Diffuse Large B-Cell Lymphoma

Hematopathology / CD10 IN DIFFUSE LARGE B-CELL LYMPHOMA

Clinicopathologic Analysis of CD10+ and CD10– Diffuse Large B-Cell Lymphoma Identification of a High-Risk Subset With Coexpression of CD10 and bcl-2 Yin Xu, MD, PhD, Robert W. McKenna, MD, Kyle H. Molberg, MD, and Steven H. Kroft, MD Key Words: Lymphoma; Diffuse large B-cell lymphoma; CD10; bcl-2

Abstract We analyzed 53 cases of diffuse large B-cell lymphoma (DLBCL) to determine whether expression of CD10 is a relevant biologic parameter. Tumor morphologic features were assessed semiquantitatively. Bcl-2 protein expression was studied by immunohistochemical analysis. The presence or absence of CD10 by flow cytometry was correlated with clinical and pathologic characteristics. CD10+ (23 cases) and CD10– (30 cases) DLBCLs were indistinguishable based on age, sex, extranodal presentation, B symptoms, clinical stage, morphologic features, or bcl-2 expression. However, cases with a CD10+ phenotype showed a significantly lower rate of complete remission. Cases expressing bcl-2 showed trends toward a lower rate of complete remission and poorer overall survival. Examination of CD10 and bcl2 interaction revealed that the prognostic effects for both of these antigens were due to a subset of CD10+ bcl-2–positive cases. Compared with cases expressing one or neither of these markers, patients with dualpositive tumors had a poorer complete response rate to initial therapy and strikingly worse overall survival. While CD10+ and CD10– DLBCLs are similar with regard to a variety of clinical and pathologic features, CD10 and bcl-2 coexpressing tumors are an extremely high-risk subset based on response to therapy and overall survival.

© American Society of Clinical Pathologists

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) in Western countries.1,2 DLBCL is a heterogeneous group from morphologic, immunophenotypic, genetic, and clinical standpoints. However, except for a few well-defined clinicopathologic entities, most efforts to dissect distinct subgroups within the DLBCL category have met with little success.3-8 CD10, a 100-kd type II cell surface glycoprotein, is expressed on a wide variety of cell lineages, including normal and neoplastic hematopoietic cells.9-12 The antigen is expressed on normal germinal center cells,13-15 as well as on the vast majority of follicular and Burkitt lymphomas,16-18 and on a high percentage of acute lymphoblastic leukemias.19 Among CD5– small B-cell lymphomas, CD10 expression distinguishes tumors of follicle center and marginal zone origin. In addition, CD10 is seen on a subset of DLBCLs. CD10 expression in DLBCL has been shown in a single study to correlate with bcl-2 gene rearrangement,20 but the latter has generally not been found to correlate with prognosis.21-24 In contrast, bcl-2 protein expression in DLBCL seems to affect response to therapy and survival.21-26 Expression of bcl-2 protein presumably prevents programmed cell death and, thus, may have an important role in survival of lymphoma cells.27-31 The present study was performed to investigate the biologic relevance of CD10 in DLBCL based on a variety of clinical and pathologic features and to examine the relationship between both CD10 and bcl-2 expression and clinical outcome. Am J Clin Pathol 2001;116:183-190

