Fig. 9.1
Classical Hodgkin Lymphoma featuring one Reed-Sternberg cell surrounded by an inflammatory background. There are five eosinophils in the field. MGG ×60
RS cells may be bi-nucleated or may present a bi-lobed nucleus with one prominent nucleolus. The size of RS cells is typically 4–5 times the surrounding lymphocytes (Fig. 9.2). Nuclei show a single, basophilic, and prominent nucleolus. Cytoplasm is well represented, vacuolated, and variable from pale-to-medium blue at May-Grünwald Giemsa (MGG) staining. RS cells are frequently multinucleated. When present, bare RS cells’ nuclei are the clue to diagnosis. RS cells appear particularly abundant and atypical in the lymphocyte-depleted variant. RS cells are accompanied by numerous inflammatory cells, which mainly comprise lymphocytes, plasma cells, eosinophils, and neutrophils (Fig. 9.1). Eosinophils can be abundant, and their presence should always raise suspicion of HL. Epithelioid cell granulomas may also be appreciated in smears from HL and enter into differential diagnosis with granulomatosis disease as tuberculosis or sarcoidosis.
Fig. 9.2
Multiple Hodgkin and Reed-Sternberg cells, some bi- and multinucleated with prominent nucleoli. Papanicolaou, ×60
9.1.2 Ancillary Techniques
RS cells are typically positive for CD30 and CD15, and are usually negative for EMA and CD45. CD20 is usually negative, but can be expressed in 10–20% of cases; other markers of B cells, such as CD19, CD22, and CD79a are usually defective, although they can be present in some cases. An immunohistochemical evaluation of the B cell transcription factors Pax5, OCT2, and Bob1 can be worthwhile in difficult cases, because classic HL usually express Pax5 with a lower intensity than normal B lymphocytes and are negative for OCT2 and/or Bob1 in most cases. Finally, in situ hybridization for EBER can demonstrate the presence of EBV genome in the nuclei of the neoplastic cells in 30–40% of the cases.
Flow cytometry is not very useful for the correct diagnosis except perhaps in cases rich in neoplastic cells or in cases with partial expression of B cell antigens such as CD20.
9.1.3 Differential Diagnosis
The differential diagnosis of classical HL includes both benign and malignant conditions. Benign conditions that may be confused with HL are mainly viral infections such as EBV, as they can show mononucleated or multinucleated immunoblasts that closely resemble Hodgkin’s and Reed-Sternberg cells.
NHL that can simulate a classic HL are mainly diffuse large B cell lymphomas, mediastinal large B cell lymphomas, and ALCL, but an accurate flow cytometric and/or immunophenotypic evaluation on cell blocks usually allows a correct diagnosis.
9.1.4 Cytomorphology of Lymphocyte Predominant HL, Nodular
Lymphocyte predominant HL, nodular (LPHL) , is much more difficult to diagnose than classical HL, since the neoplastic cells express a complete B cell differentiation program, and so are positive for CD19, CD20, CD22, CD79a, and also for all B cell transcription markers (i.e., Pax5, Bob2, and OCT2), so they are phenotypically indistinguishable from those of B cell lymphomas. Because they usually have a rich background of reactive small lymphocytes, the main differential diagnosis is with a T cell/histiocyte rich large B cell lymphoma (TCHRLBCL) . In the authors’ opinion, a differential diagnosis cannot be done with confidence by FNA, as the main requirement is the histologic evaluation of the nodularity and of the persistence of a reticulum of follicular dendritic cells in the nodules. Factors in favor of LPHL, however, are clinical parameters (limited stage) and some immunohistologic features such as a background richer in PD-1 positive follicular T-helper cells, CD4+ and CD57+ small T-lymphocytes instead of CD8+ and Tia1+ T-lymphocytes, or the positivity for the neoplastic cells for IgD, which characterizes one peculiar subgroup of LPHL.
9.2 Burkitt Lymphoma
Burkitt Lymphoma (BL) is the main subtype of mature B cell NHL in the pediatric age. It comprises 40–50% of the cases [1].
The two main types of BL are the endemic form, frequently seen in equatorial Africa, and the sporadic (non-African) form. The two forms differ in their association with the virus of Epstein-Barr (clonal EBV DNS is present in 95% of the former and in 15% of the sporadic tumors in Western countries), and in some clinical features (endemic BL have a high propensity for facial bones involvement, while sporadic BL is more common in the GI tract, in the kidney, and in the ovary). A small cohort of patients present with leukemia and extensive bone marrow involvement (Burkitt leukemia, previously termed ALL L3). Finally, a forth form typically occurs in HIV-infected immunocompromised individuals, and this form is also usually related to EBV infection.
BL is one of the most rapidly proliferating human tumors, and the doubling time has been calculated to be 12–24 h; for this reason, it can represent a medical emergency, and a rapid diagnosis has to be achieved to allow a prompt treatment that, in most cases in the pediatric population, is curative [3].
9.2.1 Cytomorphology
Cellularity is extremely high, resulting in crowding and molding of the nuclei. There is no specific organization pattern; malignant cells typically are uniformly distributed and/or arranged in clusters. “Starry sky” pattern is frequently seen (Fig. 9.3).
Fig. 9.3
Burkitt lymphoma . Highly cellular smear with monotonous tumour cell population and a few macrophages, producing the starry sky pattern. MGG ×20
Malignant cells are abundant, medium-sized (average diameter from 10 to 12 μm) and monomorphic. The nuclei are round or oval, with a smooth nuclear membrane. Chromatin appears clumped with 4–5 variably prominent nucleoli (Fig. 9.4). Cytoplasm is scant, basophilic, and typically contains small perinuclear vacuoles due to lipid droplets in smears, which are dried and stained with MGG (Fig. 9.5). Mitoses and apoptotic bodies are frequent. Tingible body macrophages are frequent and are responsible for the classic “starry sky” appearance (Fig. 9.3).
Fig. 9.4
Burkitt lymphoma cells in pleural effusion. Multiple nucleoli and cytoplasmic lipid vacuoles can be seen. MGG ×60
Fig. 9.5
Burkitt lymphoma with monotonous round nuclei , cytoplasmic vacuoles and lymphoglandular bodies in the background. MGG ×60
Necrotic and/or hemorrhagic background with lympho-glandular bodies is typically associated with BL (Fig. 9.5).
9.2.2 Ancillary Techniques
Immunocytochemistry or flow cytometry may be very useful in BL diagnosis. The typical BL immunophenotype includes positivity for CD19, CD20, CD10, and Bcl6, and negativity or only weak positivity for Bcl2. Other typical immunoreactivity is for CD38 and TCL1, whereas CD44 is negative; Sox11 (a marker expressed in mantle cell lymphomas and negative in Diffuse Large B cell Lymphoma) is also frequently positive in a high percentage of BL cases. The availability of an antibody against the cMyc protein can also be useful, as this protein is expressed in most of the cells. The proliferation activity with Ki67/MIB-1 is high, and more than 95% of the nuclei are stained (Fig. 9.6). The Cytogenetics hallmark of BL is the cMYC translocation, which is present in over 99% of cases, and the most common translocation found is t(8,22), while t(2,8) and t(8,22) are less common. The cMYC translocation can be easily demonstrated on cytologic smears with FISH analysis using break-apart probes or specific fusion probes.
Fig. 9.6
Ki-67/MIB1 positive nuclei of Burkitt lymphoma in a methanol fixed cytospin preparation, Immunocytochemistry, ×60
9.2.3 Differential Diagnosis
In BL , the differential diagnosis includes mainly diffuse large B-cell lymphoma (DLBCL), due to monomorphism that is seen in both entities. However, other morphologic features are typical of BL, and diagnosis can be easily confirmed by immunophenotypic and FISH analysis.
9.3 Diffuse Large B-cell Lymphoma, Not Otherwise Specified
DLBCL is the second most common pediatric lymphoproliferative disorder originating from mature B cells, and represents 20–30% of all pediatric NHL. Clinical presentation may be similar to BL; however, DLBCL more frequently involves lymph nodes [4].
Gene expression profiles and immunohistochemical algorithm allow separation of DLBCL into different subgroups. In the adult population, cases derived from germinal center cells (GBC) have a better survival than those resembling activated B cells (ABC). In the pediatric population, where most of the cases are GBC tumors, the relevance of this sub-classification based on the cell of origin is not clear, but it can be performed in tissues.
9.3.1 Cytomorphology
Typically, cytological preparations present high cellularity (Fig. 9.7). Malignant cells are dispersed while clusters of cells are uncommon, but may be seen and mimic carcinoma.
