Immunohistochemical profile and fluorescence in situ hybridization analysis of diffuse large B-cell lymphoma in Northern China.
Abstract: * 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)
Article Type: Report
Subject: Immunohistochemistry (Usage)
In situ hybridization (Usage)
Non-Hodgkin's lymphomas (Development and progression)
Non-Hodgkin's lymphomas (Diagnosis)
Non-Hodgkin's lymphomas (Care and treatment)
Authors: Li, Ting
Medeiros, L. Jeffrey
Lin, Pei
Yin, Hongfang
Littlejohn, Martin
Im, Whan
Lennon, P. Alan
Hu, Peter
Jorgensen, Jeffrey L.
Liang, Mei
Guo, Hua
Yin, C. Cameron
Pub Date: 05/01/2010
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
Accession Number: 230246563
Full Text: 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

Case Selection

The study was conducted according to an institutional review board-approved laboratory protocol and followed the provisions of the Helsinki accord. The files of the Department of Pathology, the First Hospital of Peking University (Beijing, China) from January 2000 through December 2005 were searched for cases of DLBCL. Seventy-two cases were identified. The diagnosis of DLBCL was based on histologic and immunophenotypic findings using the criteria of the World Health Organization classification. (1) Cases with insufficient paraffin-embedded blocks were excluded, leaving a study group of 63 cases. Clinical information was obtained by review of the hospital-based medical records. Hematoxylin-eosin-stained slides, routinely prepared from formalin-fixed, paraffin-embedded tissue sections, were reviewed.

Tissue Microarray

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

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).

Statistical Analysis

The Fisher exact test was used to compare differences between various subgroups. The results were considered statistically significant when P < .05.

RESULTS

Clinical Findings

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 Findings

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%).

Immunophenotypic Findings

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.

COMMENT

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.

References

(1.) Stein H, Chan JKC, Warnke RA, et al. Diffuse large B-cell lymphoma, not otherwise specified. In: Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France: IARC Press; 2008:233-237. WHO Classification of Tumours; vol 2.

(2.) Tibiletti MG, Martin V, Bernasconi B, et al. BCL2, BCL6, MYC, MALT1, and BCL10 rearrangements in nodal diffuse large B-cell lymphomas: a multicenter evaluation of a new set of fluorescent in situ hybridization probes and correlation with clinical outcome. Hum Pathol. 2009;40(5):645-652.

(3.) Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403(6769):503-511.

(4.) Rosenwald A, Wright G, Chan WC, 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):1937-1947.

(5.) Shipp MA, Ross KN, Tamayo P, et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nat Med. 2002;8(1):68-74.

(6.) Colomo L, Lopez-Guillermo A, Perales M, et al. Clinical impact of the differentiation profile assessed by immunophenotyping in patients with diffuse large B-cell lymphoma. Blood. 2003;101(1):78-84.

(7.) Wright G, Tan B, Rosenwald A, Hurt EH, Wiestner A, Staudt LM. A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B-cell lymphoma. Proc Natl Acad Sci U S A. 2003;100(17):9991-9996.

(8.) Rosenwald A, Staudt LM. Gene expression profiling of diffuse large B-cell lymphoma. Leuk Lymphoma. 2003;44(suppl 3):S41-S47.

(9.) Lossos IS, Jones CD, Warnke R, et al. Expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma. Blood. 2001;98(4):945-591.

(10.) Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103(1):275-282.

(11.) Van Imhoff GW, Boerma EJG, van der Holt B, et al. Prognostic impact of germinal center-associated proteins and chromosomal breakpoints in poor-risk diffuse large B-cell lymphoma. J Clin Oncol. 2006;24(25):4135-4142.

(12.) Berard CW, Greene MH, Jaffe ES, Magrath I, Ziegler J. NIH conference: a multidisciplinary approach to non-Hodgkin's lymphoma. Ann Intern Med. 1981; 94(2):218-235.

(13.) Anderson JR, Armitage JO, Weisenburger DD. Epidemiology of the non-Hodgkin's lymphomas: distributions of the major subtypes differ by geographic locations. Non-Hodgkin's Lymphoma Classification Project. Ann Oncol. 1998; 9(7):717-720.

