Abstract. Case Report

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1 Hematopathology / Composite In Situ Lymphoma Coexisting Follicular and Mantle Cell Lymphoma With Each Having an In Situ Component A Novel, Curious, and Complex Consultation Case of Coincidental, Composite, Colonizing Lymphoma Michele R. Roullet, MD, 1 Daniel Martinez, 2 Lisa Ma, 3 Melissa Halpern Fowler, MD, 4 Ellen D. McPhail, MD, 5 Alexander Judkins, MD, 2 Daniel A. Arber, MD, 3 and Adam Bagg, MD 6 Key Words: Composite; Lymphoma; In situ; Follicular; Mantle cell DOI: 1.139/AJCP5RT4MRSDGKSX Abstract A diagnosis of composite lymphoma is typically prompted by the observation of morphologic discordance. We present a case of a spleen revealing histologic features of follicular lymphoma, without any indication of a second lymphoma. Immunohistochemical stains supported this diagnosis and showed the follicular lymphoma to be BCL2. However, these studies revealed 2 additional unexpected findings: cyclin D1+ mantle zone cells surrounding neoplastic and reactive follicles (indicative of in situ mantle cell lymphoma) and BCL2-bright, histologically nonneoplastic follicles (indicative of in situ follicular lymphoma). ImmunoFISH and microdissection and polymerase chain reaction analysis documented the clonal nature of the cyclin D1+ mantle zones and illustrated clonal independence from the follicular lymphoma. This case illustrates an uncommon and unusual composite follicular and mantle cell lymphoma, with the follicular lymphoma accompanied by an in situ component, whereas the only manifestation of the mantle cell lymphoma was in situ. Composite lymphoma refers to the simultaneous occurrence of 2 morphologically and immunophenotypically different lymphomas in the same site or simultaneously in different sites. 1 The incidence of composite lymphomas ranges from 1% to 4.7%, and they occur in patients of both sexes and with a variable age range. 2 These lymphomas may or may not be clonally related, with reports of either occurrence described in the published literature. 3-7 The 2 components could possibly be any of a variety of combinations of a B-cell (non-hodgkin) lymphoma, Hodgkin lymphoma, and T-cell lymphoma. 8 Based on published reports, composite lymphomas are most often a combination of a B-cell lymphoma and Hodgkin lymphoma or a B- and T-cell lymphoma. 6,7,9-12 Composite lymphomas composed of 2 concurrent, discrete, small B-cell lymphomas have also been described, but these are less common, with mantle cell lymphoma (MCL) occurring as a component only rarely. 3,4,6,7,11-2 Although the initial consideration of a diagnosis of composite lymphoma is typically prompted by the observation of morphologic discordance, the increasing use of a battery of immunophenotypic studies in the evaluation of lymphomas may lead to unanticipated findings, as illustrated in this case study. Indeed, this case study of an unusual composite lymphoma is unique and of particular interest in that both lymphomas had in situ components within the same specimen, an observation that has not, to the best of our knowledge, been reported previously. Case Report An 84-year-old woman with recurrent rectal carcinoma underwent splenectomy for multiple radiologically detected splenic masses, presumed to represent metastatic carcinoma. The original diagnosis of rectal carcinoma was made 5 years 584 Am J Clin Pathol 21;133: Downloaded 584 from DOI: 1.139/AJCP5RT4MRSDGKSX on 2 June 218

2 Hematopathology / Case Report previously. Treatment consisted of transanal excision (with 1 lymph node reported to be benign ), followed by chemotherapy and radiotherapy. Annual surveillance computed tomography (CT) scans of the chest, abdomen, and pelvis performed before the scan that prompted splenectomy did not reveal any recurrent metastatic disease or lymphadenopathy. The spleen was submitted to one of us (A.B.) in consultation. Histologic sections of the grossly appreciated nodules in the spleen did not show carcinoma, but instead revealed features consistent with grade 1-2 follicular lymphoma (FL), with a predominance of small cleaved cells with fewer than 5 centroblasts per high-power field Image 1A. The red and white pulp in the uninvolved (nonnodular) portions of the spleen were histologically unremarkable Image 1B. The CBC was normal, apart from mild lymphopenia. The patient refused staging or therapy. A B C D Image 1 Histologic