The case of the sneaky lymphoma: solved by flow cytometry

To the Editor,

Diagnosing lymphoproliferative disorders in body cavity effusions poses a challenge for cytopathologists, as microscopy alone may not reliably distinguish reactive from neoplastic processes. Key cytomorphologic features suggesting malignancy include high cellularity, atypia, pleomorphism, monomorphic populations, and frequent apoptotic bodies. It is essential to first rule out non-hematolymphoid malignancies, then classify the effusion based on cell size dominance. Small- or medium-sized cell-predominant effusions with atypia or clinical suspicion of lymphoma require ancillary testing. Flow cytometry (FC) is particularly valuable in such cases, aiding in both the confirmation of known hematologic malignancies and the diagnosis of previously unrecognized lymphomas. This case report highlights the diagnostic utility of FC in suspected malignancies, underscoring the importance of an integrated approach that combines cytology, histopathology, and immunophenotyping to improve diagnostic accuracy and clinical understanding.

A 4.5-year-old boy presented with a 20-day history of bilateral cheek swelling, more prominent on the left side, preceded by fever and progressive abdominal distension. The child also had a history of weight loss and reduced appetite. Clinical examination revealed left-sided ptosis (Fig. 1a). No other significant medical history was noted. On examination, there was a poorly defined, diffuse swelling on both cheeks, measuring approximately 2 × 2.5 cm. The swellings were firm to hard, non-tender, and associated with left eye ptosis. Abdominal examination revealed a vague palpable mass with tenderness and the presence of ascites. Laboratory investigations showed anemia with hemoglobin at 8.1 g/dL, leukopenia (total leukocyte count 2100/µL), and thrombocytopenia (platelet count 67,000/µL). The peripheral blood smear showed pancytopenia, with normocytic, normochromic red blood cells. A chest X-ray revealed massive bilateral pleural effusion with associated atelectasis. Abdominal ultrasonography was performed to evaluate the distension and suggested a ruptured liver abscess with ascites. Based on clinical findings, a provisional diagnosis of neuroblastoma was considered. Bone marrow aspiration and biopsy showed trilineage hematopoiesis. A contrast-enhanced computed tomography (CECT) scan of the head, neck, chest, and abdomen was performed for further evaluation. The CECT of the neck revealed a well-defined mass measuring approximately 6 × 4.4 × 4 cm, with ill-defined margins located in the left masticator space. The lesion extended anteriorly into the orbit and inferiorly to the level of the mandible, with associated erosion of the maxilla (Fig. 1b). A similar mass was also identified in the right masticator space. CECT of the chest showed bilateral pleural effusions, more prominent on the left side, along with areas of lung collapse (atelectasis). Abdominal imaging demonstrated the presence of ascites and a centrally located malignant mass measuring approximately 13 × 8.4 × 11.7 cm. This mass involved the peritoneum, retroperitoneum, pancreas, duodenum, and liver and was associated with intrahepatic biliary ductal dilatation (Fig. 1c). Fine-needle aspiration cytology (FNAC) of the cheek swelling showed sparsely cellular smears composed of a monomorphic population of intermediate-sized round cells with scant cytoplasm, fine chromatin, and 1–2 inconspicuous nucleoli (Fig. 1d). Some cells exhibited fine vacuolated cytoplasm. These findings were consistent with a malignant small round blue cell tumor. To further characterize the disease, a pleural fluid tap was performed, and the sample was analyzed using FC (Beckman Coulter FC 500). The sample revealed a monotypic population of B-cells expressing bright cluster of differentiation (CD) 10, CD19, CD20, CD22, CD38, and surface kappa light chains (Fig. 1e). The cells were negative for T-cell markers (CD3, CD5), as well as for myeloid markers (CD13, CD33), and did not express terminal deoxynucleotidyl transferase (TdT) or CD34, ruling out precursor lymphoid malignancy. Notably, the population also showed a high proliferation index (Ki-67 > 95%). The immunophenotypic profile—characterized by CD10 positivity, surface immunoglobulin light chain restriction, and high proliferation rate—was strongly suggestive of Burkitt lymphoma (BL). To confirm the diagnosis, a trucut biopsy from the cheek swelling was done. Histological examination revealed sheets and nests of medium-sized, monomorphic lymphoid cells displaying a characteristic “starry sky” appearance (Fig. 1f). The mitotic activity was high, with a mitotic index of 8–9/high power field. On immunohistochemistry, these cells were diffusely positive for CD10, CD19, CD20, and c-Myc, while negative for CD3, TdT, and B-cell lymphoma/leukemia 2 (BCL2). The Ki-67 proliferation index was > 95% in the highest proliferating areas (Fig. 1g–k). Unfortunately, fluorescence in situ hybridization (FISH) analysis could not be carried out due to scant tumor tissue. These findings, in conjunction with the cytomorphology and radiologic features, confirmed the diagnosis of BL presenting with bilateral cheek masses, pleural effusion, and abdominal involvement. The patient developed central nervous system involvement during the course of the disease. He was initially started on the cyclophosphamide, vincristine [Oncovin], and prednisolone (COP) regimen, followed by the COP plus cytarabine, dexamethasone, and methotrexate (COPADM) protocol. Unfortunately, during the course of treatment, he developed febrile neutropenia and subsequently passed away.

