Immunohistochemical profile and fluorescence in situ hybridization analysis of diffuse large B-cell lymphoma in Northern China.
* Context.--Gene expression profiling of diffuse large B-cell
lymphoma using complementary DNA microarrays has revealed 2 major
prognostic groups in Western countries: germinal center B-cell-like and
nongerminal center B-cell-like lymphomas. Immunohistochemical analysis
using antibodies specific for CD10, BCL6, and MUM1 has been proposed as
a surrogate for gene expression profiling.
Objective.--To study the immunohistochemical features of diffuse large B-cell lymphoma cases from northern China because geographic differences for this disease are known to exist.
Design.--Morphologic, immunohistochemical, and fluorescence in situ hybridization analyses of 63 cases of diffuse large B-cell lymphoma from northern China.
Results.--There were 38 men and 25 women with a median age of 57 years (range, 12-87 years). CD10 was positive in 19 cases (30%), BCL6 was positive in 22 cases (35%), and MUM1 was positive in 32 cases (51%). Twenty-one (33%) cases were germinal center B-cell-like lymphoma, and 42 (67%) were nongerminal center B-cell-like lymphoma. BCL2 was expressed more often in nongerminal center B-cell-like disease versus germinal center B-cell-like disease (60% versus 24%, P = .01) and in nodal versus extranodal (64% versus 30%, P = .01) cases. Fluorescence in situ hybridization analysis showed BCL6, MYC, and BCL2 rearrangements in 11 of 32 (34%), 8 of 27 (30%), and 11 of 50 (22%) cases, respectively.
Conclusions.--These results add to what is known about the geographic variation of diffuse large B-cell lymphomas. In northern China, the frequency of the germinal center B-cell-like type and BCL6 expression and/or BCL6 rearrangement is less and the frequency of MYC rearrangement is greater than have been reported in Western countries.
(Arch Pathol Lab Med. 2010;134:759-765)
In situ hybridization (Usage)
Non-Hodgkin's lymphomas (Development and progression)
Non-Hodgkin's lymphomas (Diagnosis)
Non-Hodgkin's lymphomas (Care and treatment)
Medeiros, L. Jeffrey
Lennon, P. Alan
Jorgensen, Jeffrey L.
Yin, C. Cameron
|Publication:||Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 College of American Pathologists ISSN: 1543-2165|
|Issue:||Date: May, 2010 Source Volume: 134 Source Issue: 5|
|Geographic:||Geographic Scope: United States Geographic Code: 1USA United States|
Diffuse large B-cell lymphoma (DLBCL) constitutes 30% to 40% of
adult non-Hodgkin lymphomas in Western countries, and its frequency is
even higher in Asia.1 Diffuse large B-cell lymphoma, as a diagnostic
category, displays striking heterogeneity at the clinical, histologic,
immunophenotypic, and molecular levels. At the DNA level, abnormalities
of BCL6 at band 3q27, of BCL2 at band 18q21, and of MYC at band 8q24
have been described in 45%, 20%, and 15% of cases, respectively. (2) At
the RNA level, gene expression profiling using complementary DNA (cDNA)
microarrays has shown that DLBCL consists of molecularly distinct
subgroups. (3-7) In studies from the National Institutes of Health, 3
subgroups were reported. (4) One subgroup, termed germinal center
B-cell-like (GCB) type, expresses genes characteristic of normal
germinal center B cells. A second group, designated activated
B-cell-like type, expresses genes characteristic of activated peripheral
blood B cells and plasma cells. A third group, type C, has neither a GCB
nor an activated B-cell-like signature. These authors have further shown
that these signatures have prognostic meaning. Patients with a GCB tumor
have a relatively good prognosis following chemotherapy. In contrast,
patients with an activated B-cell-like or a type C DLBCL have a similar,
poorer prognosis and, thus, can be grouped as non-GCB.
