O R I G I N A L P A P E R
Primary Diffuse Large B-Cell Lymphoma of the Oral Cavity:
Germinal Center Classification
Indraneel Bhattacharyya•Hardeep K. Chehal• Donald M. Cohen• Samer Z. Al-Quran
Received: 19 February 2010 / Accepted: 20 May 2010 / Published online: 9 June 2010 Ó Humana 2010
Abstract Primary lymphomas of the oral cavity are rare and the most frequent type is diffuse large B-cell lymphoma (DLBCL). Recently, several reports have highlighted the value of classifying DLBCL into prognostically important subgroups, namely germinal center B-cell like (GCB) and non-germinal center B-cell like (non-GCB) lymphomas based on gene expression profiles and by immunohisto- chemical expression of CD10, BCL6 and MUM-1. GCB lymphomas tend to exhibit a better prognosis than non-GCB lymphomas. Studies validating this classification have been done for DLBCL of the breast, CNS, testes and GI tract.
Therefore we undertook this study to examine if primary oral DLBCLs reflect this trend. We identified 13 cases (age range 38–91 years) from our archives dating from 2003–09.
IHC was performed using antibodies against germinal center markers (CD10, BCL6), activated B-cell markers (MUM1, BCL2) and Ki-67 (proliferation marker). Cases were sub-classified as GCB subgroup if CD10 and/or BCL6 were positive and MUM-1, was negative and as non-GCB subgroup if CD10 was negative and MUM-1 was positive.
Immunoreactivity was noted in 2/13 cases for CD10, in 12/
13 for BCL6, in 8/13 for MUM-1, and in 6/13 for BCL2.
Therefore, 8/13 (58%) were sub-classified as non-GCB DLBCLs and 5/13 (42%) as GCB subgroup. All tumors showed frequent labeling with Ki-67 (range 40–95%). Four of the 8 patients with non-GCB subgroup succumbed to their disease, with the mean survival rate of 16 months.
Two patients in this group are alive, one with no evidence of disease and another with disease. No information was available for the other 3 patients in this group. Four of the 5 patients in the GCB subgroup were alive with no evidence of disease and one patient succumbed to complications of therapy and recurrent disease after 18 months. In conclu- sion, our analysis shows that primary oral DLBCL pre- dominantly belongs to the non-GCB subgroup, which tends to exhibit a poorer prognosis. These findings could allow pathologists to provide a more accurate insight into the potential aggressive behavior and poorer prognosis of these lymphomas.
Keywords Oral large B cell lymphoma Germinal center Immunohistochemistry CD10 BCL6 MUM-1
Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most fre- quently diagnosed type of non-Hodgkin lymphoma (NHL) and is the fifth most frequent cancer, accounting for 30–
40% of all cases reported [1]. It is frequently reported in the mediastinum, gastrointestinal tract, bone marrow, central nervous system, breast and testes. [2] Most cases are in patients in the seventh decade of life [1,2]. NHLs of the oral cavity are rare and account for only 3–5% of the lymphomas Both authors I. Bhattacharyya and H.K. Chehal have equal
contribution in the study.
I. Bhattacharyya (&) D. M. Cohen
Department of Oral and Maxillofacial Diagnostic Sciences, University of Florida College of Dentistry, PO Box 100414, Gainesville, FL 32610, USA
e-mail: ibhattacharyya@dental.ufl.edu H. K. Chehal
Department of General Dentistry, Creighton University School of Dentistry, Omaha, NE, USA
S. Z. Al-Quran
Department of Pathology, University of Florida College of Medicine, Gainesville, FL, USA
DOI 10.1007/s12105-010-0184-4
reported [3–5]. They can be primary or secondary to extension from Waldeyer’s ring [3,5]. The most frequent type of primary NHL lymphoma of the oral cavity is DLBCL [3,5], however, the incidence of primary DLBCL in the oral cavity has not been previously specified in the literature. These lesions are symptomatic and typically present as a rapidly enlarging mass [3,5]. Outside of the Waldeyer’s ring, in the oral cavity, the hard palate and the maxillary vestibule appears to be frequently involved [3,5].
DLBCL is characterized by a diffuse proliferation of large neoplastic B cells with nuclear size equal to or exceeding normal macrophage nuclei, or more than twice the size of a normal lymphocyte [1,6]. DLBCL is a het- erogenous neoplasm with variable clinical, morphologic, immunophenotypic, cytogenetic, and genetic features [1,2, 6]. This is reflected in their marked biological heterogeneity and highly variable clinical course [2]. In the past, DLBCL was subclassified based on cytomorphologic features into centroblastic, immunoblastic and anaplastic. Centroblasts are medium to large in size with oval to round nuclei and fine vesicular chromatin patterns. They have two to four nucleoli opposed toward the nuclear membrane, and rarely may have multilobated nuclei, which can predominate in extranodal disease. The tumor can be monomorphic ([90%
centroblasts) or polymorphic with admixed immunoblasts (centroblasts \ 90%) [1, 2, 6]. Immunoblasts display a uniform cytology and almost all cells exhibit a prominent central nucleoli with distinct rims of basophilic cytoplasm.
