Diffuse large B-cell lymphoma: An immunohistochemical approach to diagnosis.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin's lymphoma (NHL) that develops from the B-cells of the lymphatic system. It is the most common subtype of NHL accounting for 30%-40% of all cases. In addition to determining if NHL is indolent or aggressive and whether it is B-cell, T-cell or NK-cell lymphoma, it is very important to determine the subtype of NHL. This is because each subtype can behave differently and may require different treatments. This warrants the need for an accurate and explicit subtype-specific diagnosis that catalogs the molecular prognostic indicators. Moreover, retrospective studies have confirmed that diagnosis to treatment interval is strongly associated with prognostic clinical factors and outcome in newly diagnosed cases of DLBCL. Therefore, an expeditious histological and immunohistochemical investigation should be compassed in cases of oral lymphomas.

INTRODUCTION

Lymphomas indigenously represent a heterogeneous group of malignant diseases that range in behavior from relatively indolent to highly aggressive neoplasms.[1] These are classified into Hodgkin's and non-Hodgkin's subtypes and are believed to originate from the mutation of lymphocyte progenitor cells.[1] Lymphomas constitute nearly 14% of all head-neck malignancies, with non-Hodgkin's lymphoma (NHL) predominantly sharing the burden of 97% cases.[2]

It is alarming to note that the global incidence of NHL is on the upsurge with up to 35% rise in the last 20 years.[3] The estimated incidence of NHL is 5/100,000 (385,741 new cases), with a mortality rate of 2.5/100,000 (199,630 deaths) worldwide.[4] In India, however, the figures signify an incidence rate of 2.2/100,000 (23,801 new cases) and a mortality rate of 1.5/100,000 (16,597 deaths).[5] The most recent age-specific incidence reveals that the incidence of NHL peaks at 75 years in Indian males and 70 years in Indian females.[4]

NHL occurs commonly in the lymph nodes with extranodal sites accounting for 20%–30% of the cases.[1] Extranodal NHL is often noticed in the gastrointestinal tract, Waldeyer's ring, bone and skin. The oral cavity is an uncommon site for NHL with the incidence rate of 0.1%–5%.[6] Most head-neck NHLs belong to B-cell lineage, with diffuse large B-cell lymphoma being the most commonly seen subtype (80%–85% cases),[78] followed by small cell NHLs.[7]

Among all such cases, the most enigmatic association exists between HIV infection and NHL, with high-grade B-cell immunoblastic lymphoma as the most common. Human herpes virus 8 is associated with the development of primary effusion lymphomas, which is relatively rare in the Indian population. Lifestyle factors such as tobacco use (smoking/chewing), alcohol abuse, diet and body mass index have also been found to be associated with NHL.[5] Although no hereditary factors are hypothesized to be associated with NHL, an increased risk among persons with relatives previously diagnosed with NHLs has been reported.[5]

The clinical and histopathological features of the disease do not represent classic models. It therefore summons additional diagnostic modalities such as immunohistochemistry which warrants accurate diagnosis and an early therapeutic intervention. We hereby present a case of diffuse large B-cell NHL in the anterior mandible of a 48-year-old male patient where an immunohistochemical diagnostic work-up befriended the histopathological spectrum of the case and provided an early treatment approach.

CASE REPORT

A 48-year-old male patient reported with a chief complaint of swelling in the front region of the lower jaw for 2 months. The patient gave a history of extraction of the lower front teeth which was followed by prosthetic rehabilitation with the help of a fixed prosthesis. Extraorally, the swelling was not obvious and did not produce any facial asymmetry. Intraoral examination revealed a single swelling in the anterior region of the lower jaw. The anteroposterior and superoinferior dimensions of the swelling were 3 cm × 2.5 cm approximately and the overlying skin appeared considerably stretched with loss of paresthesia over chin region. On palpation, there was no local haul up in the temperature and the swelling was firm and tender. Radiographic examination of the lesion showed a single ill-defined unilocular radiolucency in the anterior region of the mandible extending between 33 and 45 [Figure 1]. Histopathologically, a hypercellular connective tissue stroma was seen consisting of chronic inflammatory cells, predominantly plasma cells admixed with histiocytes and lymphocytes in a collagenous background [Figure 2a and b]. Immunohistochemistry was performed using standard techniques. A panel of monoclonal antibodies was used for the following immunohistochemical markers: CD45, CD20 (a selective marker that recognizes a subpopulation of B-cells), CD3 (the marker for T-cells and NK-cells), CD30 (the marker for activated T-cells and B-cells), BCL-2 and BCL-6.

