A unique case of extranodal DLBCL sharing genetic abnormalities with a synchronous ileal lymphoma exhibiting immunoarchitectural features of in situ follicular lymphoma


  • Conflict of interest: Nothing to report.

In situ follicular lymphoma (FL) has been acknowledged as a pathological and biological entity [1, 2]. In situ FL might represent an early stage in the development of overt FL carrying the t(14;18) translocation [3]. The criteria to make a diagnosis of in situ FL include: normal follicle size with well defined mantle zones and no evidence of interfollicular infiltration. BCL2 and CD10 are required for the diagnosis. The expression of these two markers should be very strong (stronger than the mantle zone and reactive T-cells), and confined to the germinal centers (GCs) [3, 4]. We here describe a case of an extranodal diffuse large B-cell lymphoma (DLBCL) clonally related to a synchronous in situ lymphoma, displaying immunophenotypic and genotypic features of FL [1, 2, 5].

A 51-year-old man, admitted to Pordenone Hospital, presented with a 11-cm sized mass of the left iliac fossa, detected by CT studies, without lymphadenopathies. Multiple biopsies of the mass, of parietal peritoneum, and omentum showed a DLBCL with a GC B-cell-like phenotype (CD20+, CD10+, BCL6+, MUM1−, BCL2+, CD3−, CD5−, Ki67+ [90%]) (Fig. 1A). Staging procedures, including bone marrow biopsies, were negative. However, endoscopy biopsies of slightly elevated mucosal zones of the ileum revealed a lymphoma characterized by a B-cell population with immunoarchitectural features of in situ FL (CD20+, CD10+ strong, BCL6+, MUM1−, BCL2+ strong, CD3−, CD5−, Ki67+ [15%]). Morphologically, the lymphoma was exclusively localized to GCs of abnormal lymphoid follicles (Fig. 1B). The patient was then referred to CRO-Aviano, where previous histopathologic diagnoses were confirmed. After surgery, ab-extrinsic involvement of the colonic wall by DLBCL was found and Stage IVB disease was established. The patient is under treatment with rituximab-cyclophosphamide doxorubicin vincristine prednisone (R-CHOP) regimen.

Figure 1.

Morphologic, immunophenotypic, molecular, and molecular cytogenetic findings in a synchronous, clonally related, extranodal DLBCL and ileal in situ FL. A: Extranodal DLBCL cells showing a strong positivity for CD20, BCL2, and BCL6 and displaying a high proliferative index (Ki67: >90%). Inset: Hematoxylin and eosin staining showing centroblastic-like large tumor cells. B: Biopsies of the ileum showing an in situ FL. Low-power microphotographs show that CD20+, BCL2+ (very strong), and BCL6+ B-cells are mainly localized to GCs (serial sections). The proliferative index is low (Ki67: 15%). High-power microphotographs of the upper involved follicle (see the low-power microphotographs) showing that the strongly stained positive BCL2 population is confined to the center (serial sections). Typically, the BCL2 staining in this population is more intense than that exhibited by the surrounding mantle cells. The lower follicle (see the low-power microphotographs) is enlarged and shows BCL2 positive cells that expand outside the follicle without a defined mantle zone. BCL6 seems to be positive in the cells infiltrating the lamina propria outside the follicle. Therefore, for this follicle, the diagnosis of FL Grade 1 with partial or early involvement in the ileum may be alternatively considered. Images were acquired with the Olympus Dot.Slide Virtual microscopy system using an Olympus BX51 microscopy equipped with PLAN APO 2×/0.08 and UPLAN SApo 40×/0.95 objectives. C: Clonality analysis of the IGH framework 3 (FR3) region shows an identical monoclonal peak of 138 base pairs in both lesions (in situ FL and DLBCL) and the same amplicon sizes (175 base pair) for mcr region of the BCL2/JH translocation. The amplification size is shown by electropherograms. D: Extranodal DLBCL. Interphase FISH analysis on histological section using dual color split signal BCL2 probe (DakoCytomation) shows one allele with a normal colocalized signal and two alleles with a split of the red and green signals indicating BCL2 breaks in two alleles. This finding was also observed in both follicles of the ileum involved by FL (not shown). FISH images were acquired with a 100×1.70 oil-immersion objective in a Leika DMR2A fluorescence microscope equipped with the appropriate filter sets and were documented and processed using FISH imaging system (Tesimaging Milano).

Molecular studies were performed in bioptic samples from ileal lymphoma and in bioptic and surgically removed samples from extranodal DLBCL mass. Polymerase chain reaction for immunoglobulin heavy chain (PCR IGH) clonality analysis showed identical monoclonal peaks in both lymphomas (Fig. 1C). BCL2 PCR analysis showed identical peaks for mcr region (Fig. 1C). Fluorescence in situ hybridization (FISH) analysis using split signal probes (Dakocytomation, Copenhagen, Denmark) confirmed the presence of BCL2 rearrangement in both lymphomas and revealed a minor clone [6] with BCL6 rearrangement in ileal lymphoma or in extranodal DLBCL, accounting for 4% and 14% of rearranged nuclei. No MYC rearrangement was observed. The chromosome assessment evaluated using BCL2 (Fig. 1D) and BCL6 regions was trisomic in both lymphomas.

There are quite a few reports on in situ FL with associated FL and even DLBCL [7, 8]. Recently, Bonzheim et al. reported the first description of an in situ FL clonally related to a synchronous overt FL where secondary genetic alterations were demonstrated, probably representing clonal evolution as sign of disease progression [9]. We report on a unique case of lymphoma with features of in situ FL showing BCL2 rearrangement, trisomic chromosome pattern, and small clone displaying BCL6 rearrangement. The recruitment of the same chromosome pattern in addition to an expansion of the BCL6 rearranged clone demonstrates that the in situ ileal lymphoma and the extranodal DLBCL were clonally related. The lesions are clonally related based on the same clonal peak for a immunoglobulin heavy chain joining region (JH) rearrangement and the same size of the BCL2/JH PCR product. Although sequencing may strengthen the proof, the finding is already robust. We suggest that the synchronous DLBCL probably represents a clonal evolution from the in situ lymphoma with expansion (from 4% to 14%) of the clone carrying BCL6 rearrangement [6, 10]. In situ lymphoma, in turn, might represent the DLBCL in situ precursor. An origin of the in situ lymphoma from early colonization of reactive GCs by DLBCL tumor cells is less sustainable because of the lower proliferative index of the in situ lymphoma cells and for the different cell morphology displayed by the two lymphomas.

Antonino Carbone*, Maria Grazia Tibiletti†, Laura Zannier*, Antonella Selva*, Sandro Sulfaro‡, Annunziata Gloghini§, * Division of Pathology, Centro di Riferimento Oncologico Aviano (CRO), Istituto Nazionale Tumori, IRCCS, Aviano, Italy, † Unit of Pathology, University of Insubria-Ospedale di Circolo, Varese, Italy, ‡ Division of Pathology, Azienda Ospedaliera Santa Maria degli Angeli (AOSMA), Pordenone, Italy, § Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy.