is a prime example of a well-defined entity based on morphology, phenotype, genetics and also clinical features. Although most patients have an adverse clinical course, some have a better survival than others. The most consistently reported adverse prognostic parameter is a high mitotic rate. Recently, it has been shown that hypermutation in the immunoglobulin heavy-chain gene occurs in a subset of mantle cell lymphomas. It is, however, unclear whether the mutational status is stable over time within a given case, whether hypermutation might be influenced by therapy and how it is related to other relevant biological features of mantle cell lymphoma. In this study, we analyzed 23 typical mantle cell lymphoma cases with respect to mutational status and compared the results with clinicopathological and genetic data to determine whether the presence of mutation indicates a subentity with clinical or pathological relevance. We found somatic hypermutation in 26% of our cases and, interestingly, one case showed ongoing somatic hypermutation. In tumor cells of both mutated and unmutated cases, we found a preferential usage of VH3-21 (23%) and VH4-34 (19%). No significant correlations were found between mutation status and the other morphological and genetic features analyzed. In conclusion, our results provide additional evidence that mutation status in mantle cell lymphoma is better interpreted as a feature within the spectrum of disease that seems to have little clinical or pathological relevance. Modern Pathology (2009) 22, 416–425; doi:10.1038/modpathol.2008.199; published online 9 January 2009 Keywords: mantle cell lymphoma; somatic hypermutation; VH gene usage Malignant lymphomas are presently considered as a cell lymphoma has rather specific morphology with series of clinicopathological entities. Each entity is monomorphic small- to medium-sized lymphoid defined on histological, phenotypic, genetic and, to tumor cells with a nodular, diffuse or mantle zone a lesser extent, clinical features. Within a given growth pattern; however, about 20% are of the entity, there is often a spectrum of morphology, blastoid variant.2 The median survival for patients phenotype, genetics and also clinical features. These with mantle cell lymphoma is 3–5 years; the vast latter aspects can be used as prognostic factor or majority of patients cannot be cured. The most alternatively to define a subentity. Mantle cell consistently reported adverse histopathological lymphoma is a prime example of a very well defined prognostic parameter is a high mitotic rate.3 Other entity with several specific prognostic factors. The adverse prognostic factors are blastoid morphol- tumor is characterized by the t(11;14) translocation, ogy2,4 and the presence of p53 mutation accompa- which places the BCL1 locus under the influence of nied by 17p deletion.5–7 the immunoglobulin heavy-chain gene enhancer, Recently, it has been shown that hypermutation in resulting in overexpression of cyclin D1.1 Mantle the immunoglobulin heavy-chain gene occurs in a subset of mantle cell lymphomas, but the biological relevance is unclear.8–12 In analogy with the situa- Correspondence: Professor Dr JHJM van Krieken, Department of tion in chronic lymphocytic leukemia/small lym- Pathology, Radboud University Nijmegen Medical Centre, Geert phocytic lymphoma, it was suggested that mutated Grooteplein 24, Nijmegen 6500 HB, the Netherlands. cases might be considered as a relevant subentity E-mail: [email protected] 5 with different biological features and clinical beha- These authors contributed equally to this work. 13–15 Received 22 August 2008; revised and accepted 19 November vior. It is, however, at present unclear whether 2008; published online 9 January 2009 the mutational status is stable over time within a Hypermutation in mantle cell lymphoma M Schraders et al 417 given case, whether it might be influenced by 100 ng of this extracted DNA was used in each therapy and how it is related to other relevant amplification reaction. biological features of mantle cell lymphoma, such as genetic imbalances, breakpoints in the BCL1/CCND1 locus and proliferative and apoptotic rate. PCR and Sequence Analysis In this study, we analyzed a series of typical mantle cell lymphomas with respect to mutational The clonal VH gene rearrangements of the mantle status and compared the results with clinicopatho- cell