Cytologic Diagnosis of Large B-Cell Lymphoma: Diagnostic approach and differential diagnosis

Abstract

Background:

Large B-cell lymphoma (LBCL) encompasses a diverse group of aggressive lymphoid neoplasms, the most common of which is diffuse large B-cell lymphoma (DLBCL). A precise diagnosis is critical for determining prognosis and therapeutic strategies, particularly as new molecular subtypes and classification criteria have been introduced in the 5th edition of the World Health Organization (WHO) classification of hematolymphoid tumors (WHO-HAEM5, 2022). Fine needle aspiration (FNA) cytology is a safe and minimally invasive diagnostic tool for assessing lymphadenopathy and extranodal masses.

Summary:

LBCLs are typically characterized by single cells with large nuclei, vesicular chromatin pattern and prominent nucleoli. The diagnosis of LBCL and the distinction distinguishing between LBCL subtypes requires the integration of cytomorphologic features with ancillary techniques, including immunocytochemistry, flow cytometry, fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS).

Key Messages:

This review synthesizes the cytologic features, immunophenotypic profiles, molecular signatures, and clinical implications of LBCL subtypes, highlighting recent advances in cytopathology and classification.

Introduction

Large cell lymphomas represent a biologically and morphologically heterogeneous group of lymphoid neoplasms with aggressive clinical behavior. DLBCL, not otherwise specified (NOS), remains the most common subtype of non-Hodgkin lymphoma worldwide, comprising 30%–40% of adult cases [1]. The 2022 WHO-HAEM5 classification recognizes a growing spectrum of large B-cell lymphomas, integrating histological, immunophenotypic, genetic, and clinical features [2]. With advances in precision medicine, the accurate subclassification of LBCLs is increasingly vital for treatment optimization [1] [3].

Fine needle aspiration (FNA) cytology is often the initial diagnostic modality, particularly in patients with lymphadenopathy or deep-seated masses. FNA facilitates rapid triage, preparation of cell blocks, and flow cytometric evaluation [4] [5] [6]. However, the morphological overlap between LBCL subtypes and non-lymphoid malignancies necessitates a multimodal diagnostic approach. The integration of cytology with immunohistochemistry (IHC), flow cytometry, cytogenetics, and molecular profiling provides the necessary granularity to distinguish LBCL subtypes with prognostic and therapeutic relevance [7, 8]. However, variability in reporting terminology, interpretation, and diagnostic criteria has historically led to confusion and inconsistent communication between cytopathologists and clinicians. In response to this need, the Sydney System for Reporting Lymph Node Cytopathology was introduced in 2020, providing a standardized, tiered diagnostic framework like the Bethesda, Paris, Milan, and Yokohama systems for other body sites [9–11]. The Sydney System for Reporting Lymph Node Cytopathology was the basis for the currently used WHO Reporting System for Lymph Node, Spleen and Thymus Cytopathology.

The diagnostic approach to large cell lymphomas in lymph node cytology specimens, particularly for subtyping diffuse large cell lymphomas and distinguishing between diffuse large B-cell lymphoma (DLBCL), anaplastic large cell lymphoma (ALCL), and other malignancies, requires an integrated approach of different ancillary studies. This approach combines cytomorphology, immunocytochemistry, flow cytometry, and molecular diagnostics [1] [4] [12].

The cytological features of large cell lymphomas include a predominance of large lymphoid cells with vesicular nuclei, prominent nucleoli, and moderate to abundant cytoplasm. These cells often appear as cohesive clusters or as single, dispersed cells. Marked pleomorphism, frequent mitoses, and the presence of apoptotic or necrotic debris are also typical. The large B cells can have a centroblastic, immunoblastic, plasmablastic or blastoid morphology. A centroblastic morphology includes round to oval nuclei with vesicular chromatin, multiple small nucleoli, and scant to moderately abundant cytoplasm. An immunoblastic morphology shows a centrally located nuclei as opposed to multiple nucleoli seen in large cells with centroblastic morphology. The nuclei of plasmablastic cells are similar to immunoblastic cells, but they have an eccentric nuclei and more abundant cytoplasm. A pleomorphic morphology displays irregular nuclear contours and vesicular chromatin pattern. Although there are many different entities included in the large cell B cell lymphoma category, the morphological findings are not defining for each entity. As recommended by the WHO, ancillary studies, including immunocytochemistry and molecular testing, are essential for definitive classification and subtyping [10].