In the anaplastic variant, the tumor cells are variably large cells with bizarre pleomorphic nuclei. They may mimic Reed Sternberg cells or undifferentiated carcinoma, in which case, immunohistochemistry is helpful. Increased number of intermixed T cells and/or histiocytes can be seen in all variants. In rare cases, the tumor cells may comprise spindle-shaped or signet ring cell forms with a myxoid stroma. The differentiation between morphologic variants is subjective and not reproducible [1]. Moreover, the treat- ment and importantly, survival rates showed no significant differences between these morphologic variants [7–9].
Most DLBCL show somatic mutations in variable regions of their immunoglobulin heavy chain genes, and are thought to arise from B cells that have passed through the germinal center (GC)—either GC or post-GC B-cells [10–12] Various techniques have been used to sub classify DLBCL including oligonucleotide array [12], cDNA microarray [10,13], and immunohistochemical expression [14, 15]. Recently, by using the cDNA microarray tech- niques DLBCL has been subclassified into three molecu- larly distinct forms, based on their gene expression profiles:
germinal center B-cell like (GCB), non-germinal center (non-GCB) or activated B-cell like (ABC), and type 3 gene expression profiles [10, 13]. Patients in the GCB group have demonstrated better outcomes than those with
non-GCB and type 3 groups [10,13]. Subsequently, studies have shown that immunohistochemistry (IHC), which is more practical and widely utilized, is as consistent and accurate in the sub-classification of DLBCLs into these prognostically important subgroups using antibodies against CD10, BCL6 (both markers of germinal center origin) and MUM1 [9,14,15]. This sub-classification has not been attempted before for primary DLBCL of the oral cavity. The aim of this study was to apply this approach to sub-classify DLBCLs of the oral cavity into the two prognostically significant subtypes (GCB and non-GCB) using IHC. We believe that such classification will enable pathologists to provide insight into the potential behavior and prognosis of these lymphomas.
Materials and Methods
We searched for cases of primary DLBCL of the oral cavity from the University of Florida Oral and Maxillofa- cial Pathology Biopsy Service for the period of 2003–2009.
The criteria for selection of cases as primary DLBCL included absence of concomitant nodal/extranodal disease at any other site or within a 6 month period of diagnosis of oral lesion. All cases were reviewed and the diagnosis was confirmed according to the published criteria in the 2008 WHO Classification of Hematopoietic tumors [1]. Immu- nohistochemistry was performed according to standard protocol with antibodies to CD3, CD5, CD20, CD10, Bcl- 6, Bcl-2, MUM1 and Ki-67 [9] (see Table1) applied in all cases. Briefly, multiple unstained sections (5 lm) were then cut from each tissue block and mounted on plus slides.
Following deparaffinization, heat-induced antigen retrieval was performed. Rabbit antimouse amplifying kit was used to enhance staining for the above antibodies utilizing an endogenous biotin blocking kit to decrease background staining. Following antigen retrieval and primary antibody incubation, the reaction was completed in a Ventana ES automated stainer (Ventana, Tucson, AZ) using a diam- inobenzidine immunoperoxidase detection kit. Negative and positive controls in each run were adequate. All immunohistochemical studies were evaluated indepen- dently by 2 pathologists (IB and SA) and immunoreactivity for each antibody was recorded in addition to semiquanti- tative visual estimation of the percentage of tumor cells nuclei labeling with Ki-67. Disagreements were resolved by joint review on a multihead scope. For antibody against CD10, Bcl-6 and MUM1, cases were considered positive if 30% or more of the tumor cells were stained with an antibody. A uniform cutoff of 30% was used similar to what has been used in other studies [16] (see Table 2).
We classified all the cases into two subgroups: GCB and non-GCB utilizing the flow chart in Fig.1 [9]. Cases
were subgrouped as GCB if CD10 and/or BCL6 were positive and MUM-1 negative [17]. If CD10 and Bcl-6 were deemed negative, or BCL6 and MUM-1 were posi- tive, the cases were assigned to the non-GCB subgroup [17].
Results
We identified 13 cases of primary DLBCL of the oral cavity. There were six females and seven males ranging in age from 38 to 91 years of age (Table3). The symptoms in Table 1 Antibodies used in the study
Antibody Manufacturer Source Clone Type Dilution Characterization
CD20 DAKO,
Carpenteria, CA
Mouse monoclonal
L26 IgG29 1:200 Expressed by B-cell lineage
CD3 Ventana Medical Systems, Tucson, AZ
Rabbit polyclonal
PS1 Polyclonal Prediluted from manufacturer
Expressed by T-cell lineage
CD5 Ventana Medical Systems, Tucson, AZ
Rabbit monoclonal
SP19 IgG Prediluted from
manufacturer
Expressed by T cell lineage
CD10 Ventana Medical Systems, Tucson, AZ
Mouse monoclonal
56C6 IgG1 Prediluted from
manufacturer
Expressed by normal GC B-cells
Bcl-2 Ventana Medical Systems, Tucson, AZ
Mouse monoclonal
124 IgG1/K Prediluted from manufacturer
Expressed in normal lymphoid tissue but is absent in reactive GC B-cells
bcl-6 Cell Marque, Rocklin, CA
Mouse monoclonal
GI191E/A8 IgG1 Prediluted from manufacturer
Expressed in germinal center B cells and a subset of CD4? T cells. It is expressed in the majority of DLBCL’s ranging from 57 to 100%
Ki 67 DAKO,
Carpenteria, CA
Mouse monoclonal
MIB1 IgG kappa 1:30 Cell cycle marker used to assess proliferative potential of tumors
MUM1 ID Labs, Inc.