lymphoma cases were identified by PCR using logical, morphological and genetic data to determine family-specific VH framework region (FR) I primers together with a JH gene consensus primer according whether the presence of mutation indicates a 17 subentity with clinical or pathological relevance. to the BIOMED-2 protocol. PCR products were purified and sequence analysis was performed on an automatic sequencing platform (ABI 3730, Applied Materials and methods Biosystems). Sequences were analyzed using the IMGT database (http://imgt.cines.fr:8104/). The VH Case Selection gene sequences were considered mutated when the A total of 27 specimens from 23 patients were sequences showed less than 98% homology with the selected from the tissue banks of the departments of corresponding germline sequence. pathology of University Medical Centre Nijmegen, For light-chain gene rearrangement identification, University Medical Centre Groningen, Rijnstate 100 ng of genomic DNA was used in a 50 ml PCR Hospital Arnhem and PATHAN, Rotterdam, the mixture with the family specific kappa and lambda Netherlands. All cases were stained for cyclinD1 primers according to the BIOMED-2 protocol. and CD5 and were reviewed by four experienced pathologists at a multiheaded microscope. The only inclusion criterion was the diagnosis of classical Southern Blot Analysis mantle cell lymphoma and the availability of frozen 15–20 mg of genomic DNA was digested with BglII tissue. Morphological features were assessed in- and HindIII (Life Technologies, Rockville, MD, USA) cluding (vaguely) nodular growth, mantle zone and separated in 0.7% agarose gels. The DNA was growth and marginal zone or monocytoid differen- transferred by vacuum blotting to Nytran-13N tiation of the tumor cells. Mitotic figures were nylon membranes (Schleicher and Schuell, Dassel, counted per 15 high power fields (hpf). From two Germany), which were subsequently hybridized with patients, serial biopsies were available, MC50, 51, 32P-labeled probes. For the determination of the IGK 52 and 53 plus MC63 and 66, which were divided gene configuration probes specific for Jk (IGKJ5), over a period of 7 years and 1 year, respectively. Ck (IGKC)andKde (IGKDE) (DAKO Corporation, Carpinteria, CA, USA) were used.18 To examine the IGL locus, the IGLC1D and IGLJ2 probes were used.19 Immunohistochemistry From all cases, 4-mm-thick tissue sections of paraf- fin-embedded tissue were stained for CD20, CD5 The Detection of BCL1/11q13 Breakpoints (both Dako, Denmark) and cyclinD1 (Neomarkers) For the detection of breakpoints in the 800-kb BCL1/ for diagnostic purpose. The cases were also stained 11q13 region around CCND1, a previously designed for Ki-67 (mib1, DAKO, Denmark). Immunohisto- interphase FISH segregation assay was performed chemistry was performed using a standard two-step using a probe set containing cosmids cos6.7/cCl-11- immunoperoxidase method, including development 44 in combination with cos6.31/cosH1.5.20 To with the ABC-method (DAKO, Denmark) and DAB identify breakpoints immediately 30 of the CCND1 as chromogen. Furthermore, immunofluorescence gene (breakpoint cluster 4), cosmid cos6.22/CCND1 was used for the assessment of immunoglobulin in combination with cos6.31/cosH1.5 were used.21 heavy and light chains. Ki-67-positive tumor cells To map breaks within the 85-bp BCL1/MTC-region were counted by using an automated procedure as (cluster 2), we performed the t(11;14)-specific BCL1/ described previously.16 JH-PCR as described previously.17 To map breaks in the 120-kb region between MTC and CCND1 (but DNA Extraction from Tissue Sections outside the 4000-bp MTC), an additonal segregation FISH with P1 clone 9107/11q13 and cos6.22/CCND1 For DNA isolation, QIAamp DNA Mini Kit (Qiagen) was used (cluster 3).22 Breaks detected within was used. Tissue sections with thickness of the 800-kb BCL1-region but outside clusters 2–4 20–30 mm were digested overnight with 40 ml protei- are located centromeric of the MTC (cluster 1). nase K solution (20 mg/ml) and 360 ml ATL (QIAmp Probes were labeled by Kreatech’s ULS nonenzy- Tissue Lysis) buffer at 551C. The DNA was pre- matic labeling method (Kreatech Diagnostics, cipitated by the addition of 6 M NaCl and dissolved The Netherlands) with biotin-16–aUTP or digoxygen- in H2O at a concentration of 100 mg/ml. A quantity of in-11–dUTP (Roche, Basel, Switzerland). Hybridization,