Immunophenotyping is critical for distinguishing B-cell from T-cell neoplasms and for subclassifying large cell lymphomas. Flow cytometry and immunocytochemistry on cytology specimens allow for the detection of lineage-specific markers (e.g., CD20, CD3) and aberrant antigen expression, which are required for diagnosis and subtyping according to the WHO classification system. Key immunocytochemistry markers include CD19, CD20, CD22, CD79a, PAX5, CD10, BCL6, and MUM1, while evaluation of CD19, CD20, CD22, CD79b, kappa/lambda, CD10, is important in flow cytometry studies [5, 13]. Fluorescence in situ hybridization (FISH) is recommended for detecting chromosomal rearrangements, particularly MYC, BCL2, and BCL6, which define high-grade B-cell lymphomas (so-called double-hit and triple-hit lymphomas) and have major prognostic and therapeutic implications. The College of American Pathologists and the American Society for Clinical Pathology recommend FISH for MYC in all large B-cell lymphoma cases, with reflex testing for BCL2 and/or BCL6 if MYC is positive [13, 14]. Molecular genetic studies, including polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) or T-cell receptor (TCR) gene rearrangements, help establish clonality and can detect specific translocations (e.g., t(14;18), t(2;5)), further aiding in classification and sometimes obviating the need for excisional biopsy [15] [16]. Next-generation sequencing (NGS) and comprehensive genomic profiling are emerging as powerful tools for detecting somatic mutations, gene rearrangements, and copy number alterations. NGS panels add diagnostic value in up to 50% of unclassified cases and provide potential biomarkers for disease monitoring in 94% of cases. In certain scenarios, RNA-based gene expression profiling offers robust cell-of-origin classification and may outperform immunohistochemistry-based algorithms [17–19] [20]. Rapid on site evaluation is critical for optimal collection of material for the different ancillary studies.

Subtypes of Large Cell Lymphoma

Diffuse Large B-Cell Lymphoma (DLBCL)

DLBCL, NOS, is the most common subtype of DLBCL and includes morphologic variants (centroblastic, immunoblastic, and anaplastic) and molecular subgroups. FNA smears reveal large lymphoid cells arranged as single cells with vesicular chromatin pattern and prominent nucleoli. (Figure 1) The Hans algorithm divides DLBCL into germinal center B cell (GCB) and activated (ABC) subtypes, correlating with prognosis and therapeutic response [21, 22]. The GCB subtype is commonly cytologically monomorphic with centroblastic morphology. Immunohistochemical analysis revealed that they typically express CD10 and BCL6 and are MUM1 negative.[23] Molecular alterations include BCL2 translocation (t(14;18)), EZH2 mutations, and PTEN deletions [3, 24]. The ABC subtype, on the other hand, is morphologically more pleomorphic with immunoblastic/plasmacytoid features. Immunohistochemical studies revealed that these cells expressed MUM1 and were negative for CD10. The cells are often associated with MYD88 and CD79B mutations, which confer resistance to R-CHOP and necessitate targeted therapies [3, 24].

Figure 1.

Diffuse large cell lymphoma. The large lymphoid cells display round to oval nuclei, vesicular chromatin, multiple prominent nucleoli (often two to three), and a moderate amount of pale cytoplasm. A) air dried, Modified Giemsa stain, B) Alcohol fixed, Papanicolaou stain

Other variants of large B-cell lymphoma, including PMBL, large B-cell lymphoma with MYC, and BCL2 translocation, are accepted as distinct entities in the 2022 WHO classification of hematopoietic diseases. Although they may be morphologically indistinguishable, they are associated with distinct clinical, immunophenotypic, and molecular features.