Although gene expression profiling studies analyze thousands of genes, it seems highly likely that DLBCL subgroups can be distinguished with a smaller panel of genes. Rosenwald and Staudt (8) suggested that as few as 13 to 17 genes can be used to identify these prognostically different subgroups. Lossos and colleagues (9) have recommended a panel of 6 genes. Nevertheless, gene expression profiling technology is not currently available for routine clinical use in many institutions. Others have suggested that selected markers assessed by routine immunohistochemistry can be used as a surrogate for gene expression profiling. (6-10) A number of algorithms have been proposed, but the most widely used is that suggested by Hans and colleagues. (10) In this system, a GCB tumor is [CD10.sup.+] or [CD10.sup.-]/[BCL6.sup.+]/[MUM1.sup.-], and a non-GCB tumor is [CD10.sup.-]/ [BCL6.sup.-] or [CD10.sup.-]/[BCL6.sup.+]/[MUM1.sup.+]. In their study, the immunohistochemical (IHC) profile correctly predicted the gene expression profile in approximately 75% of cases. In Western countries approximately 40% to 60% of DLBCL cases are the GCB type. (7,10,11) There is considerable variation in the incidence rates of non-Hodgkin lymphomas worldwide. (12,13) For example, follicular lymphoma is more common in North America and Western Europe than in most Asian countries. (13-15) The frequency of t(14;18)(q32;q21)/BCL2-IGH is significantly higher in follicular lymphomas arising in the United States than in Asian populations. (16) These findings suggest that follicular lymphoma may be a heterogeneous disease because of geographic factors and encompasses entities with distinct molecular pathogenesis, in particular, the BCL2 rearrangement status and different etiologic or genetic factors and, potentially, distinct clinical manifestations and natural history. Similar geographic differences are likely to exist for other types of lymphomas.
In this study, we profiled 63 cases of DLBCL from northern China to compare with what has been reported in the literature for DLBCL in the United States or other Western countries. In each case, we assessed the cytologic features and used the Hans et al (10) IHC system to classify these cases into GCB and non-GCB types. We also assessed a number of other IHC markers and used fluorescence in situ hybridization (FISH) to assess BCL6, BCL2, and MYC rearrangements. To our knowledge, this is one of the largest studies on DLBCL from China.
MATERIALS AND METHODS
To construct tissue microarrays, 4 representative 0.6-mm cores of each tumor were selected from the formalin-fixed, paraffin-embedded tissue blocks and were relocated using a tissue arrayer (Beecher Instruments, Silver Spring, Maryland). Tissue sections of 5 mm thickness were then cut from the arrays using a standard microtome for immunohistochemistry and FISH.
Immunohistochemical analysis was performed using formalin-fixed, paraffin-embedded tissue sections, an avidin-biotinperoxidase complex method, and an automated immunostainer (Ventana-Biotech, Tucson, Arizona) as described previously. (17) All tissue sections underwent heat-induced antigen retrieval. The antibodies were specific for CD5 (Labvision/Neomarker, Montreal, Quebec, Canada), CD10 (Novocastra/Vision Biosystem, Benton Lane, Newcastle upon Tyne, United Kingdom), CD20 (Dako, Carpinteria, California), CD30 (Dako), CD138 (Serotec, Raleigh, North Carolina), PAX5 (Transduction Labs, San Diego, California), BCL2 (Novocastra/Vision Biosystem), BCL6 (Dako), MUM1 (Santa Cruz Biotechnology, Santa Cruz, California), ALK (Dako), MIB-1 (Ki-67, Dako), p53 (Dako), and p63 (clone 4A4, Santa Cruz Biotechnology). Clone 4A4 recognizes both the transactivating and truncated p63 isoforms.