Fig. 1.

a Clinical image of the child showing bilateral cheek swellings with associated left-sided ptosis. b Contrast-enhanced computed tomography (CECT) image of the face showing a well-defined mass in the left masticator space with ill-defined margins, extending into the orbit and mandible, with erosion of the maxilla. c CECT image of the chest and abdomen revealing bilateral pleural effusions with lung collapse, ascites, and a large central abdominal mass involving the peritoneum, retroperitoneum, pancreas, duodenum, and liver, with intrahepatic biliary ductal dilatation. d Cytology smear from cheek swelling showing sparsely cellular aspirate with monomorphic intermediate-sized round cells, scant cytoplasm, fine chromatin, and inconspicuous nucleoli (May-Grünwald Giemsa (MGG) 100×), occasional cells displaying finely vacuolated cytoplasm (inset, MGG 400×). e Flow cytometry charts showing a monotypic B-cell population with bright expression of CD10, CD19, CD20, CD22, CD38, and surface kappa light chains. f Histopathological image showing sheets of medium-sized monomorphic lymphoid cells with a classic “starry sky” appearance and high mitotic activity (hematoxylin and eosin, 400×). g–k Immunohistochemical staining showing strong positivity for CD19 (100×), CD20 (100×), CD10 (400×), and c-MYC (100×) and high proliferative Ki-67 index (~ 95%) (400×)

Body cavity effusions commonly arise as complications in a wide spectrum of both benign and malignant conditions. Among malignant effusions identified through cytological evaluation, lymphomatous involvement is not uncommon, accounting for roughly 10 to 15% of all pleural and peritoneal malignancies [1]. BL, a highly aggressive B-cell neoplasm, represents a significant proportion—around 50–70%—of pediatric lymphoid malignancies [2]. Its rapid proliferation is frequently associated with effusions, which often signal an advanced stage and carry a poor prognosis. BL is a high-grade, fast-growing non-Hodgkin lymphoma of B-cell origin, characterized by a MYC gene translocation, most often t(8;14)(q24;q32). It primarily affects children and manifests in three clinical forms: endemic, sporadic, and immunodeficiency-associated. The sporadic variant, more prevalent in developed nations, typically presents with abdominal masses but may also involve atypical sites such as facial bones, bone marrow, or serous cavities [3]. The case presented involves a 4.5-year-old child with bilateral cheek swelling, ptosis, and abdominal distension—a clinical picture that initially resembled other small round blue cell tumors like neuroblastoma or rhabdomyosarcoma. Early investigations, including bone marrow examination, were non-contributory, underscoring the complexity of diagnosis in such presentations. Imaging revealed extensive disease involving retroperitoneal structures and craniofacial regions. FNAC suggested a small round cell tumor; however, definitive diagnosis was achieved through FC performed on pleural fluid. Immunophenotyping revealed a monotypic B-cell population expressing bright CD10, surface immunoglobulin light chains, and a high proliferation index (Ki-67 > 95%), confirming the diagnosis of BL.