Primary Mediastinal Large B-cell lymphoma (PMBL) PMBL typically affects young adults and presents as a mediastinal mass. FNA samples are often sparsely cellular with large cells, clear cytoplasm, and compartmentalizing fibrosis. The immunophenotype includes immunoreactivity for CD20, CD23, CD30, PDL1, and PDL2. The differential diagnosis includes classic Hodgkin lymphoma and “gray zone lymphomas” [25, 26].

Large B-cell lymphoma with MYC and BCL2 translocation is characterized by translocations involving MYC and BCL2, and occasionally BCL6. This aggressive lymphoma is most often seen in older adults. The diagnosis requires cytogenetics studies targeted towards cMYC and BCL2. Morphologically, these tumors are similar to DLBCL in general or, might exhibit blastoid features, but cannot reliably be distinguished from other LBCLs in cytology specimens. Their high Ki-67 index, and germinal center B-cell phenotype might suggest it as potential diagnosis. Pitfalls include confusing blastoid morphology with B-ALL or Burkitt lymphoma, requiring careful immunophenotyping and genetic testing [27, 28] [29].

ALK-positive LBCL is a rare variant with plasmablastic morphology (large cells with eccentric nuclei and prominent nucleolus) and strong ALK expression due to translocation, typically affecting younger males. Key diagnostic clues include lack of CD20, strong ALK, and EMA positivity. Pitfalls include mistaking it for carcinoma or plasmacytoid neoplasms due to pseudo-cohesion, cytokeratin expression, and sinusoidal infiltration [30, 31].

Large B-cell lymphoma with IRF4 rearrangement is more common in the pediatric/young adult population with follicular or diffuse architecture and strong IRF4/MUM1 expression. A monotonous, pleomorphic intermediate/large lymphoid population was observed cytologically. The nuclei contained coarse chromatin. Scattered apoptotic debris were identified in the background or phagocytized by macrophages. Diagnosis requires confirmation of IRF4 rearrangement. Pitfalls include misclassification as pediatric follicular lymphoma or DLBCL-NOS if molecular testing is not performed [32] [33] [34].

High grade B cell lymphoma with 11q aberration lacks MYC rearrangement but carries a specific 11q aberration. It mimics Burkitt lymphoma cytologically but shows more pleomorphism. Pitfalls arise if only morphology is used without genetic confirmation [35] [36].

Lymphomatoid granulomatosis is an angiocentric EBV-driven B-cell lymphoproliferative disorder. Lymphomatoid granulomatosis is characterized cytologically by a polymorphous cellular infiltrate with a mixed inflammatory background. The aspirate typically shows a mixture of small lymphocytes (predominantly T cells), histiocytes, plasma cells, and occasional neutrophils [37] [38]. Scattered large, atypical cells may be infrequent, especially in lower-grade lesions, but become more prominent in higher-grade disease [37] [38] [39]. Cytologic preparations may show clusters of atypical lymphoid cells associated with vascular structures, reflecting the disease’s angiocentric nature [40]. Necrosis foci are common, especially in higher-grade lesions, and may be seen as background debris in cytological specimens. The large, atypical cells are positive for B-cell markers (CD20, PAX5) and activation markers (CD30, MUM1), and are typically EBV-positive by in situ hybridization for EBER [37] [38]. Pitfalls include reactive lesions due to its polymorphic inflammatory background, which may lead to under-recognition as a lymphoma unless EBER ISH is performed [41] [37] [42].

EBV-positive DLBCL occurs mostly in the elderly and immunocompetent individuals. Polymorphic and monomorphic histology may resemble T-cell/histiocyte-rich LBCL or conventional DLBCL. Diagnosis requires EBER positivity and clinical correlation. Pitfall: misclassification due to overlapping morphology is a potential problem [43] [44] [45].

DLBCL associated with chronic inflammation (CI-DLBCL) is often associated with EBV. This type of lymphoma occurs in patients with long-term inflammation (e.g., pyothorax). It shows plasmablastic features and requires a clinical history to distinguish it from other lymphomas. Pitfalls include misdiagnosing fibrin-associated or primary effusion lymphomas [46] [47].