Figure 1

Ill-defined radiolucency in the anterior mandible extending between 33 and 45

Figure 2

Diffuse large B-cell lymphoma. (a and b) Centroblastic variant presenting large cells with moderate amount of cytoplasm and round-to-oval vesicular nuclei (H and E, ×40 and × 100), (c and d) membranous and cytoplasmic expression of CD45 (IHC, ×40 and × 100), (e) CD20-positive cells in the stroma (IHC, ×40), (f) scant cytoplasmic CD3 expression (IHC, ×40), (g) diffuse membranous and cytoplasmic expression of BCL-2 (IHC, ×40), (h) mild nuclear positivity of BCL-6 in scattered lymphocytes (IHC, ×40)

DISCUSSION

The pathogenesis of B-cell NHLs begins with naive B-cells that undergo rearrangement of their immunoglobulin gene segments in the bone marrow. Immature B-cells encounter multiple antigens post their egress from the marrow and form primary and secondary lymphoid follicles in the secondary lymphoid organs, such as the lymph nodes and spleen with the help of T-cells. The various subtypes of B-cell NHLs are characterized on the basis of the differentiation status of B-cells that in turn depend on genetic processes, such as somatic hypermutation and class switch recombination. Under normal conditions, such genetic events play crucial roles in the generation of heterogeneous antibodies; however, they may become errant and give rise to lymphomas.[9]

Diffuse large B-cell lymphoma (DLBCL) is the most common type of NHL, worldwide. The histopathological picture of DLBCL defines it as a neoplasm of large B-cells arranged in a diffuse pattern. The large size of lymphoma cells is determined when they are larger than the nuclei of benign histiocytes in the same tissue section.[10] However, in view of the genetic etiopathogenesis of the lesion, the diagnosis of DLBCL should be based on more than just the morphological characteristics. The updated 2016 classification of DLBCL has made a few requisite modifications for the diagnosis of DLBCLs.[11] It has been suggested that the cell-of-origin classification, i.e., germinal center B-cell (GCB) versus activated B-cell or non-GCB type, should be included in the pathology report. The immunohistochemical expression of MYC and BCL-2 has been ascertained as adverse prognostic factors. Furthermore, the prognostic value of CD5 (expressed on the surface of all T-cells bearing the αβ heterodimer T-cell receptor) should be assessed. Finally, the category “B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma” has been eliminated and replaced by two new categories: (a) high-grade B-cell lymphoma, with MYC and BCL-2 and/or BCL-6 translocations (also known as double-hit or triple-hit lymphoma); (b) high-grade B-cell lymphoma, not otherwise specified.

CD45/LCA is a glycoprotein marker expressed in lymphohematopoietic cells. The expression of this antigen has been known to increase during B-lymphocyte ontogeny, with low levels of CD45 reported in pathologic lymphocytes from typical chronic lymphocytic leukemia (CLL) and higher levels observed in cases of atypical CLL and in some cases of NHL. In the present case, diffuse cytoplasmic expression of CD45 was noted in the lymphocytes throughout the stroma [Figure 2c and d]. CD20 is another widely used pan-B-cell marker and is expressed from the naive B-cell until the final stages of B-cell development just before plasmacytic differentiation. CD20 revealed the marked predominance of sheets of B-cells in the stroma in the present case, suggestive of NHL [Figure 2e]. Furthermore, scanty immunoexpression of CD3 [Figure 2f] and absence of CD30 staining pointed toward the absence of T-cells in the stroma.

BCL-2 is an anti-apoptotic molecule normally expressed in pre-B-cells, resting B-cells including normal mantle zone lymphocytes and certain types of proliferating B-cells. BCL-2 is downregulated in normal GCBs. Virtually, all of the small B-cell neoplasms express BCL-2. It is most useful in distinguishing reactive follicular hyperplasia from follicular lymphoma. B-cells in reactive follicle centers lack BCL-2 expression, whereas the normal mantle zone and primary follicles are BCL-2 positive. Figure 2g shows the expression of BCL-2 in the present case.

BCL-6 is involved in germinal center function and is downregulated as the B-cell undergoes apoptosis or as it exits the germinal center destined to become a memory B-cell or a plasma cell. Few isolated BCL-6-positive cells may normally be present in the interfollicular areas of lymph nodes (predominantly T-cells). BCL-6 may be helpful in identifying a follicular origin. Mild expression of BCL-6 was noted in the nucleus in few lymphocytes in the current case [Figure 2h].

The aforementioned histopathological criteria and panel of immunohistochemical markers facilitated the diagnosis of DLBCL. Oral intraosseous DLBCLs are rare to be reported in the anterior jaw segments, and a thorough PubMed search in relation to primary NHL in the mandibular anterior segment could reveal only three cases which have been summarized in Table 1. Angiero et al. reported an erythematous nodular lesion in anterior mandibular gingiva in a 56-year-old male patient. Although the mandible showed no bony changes on panoramic radiograph in their case, an immunohistochemical investigation with CD20, CD10, CD3, CD30, ALK-1 and Ki-67 found the biopsy specimens to be positive for CD20 and Ki-67. A final diagnosis of DLBCL was put forth with an indication of the role of chronic antigenic stimulation by hepatitis C virus in the development of the primary lesion as well as the recurrent tumor. A case of postextraction lymphoproliferative disorder in the anterior mandible was reported by Munir et al. The clinical and radiographic picture of the case presenting irregular margins with wide destruction of buccal cortex, alveolar ridge and medullary area of anterior mandible led the authors to a differential diagnosis of chronic osteomyelitis, Ewing's sarcoma, infected odontogenic cyst, ameloblastoma and intraosseous hemangioma. However, the final diagnosis was confirmed upon histopathological analysis and immunohistochemical positivity for leukocyte common antigen (LCA). In contrast, Reddy et al. found immunopositivity for CD34, Tdt and Pax5 and negative for CD20, CD3, CD10 and LCA to conclusively designate their case as a B-cell lymphoblastic lymphoma. In conclusion, on comparison of previously published cases with the present case, it may be suggested that an immunohistochemical panel consisting of CD45, CD20, CD3, CD10, CD138, ALK-1, BCL-2 and BCL-6 supplemented with prognostic markers is a prime requisite for a patient-center subtype-specific final diagnosis in lymphomas.

Table 1

Diffuse large B-cell lymphoma cases in the anterior mandible

Author information	Age/gender	Chief complaint	Clinical features	Radiographic features	Histopathology	IHC (+/−)	Diagnosis
Reddy and Diwakar[12]	13/female	Pain for 1 year and swelling in lower cheek region for 6 months	E/O, diffuse swelling present in anterior region of mandible, approximately 4 cm × 5 cm in size. Firm to hard on palpation. Bilateral submandibular, submental lymph nodes palpable, tender and firm and not fixed to underlying structures I/O, swelling extended from 36 to 46. Expansion of buccal and lingual cortical plates. Mobility of teeth	Absence of follicular space and multiple unerupted permanent teeth. Sparse trabecular pattern and ground glass appearance	In lymph nodes, medium-sized monomorphic cells admixed with tingible body macrophages giving starry-sky pattern Foci of necrosis with malignant cells invading the adjacent skeletal muscles	+ CD34, Tdt, Pax5 − CD20, CD3, CD10, LCA	B-cell lymphoblastic lymphoma
Angiero et al.[13]	56/male	Rapidly progressive swelling of left anterior mandibular buccal gingiva	Soft tissue swelling approximately 1.5 cm × 1.0 cm No palpable cervical lymph nodes	-	Diffuse large lymphocytes with clear cytoplasm Mitotic figures were frequent The cells were large, isolated or in small groups, demonstrating a moderate amount of cytoplasm with large, round-to-oval nuclei containing coarse chromatin and varied sized nucleoli. Prominent nuclear irregularity and pleomorphism were evident. Some cells were binucleate	+ CD20, Ki-67−CD3, Cd10, CD30 and ALK-1	Diffuse large B-cell lymphoma
Munir et al.[14]	61/male	Erythematous swelling in anterior mandible	Erythematous swelling and multiple draining sinuses extra orally in anterior mandible. Teeth extracted for pain and mobility 3 months back	Radiolucent area in anterior mandible from where teeth were extracted for pain and mobility 3 months back with wide destruction of buccal cortex, alveolar ridge and medullary area of anterior mandible	Proliferative sheets of intermediate to small size cells with high nuclear cytoplasmic ratio infiltrating into adjacent muscles, fat and dermal tissue	+ LCA−Cytokeratin (cocktail)	Non-Hodgkin’s lymphoma

IHC: Immunohistochemistry, LCA: Leukocyte common antigen, E/O: Extraoral, I/O: Intraoral

CONCLUSION

Intraoral lymphomas mimic inflammatory conditions, but each lymphoid malignancy is unique and possesses a characteristic immunophenotype. It may therefore be emphasized that correct immunophenotyping in the context of a panel is indispensable in the diagnosis of lymphoid neoplasms. In addition, the familiarity of a dentist with the diagnostic criteria of each lymphoid tumor and ultimately its correlation with the clinical course of the disease, histopathology and molecular interpretation is essential to confirm the diagnostic impression and avoid potential pitfalls.

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Conflicts of interest

There are no conflicts of interest.