London, ON, Canada
Mouse monoclonal
MUM 2p IgG1 1:20 Expression denotes the final step of intra- GC B-cell
Visualization for all antibodies done by UltraView DAB kit Ventana Medical Systems, Tucson, AZ
Table 2 : Summary of results of immunohistochemical staining
Case CD 10 CD 20 CD 3 CD 5 Ki 67 (%) Bcl-2 Bcl-6 MUM1 Sub classification Outcome
1 2 1 2 2 40–50 2 1 1 Non- GCB DOD
2 2 1 2 2 80–95 1 2 1 Non- GCB DOD
3 2 1 2 ND [75 1 1 1 Non- GCB DOD
4 2 1 1 2 60–70 2 1 1 Non- GCB NIA
5 1 1 2 2 40–50 2 1 2 GCB Alive, NOD
6 2 1 2 2 90 1 1 2 GCB Alive, NOD
7 2 1 2 ND 50–70 2 1 1 Non- GCB NIA
8 1 1 2 2 [80 1 1 2 GCB DOD
9 2 1 2 2 [80 2 1 2 GCB Alive, NOD
10 2 1 2 2 80 2 1 1 Non-GCB NIA
11 2 1 2 2 40–50 1 1 1 Non-GCB DOD
12 2 1 2 2 [80 1 1 2 GCB Alive, NOD
13 2 1 2 2 40–60 1 1 1 Non-GCB Alive, with disease
ND not done, NIA no information available, DOD died of disease, NOD no evidence of disease
the cases presented here ranged from none to generalized pain and numbness of chin and lower lip. Varied working diagnoses were provided by the clinicians and included fibroma, pyogenic granuloma, odontogenic cyst, giant cell lesion, myxoma, pleomorphic adenoma, metastatic carci- noma, squamous cell carcinoma and lymphoma. None of these patients had HIV or evidence of immunosuppression.
Many of the cases in this study arose in intrabony locations with five in the maxilla around the alveolus or sockets of extracted teeth and case in the right body of the mandible (Fig.2). The maxillary vestibule was a common site for the occurrence of soft tissue tumors. Seven cases presented as soft tissue swellings with six in the maxilla and one in the mandible. The vestibule was a common site for the occurrence of this lesion as seen in the clinical image from case 11 (Fig.3). One patient had a recurrence in the ves- tibule (Fig.3b).
All cases were initially diagnosed as DLBCL and all demonstrated a diffuse infiltration of medium to large neoplastic B cells (Fig.4a) that had large nuclei with nuclear size equal to or exceeding normal macrophage nuclei, or more than twice the size of a normal lympho- cyte. (Figs4b, d). Variable proportions of centroblasts and immunoblasts were observed (Fig.4d) and tumor cells with multilobated nuclei were occasionally seen. There were variable numbers of intermixed histiocytes and/or T cells. These infiltrates demonstrated a diffuse destructive growth pattern and areas of perivascular infiltrate (Fig4c).
In all cases, the tumor cells demonstrated strong immu- noreactivity with anti-CD20 antibody (Fig.5), and were
negative for CD3 and CD 5 (cyclinD1 and CD138 in three cases). Immunoreactivity for Bcl-6 was seen in (12/13) cases, for CD10 in 2/13cases, 8/13 for MUM1 and 6/13 for Bcl-2 (Fig. 6). As a result eight cases (58%) were classified in the non-GCB subgroup and five cases (42%) in the GCB subgroup (Fig. 6). All the tumors showed frequent labeling with Ki-67 (40–90%) (Fig.7; Table2).
Limited clinical data was available for all the cases including status of staging bone marrow biopsy and imaging studies.
Reported treatment included: combination chemother- apy alone (3), combination chemotherapy and radiotherapy (5), treatment declined (2) and unknown treatment status (3). Four of the eight patients with non-GCB subgroup succumbed to their disease, with the mean survival of 16 months. One patient was alive with no evidence of disease and one patient was alive with disease. No infor- mation was available for 3 patients in the non-GCB group.
In the GCB subgroup four of the five patients were alive with no evidence of disease and one patient succumbed to complications arising from therapy and recurrent disease 18 months after diagnosis.
Discussion
DLBCL is the most common type of NHL in the US and Western world and accounts for 30–40% of all cases [2].
Up to 40% of the cases present with only extranodal involvement, and isolated bone marrow involvement is extremely rare. They are associated with the presence of large cells that express a mature B-cell immunophenotype and have a high proliferative index [1]. Apart from being related to AIDS in a few cases, the etiology of the primary DLBCLs of the oral cavity is unknown [3,4]. Oral mani- festations are seen in 3–5% of the cases [3–5] DLBCL rarely manifests as a primary malignancy in the head and neck region (\1%) [5]. The presence of DLBCL in the head and neck may be associated with undiagnosed HIV infection since they account for 2% of oral neoplasms in patients with AIDS [18]. DLBCL has been reported in the oral cavity in the buccal mucosa, hard palate and gingiva [3,5]. Many of the cases in our study arose in intrabony locations and the maxillary vestibule was a common site for the occurrence of soft tissue tumors.
The most common presenting symptoms of extranodal NHL in the head and neck region include local swelling, pain and discomfort in the region of involvement [19].