Fibrin-associated DLBCL occurs incidentally in fibrinous exudates (e.g., around prostheses or myxomas). The absence of mass lesion and EBV association are distinguishing features. The cells are large with narrow to broad basophilic cytoplasm, round or irregular nuclei with coarse chromatin pattern. If cytologic atypia is overlooked, it can be mistaken for a reactive fibrin-associated process. The cells are positive for CD20, MUM1 and EBV LMP [48] [49].

Fluid Overload-Associated Large B Cell Lymphoma occurs in serous effusions in elderly patients with heart failure and renal or liver disease. Cytology shows variable morphology with centroblastic or immunoblastic morphology but often no mass. Misdiagnosis as primary effusion lymphoma or metastatic carcinoma is a potential pitfall if HHV8 and immunophenotype are not assessed. The cells are commonly positive for CD20 and express MUM1. [50] [51].

Plasmablastic lymphoma (PBL) can present in nodal or extranodal sites. Initially associated with HIV infection, although non-HIV cases have been described. It is characterized by a population of large single cells with excentric nuclei, prominent nucleoli and vesicular chromatin pattern (Fig. 2), but might also present with immunoblastic morphology. Immunophenotypically, it is positive for CD138 and negative for most B cell markers, including CD20 and PAX5. It is usually negative for CD45. Misdiagnosis as plasma cell myeloma due to CD138 expression and morphological features are key pitfalls. The presence of EBV positivity and the absence of M spike and lytic lesions can help in the diagnosis of EBV [52] [53] [54] [55].

Figure 2.

Plasmablastic lymphoma. The large neoplastic cells have plasmablastic or immunoblastic features, including eccentrically placed nuclei, prominent central or multiple nucleoli, and abundant basophilic to amphophilic cytoplasm. A) air dried, Modified Giemsa stain B) Alcohol fixed, Papanicolaou stain

Primary large B-cell lymphoma of immune-privileged sites occurs in non-nodal sites, specifically in the central nervous system, testis, and eye. They usually exhibit a non-GCB phenotype. Cytology (e.g., cerebrospinal fluid or vitreous) may be the only sample. Inflammatory lesions are the main potential misdiagnosis, which can be attributed to limited sampling [56] [57–59] [60, 61] [62].

Primary Cutaneous LBCL, Leg Type (PCDLBCL, LT) presents as leg nodules, mostly in elderly women. It is characterized by a monotonous proliferation of centroblasts and immunoblasts. These lymphomas particularly affect elderly women and present with generally rapidly growing tumors on 1 or both (lower) legs, or in 15%–20% of patients at sites other than the legs. It strongly expresses BCL2, IRF4/MUM1, and IgM. MYC and BCL2 double expression is also present in two-thirds of patients with PCDLBCL LT. Rearrangements of the MYC gene have been detected in 30% of PCDLBCL, LT cases, with a second rearrangement in the BCL6 gene in rare cases. Both loss of CDKN2A either by gene deletion or promoter methylation and the presence of MYD88 L265P mutations, are observed in approximately two-thirds of patients. Cytology may be low in cellularity but shows large, monotonous cells. It may be misclassified as a systemic DLBCL without staging [63–65]

High Grade B-cell Lymphoma, NOS is a category for high-grade B-cell lymphomas not fitting Burkitt lymphoma or DLBCL, without MYC/BCL2/BCL6 rearrangements. It is a diagnosis of exclusion. A misdiagnosis may be due to inadequate molecular workup, which may miss defining features [66].

Primary Effusion Lymphoma (PEL) HHV8/KSHV-associated lymphoma in serous cavities, which is most common in HIV+ patients, requires HHV8/LANA. Pitfalls include misidentification of this LBCL as fluid-overload LBCL or carcinoma due to lack of CD20 and presence of large cells [67, 68] [69]. A summary of the main morphological, immunohistochemical and molecular findings seen in large B cell lymphomas can be seen in Table 1.