183

Xu et al / CD10 IN DIFFUSE LARGE B-CELL LYMPHOMA

Materials and Methods Patients All cases of CD5– DLBCL were identified in the clinical flow cytometry database at the University of Texas Southwestern Medical Center, Dallas, from April 1994 to July 1999. Among 74 identified cases with histologic material available for review, excluded were 15 cases with evidence of previous or coexistent small B-cell lymphoma, 4 HIV-associated cases, and 1 case in which CD10 expression could not be assessed by flow cytometry because of a lack of isotypic control. One case of cutaneous DLBCL also was excluded based on the indolent clinical behavior of this entity compared with DLBCLs in other sites. 32 The remaining 53 cases demonstrated morphologic features consistent with DLBCL. Patient follow-up information was obtained by review of medical records and contact with hospital tumor registries and patients’ physicians. Patients were staged according to the Ann Arbor staging system. Among 40 patients with clinical follow-up, 31 were treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP); 6 received combined CHOP with radiation; 3 with advanced clinical stage received chemotherapy with prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, and methotrexate (ProMace-CytaBom). Complete remission was defined as no clinical evidence of disease. Overall survival was calculated from the date of diagnosis. Flow Cytometry Fresh biopsy tissue was sliced and disaggregated through a mesh smaller than 100 µm, and cells were suspended in 5% newborn calf serum in RPMI 1640 tissue culture medium. Cell counts were performed manually, and 500,000 cells per tube were washed with a phosphate buffered saline (PBS)/0.0455% sodium azide/0.1% bovine serum albumin solution (PAB) and then incubated with a 3or 4-color combination of antibodies. Antibodies against CD2 (55.2), CD3 (SK7), CD4 (SK3), CD5 (L17F12), CD7 (4H9), CD8 (SK1), CD10 (W8E7), CD19 (SJ25C1), CD20 (L27), CD38 (HB7), CD45 (2D1), CD45RO (UCHL-1), and monoclonal kappa (TB28-2) and lambda (I-155-2) immunoglobulins were obtained from Becton Dickinson (San Jose, CA). Antibodies against FMC7 (FMC7), CD23 (B6), and polyclonal immunoglobulins kappa (goat) and lambda (goat) were obtained from Coulter-Immunotech (Hialeah, FL). Anti-CD30 (BerH2) was obtained from DAKO (Carpinteria, CA). These antibodies were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC). CD10 expression was determined using one of the 184

Am J Clin Pathol 2001;116:183-190

following antibody combinations: CD10-FITC/CD38PE/CD20-PCP, or CD10-FITC/CD19-PE/CD20-PCP/CD38APC. Twenty microliters of CD10 antibody were used; this amount was determined by previous titration studies. Specimens were incubated at 2°C to –8°C in the dark for 20 minutes, washed with PAB, and resuspended in 1% paraformaldehyde in PBS. Flow cytometric data were acquired using a 3-color FACScan (26 specimens) or 4-color FACSCalibur (27 specimens) flow cytometry instrument with CELLQuest software (Becton Dickinson). Data analysis was performed using Paint-a-Gate Software (Becton Dickinson). Nonviable cells and debris were excluded based on forward and orthogonal light scatter properties. To assess antigen expression by the tumor population, the same population was identified by scatter characteristics in an isotypic control tube, and a 2% threshold was set on the tumor population. CD10 positivity was assessed as an overt qualitative shift of the tumor cell population (cluster) relative to the same population beyond the isotypic control threshold. For equivocal cases with dim or partial CD10 expression, a cutoff of 10% of tumor cells beyond the isotypic control threshold was considered positive. Histologic and Immunohistochemical Studies Initial diagnostic biopsy specimens were fixed in B-5 fixative and/or 10% neutral buffered formalin, embedded in paraffin, and sectioned at 3 µm. Sections were stained with H&E and evaluated without previous knowledge of results of CD10 phenotype. Tumor morphologic features were assessed for nuclear size (grade I, majority of cells with nuclei approximating those of histiocyte nuclei; grade II, majority of cells with nuclei at least 1.5-2 times the diameter of histiocyte nuclei), pleomorphism (uniform vs pleomorphic), cytoplasm (scant vs moderate/abundant), nuclear irregularity (slightly vs markedly irregular), and prominence of nucleoli (inconspicuous/small vs prominent). Morphologic subtypes are illustrated in ❚Image 1❚. Morphologic scoring was performed at a multiheaded microscope, and consensus was achieved among observers (Y.X., R.W.M., K.H.M., and S.H.K.). Immunohistochemical analysis was performed on the paraffin sections of 48 cases with available blocks with anti–bcl-2 monoclonal antibody (Dako, Glostrup, Denmark) using a TechMate automated immunostainer (Ventana Biotek, Tucson, AZ) and a standard streptavidin-biotin peroxidase detection system. The sections were scored as positive if more than 5% of the tumor cell population exhibited cytoplasmic staining and as strongly positive if more than 50% of tumor cells were stained. Statistical Analysis The categorical variables were analyzed with 2-tailed chi-square tests. Continuous variables were analyzed with © American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE

A

B

C

D

❚Image 1❚ Tumor morphologic features. A, Uniform lymphoma cells with grade I nuclear size, round to slightly irregular nuclei, prominent nucleoli, and scant cytoplasm. B, Uniform lymphoma cells with grade II nuclear size, irregular nuclei, prominent nucleoli, and abundant cytoplasm. C, Uniform lymphoma cells with grade I nuclear size, markedly irregular nuclei, and small nucleoli. D, Pleomorphic lymphoma cells with grade II nuclear size, prominent nucleoli, and moderately abundant cytoplasm. (H&E, ×250)

independent-sample t tests. Survival curves were plotted by the method of Kaplan and Meier and compared by log-rank tests.

Results Patient Characteristics The mean age of the 53 patients with de novo DLBCL was 53 years (range, 3-92 years). There were 23 males and 30 females. Sixteen patients (30%) had B symptoms at the time of the diagnosis. Twelve patients were diagnosed as having clinical stage I disease, 11 as stage II, 6 as stage III, © American Society of Clinical Pathologists

and 16 as stage IV. In 8 cases staging information was not available. Twenty-seven (51%) had a primary extranodal presentation. There were no significant differences in CD10 expression (P = .21), bcl-2 expression (P = .68), CD10/bcl-2 coexpression (P = .32), clinical stage (P = .65), complete remission (P = .65), or overall survival (P = .42) between nodal and extranodal DLBCLs. Comparison of CD10+ and CD10– DLBCL Of the 53 cases, 23 cases (43%) were CD10+ and 30 (57%) were CD10– by flow cytometric immunophenotyping. The clinical characteristics of both groups of patients are compared in ❚Table 1❚. There were no significant Am J Clin Pathol 2001;116:183-190

185


differences in age, sex, extranodal presentation, B symptoms, or clinical stage between CD10+ and CD10– DLBCLs. Morphologic Features Cases in both groups were morphologically heterogeneous. The tumor cells were generally large with vesicular nuclei and variable degrees of nucleolar prominence, cellular pleomorphism, and nuclear irregularity. There were no significant differences in cell size, pleomorphism, cytoplasm, nuclear irregularity, and prominence of nucleoli between CD10+ and CD10– cases by semiquantitative assessment (Table 1). Bcl-2 Expression The expression of bcl-2 was determined by immunohistochemical analysis in 48 cases. Of the 48 cases, 32 (67%) showed expression of bcl-2 protein in tumor cells, and 22 (46%) of 48 exhibited high levels (>50% of tumor cells staining) of bcl-2 protein. The number of bcl-2–positive cases was similar in CD10+ and CD10– groups; the comparison of bcl-2 protein expression by CD10 phenotype is shown in Table 1. ❚Table 1❚ Clinicohistologic Characteristics of Diffuse Large B-Cell Lymphomas by CD10 Expression* CD10+ Mean age (y) Sex Male Female B symptoms Absent Present Clinical stage I-II III-IV Site of primary presentation Nodal Extranodal Nuclear size I II Pleomorphism Uniform Pleomorphic Cytoplasm Scant Moderate/abundant Nuclear irregularity Slightly irregular Markedly irregular Nucleoli Inconspicuous/small Prominent Bcl-2 protein expression Positive (5% or more) Strongly positive (50% or more)

50.3

CD10– 54.9

12/23 (52) 11/23 (48)

11/30 (37) 19/30 (63)

10/18 (56) 8/18 (44)

20/28 (71) 8/28 (29)

9/18 (50) 9/18 (50)

14/27 (52) 13/27 (48)

14/23 (61) 9/23 (39)

13/30 (43) 17/30 (57)

6/23 (26) 17/23 (74)

11/30 (37) 19/30 (63)