(14.) Ho FC, Todd D, Loke SL, Ng RP, Khoo RK. Clinico-pathological features of malignant lymphomas in 294 Hong Kong Chinese patients: retrospective study covering an eight-year period. Int J Cancer. 1984;34(2):143-148.

(15.) Harrington DS, Ye YL, Weisenburger DD, et al. Malignant lymphoma in Nebraska and Guangzhou, China: a comparative study. Hum Pathol. 1987;18(9): 924-928.

(16.) Biagi JJ, Seymour JF. Insights into the molecular pathogenesis of follicular lymphoma arising from analysis of geographic variation. Blood. 2002;99(12): 4265-4275.

(17.) Yin CC, Medeiros LJ, Cromwell CC, et al. Sequence analysis proves clonal identity in five patients with typical and blastoid mantle cell lymphoma. Mod Pathol. 2007;20(1):1-7.

(18.) Yin CC, Medeiros LJ, Glassman AB, Lin P. t(8;21)(q22;q22) in blast phase of chronic myelogenous leukemia. Am J Clin Pathol. 2004;121(6):836-842.

(19.) Kusumoto S, Kobayashi U, Sekiguchi N, et al. Diffuse large B-cell lymphoma with extra Bcl-2 gene signals detected by FISH analysis is associated with a "non-germinal center phenotype." Am J Surg Pathol. 2005;29(8):10671073.

(20.) Tagawa H, Suguro M, Tsuzuki S, et al. Comparison of genome profiles for identification of distinct subgroups of diffuse large B-cell lymphoma. Blood. 2005;106(5):1770-1777.

(21.) Carreon JD, Morton LM, Devesa SS, et al. Incidence of lymphoid neoplasms by subtype among six Asian ethnic groups in the United States, 1996-2004. Cancer Causes Control. 2008;19(10):1171-1181.

(22.) Pasqualucci L, Bereschenko O, Niu H, et al. Molecular pathogenesis of non-Hodgkin's lymphoma: the role of Bcl-6. Leuk Lymphoma. 2003;44(suppl 3): S5-S12.

(23.) Cattoretti G, Chang CC, Cechova K, et al. BCL-6 protein is expressed in germinal-center B cells. Blood. 1995;86(1):45-53.

(24.) Ye BH, Cattoretti G, Shen Q, et al. The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation. Nat Genet. 1997;16(2): 161-170.

(25.) Barrans SL, Carter I, Owen RG, et al. Germinal center phenotype and bcl-2 expression combined with the international prognostic index improves patient risk stratification in diffuse large B-cell lymphoma. Blood. 2002;99(4):1136-1143.

(26.) Iqbal J, Neppalli VT, Wright G, et al. BCL2 expression is a prognostic marker for the activated B-cell-like type of diffuse large B-cell lymphoma. J Clin Oncol. 2006;24(6):961-968.

(27.) Muris JJ, Meijer CJ, vos W, et al. Immunohistochemical profiling based on Bcl-2, CD10 and MUM1 expression improves risk stratification in patients with primary nodal diffuse large B cell lymphoma. J Pathol. 2006;208(5):714-723.

(28.) Park CK, Oh YH. Expression of p63 in reactive hyperplasias and malignant lymphomas. J Korean Med Sci. 2005;20(5):752-758.

(29.) Hedvat CV, Teruya-Feldstein J, Puig P, et al. Expression of p63 in diffuse large B cell lymphoma. Appl Immunohistochem Mol Morphol. 2005;13(3):237242.

(30.) Di Como CJ, Urist MJ, Babayan I, et al. p63 expression profiles in human normal and tumor tissues. Clin Cancer Res. 2002;8(2):494-501.

(31.) Hallack Neto AE, Siqueira SAC, Dulley FL, Ruiz MA, Chamone DAF, Pereira J. p63 protein expression in high risk diffuse large B-cell lymphoma. J Clin Pathol. 2009;62(1):77-79.

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: cyin@mdanderson.org).
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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