Clinical signs included unilateral enlargement of face, localized swelling, destruction of hard and soft tissue, cavitation and ulceration and/or sessile soft tissue mass formation. This diverse presentation in our study and var- ied working diagnoses provided, are consistent with the Fig. 1 Flow chart for GCB classification. Adapted from [9]
heterogenous clinical presentation of DLBCLs reported in the literature [3].
In spite of the hematologic and biochemical profiles of affected patients being often normal, patients may have a decreased number of peripheral blood lymphocytes or decreased serum albumin levels and elevated LDH, interleukin-6 (IL-6), IL-10, and IL- 2 receptor levels which have been shown to correlate with a poor prognosis [20].
Most DLBCL have been shown to undergo somatic mutations in the variable regions of their immunoglobulin
heavy chain genes, and hence, are thought to be derived from B cells that have passed through the germinal center (GC)-representing either GC or post-GC B-cells [13]. With the help of cDNA microarray techniques, the gene expression profiles of DLBCL have been evaluated and the two distinct molecular forms, the GC-like (GCB) and the activated B-cell like (non-GCB) DLBCL were identified [10]. GCB lesions express genes normally expressed by germinal center B cells and the non-GCB DLBCL express genes normally induced during in vitro activation of peripheral blood B cells [10].
Table 3 Summaryof clinical findings Case Age
(in years)
Sex Race Site Duration Clinical presentation Working diagnosis Diagnosis Treatment
&
Prognosis 1 91 F C Left crest of maxillary
alveolus
[1 month Ulcerated, soft tissue mass SCCa Non- GCB
Declined DOD 2 76 M C Socket of the extracted
mand left 2nd molar
[3 weeks Pain in extraction socket, numbness in chin & left side of lower lip
PG, Met Ca Non-
GCB
Ch ? Xrt DOD
3 80 M C Facial aspect of right maxillary tuberosity
2.5 weeks Swelling None Non-
GCB
Declined DOD 4 77 F C Sockets of extracted max
left 2nd & 3rd molars
Unknown Soft tissue mass GC lesion, Myxoma
Non- GCB
NIA
5 38 M H Maxillary vestibule, buccal to tooth # 9
1.5 month Painful swelling Fibroma GCB Ch ? Xrt
NOD 6 51 F Unk Right maxillary buccal
vestibule
[6 months Large sessile soft tissue mass SCCa, Lymphoma, Minor salivary gland tumor
GCB Ch NOD
7 60 M C Right mandibular buccal vestibule
2.5 months Swelling and numbness in area Neoplasia Non- GCB
NIA
8a 56 M C Maxilla, buccal to edentulous space in area of # 6
[2 weeks Generalized pain in oral cavity ‘‘Not just necrotic tissue’’
GCB Ch ? Xrt
DOD
9a 45 F C Right body of mandible 3 months Enlargement of right side of face
Myxoma, Pleomorphic Adenoma, a
GCB Ch NOD
10 54 M H Maxilla, area of right first molar
Unknown Soft tissue swelling over bony exostosis
None Non-
GCB NIA
11b 72 M C Maxillary alveolus, left central incisor to right first premolar
Unknown Swelling with obliteration of vestibule with bone/soft tissue destruction
Odontogenic cyst/
Infection
Non- GCB
DOD
12b 89 F C Right maxillary vestibule 1 week Swelling with discomfort in denture wearing, small ulceration on surface
None GCB Ch NOD
13 74 F C Right maxillary tuberosity
[1 month Swelling with mass sensation Lymphoma Non- GCB
Ch ? Xrt Alive with disease C caucasian, H hispanic, NIA no information available, NOD alive with no evidence of disease, DOD died of disease, Ch chemotherapy, Xrt radiation therapy
a radiographs (Fig.6)
b clinical images (Fig.7)
DLBCLs express CD 45 (leukocyte common antigen) and pan-B-cell antigens (CD19, CD20 and CD79). In some cases, expression of one or more of these antigens may be lacking [20]. CD20 is expressed on B-cells from the mature precursor B cell (hematogone) until the pre plasma cell stage of differentiation [21]. It is a highly specific marker for B-cells lineage and most DLBCL show homogenously bright staining for CD20 [22]. In termi- nally differentiated DLBCLs with immunoblastic or plasmablastic morphology, CD20 expression can be lost [22, 23]. CD10 is a membrane metalloproteinase that is detected in early lymphoid progenitors. CD10 expression is restricted to germinal centers of secondary follicles [24]. Majority of follicular lymphomas are CD10 positive while other small B-cell lymphomas are CD10 negative [17]. It is expressed in 20–40% of DLBCLs. The staining is membranous and usually homogenous throughout the tumor [8,25].
CD3 is a T cell marker that is associated with the T-cell receptor and transmits the activation signal to the cyto- plasm. It is highly restricted in its expression to T lymphoid cells and is an excellent marker since it is retained fol- lowing neoplastic transformation [26].
Ki67 is a cell cycle marker used as a proliferation marker. Ki67 defines growth fraction of actively cycling
cells. In DLBCL, the expression varies from 30 to 100%
but is usually high [27]. Most studies show that a high proliferative index is an adverse prognostic factor. Tumors with a Ki 67 expression of [80% are termed as ‘‘highly proliferative’’ or aggressive tumors [8].