P .40 .26 .27 .90 .21

Response to Therapy and Survival Survival data were available for 40 cases with a median follow-up period of 29 months (range, 2-40 months) for the 27 surviving patients. Information related to therapeutic response was available for 39 cases. Of 15 patients with CD10+ tumors, 7 (47%) achieved a complete remission. In contrast, of 24 patients with CD10– tumors, 19 (79%) experienced a complete remission (P = .036) ❚Table 2❚. Patients who did not achieve remission were either dead or had persistent disease at last follow-up. Patients with CD10+ tumors showed a poorer overall survival, but this was not statistically significant (P = .19), as shown in ❚Figure 1❚. Patients with bcl-2–expressing tumors showed a trend toward a lower rate of complete remission (58% vs 85%; P = .09; Table 2). These patients also showed a trend toward a poorer overall survival (P = .12) ❚Figure 2❚. When the interaction of CD10 and bcl-2 expression was explored, patients with dual expression of CD10 and bcl-2 were found to achieve a strikingly lower rate of complete remission (12% vs 81%; P = .0003) and demonstrated a significantly poorer overall survival (P = .007; median survival: 8 months vs not yet reached) compared with those with CD10+bcl-2–negative, CD10–bcl-2–positive, or CD10–bcl-2–negative phenotypes (Table 2) ❚Figure 3❚; the latter 3 groups demonstrated virtually no difference in response to therapy or survival (data not shown). Patients with tumors coexpressing CD10 and bcl-2 showed a trend toward a higher clinical stage (III-IV) (P = .06). There was no significant difference in other clinicohistologic parameters between CD10+bcl-2–positive and other DLBCL groups ❚Table 3❚. It was recognized that patients with stage IV disease were the largest group of patients in the study. Analysis of stage IV disease revealed no association with CD10 (P = .31). However, stage IV cases were more likely to express bcl-2 protein (P = .02) than stages I through III and to

.41 .74 12/23 (52) 11/23 (48)

17/30 (57) 13/30 (43)

12/23 (52) 11/23 (48)

11/30 (37) 19/30 (63)

❚Table 2❚ Comparison of Therapy Response by CD10 and/or bcl-2 Expression*

.26 .53 15/23 (65) 8/23 (35)

17/30 (57) 13/30 (43) .88

11/23 (48) 12/23 (52)

15/30 (50) 15/30 (50)

12/21 (57) 10/21 (48)

20/27 (74) 12/27 (44)

.22 .83

CD10+ CD10– P bcl-2 Positive bcl-2 Negative P CD10+bcl-2 positive CD10+bcl-2 negative/CD10–bcl-2 positive/CD10–bcl-2 negative† P *

*

Data are given as number of affected cases/total number (percentage) unless otherwise indicated.

186

Am J Clin Pathol 2001;116:183-190

†

Complete Remission

Relapse

7/15 (47) 19/24 (79) .036 15/26 (58) 11/13 (85) .09 1/8 (12) 25/31 (81)

0/7 (0) 2/19 (11) 1/15 (7) 1/11 (9) 0/1 (0) 2/25 (8)

.0003

Data are given as number of affected cases/total number (percentage) unless otherwise indicated. Combined because of no difference in treatment response among these 3 groups.


1.0

1.0

0.8

0.8

Cumulative Survival

Cumulative Survival


0.6

0.4

0.2

0.6

0.4

0.2

CD10– CD10+

bcl-2 negative bcl-2 positive

0.0

0.0 0

10

20

30

40

Time (Months)

0

10

20

30

40

Time (Months)

❚Figure 1❚ Overall survival for CD10+ (n = 16) and CD10– (n = 24) diffuse large B-cell lymphomas. P = .19.