and immunophenotypic findings demonstrate the coexistence of mantle cell lymphoma and follicular lymphoma (FL) in the spleen. The numbers in brackets refer to the 5 regions that were microdissected for molecular studies (see Image 3). A, H&E stain showing FL with back-to-back follicles [1] and attenuated mantle zones [2] ( 1). There is a monotonous population of small lymphocytes with cleaved nuclei, with rare centroblasts (inset, 1,). B, H&E stain showing FL nodule and uninvolved spleen ( 1). Outside of the nodule of FL the histologic features are quite different, showing essentially normal, histologically uninvolved spleen with reactive germinal centers [3], but with somewhat expanded mantle zones [4]. C, Cyclin D1 immunohistochemical stain ( 1) reveals strong nuclear positivity in the mantle zone cells surrounding the FL [2] and in the histologically uninvolved spleen [4]. D, BCL2 immunohistochemical stain ( 1) is negative in the FL [1]. In the histologically uninvolved spleen, BCL2 is also negative in most reactive germinal centers [3] but is brightly positive in other normal-appearing germinal centers [5]. Downloaded from on 2 June 218 Am J Clin Pathol 21;133: DOI: 1.139/AJCP5RT4MRSDGKSX 585

3 Roullet et al / Composite In Situ Lymphoma A CT scan performed 9 months after splenectomy revealed mildly prominent intra-abdominal lymphadenopathy. The patient died of unknown causes 13 months after the initial diagnosis; however, there was never any documented evidence of the development of lymphoma elsewhere, other than what was originally noted in the spleen. Materials and Methods Immunohistochemical Studies These studies were performed using our standard small B-cell lymphoma panel, which consists of CD3, CD5, CD1, CD2, CD23, CD43, CD79a, cyclin D1, BCL6, and BCL2. Antibodies used were all prediluted and obtained from Ventana Medical Systems (Tucson, AZ) and included the following clones (in parentheses): CD3 (2VG6), CD5 (SP19), CD1 (56C6), CD2 (L26), CD23 (1B12), CD43 (L6), CD79a (11E3), cyclin D1 (SP4), BCL6 (GI191E/ A8), and BCL2 (124). Immunostaining was performed on the Ventana BenchMark XT (Ventana Medical Systems) using standard procedures per the manufacturer s instructions. All immunostains were counterstained with bluing and hematoxylin. Fluorescence In Situ Hybridization and ImmunoFISH Fluorescence in situ hybridization (FISH) and immuno- FISH experiments were performed using protocols previously described. 21,22 For FISH studies, dual-fusion DNA probes for IGH@-BCL2 and IGH@-CCND1 and a break-apart probe for BCL6 (Vysis, Downers Grove, IL) were used. For immunofish analysis, immunofluorescence was performed using a streptavidin method with antibodies against BCL2, CD1, or CD2 (all from DAKO, Carpinteria, CA) and Alexa Fluor 35 goat antirabbit IgG(H+L) (Molecular Probes, Invitrogen, Carlsbad, CA). Slides were then hybridized with BCL2, CCND1, or IGL@ break-apart probes or dual-color IGL@-BCL2 FISH probes (CCND1 break-apart, Vysis; BCL2 break-apart, Vysis; IGL@, homebrew; and IGL@-BCL2 dual fusion, homebrew). Laser Capture Microdissection and Polymerase Chain Reaction Analysis We cut 5-μm sections of formalin-fixed, paraffinembedded tissue onto PEN-membrane slides (Carl Zeiss Microimaging, Thornwood, NY) and stained them with H&E. Laser capture was performed using the PALM Microlaser System (Carl Zeiss Microimaging). Five distinct regions were isolated, based on the histologic and immunohistochemical findings (see the Results section), and collected into separate collection tubes. DNA was also isolated from these regions using the Qiagen Tissue (Qiagen, Valencia, CA) protocol. IGH@ and IGK@ polymerase chain reaction (PCR) was performed using the IGH and IGK Gene Clonality assays (Invivoscribe Technologies, San Diego, CA), containing BIOMED-2 primers, according to the manufacturer s protocol. 