Bcl-2 protein functions as an anti-apoptotic protein protecting cells from programmed cell death [28]. In DLBCL, Bcl-2 protein expression is found in 30–60% of cases with ill understood prognostic value [28]. However, some studies suggest that Bcl-2 expression is associated with a significantly worse overall survival rate [29].
Bcl-6 is selectively expressed by GC B cells in normal lymphoid tissues and a subset of CD4?YT cells [9,30,31].
Their expression in DLBCLs has been found in a majority of the cases ranging from 57 to 100% [32]. Bcl-6 is reported in about 40% of DLBCL but the biological sig- nificance of this expression is not clear [8,10,31,33].
MUM 1 is a lymphoid-specific member of the interferon regulatory factor family of transcription factors [9]. The staining is nuclear and may be associated with cytoplasmic positivity. In DLBCLs MUM1 is expressed in 50–75% of cases and is seen both with and without Bcl-6 expression [34]. Hans et al. report that expression of MUM1 in at least 30% of tumor cells in their series of DLBCL and were associated with a significantly worse overall survival rate Fig. 2 a, b. Periapical
radiographs demonstrating irregular radiolucencies with indistinct margins seen in a 56- year-old male with swelling in area of right maxillary canine to molars (Case #8 Table3). c Section from panoramic radiograph demonstrating lesion arising within the mandible in the right body of the mandible (Case #9 Table3)
[9]. MUM1 is considered to be a marker of the non-GCB phenotype especially when used in conjunction with CD10 and Bcl-6 [8–10,35,36].
In normal B-cell differentiation, the B cells go through the pre-germinal center, GC and post GC stages. Pre GC B cells are virgin B cells. Cells comprising the GC consist of small blast cells, centroblasts, centrocytes and occasionally plasma cells. The B cells that leave the GC and enter the post GC phase, differentiate either towards the memory cells or plasma cells [8]. Normal GC B cells express CD10 and Bcl-6 [17]. Bcl-6 may be lost by late GC B cells, which in turn acquire MUM-1 expression [34]. MUM-1, and CD138 are expressed by post GC B cells while MUM-1 may be acquired late in the GC reaction, CD138 expression is usually restricted to cells exhibiting plasmablastic dif- ferentiation and to plasma cells [8].
It has now been accepted that the distinct gene expres- sion profiling subtypes, the GCB and non-GCB of DLBCL can be predicted using a panel of antibodies by immuno- histochemistry that include CD10, Bcl-6 and MUM1 [9, 35, 37] If both CD10 and Bcl-6 are positive or even if CD10 alone is positive the tumor can be assigned in the GCB subgroup. If CD10 is negative and Bcl-6 is positive, the expression of MUM-1 determines the subgroup. If MUM-1 is negative the tumor represents a GCB subgroup whereas if MUM-1 is positive, a non-GCB designation is given. These antibodies recognize molecules whose mRNA expression is highly associated with the GCB and non- GCB subtypes in cDNA microarray studies [10, 13].
Cytogenetic abnormalities in DLBCL point to genes that are responsible for the early steps of lymphomagenesis [10]. Majority of the genes identified effect either the apoptotic pathway and/or the cellular proliferation
pathways [38]. Two of the most common translocations found in DLBCL are t(14;18) and t(3;14). t(14;18) juxta- pose Bcl-2 which encodes an antiapoptotic protein on 18q21(apoptic pathway) [39, 40]. Whereas t(3;14) juxta- pose Bcl-6 which encodes a proliferative transcription factor on 3q27 (cellular proliferation pathway), with the immunoglobulin heavy chain on 14q32, leading to upreg- ulation [41]. Chromosomal gains such as 8q and 2p are correlated with upregulation of c-myc, a proliferative protein and rel, which plays a role in preventing apoptosis [42, 43]. Chromosomal loss of 17p correlates with the deletion of tumor suppressor gene encoding for the p53 protein [42,44]. Gene expression profiling techniques are very powerful and probably predictive of prognosis but are not easily available in clinical settings.
In the early 1990s, based on five clinical parameters the International Prognostic Index IPI) was developed [45].
This has developed into the most successful tool in pre- dicting survival in lymphoma patients to date. The parameters included are age, stage of disease, extranodal site, performance status and serum lactate dehydrogenase (LDH) level [45]. Patients with a low risk IPI score had an approximate 5-year survival rate of 73% compared to 26%
for high risk group [45]. Efforts to treat poor risk DLBCL (based on IPI) has produced inconsistent results and this has been attributed to the biologic heterogenicity of this entity [38,46]. However IPI remains a very valuable tool but recent studies analyzing biologic differences, which are not taken into account by the IPI, significantly modify the clinical outcome [10,12]. Multiple studies have shown that GCB subgroup lymphomas tend to exhibit statistically significant better prognosis when compared to the non- GCB group [8,20,35,37,38]. Distinctly poorer prognosis Fig. 3 a72-year-old male with swelling and obliteration of the anterior maxillary vestibule presented with bone and soft tissue destruction (Case
#11 Table3). b 89 year old female with swelling exhibiting a small ulceration on the surface in the right maxillary vestibule presented with discomfort in denture wearing. (Case #12 Table3)
has been reported in non-GCB DLBCLs in many diverse locations such as the central nervous system, gastrointes- tinal tract, breast, testes, among others [7, 13,32, 35–37, 47] Our results appear to indicate a similar trend. A sig- nificant proportion ([50%) of the patients with non-GCB DLBCL succumbed to their disease, though limited infor- mation was available on the treatment these patients underwent. Importantly, the GCB group patients fared better and four of the five patients are alive and disease free as of submission of the manuscript.