❚Figure 2❚ Overall survival for bcl-2–positive (n = 27) and bcl2–negative (n = 13) diffuse large B-cell lymphomas. P = .12.

express both CD10 and bcl-2 (P = .02). The small number of cases precludes meaningful multivariate analysis of the effects of stage and CD10/bcl-2 coexpression on clinical outcome.

lymphoma and high-grade Burkitt-like B-cell lymphoma. With modern treatment regimens, many patients with DLBCL experience a long-term, disease-free remission,2,33,34 but a subset of DLBCLs seems to be more resistant to chemotherapy, and patients who do not achieve complete remission rapidly die. While clinical and laboratory characteristics such as age, tumor stage, serum lactate dehydrogenase level, performance status, and number of extranodal sites as defined by the International Prognostic Index may be used for prognostic stratification,35 they yield little insight into underlying biologic factors. Therefore, efforts to identify variables to dissect DLBCL into distinct biologic categories continue.36-39 CD10, a cell surface molecule used in the classification of several types of NHL, was studied for potential usefulness in the immunophenotypic subgrouping of DLBCLs in the present study. CD5+ DLBCL was excluded based on previous data indicating that CD5+ DLBCLs may behave more aggressively and thus may represent a distinct clinicopathologic entity.36,39 Based on analysis of 53 CD5– de novo DLBCL cases, we determined that CD10+ and CD10– DLBCLs were indistinguishable on the basis of presenting clinical features, morphologic features, and expression of bcl2 protein. However, cases with CD10 expression showed a significantly poorer response to therapy, although no significant impact on overall survival was noted in our cohort. There has been little information on the clinical relevance of CD10 expression in DLBCL reported in the literature. A study of 28 cases presented in abstract form demonstrated a shortened overall survival duration in patients with CD10+ DLBCL.38 In contrast, another study reported that CD5–CD10– (33 cases) and CD5–CD10+ (19 cases) DLBCLs did not differ in overall survival.33 However, a lower portion of CD10+ cases was present in the latter study, suggesting the possibility of methodologic differences in the

Discussion DLBCL is a clinically, morphologically, immunophenotypically, and genetically heterogeneous group of NHLs. Although these tumors have been morphologically subclassified in the past, the subclassifications yielded low reproducibility, and the morphologic variants have not shown consistent differences in clinical behavior. Therefore, the Revised European-American Lymphoma classification1 proposed the consolidation of the tumors into a single category with the exception of primary mediastinal large B-cell

Cumulative Survival

1.0

0.8

0.6

0.4

0.2 CD10+bcl-2–/CD10–bcl-2+/CD10–bcl-2– CD10+bcl-2+

0.0 0

10

20

30

40

Time (Months)

❚Figure 3❚ Overall survival for diffuse large B-cell lymphomas (DLBCLs) coexpressing CD10 and bcl-2 (n = 9) and DLBCLs expressing one or neither of these markers (n = 31). P = .007.


Am J Clin Pathol 2001;116:183-190

187


❚Table 3❚ Clinicohistologic Characteristics of Diffuse Large B-Cell Lymphomas by CD10 and bcl-2 Expression* CD10+bcl-2 Positive Mean age (y) Sex Male Female B symptoms Absent Present Clinical stage I-II III-IV Site of primary presentation Nodal Extranodal Nuclear size I II Pleomorphism Uniform Pleomorphic Cytoplasm Scant Moderate/abundant Nuclear irregularity Slightly irregular Markedly irregular Nucleoli Inconspicuous/small Prominent *

CD10+bcl-2 Negative/CD10–bcl-2 Positive/CD10–bcl-2 Negative

58.4

51.6

5/12 (42) 7/12 (58)

16/36 (44) 20/36 (56)

6/11 (55) 5/11 (45)

23/33 (70) 10/33 (30)

3/11 (27) 8/11 (73)

20/33 (61) 13/33 (39)

8/12 (67) 4/12 (33)

18/36 (50) 18/36 (50)

2/12 (17) 10/12 (83)

12/36 (33) 24/36 (67)

6/12 (50) 6/12 (50)

20/36 (56) 16/36 (44)

5/12 (42) 7/12 (58)

16/36 (44) 20/36 (56)

7/12 (58) 5/12 (42)

21/36 (58) 15/36 (42)

6/12 (50) 6/12 (50)

16/36 (44) 20/36 (56)

P .38 .87 .36 .06 .32 .27 .74 .87 1.00 .74

Data are given as number of affected cases/total number (percentage) unless otherwise indicated.