23 The PCR products were analyzed by capillary electrophoresis on an ABI PRISM 31 Genetic Analyzer (Applied Biosystems, Foster City, CA). The specimen was received in fixative; hence, neither flow cytometry nor conventional karyotypic analysis was performed. Results Immunohistochemical Studies Immunohistochemical stains revealed that the nodules of histologically evident FL contained CD2+/CD79a+ B cells that were positive for CD1, CD23, and BCL6 but negative for BCL2, CD5, and CD43. Unexpectedly, the mantle zone lymphocytes, those surrounding the follicles of FL and in the histologically uninvolved white pulp of the spleen were cyclin D1+ Image 1C. These cyclin D1+ cells were confined to the mantle zones and did not colonize the germinal centers. Of note, these cyclin D1+ cells did not coexpress CD5. Although most of the CD1+/BCL6+ germinal centers in the uninvolved areas were BCL2, a subset contained variable numbers of brightly BCL2+ cells Image 1D. These cells were cyclin D1. The T cells in the background were positive for CD3 and CD5. FISH and ImmunoFISH FISH (on whole, nonmicrodissected tissue) using fusion probes was positive for a t(11;14)/ccnd1/igh@ fusion but negative for t(14;18)/bcl2/igh@. This tissue was also negative for a BCL6 translocation, using the break-apart probe. ImmunoFISH analysis, using break-apart probes for BCL2 and CCND1, was positive for split signals with BCL2 probes in CD1+ cells Image 2A and with the CCND1 probes in CD1 cells Image 2B. Conversely, no BCL2 break apart was evident in the CD1 cells, and no CCND1 break apart was present in the CD1+ cells. The positive (split signal) BCL2 result was present in the BCL2 FL and the brightly BCL2+ germinal center cells in the histologically uninvolved spleen. Similarly, IGL@ break-apart probes also showed a split signal in the FL nodules and the BCL2+ germinal center cells in the uninvolved spleen, and dual FISH using the IGL@ and BCL2 probe was positive for IGL@/BCL2 fusion Image 2C. However, abnormal signals were not detected in the BCL2 germinal centers in the uninvolved spleen. 586 Am J Clin Pathol 21;133: Downloaded 586 from DOI: 1.139/AJCP5RT4MRSDGKSX on 2 June 218

4 Hematopathology / Case Report A B C Image 2 Fluorescence in situ hybridization (FISH) analysis reveals the presence of 2 different translocations. A, BCL2 split signal (red-green separation) in a CD1+ (blue membranous staining) cell within the histologically evident follicular lymphoma. B, CCND1 split signal (red-green separation) in a CD1 cell in the histologically normal mantle zone surrounding the follicular lymphoma. C, Abnormal nucleus from the follicular lymphoma nodules showing IGL@-BCL2/t(18;22)(q21;q11.2) (1 red, 1 green, 2 fusion signal pattern using a dual fusion IGL@-BCL2 FISH probe). Laser Capture Microdissection and PCR Analysis A minimum of 94,358 μm 2 (range, 94,358-1,959,468 μm 2 ) of tissue was collected for each of the 5 distinct regions by laser capture microdissection Image 3, corresponding to approximately 1,14 to 22,926 cells per region. PCR analysis was subsequently performed on each of these regions using BIOMED-2 IG primers. IGH@ PCR revealed identical monoclonal peaks using V-FR2 primers, as well as with the DH1-6 primers in 2 regions: tissues dissected from the mantle zones within the nodules of FL and also in the mantle zones of the uninvolved white pulp; these peaks were not detected in 3 other regions: the BCL2 FL; the normal, BCL2 germinal centers in the uninvolved white pulp; and in the histologically normal but BCL2+ germinal centers in the white pulp Image 4. IGK@ PCR (VK-Kde) revealed the same monoclonal rearrangement in all 5 regions. Discussion In this consultation case, the initial clinical concern for carcinoma in the spleen was excluded. Rather, the nodules, which were indeed neoplastic, reflected a coincidentally discovered case of composite lymphoma, consisting of FL and MCL, with the latter evident in the in situ form only. Cases of composite FL and MCL are rare, with only 6 cases reported in the literature, to the best of our knowledge. 3,4,13-16 These reported cases involved nodal and extranodal sites, including the orbit 14 and small bowel 3 ; none, however, involved the spleen exclusively. The FL component typically appeared to be low grade and BCL2+ by immunohistochemical analysis and to harbor a t(14;18). The MCL component most commonly displayed a mantle zone growth pattern with diffuse and in situ patterns also observed. Unlike the present case, the MCL was morphologically visible in all reported cases, before immunohistochemical stains. Downloaded from on 2 June 218 Am J Clin Pathol 21;133: DOI: 1.139/AJCP5RT4MRSDGKSX 587

5 Roullet et al / Composite In Situ Lymphoma A B Image 3 Laser capture microdissection and immunoglobulin polymerase chain reaction from microdissected regions is supportive of 2 distinct clonal processes. The numbers in brackets correspond to regions 1 through 5 in Image 1. A, BCL2 follicular lymphoma (FL) [1] and mantle zones within the nodules of FL (mantle cell lymphoma [MCL] in situ) [2] before capture ( 4). B, Germinal center (GC) in uninvolved spleen [3] ( 4) and mantle zones of the uninvolved white pulp (MCL in situ) [4] ( 2) before and after capture. Not pictured, BCL2+ GCs in uninvolved spleen (FL in situ) [5]. IGH FR2 IGH D1-6 IGK (VK-Kde) , Image 4 IGH@ polymerase chain reaction (PCR) demonstrates monoclonality in the mantle cell lymphoma (MCL) and IGK@ PCR demonstrates a common clone in all 5 regions. The numbers in brackets correspond to regions 1 through 5 in Image 1. IGH@ PCR showing peaks of 257 base pairs (bp) using V-FR2 primers and 159 bp using DH1-6 primers in tissues dissected from the mantle zones within the nodules of follicular lymphoma (MCL in situ [2]) and in the mantle zones of the uninvolved white pulp (MCL in situ [4]); IGK@ PCR (VK-Kde) showing the same monoclonal rearrangement of approximately bp in all 5 regions. Arrows show monoclonal peaks. 588 Am J Clin Pathol 21;133: Downloaded 588 from DOI: 1.139/AJCP5RT4MRSDGKSX on 2 June 218

6 Hematopathology / Case Report Immunophenotypically, the MCL component may be CD5 positive or CD5 negative. These composite lymphomas composed of FL and MCL are typically clonally unrelated, but, on occasion, appear to be derived from the same clone. However, the techniques used to make this determination have been heterogeneous and, thus, difficult to compare. In the present case, the common monoclonal IGK@ rearrangement suggests that the FL and MCL are clonally related. However, other data support clonal independence at 2 levels. First, immunofish showed evidence of 2 separate translocations in immunophenotypically different cell populations, namely a BCL2 translocation only in the CD1+ FL and a CCND1 translocation only in the CD1 MCL. Second, IGH@ PCR documented monoclonality in the MCL but not the FL. The failure to detect clonality at this locus in the FL is likely related to heavy somatic hypermutation. To reconcile these seemingly conflicting data, one could speculate (but not easily prove) that there is a common (ancestral) clonal origin to the FL and MCL (based on the IGK@ findings) with subsequent divergence (based on the different and independent translocations). The BCL2 translocation evident by FISH in the FL but not the MCL was an unusual t(18;22), resulting in the fusion of the BCL2 gene with the IGL@ gene, rather than the much more common t(14;18), which affects the IGH@ gene. There are 2 additional intriguing findings in this case, perhaps even more noteworthy than its reflecting an uncommon composite lymphoma. First, the cyclin D1 immunohistochemical studies unexpectedly unmasked the presence of a second lymphoma, MCL, which was (and indeed still is, even after subsequent and independent expert hematopathologic review) histologically inapparent. These morphologic and immunophenotypic findings are thus best interpreted to reflect MCL in situ, or colonization of follicles, that happen to contain overt or in situ FL, by clonal MCL cells. The concept of MCL in situ has been recently proposed. 13,24 In situ MCL occurs without disruption of normal architecture and involves mantle zones that are not expanded or only minimally so. The apparent colonization of the neoplastic follicles in our case perhaps illustrates how MCL [which might have its oncogenic origins in the marrow, since that is where the t(11;14) translocation is believed to occur] preferentially localizes to the mantle zones. It is interesting that in our case and in a report that included a case of FL and an in situ component to the MCL, the MCL was negative for CD5. 13,15 This could support the hypothesis that CD5 acquisition occurs later in the course of events resulting in MCL. Of note, the unusual lack of CD5 expression has also been reported in a subset of cases of overt MCL that were associated with an indolent clinical course. 25,26 The second additionally interesting facet is the presence of the focally bright BCL2+ germinal centers in the histologically uninvolved portions of the spleen. Similar to the mantle zones described, these germinal centers were somewhat unexpectedly morphologically unremarkable (even on expert rereview) and likely reflect FL in situ, analogous to MCL in situ. 27,28 FL in situ is also referred to as intrafollicular neoplasia because it is not uniformly accepted that this in situ process is biologically comparable with, for example, in situ carcinoma. In studies performed by others, the BCL2+ germinal centers were shown, in the majority of cases examined, to be monoclonal and harbor BCL2 translocations. 27 In our study, immunofish revealed that a BCL2 translocation was indeed evident in the FL in situ and in the overt FL. An apparently identical monoclonal IGK@ rearrangement was also evident in both of these regions. The additional finding of this PCR-detectable clone in a third region, namely in germinal centers that were histologically and immunohistochemically unremarkable and negative by immunofish, is of interest. These data suggest that these germinal centers might have pre in situ FL, below the level of sensitivity of immunohistochemical and immunofish detection but detectable by more sensitive PCR. As is the case with ML in situ, patients with FL in situ may or may not have disease elsewhere. The patient described herein had no other clinical evidence of lymphoma, and there was only mildly prominent intra-abdominal lymphadenopathy, detected by subsequent CT scan. One may speculate that the in situ FL noted in this report (and perhaps preceded by a pre in situ component) progressed into overt FL in an adjacent area of the spleen. It is likely that an acquired mutation in the BCL2 gene accompanied this progression, leading to an altered epitope no longer recognized by the antibody used for immunohistochemical analysis, 29 accounting for the BCL2 positivity by immunohistochemical studies in the FL in situ but the failure to detect BCL2 expression in the overt FL. This FL (and indeed the MCL) in this patient was seemingly confined to the spleen at the time of diagnosis. (Radiologic studies performed 9 months following splenectomy did reveal mild intra-abdominal lymphadenopathy; however, these lymph nodes were never biopsied.) The spleen may be involved by FL primarily or secondarily as part of systemic disease. Approximately 3% to 75% of splenectomy cases containing FL reflect primary presentation. 3,31 Recently, the characteristics of splenic FL have been described in 2 series. 3,31 Morphologically, FL in the spleen may demonstrate architectural abnormalities similar to those seen in nodal FL. In addition, there may be architectural preservation or an intrafollicular or in situ growth pattern. 3 All of these patterns were observed in cases of splenic FL in which the spleen was the primary site of presentation. 3 Compared with nodal FL, a higher percentage of cases occurring in the spleen was observed to be BCL2 by one group but not the other. Downloaded from on 2 June 218 Am J Clin Pathol 21;133: DOI: 1.139/AJCP5RT4MRSDGKSX 589

7 Roullet et al / Composite In Situ Lymphoma This consultation case is noteworthy for the several unanticipated (ie, coincidental) findings: composite FL and MCL, both with an apparent in situ component, with one component (MCL in situ) perhaps colonizing the other (FL, the overt and in situ components). Furthermore, this case also illustrates the potential added (but also perhaps confounding) value of using an extended panel of immunohistochemical studies in an otherwise histologically clear-cut case of FL. Without the BCL2 and cyclin D1 stains, which are both part of our routine panel for the diagnosis of small mature B-cell lymphomas, the in situ MCL and in situ FL would have gone undetected. And had these stains not been performed, you would not be reading this report! From the 1 Pathology Sciences Medical Group and Eastern Virginia Medical School, Norfolk; 2 Department of Pathology and Pathology Core Laboratory, Children s Hospital of Philadelphia, University of Pennsylvania Medical Center, Philadelphia; 3 Department of Pathology, Stanford University Medical Center, Stanford, CA; 4 Saint Vincent Health Center Laboratory, Erie, PA; 5 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN; and 6 Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia. Supported by a grant from the Leukemia & Lymphoma Society, White Plains, NY (Dr Bagg). Address reprint requests to Dr Bagg: Dept of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, 7.13 Founders Pavilion, 34 Spruce St, Philadelphia, PA References 1. Kim H. Composite lymphoma and related disorders. Am J Clin Pathol. 1993;99: Ioachim HM, Medeiros LJ, eds. Ioachim s Lymph Node Pathology. 4th ed. 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