Large B-Cell Lymphomas Cytology: Diagnostic Features

Entity	Key Cytomorphologic Features	Most Informative immunocytochemistr y on Cell Block	Critical Molecular / FISH Tests
DLBCL, NOS	Discohesive large cells; vesicular chromatin; single/multiple nucleoli; variable pleomorphism; necrosis/apoptosis common	CD20, PAX5, CD3 (background); CD10, BCL6, IRF4/MUM1 for COO	FISH: MYC ± BCL2/BCL6 if high-grade features;
Primary Mediastinal LBCL	Large cells with pale cytoplasm; frequent sclerosis fragments; cell clustering	CD20, CD23, CD30 (weak), PDL1, PDL2	9p24.1 gain/amplification
LBCL with MYC and BCL2 rearrangemen ts	Highly atypical large or blastoid cells; frequent apoptosis; Burkitt-like or DLBCL-like cytology	CD20, PAX5; MYC, BCL2, BCL6; Ki-67 often very high	FISH: MYC + BCL2 +
ALK-positive LBCL	Immunoblastic/plasmablastic cells; abundant cytoplasm	ALK, CD138; CD20 usually negative	FISH for ALK rearrangement
LBCL with IRF4 rearrangement	Large centroblastic/immunoblastic cells; sometimes follicular fragments	IRF4/MUM1 strong; CD10 and BCL6 often positive	FISH for IRF4 rearrangement
LBCL with 11q aberration	Burkitt-like cytology but often slightly more pleomorphic	CD20, CD10, BCL6; BCL2 negative; LMO2 in ~50%	FISH for 11q gain/loss; MYC FISH negative
Lymphomatoid Granulomatosis	Polymorphous background; necrosis; angiocentric fragments	EBER highlights atypical B cells	EBV-driven; graded by EBV+B-cell number
EBV-positive DLBCL, NOS	Polymorphic or monomorphic large cells; Hodgkin-like cells may be present	EBER ISH; CD20/PAX5 variable	EBV-driven
Chronic inflammation DLBCL	Centroblastis or immunoblastic morphology	CD20, MUM1 and EBV LMP	EBV driven
Fibrin associated DLBCL	Clusters of atypical large B cells in a background of fibrinous debris	CD20, MUM1 and EBER-ISH	EBV driven
Fluid overload associated DLBCL	Centroblastic or immunoblastic morphology	CD20 and MUM1 positive, HHV8 negative	Clonal IgH rearrangement
Plasmablastic Lymphoma	Large cells with eccentric or centrally located nuclei; prominent nucleoli	CD138, CD38, IRF4/MUM1; CD20 negative, EBER-ISH	MYC rearrangement common
Primary DLBCL of Immune-Privileged Sites	Large atypical cells; necrosis; angiocentric features	CD20, PAX5; IRF4/MUM1	MYD88 and CD79B mutations
Primary Cutaneous DLBCL, Leg Type	Discohesive large cells; frequent mitoses; necrosis possible	CD20, PAX5; IRF4/MUM1; BCL2 often positive	MYD88 mutation
HGBCL, NOS	Monomorphic to pleomorphic high-grade cells; Burkitt-like without classic uniformity	CD20, PAX5; CD10/BCL6/MUM1 variable	FISH to exclude MYC/BCL2/BCL6 rearrangements
Primary Effusion Lymphoma	Large atypical cells in effusion fluid; no tissue mass	HHV8 LANA-1; plasma-cell phenotype	HHV8-driven

DLBCL: Diffuse Large B cell Lymphoma; LBCL: Large B cell lymphoma; HGBCL: High grade B cell lymphoma; ISH: In situ hybridization; FISH: Fluorescence in-Situ hybridization

Lymph node metastasis of malignant melanoma showing the presence of large epithelioid cells with prominent nucleoli, coarse chromatin pattern (Figure 3) can occasionally pose diagnostic challenges, with large cell lymphoma being one of the differential diagnoses [70]. The most typical cytopathological pattern consists of dissociated polygonal cells with abundant, well-defined cytoplasm and round to ovoid nuclei with prominent, round nucleoli. Intranuclear inclusions and binucleated cells are frequently observed. Although the intracytoplasmic melanin pigment is characteristic, it may be sparse or completely absent. In addition, metastatic melanoma cells can exhibit a uniformly small, spindle-shaped morphology with elongated nuclei, or display marked pleomorphism, including multinucleated forms. Occasionally, large cell lymphoma cells with immunoblastic morphology (scattered large cells with round, nuclei, each containing a prominent nucleolus and abundant pale bluish cytoplasm) may be mistaken for melanoma cells, especially in patients with a history of melanoma (Figure 4). ICC will help to make the distinction in difficult cases including stains such as S-100, SOX10, Melan-A and HMB-45.