The IHC results for CD10, BCL2, BCL6 and MUM1 were designated as positive or negative according to the criteria defined by Hans et al, (10) with a cutoff level of 30%. Results of IHC studies for other antibodies were semiquantitatively scored as negative (-, <5% cells positive), weakly positive ([1.sup.+], 5% to <25% cells positive), moderately positive ([2.sup.+], 25%275% cells positive), or strongly positive ([3.sup.+], >75%-100% cells positive). The percentage of [Ki-67.sup.+] cells was estimated to the nearest 10% (ie, 10%, 20%, 30%, etc). Only nuclear staining was regarded as positive for Ki-67, PAX5, BCL2, p53, and p63. For all cases, both hematoxylin-eosin-stained and immunohistochemical slides were reviewed independently by 2 pathologists. Discordant interpretations were resolved by review at a multiheaded microscope.
Using the algorithm of Hans et al, (10) cases were designated as GCB if CD10 was positive, or as non-GCB if both CD10 and BCL6 were negative. If CD10 was negative and BCL6 was positive, the expression of MUM1 determined the subclassification: if MUM1 was negative, the case was assigned to the GCB subgroup; if MUM1 was positive, the case was assigned to the non-GCB subgroup.
In Situ Hybridization
In situ hybridization analysis for Epstein-Barr virus-encoded small RNA (EBER) was performed using formalin-fixed, paraffin-embedded tissue sections, a fluorescein-labeled peptide nucleic acid probe, and the Novocastra in situ hybridization kit (NCL-EBV-K) according to the manufacturer's instructions, with the appropriate positive and negative control samples.
Fluorescence In Situ Hybridization
Interphase FISH analysis was performed on tissue microarrays sections using dual-color, break-apart probes specific for BCL2, BCL6, and MYC (Abbott Laboratories, Des Plaines, Illinois) as previously reported. (18) Briefly, formalin-fixed, paraffin-embedded tissue microarrays sections were deparaffinized using the Paraffin Pretreatment Kit II (Abbott) according to the manufacturer's instructions. Following hybridization with the above FISH probes, the slides were analyzed using a Zeiss Axiophot fluorescent microscope including single- and triple-band pass filters (Welwyn, Garden City, Herts, United Kingdom). One hundred intact, nonoverlapping nuclei were assessed by 2 independent investigators (200 total), and the percentages of positive nuclei were averaged. The positive cutoff value used was 8% for all FISH probes. Digital images were captured by a Power Macintosh G3 System and MacProbe version 4.4 (Applied Imaging, San Jose, California).
The Fisher exact test was used to compare differences between various subgroups. The results were considered statistically significant when P < .05.
The study group included 38 men and 25 women with a median age of 57 years (range, 12-87 years). The series included 33 nodal (52%) and 30 extranodal (48%) DLBCLs. The distribution of extranodal sites was stomach (n = 9, 30%), small intestine (n = 6, 20%), brain (n = 4, 13%), colon (n = 3, 10%), tonsil (n = 3, 10%), thyroid gland (n = 2, 7%), spleen (n = 1, 3%), breast (n = 1, 3%), and testis (n = 1, 3%).
Histologic sections of all 63 cases showed diffuse infiltration by large lymphoid cells. According to the World Health Organization classification, (1) these cases were subclassified as follows: (1) centroblastic (n = 50, 79%), composed of medium-sized to large lymphoid cells with round to oval, vesicular nuclei, fine chromatin, 2 to 4 membrane-bound nucleoli, and scant amphophilic cytoplasm (Figure 1, A); (2) immunoblastic (n = 4, 6%), in which greater than 90% of the neoplastic cells were immunoblasts with prominent, centrally located nucleoli and appreciable basophilic cytoplasm (Figure 1, B); (3) anaplastic (n = 3, 5%), characterized by very large round, oval, or polygonal cells with bizarre pleomorphic nuclei, some of which resembled Reed-Sternberg cells (Figure 1, C). In addition, 6 cases (10%) of DLBCL in this study were large-cell transformation of extranodal (gastrointestinal tract) marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). These neoplasms were characterized by the presence of sheets of large cells with irregular nuclear contours and abundant eosinophilic cytoplasm in a background of low-grade MALT lymphoma (Figure 1, D). The distribution of these variants is summarized in Table 1.
