Composite mantle cell and follicular lymphoma. A case report

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1 Human Pathology (2009) 40, Case study Composite mantle cell and follicular lymphoma. A case report Raquel B. Ilgenfritz MD a,, Agnès Le Tourneau MD a, Michel Arborio MD b, Thierry J. Molina MD, PhD a, Jacques Diebold MD a, Diane Damotte MD, PhD a, Josée Audouin MD a a Department of Pathology, Hotel Dieu, Paris Cedex 04, France b Laboratoire Medibio, Melun, Seine-et-Marne, France Received 8 February 2008; revised 7 May 2008; accepted 19 May 2008 Keywords: Composite lymphoma; Mantle cell lymphoma; Follicular lymphoma; Immunohistochemistry Summary We describe the association of 2 types of small B-cell lymphomas with different morphologic and immunophenotype patterns inside the same lymph node. Morphologically distinct zones were detected and studied with immunohistochemistry analyses. Most of the areas examined were characteristic of classic mantle cell lymphoma (CD20+, CD5+, cyclin D1+) with nodular and mantle zone areas. However, other areas had the morphologic and immunohistochemistry pattern of follicular lymphoma (CD20+, CD10+, Bcl2+). The diagnosis of both lymphomas was confirmed by polymerase chain reaction detection of both Bcl-1 MTC and Bcl-2 MBR rearrangements. DNA degradation in fixed tissue prevented a complete polymerase chain reaction analysis of immunoglobulin heavy chain rearrangements, but a single immunoglobulin H rearrangement was detected at the FR3 locus. These findings confirm the presence of a monoclonal cell population but do not demonstrate the same clonal origin for both lymphoma populations Elsevier Inc. All rights reserved. 1. Introduction 2. Case report A composite lymphoma is defined as the presence of 2 (or more) distinct lymphomas in the same tissue or organ [1].We report the association of a mantle cell lymphoma (MCL) and a follicular lymphoma (FL), each with its typical morphology. We highlight the importance of a thorough examination of histologic slides using the recent World Health Organization classification criteria [2] and the use of immunohistochemistry (IHC) and molecular biology studies to assess diagnosis. We compared our case to 3 other previously described composite lymphomas of the same type [3-5]. Corresponding author. address: raquel.fritz@gmail.com (R. B. Ilgenfritz). A 66-year-old woman presented with dyspnea and a full throat sensation. Clinical examination and imaging showed Waldeyer ring lesions morphologically consistent with the diagnosis of lymphoma. Multiple biopsies were performed on the right tonsil, left pharyngeal wall, left lingual tonsil, cavum, and nasal cavity. Bone marrow biopsy was normal. Intense uptake zones in the mediastinum, peribronchial, and subdiaphragmatic regions were detected by positron emission tomography (PET) scan. Corticoid treatment was started, and follow-up involving physical examinations and PET scan was performed every 3 to 6 months. Several small fragments were sampled. The diffuse and partially nodular patterns found were suggestive of a small B-cell lymphoma. Neoplastic cells were positive for CD20, /$ see front matter 2009 Elsevier Inc. All rights reserved. doi: /j.humpath