Figure 3.

Metastastic melanoma. A) This field is predominantly composed of melanoma cells of varying sizes (air dried -MGG stain) B) Alcohol fixed, Papanicolaou stain C) S-100 immunostaining on the cell block demonstrates positivity in the melanoma cells, while the surrounding small lymphocytes are negative.

Figure 4.

Diffuse large B-cell lymphoma, immunoblastic subtype. Scattered large cells with round, eccentrically placed nuclei, each containing a prominent nucleolus and abundant pale bluish cytoplasm. A) air dried, Modified Giemsa stain B) Alcohol fixed, Papanicolaou stain C) Immunocytochemistry on ThinPrep specimens demonstrated a few large CD45-positive cells.

The scattered large cells with prominent nucleoli seen in anaplastic large cell lymphoma (Figure 5) and the immunoblastic cells of diffuse large B-cell lymphoma may closely resemble seminoma cells, which also present as single cells large cells with prominent nucleoli (Figure 6).

Figure 5:

Anaplastic large cell lymphoma. FNA from a 4 cm large lymph node in the right axilla. Numerous pleomorphic cells with abundant cytoplasm A) air dried, Modified Giemsa stain B) Alcohol fixed, Papanicolaou stain

Figure 6.

Metastatic seminoma. Ultrasound-guided aspirate from an enlarged retroperitoneal lymph node. Numerous large seminoma cells are present, some exhibiting fragile cytoplasm and large round nuclei with prominent nucleoli, set against a background of small lymphocytes A) air dried, MGG B) Alcohol fixed, Papanicolaou stain.

However, most tumor cells in seminoma smears typically appear as naked nuclei against a disrupted cytoplasm background, producing the characteristic “tigroid” background. Some neoplastic cells seen in the anaplastic large cell lymphoma can be multinucleated. Immunostaining for OCT3/4, CD117, PLAP, and SALL4 typically highlights seminoma cells.

Lymph node metastases of sarcoma are exceedingly rare but have been reported in patients with Ewing sarcoma, rhabdomyosarcoma, synovial sarcoma, clear cell sarcoma, epithelioid sarcoma, angiosarcoma, and Kaposi’s sarcomas [71]. Cytomorphology may provide some clues, and an immunocytochemistry ICC panel, including markers such as CD99, FLI-1, desmin, myogenin, myoD1, TLE1, EMA, CD31, ERG, CD34, and HHV-8, can be performed on a cell block. Due to the rarity of lymph node sarcoma metastases, it is essential to include markers that help exclude lymphoma, carcinoma, and melanoma in the ICC panel.

The diagnosis of LBCL using fine-needle aspiration and cytology specimens can be challenging and require collection of material with proper fixation for reliable ancillary studies such as immunohistochemistry, FISH, and molecular assays. Rapid on site evaluation is critical in cases of fine needle aspiration specimens for optimal triage for necessary ancillary studies and it is important to perform additional passes to create a suitable cell block. A practical diagnostic approach to specimens composed of large cells can be seen in Figure 7. An inconclusive result or diagnosis should lead to repeat biopsy for proper classification.

Figure 7.

Practical diagnostic approach to specimens composed of large cells

Conclusion

An integrative approach that combines cytomorphology, immunophenotyping, cytogenetics, and molecular data is required for the differential diagnosis of large cell lymphoma. Recent updates in WHO classification and advances in cytopathology techniques enhance diagnostic accuracy and guide precision oncology. Cytology, particularly FNA with ancillary testing, continues to evolve as a frontline diagnostic modality in the era of personalized lymphoma therapy.