A suspicious or persistent lymphadenopathy in children is a frequent situation that the paediatrician has to cope with routinely. The challenge is to reassure the parents' and the physician's fears of malignancy and to do so in a safe, timely and cost-effective manner. In spite of this, FNB should preferably be restricted to cases with a persistent enlarged lymph node or a strong clinical suspicion of a specific infection or neoplasia. Performing an adequate clinical and physical examination is extremely important. Most children have palpable small cervical, axillary and inguinal nodes, but in some locations these are considered definitely abnormal, namely lymphadenopathies in the posterior auricular, epitrochlear and supraclavicular areas.
Most lymphadenopathies in children are benign and self-limited, and are frequently as a result of viral or bacterial (Staphylococcus aureus and β-haemolytic Streptococcus) or mycobacterium infections. Other less common causes include fungal infection (Figure 3a) and primary or metastatic malignancies. Mycobacteria, fungi or a virus can be identified in cytological preparations with haematoxylin and eosin staining (Figure 3b) or with the assistance of histochemical techniques such as Ziehl Neelsen, Grocott, (Figure 3c), Gram, Gomori's methenamine silver (GMS), periodic acid-Schiff (PAS), alcian blue and even more specific immunostains. Fine needle samples can also be rinsed into a specific culture medium and sent to a microbiology laboratory for bacterial cultures or polymerase chain reaction (PCR).
Figure 3. (a) Cervical nodules in a 12-year-old African boy. (b) Cytological preparation shows numerous spherical to oval fungal cells (haematoxylin and eosin ×600). (c) A thick double wall on Grocott staining permits the diagnosis of Histoplasma Capsulatum (Duboisii) (Grocott ×600).
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Lymphomas and leukaemia
The primary malignant tumours that involve the head and neck in children are most often lymphomas. The child's age conveys important diagnostic information evaluating enlarged nodes. In children younger than 6 years of age, the most common malignancies are non-Hodgkin lymphoma and leukaemia. From 7 to 13 years of age, Hodgkin lymphoma and non-Hodgkin lymphoma are the most common and over 13 years of age, the former predominates. Nevertheless, although unusual, solid tumours such as neuroblastoma and RMS can present as a lymph node metastasis.
FNB is an excellent technique for addressing lymphomas; however, to perform a diagnosis of lymphoma, nowadays it is no longer acceptable to carry out FNB without using ancillary techniques, namely flow cytometry. The combination of flow cytometry and classic cytological features represent a powerful combination of methods in the accurate diagnosis and subclassification of lymphomas. According to WHO, lymphoma subtyping is based on morphology, immunophenotype and molecular characterization of the lymphoid cells. Most non-Hodgkin lymphomas can be characterized with this multidisciplinary approach. Immunophenotype characterization of lymphocytes can be accomplished by immunocytochemistry (ICC) (preferably in cytospins or cell blocks) or by flow cytometry (Table 1). Immunostains on cell blocks or cytospins require a sample of additional quantity and quality, without blood contamination; a condition that is not always feasible when dealing with children. FNB samples can also be used to perform molecular studies, such as PCR or fluorescence in situ hybridization (FISH), in order to detect translocations, deletions and gene amplifications in the DNA, which are specific of several subtypes of lymphomas (Table 2).