2 260 R. B. Ilgenfritz et al. CD5, immunoglobulin (Ig) D, and Bcl-2; cyclin D1 was only detected in some cells, at a low level. The initial diagnosis was that of a partially nodular small B-cell lymphoma, probably of mantle cell origin. Follicular hyperplasia or FL was not identified. Polymerase chain reaction (PCR) analysis of the FR3 locus revealed a clear monoclonal band. Analyses of the IgH FR1 and FR2, Bcl-1, and Bcl-2 loci were not interpretable because of DNA degradation. One year later, the patient complained of the same symptoms. A discrete right cervical lymphadenopathy was found, but oropharyngeal examination and regional PET scan did not reveal any other lesion. Imaging studies showed a reduction of the subdiaphragmatic and peribronchial masses. The lymph node biopsy was then performed and sent to our department. 3. Materials and methods 3.1. Histologic and IHC studies The small lymph node measured 0.8 cm and was totally included for study. Tissue sections were stained with hematoxylin and eosin, Giemsa, and Gordon and Sweet's silver impregnation method. IHC was performed in paraffin-embedded sections using antigen microwave retrieval and avidin-biotin-peroxidase complex, according to standard methods. The primary antibodies used were directed against CD20, CD79a, Bcl-2, Bcl-6, Ki-67, polyclonal IgD (Dako, Carpinteria, CA), CD3 (Neomarkers, Fremont, CA), cyclin D1 (Neomarkers), CD5, CD10, CD21, and CD23 (Tebu, Paris, France) with dilutions between 1/100 and 1/ Polymerase chain reaction DNA for polymerase chain reaction (PCR) was obtained from three 10-μm sections of formalin-fixed, paraffinembedded tissue using the Illustra blood and cell culture DNA extraction Kit (GE Inc., Lille, France). IgH rearrangement at the FR1, FR2, FR3, and JH regions was performed following protocol detailed elsewhere [6], using negative and positive controls. Partial DNA degradation due to fixation and paraffin inclusion prevented FR1 and FR2 locus interpretations. The Bcl-1/MTC and Bcl-2/MBR rearrangements were detected with the use of standardized methods and primers of the BIOMED-2 protocol [7]. 4. Results Histologic studies showed a small lymph node with a preserved, non-infiltrated capsule. The subcapsular sinus and segments of cortical sinuses were recognizable and filled Fig. 1 Follicular and MCL (hematoxylin and eosin). A, Multiple nodules of different sizes can be recognized; comparison with IHC (Figs. 2 and 3) allows a better understanding of each nodule (original magnification 5 ). B, In the encircled zone, large nodules represent the FL surrounded by MCL. Other areas show nodular MCL (thin arrow) and mantle zone pattern MCL around reactive GC (small arrow). Detail from area inside the circle, showing the morphology of FL cells surrounded by the MCL (original magnification 100 ). with numerous small lymphoid cells, associated with histiocytes and neutrophils. The lymph node architecture was mostly replaced by numerous nodules of various sizes, round or oval, some with an irregular contour and others with a back-to-back or confluent appearance (Fig. 1A). In about 60% of the lymph node area, 2 types of nodules could be recognized. Some of them, very homogeneous, were constituted by small- and medium-sized lymphocytes with irregular nuclei, open chromatin, small nucleoli, and scanty cytoplasm, suggestive of MCL. Mitoses were rare, and no large cells (centroblasts or immunoblasts) or tingible body macrophages were seen. These lymphocytes were positive for CD20, CD5 (Fig. 2B and D), IgD, cyclin D1 (Fig. 2C), and Bcl-2 (Fig. 3B), and negative for CD3 (Fig. 2A), CD10 (Fig. 3A), and Bcl-6. This immunophenotype is diagnostic of MCL and was corroborated by the finding of Bcl-1 (MTC locus) rearrangement in PCR study, related to the t(11; 14) translocation. Less than 20% of these cells were positive for Mib-1 (Ki67), excluding the diagnosis of a blastic

3 Composite mantle cell and follicular lymphoma 261 Fig. 2 MCL (immunoperoxidase). A and B, Detail from left part of Fig. 1A (original magnification 5 and 100 ). Comparison between CD3 (A) and CD5 (B) shows that neoplastic CD5+ CD3 cells are organized either in a nodular pattern or in a mantle cell pattern, around the FL (large pale nodules) or around reactive GC (small pale nodules). C and D, Same area as Fig. 1B showing (C) nuclear staining for cyclin D1 in the mantle zone around the FL and (D) weak staining of MCL with CD5, contrasting with the strong marking of reactive T lymphocytes.

4 262 R. B. Ilgenfritz et al. CD20, CD10, Bcl-2, and Bcl-6 (Fig. 3) but not for CD5 (Fig. 2B) or cyclin D1, confirming the diagnostic of a FL. A meshwork of CD21- and CD23-positive FDC was also present. The t(14:18) translocation was demonstrated by the rearrangement of Bcl-2 at the MBR locus. The nodules of FL were surrounded by neoplastic mantle cells. Detection of immunoglobulin heavy chain (IgH) rearrangements was only possible at the FR3 locus because of DNA degradation in fixed tissue. We detected the presence of the same FR3-IgH PCR band found in the Waldeyer ring biopsies, clearly related to the MCL component. Further analyses such as microdissection, gene sequencing techniques, and Fluorescence in situ hybridization (FISH) would be necessary to demonstrate a common clonal origin for both FL and MCL populations. However, more studies of the material were not possible due to tissue consumption. The interpretation of this complex association of 2 different lymphomas was difficult based only on morphology, and IHC was indispensable to clearly define both components and their relationship. PCR studies of Bcl-1 and Bcl-2 loci were also critical to confirm at the molecular level the presence of 2 different rearrangements. 5. Discussion Fig. 3 Follicular lymphoma (immunoperoxidase, original magnification 5 ). A, Strong staining for CD10 in the FL (large dark nodules in the upper left) and weak staining in the small reactive GC (center). B, Bcl-2 shows staining of both lymphomas in the left part of the figure and staining of MCL with nodular or mantle zone pattern around negative small GC in the other areas. transformation. A few typical follicular dendritic cells (FDCs) were observed and stained in a meshwork pattern by CD21 and CD23, which established that these nodules were former benign follicles colonized by the MCL. They represent the nodular pattern of MCL, and although they were sometimes confluent, resembling a diffuse pattern, IHC and the Gordon-Sweet method were able to confirm their nodular nature. The second type of nodules represented MCL with a mantle zone pattern. The mantle cells showed the same morphology and immunophenotype described above and were organized in a thick band surrounding small reactive germinal centers (GCs). These were positive for CD20, CD10, and Bcl-6 but negative for Bcl-2, cyclin D1, and CD5 (Fig. 3). Numerous cells expressed Ki67. In some areas, the reactive GC were partially colonized by neoplastic mantle cells. Most of the lymph node was occupied by the MCL with these 2 different patterns. Three large nodules with the typical findings of FL were also detected, using morphologic and IHC studies. They showed a marked preponderance of centrocytes and only a few centroblasts (Fig. 1A). These cells were positive for We detected 2 different small B-cell lymphomas in the same lymph node mantle cell and follicular lymphomas closely associated to produce a complex morphologic pattern. Such an association corresponds to the definition of composite lymphoma, initially proposed by Kim [1]. Cachia et al [8] reported the association of a MCL and a plasmacytoma, emphasizing the need to restrict this definition to cases involving lymphomas that show clearly distinct immunophenotypes, as well as different molecular markers. Recently, 3 other similar cases have been published: 2 in 1999 by Fend et al [3] and by Tsang et al [4], and another in 2006 by Zamò et al [5]. Our patient had 2 different B-cell lymphomas with distinct morphologic patterns and immunophenotypes, consistent with 2 of these previously reported cases. In addition, we detected the t(11:14) translocation involving Bcl-1 rearrangement, typically found in MCL, and the t(14:18) translocation involving Bcl-2 rearrangement, typically found in follicular lymphoma. An important consideration in all these cases of composite lymphoma concerns their clonal origin. In the first previously published case [3], PCR analysis of DNA obtained from a whole tissue section failed to demonstrate biclonality. The laser capture microdissection of both different cell populations revealed a molecular microheterogeneity suggestive of a biclonal origin of both lymphomas; however, the immunohistochemical and molecular properties of the 2 lymphoma populations were not

5 Composite mantle cell and follicular lymphoma clearly distinct. Bcl-2 rearrangement was detected in both populations, and the IHC antigen cyclin D1, suggestive of a MCL, was not detected. Tsang et al [4], using flow cytometry, PCR, DNA cloning, and sequencing analysis, demonstrated that both lymphomas seemed to develop from a common malignant B-cell clone. More recently, Zamò et al [5] used various molecular techniques to demonstrate 2 distinct and clonally unrelated cell populations. In our study, PCR analysis revealed in both Waldeyer ring and lymph node biopsies a single band rearrangement of IgH at the FR3 locus. This rearrangement was certainly present in the MCL, but in the absence of microdissection, this was not sufficient to prove the common clonality of the MCL and FL lymphomas. The frequent occurrence of ongoing somatic mutations of the FR3 locus in follicular lymphomas is well known, and its presence often renders the results of PCR at the FR3 locus not suitable because of false-negative findings. So, a potential unrelated clone linked to the FL may not have been detected. However, using whole tissue PCR on laser microdissected tissue, Fend et al [3] separated their 2 morphologically distinct entities and confirmed the presence of 2 unrelated clones at the FR3 locus, demonstrating the different immunophenotypes of the 2 populations. References 263 [1] Kim H. Composite lymphoma and related disorders. Am J Clin Pathol 1993;99: [2] Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. World Health Organization classification of tumors. Pathology and genetics of tumors of haematopoietic and lymphoid tissues. Lyon: IARC Press; p [3] Fend F, Quintanilla-Martinez L, Kumar S, et al. Composite low grade B- cell lymphomas with two immunophenotypically distinct cell populations are true biclonal lymphomas. A molecular analysis using laser capture microdissection. Am J Pathol 1999;154: [4] Tsang P, Pan L, Cesarman E, Tepler J, Knowles DM. A distinctive composite lymphoma consisting of clonally related mantle cell lymphoma and follicle center cell lymphoma. HUM PATHOL 1999;30: [5] Zamò A, Zanotti R, Lestani M, et al. Molecular characterization of composite mantle cell and follicular lymphoma. Virchows Arch 2006; 448: [6] Camilleri-Broët S, Devez F, Tissier F, et al. Quality control and sensitivity of polymerase chain reaction techniques for the assessment of immunoglobulin heavy chain gene rearrangements from fixed- and paraffin-embedded samples. Ann Diagn Pathol 2000;4:71-6. [7] Van Dongen JJ, Langerak AW, Brüggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT Leukemia 2003;17: [8] Cachia AR, Diss TC, Isaacson PG. Composite mantle-cell lymphoma and plasmacytoma. HUM PATHOL 1997;28: