Case 7 -

Burkitt Lymphoma, Atypical Variant D. Ashley Hill, M.D. Mihaela Onciu M.D.

Case History: 18-year-old boy with a history of blunt trauma to the abdomen followed by persistent abdominal pain. Physical examination prompted a clinical diagnosis of appendicitis and the patient underwent appendectomy. Introperative findings included purulent ascites, a massively enlarged appendix (18 cm in length and approximately 4 cm in diameter), and extensive small bowel involvement by tumor. Further staging work-up revealed extensive bone marrow involvement (ALL, L3) and many blasts present in the peritoneal fluid. Microscopic Findings: Histologic sections of the appendix showed extensive infiltration of the appendiceal wall by a malignant lymphoid neoplasm with diffuse growth pattern and a prominent "starry-sky" appearance. On high power examination, the tumor was predominantly composed of monotonous intermediate-sized lymphoid cells with coarsely clumped nuclear chromatin and 1-3 small nucleoli. Scattered large tumor cells with pleomorphic nuclei were present throughout the tumor. A brisk mitotic rate was noted. Differential Diagnosis: Burkitt lymphoma, atypical variant Diffuse large B-cell lymphoma Precursor B-cell/T-cell lymphoblastic lymphoma/leukemia Immunophenotypic Findings (Immunohistochemistry and Flow Cytometry): The tumor cells were strongly positive for CD45, CD20, CD10, and Ki-67 (the latter expressed by >99% of the lymphoma cells). By in situ hybridization, the lymphoma cells showed immunoglobulin kappa light chain restriction. By flow cytometry, the lymphoma cells showed surface immunoglobulin expression with kappa light chain restriction (Ig Mu/kappa). The tumor cells were negative for CD3 and Tdt (the former highlighted rare mature T-lymphocytes scattered throughout the tumor). By flow cytometry (cell cycle analysis), the DNA index was 1.00 (normal DNA content) and the S-phase fraction was 45%. Molecular/Cytogenetic Analysis: Molecular analysis was not performed. Conventional cytogenetic analysis demonstrated the t(8;14)(q24;q32) chromosomal translocation. Metaphase FISH showed that this translocation juxtaposed the 8q24.1 [C-MYC] and 14q32.3 [IGH] regions. Diagnosis: Burkitt lymphoma, atypical (Burkitt-like) variant Burkitt Lymphoma Definition: Burkitt lymphoma is defined by the WHO criteria as a highly aggressive B-cell lymphoma composed of monomorphic medium-sized cells with numerous mitotic figures and constant association with chromosomal translocations involving the MYC gene. The tumor is variably associated with the Epstein-Barr virus (EBV). The presence of a very high growth fraction ("nearly 100%") as measured by percentage of Ki-67-positive cells is required for this diagnosis and accepted as a surrogate for MYC gene translocations, if all morphologic and immunophenotypic features are supportive of Burkitt lymphoma. Epidemiology and Clinical Features: Burkitt lymphoma accounts for 30-50% of childhood lymphoma in the Western countries. This highly aggressive neoplasm was initially described by Dennis Burkitt in children from Ecuatorial Africa, where a possible association with immunosuppression secondary to malaria infection was thought to account for the high prevalence of this disease. The tumors occurring within this area have been designated as endemic Burkitt lymphoma and are similar to those observed in other geographic areas with similar climate (e.g. Papua, New Guinea). Endemic Burkitt lymphoma is consistently associated with the classic morphologic variant and with the presence of EBV. In fact, EBV was discovered in one of the first tumor cell lines established from an African Burkitt lymphoma. Burkitt lymphoma also occurs throughout the world outside these endemic areas, in which case most tumors are designated as sporadic Burkitt lymphoma. A somewhat different subset of tumors appear to affect children in South America and North Africa, where the clinical features and response to therapy