[FIGURE 1 OMITTED]
Among the 33 cases of nodal DLBCL, 29 (88%) were centroblastic, 3 (9%) were immunoblastic, and 1 (3%) was anaplastic. Among the 30 cases of extranodal DLBCL, 21 (70%) were centroblastic, 2 (7%) were anaplastic, and 1 (3%) was immunoblastic. The other 6 (20%) cases were associated with MALT lymphoma. A starry sky pattern was observed in 4 DLBCLs (6%); all were centroblastic. Marked apoptosis or necrosis was noted in 4 cases (6%), and prominent sclerosis was identified in 2 cases (3%).
All cases showed moderate ([2.sup.+]) to strong ([3.sup.+]) expression of CD20 and PAX5 and were negative for CD3, supporting B-cell lineage. Three cases (5%) of anaplastic DLBCL demonstrated moderate to strong positivity for CD30. CD5 was positive in 3 centroblastic cases (5%). Scattered CD138-immunoreactive cells were seen in 1 (2%) centroblastic tumor. All cases were negative for ALK.
The results of IHC staining for CD10, BCL2, BCL6, and MUM1 are summarized in Table 2. MUM1 was expressed in 32 (51%), BCL6 in 22 (35%), and CD10 in 19 (30%) of the cases. Based on the results of IHC studies for the above 3 markers, the cases were subclassified into 2 groups: GCB and non-GCB. Twenty-one (33%) were GCB, and 42 (67%) were non-GCB. Among the 21 GCB cases, 10 (48%) expressed both CD10 and BCL6 ([CD10.sup.+]/[BCL6.sup.+]/[MUM1.sup.+], n = 6; or [CD10.sup.+]/[BCL6.sup.+]/[MUM1.sup.-], n = 4); 9 (43%) expressed CD10 alone ([CD10.sup.+]/[BCL6.sup.-]/[MUM1.sup.-]); and 2 (10%) expressed BCL6 alone ([CD10.sup.-]/[BCL6.sup.+]/[MUM1.sup.-]). Among the 42 non-GCB cases, 16 (38%) expressed MUM1 alone ([CD10.sup.-]/[BCL6.sup.-]/[MUM1.sup.+]), 16 (38%) were negative for all 3 markers ([CD10.sup.-]/[BCL6.sup.-]/[MUM1.sup.-]), and 10 (24%) expressed both MUM1 and BCL6 ([CD10.sup.-]/[BCL6.sup.+]/ [MUM1.sup.+]). MUM1 was expressed more often in nodal (22 of 33; 67%) than in extranodal (10 of 30; 30%; P = .01) DLBCLs. No significant differences in the expression of BCL6 or CD10 between nodal and extranodal cases were observed (Table 2).
[FIGURE 2 OMITTED]
BCL2 expression was observed in 30 of 63 cases (48%), of which, 25 (60%) were non-GCB and 5 (24%) were GCB (P = .01). Twenty-one of 33 nodal cases (64%) were positive, whereas 9 of 30 of the extranodal cases (30%) were positive (P = .01).
Staining for p53 and p63 was predominantly nuclear with occasional cytoplasmic staining. Thirty DLBCL cases (48%) were positive for p53: 17 (27%) showed weak staining, 8 (13%) had moderate staining, and 5 (8%) had strong staining. Thirty-six DLBCL cases (57%) were positive for p63: 15 (24%) showed weak staining, 18 (29%) had moderate staining, and 3 (5%) had strong staining (Figure 2, A through D). Expression of both p53 and p63 was observed in 21 DLBCL cases (33%), including 10 GCB and 11 non-GCB types. Ki-67 showed a low (<30%), moderate (30%-70%), or high (>70%) proliferation index in 22 (35%), 34 (54%), and 7 (11%) of cases, respectively (Table 3).
In Situ Hybridization for EBER
Epstein-Barr virus-encoded small RNA (EBER) was detected in 3 cases (5%) of DLBCL: 2 were nodal non-GCB and 1 was an extranodal GCB type. In 2 of these tumors, most cells were positive for EBER. In 1 case, a nodal non-GCB DLBCL, only 5% of tumor cells were positive.
Fluorescence In Situ Hybridization for MYC, BCL2, and BCL6
Fluorescence in situ hybridization analysis revealed BCL6 rearrangement in 11 of 32 cases (34%) of DLBCL (range of signals, 12%-55%; median, 46%). MYC was rearranged in 8 of 27 cases (30%; range of signals, 8%-64%; median, 27%), and BCL2 was rearranged in 11 of 50 cases (22%; range of signals, 8%-56%; median, 11%) (Table 4; Figure 3). No significant difference was noted in BCL6, MYC, or BCL2 rearrangements between GCB and nonGCB tumors or between nodal and extranodal DLBCL. Three cases (2 non-GCB, 1 GCB) were positive for both BCL2 and BCL6 rearrangements. One GCB case of DLBCL was positive for both MYC and BCL2 rearrangements.
We studied the morphologic features, immunophenotypic profile, and FISH results in 63 cases of DLBCL from northern China. Using the same algorithm proposed by Hans et al, (10) 21 tumors (33%) were of GCB and 42 (67%) were of non-GCB type. The percentage of DLBCL cases with a GCB immunophenotype in our series from China is somewhat less than that reported in Western countries. In various studies, the percentage of cases of GCB type has ranged from 40% to 60%. (7,10,11) Similar to our results, 2 groups from Japan reported that the proportion of GCB DLBCL ranged from 32% to 39%. (19,20) These results in DLBCL from China are in keeping with the results in Japanese patients. As many cases of DLBCL may be preceded by, or are related to, follicular lymphoma, our results are also in parallel with the lower frequency of follicular lymphoma in Asian countries. (21)
The frequency of CD10 (30%) and MUM1 (51%) expression in this study fell within ranges reported by others of 25% to 50% and 47% to 54%, respectively. (10,11) However, only 35% of lymphomas in our series expressed BCL6, which is less than the lower end of the reported range of 57% to 75% from Western countries. (10,22) Furthermore, only 34% of cases assessed showed BCL6 gene rearrangement, which is also lower than the reported frequency of 45%. (2) BCL6 is a zinc-finger protein that acts as a transcriptional repressor. It is expressed in germinal center B cells and in a subset of [CD4.sup.+] T cells (23) and plays a critical role in germinal center formation. (24) We do not have an explanation for the lower frequency of BCL6 rearrangement and expression in cases of Chinese DLBCL, except to implicate geographic and genetic factors. Because BCL6 gene abnormalities are common in DLBCL seen in Western countries, the fewer BCL6 cases of rearrangement and expression in Chinese DLBCL may suggest that therapies targeted at the BCL6 pathway may be less effective in Chinese patients.
No significant differences in BCL2 expression between GCB and non-GCB types of DLBCL have been reported in previous studies (50%-67% in GCB cases versus 45%-62% in non-GCB cases). (6,25) Although the overall frequency of BCL2 expression in our study (48%) fell within the reported range, BCL2 was expressed significantly more often in non-GCB (60%) than in GCB cases (24%, P = .01) and in significantly more nodal (64%) versus extranodal DLBCL (30%, P = .01). However, no significant difference in the frequency of BCL2 gene rearrangement was observed between GCB and non-GCB cases. It has been suggested that mechanisms other than t(14;18)(q32;q21), such as 18q21 amplification (where BCL2 gene resides) or activation of the nuclear factor-[kappa]B pathway, may be primarily responsible for the upregulation of BCL2 expression in the non-GCB subgroup. (26) The prognostic role of BCL2 overexpression in DLBCL is still controversial. Some groups using multivariate analysis have suggested that BCL2 is the most predictive prognostic marker. (27) Other groups have reported that there is no significant correlation between BCL2 expression and overall survival within the GCB subgroup but that BCL2 expression has a significant adverse effect on overall survival within the non-GCB subgroup. (10,26) Unfortunately, most of the cases in this study group were received in consultation from other institutions, and we do not have adequate clinical or survival data to comment on prognosis.