Rymkiewicz et al. conducted one of the most extensive studies on the diagnosis of BL using FC, evaluating 78 cases from fine-needle aspiration biopsy (FNAB)–derived samples taken from diverse sites such as lymph nodes, pleural effusions, and abdominal masses [4]. This study stands out for its inclusion of both MYC-positive and MYC-negative subtypes, comprehensive immunophenotypic analysis, and the combined use of FNAB, FC, cytogenetics, and FISH. It highlights a fast, minimally invasive, and cost-effective diagnostic approach, showcasing the utility of FC in both conventional and non-traditional specimens for early and accurate identification of BL, especially in urgent clinical scenarios. Tsagarakis et al. showed that BL can be distinguished from other MYC-rearranged B-cell lymphomas, like double-hit lymphoma (DHL) and MYC-positive diffuse large B-cell lymphoma (MYC-DLBCL) using key immunophenotypic and morphologic features. BL typically shows CD38 > 90%, CD10 > 80%, CD10 median-fluorescence intensity ratios (MFIR) > 10, Ki-67 > 70%, Bcl-2 < 30%, and classic Burkitt-type cytology. In contrast, DHL and MYC-DLBCL often have lower CD10 and Ki-67 and higher Bcl-2, and DHL shows blastoid features with single-stranded DNA-binding protein/T antigen (SS B/T) cells < 1.5. These markers support a FC-guided approach to prompt targeted FISH analysis and inform management [5]. High-grade B-cell lymphoma with 11q aberration (HGBL-11q) closely resembles classical BL in morphology and immunophenotype but is genetically distinct. Unlike BL, which is defined by a MYC rearrangement and often shows uniform medium-sized cells with cytoplasmic vacuoles, HGBL-11q lacks MYC translocation and instead features a characteristic 11q gain/loss pattern. It may show more cellular pleomorphism and blastoid features. Molecularly, HGBL-11q lacks the typical BL-associated inhibitor of DNA binding 3 (ID3) and transcription factor 3 (TCF3) mutations and instead often harbors guanine nucleotide-binding protein subunit alpha-13 (GNA13) mutations, aligning it more with diffuse large B-cell lymphoma. Despite overlapping features, these genetic and molecular differences establish HGBL-11q as a separate entity from BL.

The present case underscores the diagnostic value of FC in serous fluids, particularly when traditional approaches yield inconclusive results. It highlights the importance of considering BL in the differential diagnosis of pediatric patients presenting with facial or abdominal masses accompanied by effusion. FC, especially when applied to non-traditional specimens such as body fluids, proves to be a powerful, minimally invasive tool for the early and accurate detection of high-grade lymphomas.

Author contribution

Conceptualization: RS, MAO, RM, SS, JC.

Data curation: RS, MAO, RM.

Project administration: RM, SS, JC.

Writing—original draft: RS, MAO, RM.

Writing—review and editing: RS, MAO, RM, SS, JC.

Approval of final manuscript: RS, MAO, RM, SS, JC.

Funding

None.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethical committee approval

For this case report, formal consent from a local ethics committee is not required.

Declaration of patient consent

The authors certify that they have obtained the appropriate consent from the patient’s parents. The patient’s parents have given consent for the images and other clinical information to be reported in the journal. The parents understand that the name and initials will not be published, and due efforts have been made to conceal the same.

Conflict of interest

The authors declare that there is no conflict of interest.

Statement of human and animal rights

This study involved human participants and was conducted in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments. No animal studies were conducted.

Guarantor of submission

The corresponding author is the guarantor of submission.