Current treatment of DLBCL usually begins with multi- agent chemotherapy, typically CHOP (cyclophosphamide, hydroxydoxorubicin, oncovin and prednisone) [48]. Inspite of the initial response to therapy, more than half the patients
succumb to the disease [48]. Early stage disease care involves either chemotherapy alone or a combination of chemotherapy and radiotherapy. The chemotherapy usually involves 3 cycles of CHOP [49].
Patients are considered for bone marrow transplant if remission is not maintained. The role of surgery is severely limited in treatment of DLBCL. Other drugs used in multiagent chemotherapy for advanced stage disease usu- ally involve various combinations of methotrexate, bleo- mycin, doxorubicin, vincristin, dexamethasone and leukcovorin, etoposide, mechlorethamine, procarbazine, cytarabine [50,51].
In a recent study, Natkunam et al. [52], demonstrated that the Han’s classification cannot predict outcome in a Fig. 4 Composite photomicrograph (hematoxyllin and eosin stain) of
primary Diffuse large B cell lymphomas in the oral cavity (a and b, c and d). a Diffuse infiltrate of abnormal lymphoid cells (original magnification 9200). b Higher magnification showing that the nuclear size of the abnormal lymphocytes (black arrows) is more than two times the size of normal small lymphocytes (white arrows).
Neoplastic cells demonstrating an immunoblastic morphology with
prominent central nucleoli are noted in lower left corner (original magnification 9500). c Both diffuse and angiocentric abnormal lymphocytic infiltrates of large cells (original magnification 9200).
dHigher magnification showing that typical appearance of centrob- lastic variant (arrow heads highlight mitotic figures). The nuclear size of the abnormal lymphocytes is more than twice the size of a normal lymphocyte (original magnification 91000)
large cohort study of DLBCL patients treated with ritux- imab. Significant differences in protein expression have been found for DLBCL and have yielded conflicting results [52,53]. Since many antibodies are not available for many germinal center specific genes, validation of these results in independent groups of patients are necessary. However, treatment for both GCB and non-GCB subgroups is the
same. Therefore newer studies should be directed toward developing better predictors of survival in DLBCL patients treated with rituximab.
Conclusions
Similar studies analyzing GCB and non-GCB subgroups of DLBCL have been conducted in tumors of the breast [36], CNS[47] and testes [35] and have revealed that non-GCB tumors exhibit a high proliferative rate and poor prognosis compared to GCB lesions. Our study of 13 primary DLBCLs shows that non-GCB lesions predominate and may exhibit a poorer prognosis with half of the patients succumbing to their disease while majority of the GCB group continue to be disease free. Pathologists and clini- cians should be aware of this trend and may consider adding GCB classification to their reports when referring patients with oral DLBCL for further treatment and eval- uation. The relatively small number of cases and short follow up periods (less than 5 years) are a limitation of the current study and additional investigation is warranted.
However, stratification of DLBCL by immunohistoche- mitry is possible and is a relatively inexpensive, readily available and effective method of delineating the subtypes of DLBCL.
Fig. 6 Immunohistochemical panel (magnification 940) demonstrating positive reactions Fig. 5 Immunohistochemical study for CD20. All case demonstrated
positive reactivity with this antibody. (Original magnification 9400)
Acknowledgments The authors would like to thank Ms. Elaine Dooley for her technical support.
References
1. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC Press; 2008.
2. Coiffier B. Diffuse large cell lymphoma. Curr Opin Oncol.
2001;13(5):325–34.
3. Kolokotronis A, Konstantinou N, Christakis I, Papadimitriou P, Matiakis A, Zaraboukas T, et al. Localized B-cell non-Hodgkin’s lymphoma of oral cavity and maxillofacial region: a clinical study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod.
2005;99(3):303–10.
4. Solomides CC, Miller AS, Christman RA, Talwar J, Simpkins H.
Lymphomas of the oral cavity: histology, immunologic type, and incidence of Epstein-Barr virus infection. Hum Pathol. 2002;
33(2):153–7.
5. Wolvius EB, van der Valk P, van der Wal JE, van Diest PJ, Huijgens PC, van der Waal I, et al. Primary extranodal non- Hodgkin lymphoma of the oral cavity. an analysis of 34 cases.
Eur J Cancer B Oral Oncol. 1994;30B(2):121–5.
6. Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML, et al. A revised European-American classification of lymphoid neoplasms: a proposal from the international lymphoma Study Group. Blood. 1994;84(5):1361–92.
7. Alacacioglu I, Ozcan MA, Ozkal S, Piskin O, Turgut N, Demirkan F, et al. Prognostic significance of immunohisto- chemical classification of diffuse large B-cell lymphoma.
Hematology. 2009;14(2):84–9.
8. de Leval L, Harris NL. Variability in immunophenotype in dif- fuse large B-cell lymphoma and its clinical relevance. Histopa- thology. 2003;43(6):509–28.
9. Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103(1):275–82.
10. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma
identified by gene expression profiling. Nature. 2000;403(6769):
503–11.
11. Lossos IS, Okada CY, Tibshirani R, Warnke R, Vose JM, Greiner TC, et al. Molecular analysis of immunoglobulin genes in diffuse large B-cell lymphomas. Blood. 2000;95(5):1797–803.