[FIGURE 3 OMITTED]
Rearrangement of MYC at band 8q24, a characteristic event in Burkitt lymphoma, has been reported in approximately 15% of DLBCL cases in Western countries and has been associated with a poor clinical outcome. (2) MYC plays a role in the pathogenesis of DLBCL by promoting cell-cycle progression and tumor proliferation. In this study, the frequency of MYC rearrangement was 30%, double of that reported in Western populations. These are additional data to implicate geographic or genetic differences between DLBCL in northern China and in Western countries. Our study group (n = 27) is small, however, and this observation needs to be confirmed in larger studies. In our group of Chinese patients with DLBCL, there was no significant difference in the frequency of MYC rearrangement between the GCB and non-GCB subgroups.
Inactivation of the p53 tumor suppressor gene is recognized as a key step in the development of 50% to 60% of human malignancies. p53 gene mutations have been observed in 20% to 45% of patients with DLBCL. (6,28,29) p63, a member of the p53 tumor suppressor gene family, with structural homology to p53, is essential for healthy embryonic development and is thought to behave as an oncogenic molecule when overexpressed. (28,30) In contrast with p53, which is immunohistochemically undetectable or very weakly expressed in healthy tissues and which reaches detectable levels only after mutational inactivation or genotoxic stress, p63 exhibits a consistent expression pattern in certain normal tissues. (30) p63 is expressed at high levels in stratified epithelial cells and in their corresponding tumors, and p63 is also expressed at low to moderate levels in a subset of germinal center cells in lymph nodes. (30) Only small amounts of data are available on p63 expression in malignant lymphomas. One study from the United States reported that p63 was expressed in 32% of DLBCLs. (29) Other groups from Brazil and Korea found that p63 was expressed in 15% and 53% of DLBCLs, respectively. (28,31) The Korean group further noted that p53 and p63 were both expressed in 30% of cases and that patients with p63 overexpression showed significantly poorer rates of survival. (28) They speculated that p63 could act as an oncogene by inhibiting p53 function in DLBCL. Similar to what was reported from Korea, we found that 48% of the patients in our case study had p53 overexpression, 57% had p63 overexpression, and 33% expressed both p53 and p63. The higher expression and co-expression of these 2 proteins in Chinese patients with DLBCL may suggest a role in pathogenesis.
In conclusion, we have identified differences between cases of DLBCL in northern China and that reported for DLBCL cases in Western countries. In particular, the frequency of the GCB type is less, BCL6 is less frequently expressed or is rearranged, and MYC is more often rearranged in patients with DLBCL in China compared with Western countries. The relatively high frequency of overexpression of p53 and p63 in this case series, compared with DLBCL cases in other countries, is also of interest, raising the possibility of a role for this pathway in the pathogenesis of DLBCL from China.
This study was supported by a scholarship from the Chinese Scholarship Council.
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Ting Li, MD; L. Jeffrey Medeiros, MD; Pei Lin, MD; Hongfang Yin, MD, PhD; Martin Littlejohn, BA; Whan Im, BA; P. Alan Lennon, PhD; Peter Hu, PhD; Jeffrey L. Jorgensen, MD, PhD; Mei Liang, MD, PhD; Hua Guo, MD; C. Cameron Yin, MD, PhD
Accepted for publication July 8, 2009.
From the Department of Pathology, First Hospital of Peking University, Beijing, China (Drs Li, H. Yin, and Guo); and the Department of Hematopathology (Drs Medeiros, Lin, Jorgensen, Liang, and C. C. Yin) and the Molecular Genetic Technology Program (Messrs Littlejohn and Im and Drs Lennon and Hu), University of Texas M. D. Anderson Cancer Center, Houston.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: C. Cameron Yin, MD, PhD, Department of Hematopathology, Unit 72, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030 (e-mail: email@example.com).