appear to be intermediate between the endemic and sporadic subtypes. Sporadic Burkitt lymphomas are variably associated with EBV and may present as any of the histologic variants. Burkitt lymphoma can also be seen in association with immunodeficiency, whether acquired (post-solid organ transplantation, AIDS) or congenital. Immunodeficiency associated Burkitt lymphoma is positive for EBV in 25-40% of the cases. It often presents with lymph node localization and bone marrow involvement. It is frequently associated with the atypical and plasmacytoid histologic variants. An unusually high incidence of Burkitt lymphoma has been found in patients with the X-linked lymphoproliferative disorder (Duncan disease). This disorder affects males who have loss-of-function mutations in the SAP (signaling lymphocyte activation molecule [SLAM]-associated protein) gebe, also known as SH2D1A. This leads to T-cell activation defects that result in an unusually severe and often fatal clinical course for EBV infections and Burkitt lymphomas (the latter occurring in both EBV-positive and EBV-negative patients). Clinical features and epidemiological data as seen in the endemic and sporadic variants are summarized in Table 7.1. Molecular Biology and Cytogenetics: Burkitt lymphoma is highly associated with chromosomal translocations that juxtapose the c-myc gene to the immunoglobulin genes normally expressed in mature B-lymphocytes. These translocations are thought to lead to the dysregulation of c-myc, a highly pleiotropic gene that encodes for a transcription factor with roles in cell proliferation and apoptosis. In turn, c-myc dysregulation is believed to play a central role in tumorigenesis and tumor growth. EBV is thought to act as a co-factor in malignant transformation. Burkitt lymphoma is the fastest growing human neoplasm, feature reflected in the high proliferation rate seen by immunohistochemistry for Ki-67 and high S-phase evaluated by cell cycle analysis (flow cytometry). The most common chromosomal translocation associated with Burkitt lymphoma (75-90%) is the t(8;14)(q24;q32) which translocates the c-myc on 8q24 to the 14q32 region, thus bringing it under the control of the regulatory regions for IgH (the immunoglobulin heavy-chain gene). Less commonly, these translocations involve 8q24 and the immunoglobulin light chain genes, as t(8;22)(q24;q11) or t(2;8)(q11;q24), which involve the Ig lambda and Ig kappa genes, respectively. Notably, in these cases c-myc remains most often on 8q and the light chain genes are translocated to this chromosome, thus leading to myc dysregulation. All these abnormalities can be demonstrated by conventional cytogenetics and FISH. Histologic Features: The entity currently recognized by the WHO as Burkitt lymphoma may show a range of morphologic features. Entities previously designated as Burkitt lymphoma and Burkitt-like lymphoma have been gathered under the common designation of Burkitt lymphoma, with three corresponding histologic variants: classical, atypical (Burkitt-like), and Burkitt lymphoma with plasmacytoid differentiation. All variants are characterized by a diffuse growth pattern and a prominent "starry-sky" appearance typically associated with a very high mitotic rate. An interesting phenomenon described in association with Burkitt lymphoma is that of "follicular colonization" seen in cases where the underlying lymphoid tissue is only subtotally effaced. In the latter cases the germinal centers of the colonized follicles contain a homogeneous population of cells cytologically similar to the diffuse component. Burkitt lymphomas often show aggressive infiltration of the tissues that they involve. However, in a number of cases lymph node or small intestinal wall tumors may be relatively circumscribed, such that regional lymph nodes dissected during the same surgical procedure might show no morphologic evidence of lymphoma. In the classical variant the lymphoma cells are uniform in appearance, with