12. Shipp MA, Ross KN, Tamayo P, Weng AP, Kutok JL, Aguiar RC, et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nat Med. 2002;8(1):68–74.
13. Rosenwald A, Wright G, Chan WC, Connors JM, Campo E, Fisher RI, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med. 2002;346(25):1937–47.
14. Barrans SL, O’Connor SJ, Evans PA, Davies FE, Owen RG, Haynes AP, et al. Rearrangement of the BCL6 locus at 3q27 is an independent poor prognostic factor in nodal diffuse large B-cell lymphoma. Br J Haematol. 2002;117(2):322–32.
15. Linderoth J, Jerkeman M, Cavallin-Stahl E, Kvaloy S, Torlakovic E. Immunohistochemical expression of CD23 and CD40 may identify prognostically favorable subgroups of diffuse large B- cell lymphoma: a nordic lymphoma group study. Clin Cancer Res. 2003;9(2):722–8.
16. Milanes-Yearsley M, Hammond ME, Pajak TF, Cooper JS, Chang C, Griffin T, et al. Tissue micro-array: a cost and time- effective method for correlative studies by regional and national cancer study groups. Mod Pathol. 2002;15(12):1366–73.
17. Dogan A, Bagdi E, Munson P, Isaacson PG. CD10 and BCL-6 expression in paraffin sections of normal lymphoid tissue and B- cell lymphomas. Am J Surg Pathol. 2000;24(6):846–52.
18. Lozada-Nur F, de Sanz S, Silverman S Jr, Miranda C, Regezi JA.
Intraoral non-Hodgkin’s lymphoma in seven patients with acquired immunodeficiency syndrome. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996;82(2):173–8.
19. Shima N, Kobashi Y, Tsutsui K, Ogawa K, Maetani S, Naka- shima Y, et al. Extranodal non-Hodgkin’s lymphoma of the head and neck. A clinicopathologic study in the Kyoto-Nara area of Japan. Cancer. 1990;66(6):1190–7.
20. de Leval L, Braaten KM, Ancukiewicz M, Kiggundu E, Delaney T, Mankin HJ, et al. Diffuse large B-cell lymphoma of bone: an analysis of differentiation-associated antigens with clinical cor- relation. Am J Surg Pathol. 2003;27(9):1269–77.
21. Chang KL, Arber DA, Weiss LM. CD20: a review. Appl Immunohistochem. 1996;4(1):1–15.
Fig. 7 Immunohistochemical study for Ki-67 (magnification 920). All tumors showed frequent labeling with Ki-67 (ranging from low 40% to high 95%). a low percentage of positivity; b high percentage of staining
22. Mohrmann RL, Arber DA. CD20-Positive peripheral T-cell lymphoma: report of a case after nodular sclerosis Hodgkin’s disease and review of the literature. Mod Pathol. 2000;13(11):
1244–52.
23. Yao X, Teruya-Feldstein J, Raffeld M, Sorbara L, Jaffe ES.
Peripheral T-cell lymphoma with aberrant expression of CD79a and CD20: a diagnostic pitfall. Mod Pathol. 2001;14(2):105–10.
24. Kaufmann O, Flath B, Spath-Schwalbe E, Possinger K, Dietel M.
Immunohistochemical detection of CD10 with monoclonal anti- body 56C6 on paraffin sections. Am J Clin Pathol. 1999;111(1):
117–22.
25. Fabiani B, Delmer A, Lepage E, Guettier C, Petrella T, Briere J, et al. CD10 expression in diffuse large B-cell lymphomas does not influence survival. Virchows Arch. 2004;445(6):545–51.
26. Mason DY, Cordell J, Brown M, Pallesen G, Ralfkiaer E, Rothbard J, et al. Detection of T cells in paraffin wax embedded tissue using antibodies against a peptide sequence from the CD3 antigen. J Clin Pathol. 1989;42(11):1194–200.
27. Hall PA, Richards MA, Gregory WM, d’Ardenne AJ, Lister TA, Stansfeld AG. The prognostic value of Ki67 immunostaining in non-Hodgkin’s lymphoma. J Pathol. 1988;154(3):223–35.
28. Pezzella F, Tse AG, Cordell JL, Pulford KA, Gatter KC, Mason DY. Expression of the bcl-2 oncogene protein is not specific for the 14;18 chromosomal translocation. Am J Pathol. 1990;137(2):
225–32.
29. Rantanen S, Monni O, Joensuu H, Franssila K, Knuutila S.
Causes and consequences of BCL2 overexpression in diffuse large B-cell lymphoma. Leuk Lymphoma. 2001;42(5):1089–98.
30. Flenghi L, Bigerna B, Fizzotti M, Venturi S, Pasqualucci L, Pileri S, et al. Monoclonal antibodies PG-B6a and PG-B6p recognize, respectively, a highly conserved and a formol-resistant epitope on the human BCL-6 protein amino-terminal region. Am J Pathol.
1996;148(5):1543–55.
31. Ye BH, Lista F, Lo Coco F, Knowles DM, Offit K, Chaganti RS, et al. Alterations of a zinc finger-encoding gene, BCL-6, in dif- fuse large-cell lymphoma. Science. 1993;262(5134):747–50.