Table 1. Morphologic Features of 63 Cases of Diffuse Large B-Cell Lymphoma From China CB, No. IB, No. Anaplastic, MALT-L, Total, Type (%) (%) No. (%) No. (%) No. (%) Nodal 29 (46) 3 (5) 1 (2) 0 (0) 33 (52) Extranodal 21 (33) 1 (2) 2 (3) 6 (10) 30 (48) Total 50 (79) 4 (6) 3 (5) 6 (10) 63 (100) Abbreviations: CB, centroblastic; IB, immunoblastic; MALT-L, diffuse large B-cell lymphoma associated with low-grade marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue. Table 2. Immunophenotypic Features of 63 Cases of Diffuse Large B-Cell Lymphoma From China GCB, Non-GCB, Total, No. n = 21, n = 42, Antibody (%) No. (%) No. (%) P CD10 19 (30) 19 (91) 0 (0) <.001 BCL6 22 (35) 12 (57) 10 (24) .01 MUM1 32 (51) 6 (29) 26 (62) .01 BCL2 30 (48) 5 (24) 25 (60) .01 Nodal, Extranodal, n = 33, n = 30, Antibody No. (%) No. (%) P CD10 10 (30) 9 (30) .96 BCL6 14 (42) 8 (27) .22 MUM1 22 (67) 10 (33) .01 BCL2 21 (64) 9 (30) .01 Abbreviation: GCB, germinal center B-cell-like type. Table 3. Staining Pattern of p53, p63, and Ki-67 p53, No. (%) Type No. - 1+ 2+ 3+ GCB 21 9 (14) 9 (14) 2 (3) 1 (2) Non-GCB 42 24 (38) 8 (13) 6 (10) 4 (6) Total 63 33 (52) 17 (27) 8 (13) 5 (8) Nodal 33 18 (29) 7 (11) 5 (8) 3 (5) Extranodal 30 15 (24) 10 (16) 3 (5) 2 (3) Total 63 33 (52) 17 (27) 8 (13) 5 (8) p63, No. (%) Type - 1+ 2+ 3+ GCB 8 (13) 7 (11) 5 (8) 1 (2) Non-GCB 19 (30) 8 (13) 13 (21) 2 (3) Total 27 (43) 15 (24) 18 (29) 3 (5) Nodal 17 (27) 5 (8) 11 (17) 0 (0) Extranodal 10 (16) 10 (16) 7 (11) 3 (5) Total 27 (43) 15 (24) 18 (29) 3 (5) Ki-67, No. (%) Type - 1+ 2+ 3+ GCB 2 (3) 3 (5) 10 (16) 6 (10) Non-GCB 5 (8) 12 (19) 24 (38) 1 (2) Total 7 (11) 15 (24) 34 (54) 7 (11) Nodal 2 (3) 9 (14) 19 (30) 3 (5) Extranodal 5 (8) 6 (10) 15 (24) 4 (6) Total 7 (11) 15 (24) 34 (54) 7 (11) Abbreviations: 2, negative; 1+, weakly positive; 2+, moderately positive; 3+, strongly positive; GCB, germinal center B-cell-like type. Table 4. Fluorescence In Situ Hybridization Results of MYC, BCL2, and BCL6 Rearrangement Gene Total GCB Non-GCB P MYC 8/27 (30) 1/8 (13) 7/19 (37) .36 BCL2 11/50 (22) 5/19 (26) 6/31 (19) .73 BCL6 11/32 (34) 5/14 (36) 6/18 (33) >.99 Gene Nodal Extranodal P MYC 3/13 (23) 5/14 (36) .68 BCL2 4/22 (18) 7/28 (25) .73 BCL6 5/14 (36) 6/18 (33) >.99 Abbreviation: GCB, germinal center B-cell-like type.
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