medium-size (equal to that of the tingible-body macrophage nuclei), round nuclei with coarsely clumped chromatin and several small basophilic nucleoli. In touch imprints and in aspirate samples these cells have a deeply basophilic cytoplasm containing prominent clear (lipid-containing) vacuoles. When involving bone marrow extensively, lymphoma cells with this morphology have been designated by the French-American-British (FAB) classification as "acute lymphoblastic leukemia – ALL, L3 subtype." In the atypical (Burkitt-like) variant the predominant cell population is similar to that of the classical variant, however the lymphoma cells are more pleomorphic, with frequent larger cells (exceeding the size of the macrophage nuclei), irregular nuclei and more prominent often single nucleoli. Finally, the Burkitt lymphoma with plasmacytoid differentiationalso shows more cellular pleomorphism than the classical variant, with a morphologic range that includes more cells with eccentric nuclei, basophilic cytoplasm and single central prominent nucleoli. In the latter variant lymphoma cells contain more cytoplasmic immunoglobulin than the other variants, easy to visualize using immunohistochemical staining. Immunophenotype: Burkitt lymphoma cells of all variants typically exhibit a mature B-cell phenotype, that includes strong expression of CD45, CD20 and monotypic (light-chain-restricted) surface immunoglobulin. Other B-cell specific antigens (CD19, CD22, CD24, CD79a) are typically strongly positive. In addition, these tumors express CD10 and BCL-6, and are negative for CD5, CD23, BCL-2 and Tdt. Rare cases of Burkitt lymphoma exhibiting the characteristic chromosomal abnormalities may have a precursor B-cell immunophenotype (see below) that may include expression of Tdt, weak or absent expression of CD20 and lack of monotypic immunoglobulin. These cases are best diagnosed and treated as Burkitt lymphoma. Staining for proliferation markers (Ki-67) highlights a growth fraction of nearly 100% in all the histologic variants. Demonstration of this high proliferation rate is required for the diagnosis of Burkitt lymphoma. Also, when analyzed by flow cytometry (cell cycle analysis) this feature correlates with a very high S-phase (40-60%). Immunohistochemical staining for T-cell antigens (e.g. CD3, CD5) typically highlights only rare scattered infiltrating T-lymphocytes. Differential Diagnosis: Diffuse large B-cell lymphoma (especially for the atypical variant) Diffuse large B-cell lymphoma, immunoblastic (especially for the plasmacytoid variant) Precursor B-cell lymphoblastic lymphoma T-cell lymphoma (ALCL, monomorphic variant) Helpful immunophenotypic and morphologic features for this differential diagnosis are summarized in Table 7.2. Table 7.1: Clinical Features and Epidemiology in Endemic and Sporadic Burkitt Lymphoma Feature Endemic Burkitt Lymphoma Sporadic Burkitt Lymphoma Sites of involvement (%) - Abdomen/Pelvis 58 67 - Ileo-cecal 0 20 - Bone marrow 7-13 12 - CNS 19-30 5 - Epidural 17 0-3 - Jaw 58 (one/several quadrants) 7 (one quadrant) - Lymph nodes 4 17 - Ovary 0 60 Peak age (years) 4-7 4-7 (adult median, 30) Male : Female ratio 2:1 2-3:1 EBV (%) 95-98 15-20 Table 7.2: Morphologic and Immunphenotypic Features Helpful in the Differential Diagnosis of B-lineage Pediatric Lymphomas Type of lymphoma Morphology Immunophenotype CD20 CD79a CD10 BCL-6 BCL-2 Ig Ki-67 T-cells Burkitt lymphoma Monotonous medium-sized cells; smaller component of large, pleomorphic cells ++ ++ ++ + - sIg, monotypic + (100%) Rare Diffuse large B-cell lymphoma Predominantly large cells with irregular nuclei ++ ++ +/- +/- +/- sIg or cyIg, monotypic + (70-90%) Frequent, may be numerous Precursor B-cell lymphoblastic lymphoma Monotonous intermediate-sized blasts with fine chromatin and inconspicuous nucleoli +/- ++ (cyto-plasmic) ++/- - + cy/s Ig Mu (heavy chain only) + (60-70%) Rare Abbreviations: Ig – Immunoglobulin, s – surface, cy – cytoplasmic.