Table 2. Most common molecular alterations found in frequent paediatric lymphomas
|Anaplastic large cell lymphoma||t(2;5)(p23;q25)|
|Lymphoblastic T-cell lymphoma||t(1;14)(p32;q11)|
Sporadic Burkitt lymphoma frequently presents as a large and rapidly growing abdominal mass with spontaneous tumour lysis syndrome, creating a therapeutic emergency. In Europe and North America, Burkitt lymphoma represents around half of all malignant non-Hodgkin lymphoma in children. In spite of its characteristic round-to-oval nuclei, multiple small nucleoli, speckled chromatin (Figure 4a) and tiny cytoplasmic lipid vacuoles (Figure 4b), other entities such as lymphoblastic lymphoma, diffuse large B-cell lymphoma (DLBCL) and plasmablastic lymphoma can mimic this cytological pattern. Burkitt lymphoma can exhibit an atypical morphological appearance, raising some doubts in the differential diagnosis even with other small round cell tumours. Evaluation of the tumour immunophenotype is crucial in the differential diagnosis with other entities (Figure 4c and Table 1). However, an unusual immunophenotype is not uncommon in Burkitt lymphoma and detection of the typical t(8;14), c-myc translocation is sometimes a determining factor for the correct diagnosis (Figure 4d). A more superficial idea of all these techniques may give us the misconception that they represent a solution for the diagnosis. MYC translocation for instance, although a consistent feature of Burkitt lymphoma, is not specific and may be shared with DLBCL. Once again we emphasize the importance of the multidisciplinary approach in the evaluation of the appropriate techniques.
Figure 4. Burkitt lymphoma fine needle biopsy. (a) A monotonous population of intermediate-sized cells: nuclei have a characteristic speckled chromatin (haematoxylin and eosin ×600). (b) Deep blue cytoplasm with tiny lipid vacuoles can be seen on Giemsa staining (×600); note the close resemblance to rhabdomyosarcoma in Figure 5c. (c) Demonstration on flow cytometry of a monoclonal (K expression) B-cell population with intense staining with CD19; CD10 and CD38, in the absence of leukocyte-associated IG-like receptor (LAIR) expression, point to the diagnosis of Burkitt lymphoma. (d) Fluorescence in situ hybridization analysis using Vysis LSI IGH/MYC, CEP 8 Tri-color, Dual Fusion Translocation Probe shows a classic MYC/IgH rearrangement pattern with two fused signals (IGH-MYC fusions on der(8), t(8;14) and der(14), t(8;14)), one red signal (normal MYC allele), one green signal (normal IGH allele) and two blue signals for the two centromeric probes for chromosome 8.
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Hodgkin lymphoma comprises about 6% of childhood cancers according to the National Cancer Institute (NCI) data. An accurate cytological diagnosis of Hodgkin lymphoma relies on a combination of morphological findings: Reed–Sternberg (RS) cells in a background of small mature T cells, (CD57-positive) and immunostains (CD30 and CD15 immunophenotype in RS cells). To date, flow cytometry has not been considered useful to confirm this diagnosis. In most situations, RS cells represent less than 1% of the total cells present in the sample, and fall outside the area that is usually used for recognition of individual (non-aggregated) cells, or remain located at the very edge or beyond the high end of the measurable range of forward scatter parameters set of analysis. Besides this, the rosette complex, between RSC and T-cell lymphocytes, which are generally produced in Hodgkin lymphoma, display a composite immunophenotype combining these two cell immunophenotypes, thus effacing the CD30 and CD15 that typically characterizes RS cells.[16, 17] Recently, modern cytometers have increased sensitivity, as well as the capacity to identify these minor populations with increased confidence.
Hodgkin lymphoma should also be differentiated from other non-Hodgkin lymphomas that may involve the mediastinum of children, namely mediastinal large B-cell lymphoma (MLBCL) and anaplastic large cell lymphoma, mainly the large cell subtype (ALCL). In both these lymphomas cytological features can be similar; however, both in MLBCL and in ALCL neoplastic cells are CD45-positive, which differentiates them from RS cells, and in ALCL neoplastic cells can express T-cell markers (CD2 and CD4). In a great majority of the cases neoplastic lymphocytes of ALCL also express ALK protein and in FISH analysis a t(2;5) or t (1;2) can be detected (Table 2). The differentiation between Hodgkin and anaplastic lymphoma is critical, owing to the fact that they have a different prognosis and anaplastic lymphoma implies a different and more intensive treatment. Sometimes in reactive lymph nodes, sustentacular reticular or endothelial cells assume atypical shapes, similar to popcorn or RS cells. The differential diagnosis lays in the careful observation of the remaining lymphoid population which is polymorphic as well as the presence of macrophages with tangible bodies, which are more frequent in a reactive lymph node.