32. Braaten KM, Betensky RA, de Leval L, Okada Y, Hochberg FH, Louis DN, et al. BCL-6 expression predicts improved survival in patients with primary central nervous system lymphoma. Clin Cancer Res. 2003;9(3):1063–9.
33. Capello D, Vitolo U, Pasqualucci L, Quattrone S, Migliaretti G, Fassone L, et al. Distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia. Blood. 2000;95(2):
651–9.
34. Falini B, Fizzotti M, Pucciarini A, Bigerna B, Marafioti T, Gambacorta M, et al. A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells. Blood.
2000;95(6):2084–92.
35. Al-Abbadi MA, Hattab EM, Tarawneh MS, Amr SS, Orazi A, Ulbright TM. Primary testicular diffuse large B-cell lymphoma belongs to the nongerminal center B-cell-like subgroup: a study of 18 cases. Mod Pathol. 2006;19(12):1521–7.
36. Yoshida S, Nakamura N, Sasaki Y, Yoshida S, Yasuda M, Sagara H, et al. Primary breast diffuse large B-cell lymphoma shows a non-germinal center B-cell phenotype. Mod Pathol. 2005;18(3):
398–405.
37. Al-Quran SZ, Hameed O, Cardona DM, Jhala D, Li Y. Primary large B-Cell lymphomas of the breast: subclassification by immunophenotyping. Am J Clin Pathol. 2007;128(4):693–4.
38. Wu G, Keating A. Biomarkers of potential prognostic significance in diffuse large B-cell lymphoma. Cancer. 2006;106(2):247–57.
39. Jacobson JO, Wilkes BM, Kwaiatkowski DJ, Medeiros LJ, Aisenberg AC, Harris NL. bcl-2 rearrangements in de novo dif- fuse large cell lymphoma. association with distinctive clinical features. Cancer. 1993;72(1):231–6.
40. Weiss LM, Warnke RA, Sklar J, Cleary ML. Molecular analysis of the t(14;18) chromosomal translocation in malignant lym- phomas. N Engl J Med. 1987;317(19):1185–9.
41. Shaffer AL, Yu X, He Y, Boldrick J, Chan EP, Staudt LM. BCL-6 represses genes that function in lymphocyte differentiation, inflam- mation, and cell cycle control. Immunity. 2000;13(2):199–212.
42. Bea S, Colomo L, Lopez-Guillermo A, Salaverria I, Puig X, Pinyol M, et al. Clinicopathologic significance and prognostic value of chromosomal imbalances in diffuse large B-cell lym- phomas. J Clin Oncol. 2004;22(17):3498–506.
43. Berglund M, Enblad G, Flordal E, Lui WO, Backlin C, Thunberg U, et al. Chromosomal imbalances in diffuse large B-cell lym- phoma detected by comparative genomic hybridization. Mod Pathol. 2002;15(8):807–16.
44. Tagawa H, Tsuzuki S, Suzuki R, Karnan S, Ota A, Kameoka Y, et al. Genome-wide array-based comparative genomic hybrid- ization of diffuse large B-cell lymphoma: comparison between CD5-positive and CD5-negative cases. Cancer Res. 2004;64(17):
5948–55.
45. A predictive model for aggressive non-Hodgkin’s lymphoma.
The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. N Engl J Med. 1993;329(14):987–94.
46. Shipp MA, Abeloff MD, Antman KH, Carroll G, Hagenbeek A, Loeffler M, et al. International consensus conference on high- dose therapy with hematopoietic stem cell transplantation in aggressive Non-Hodgkin’s lymphomas: report of the jury. J Clin Oncol. 1999;17(1):423–9.
47. Hattab EM, Martin SE, Al-Khatib SM, Kupsky WJ, Vance GH, Stohler RA, et al. Most primary central nervous system diffuse large B-cell lymphomas occurring in immunocompetent indi- viduals belong to the nongerminal center subtype: a retrospective analysis of 31 cases. Mod Pathol. 2010;23(2):235–43.
48. Vose JM. Current approaches to the management of non-Hodg- kin’s lymphoma. Semin Oncol. 1998;25(4):483–91.
49. Connors JM, Klimo P, Fairey RN, Voss N. Brief chemotherapy and involved field radiation therapy for limited-stage, histologi- cally aggressive lymphoma. Ann Intern Med. 1987;107(1):25–30.
50. Cabanillas F, Hagemeister FB, McLaughlin P, Velasquez WS, Riggs S, Fuller L, et al. Results of MIME salvage regimen for recurrent or refractory lymphoma. J Clin Oncol. 1987;5(3):407–12.
51. Fisher RI, Gaynor ER, Dahlberg S, Oken MM, Grogan TM, Mize EM, et al. Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin’s lymphoma. N Engl J Med. 1993;328(14):1002–6.
52. Natkunam Y, Farinha P, Hsi ED, Hans CP, Tibshirani R, Sehn LH, et al. LMO2 protein expression predicts survival in patients with diffuse large B-cell lymphoma treated with anthracycline- based chemotherapy with and without rituximab. J Clin Oncol.
2008;26(3):447–54.
53. Seki R, Ohshima K, Fujisaki T, Uike N, Kawano F, Gondo H, et al. Prognostic impact of immunohistochemical biomarkers in diffuse large B-cell lymphoma in the rituximab era. Cancer Sci.
2009;100(10):1842–7.