detection of CD10 expression. While previous studies have suggested good concordance between CD10 by paraffinsection immunohistochemical and flow cytometric analyses,40,41 Xu et al42 found that paraffin-section immunohistochemical analysis for CD10 has a lower sensitivity (75%) compared with multiparameter flow cytometry with cluster analysis in DLBCL. The effects of bcl-2 on clinical course have been widely studied within many lymphoma types in the past. The incidence of bcl-2 expression in DLBCL varies between 45% and 55% and overexpression of bcl-2 protein in DLBCL is associated with a decreased overall survival.21-26 It has been demonstrated that bcl-2 overexpression is not always dependent on bcl-2 gene rearrangement, but also is associated with a number of different mechanisms.43,44 Consistent with the findings of previous studies, we demonstrated that 60% of cases expressed bcl-2 protein, with a high level of bcl-2 protein present in 46% of cases. There were trends toward decreased response to therapy and overall survival in the bcl2–expressing DLBCLs (P = .09 and P = .12, respectively). The lack of statistical significance for this relation may be explained by the small number of cases and limited follow-up period in our study. The bcl-2 expression in these DLBCL cases was not related to the CD10 expression. However, when the interaction of CD10 and bcl-2 was examined, it was seen that a subset of CD10+bcl-2–positive cases demonstrated an 188

Am J Clin Pathol 2001;116:183-190

extremely poor response to therapy and overall survival. Thus, while CD10 alone failed to demonstrate a significant influence on overall survival, its role appeared clearly when combined with bcl-2. It should be noted, however, that the coexpression of CD10 and bcl-2 was associated significantly with stage IV disease, possibly explaining the effect on prognosis. A multivariate analysis of a large number of cases would be required to assess whether the effect of coexpression of CD10 and bcl-2 is independent of clinical stage. Recently, a subdivision of DLBCLs into germinal center (GC) B-cell–like DLBCL and activated B-cell–like DLBCL has been proposed using gene expression profiling,37 demonstrating a better overall survival in patients with GC Bcell–like DLBCL. CD10 was one of the selected genes defining GC B-cell–like DLBCL, and CD10 messenger RNA (mRNA) was expressed to a varying degree in all of the GC B-cell–like DLBCL cases. Thus, the correlation of GC B-cell–like DLBCL with a better overall survival reported in that study seems to contradict the findings in the present study. Since CD10 expression in gene expression profiling was determined indirectly by mRNA level in cytoplasm, it may not correlate completely with the CD10 protein level on the cell surface detected by flow cytometry. Furthermore, that study did not specifically address whether CD10 mRNA was expressed to a significant extent in the activated B-cell–like group, although it was generally stated © American Society of Clinical Pathologists


that expression of no single gene completely segregated into one DLBCL group or the other. It is unclear at present, therefore, whether CD10 is strictly a marker of GC differentiation on DLBCL. Regardless, it seems to have biologic significance in this subset of tumors. Our study demonstrated that although CD10+ and CD10– DLBCLs are similar with regard to a variety of clinical and pathologic features, CD10 expression in de novo DLBCL may have a negative impact on treatment response and overall survival. Furthermore, we identified CD10 and bcl-2 coexpressing DLBCL as an extremely high-risk subset based on poor response to therapy and overall survival. These findings suggest that further studies are warranted to confirm the prognostic importance of these molecular markers. From the Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX. Address reprint requests to Dr Kroft: Dept of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9072. Acknowledgment: We thank William H. Frawley, PhD, for assistance with statistical analysis.

References 1. Harris NL, Jaffe SJ, Stein H, et al. A Revised EuropeanAmerican Classification of Lymphoid Neoplasms: a proposal from the International Lymphoma Study Group. Blood. 1994;84:1361-1392. 2. The Non-Hodgkin’s Lymphoma Classification Project: A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin’s lymphoma. Blood. 1997;89:3909-3918. 3. Engelhard M, Brittinger G, Huhn D, et al. Subclassification of diffuse B-cell lymphomas according to the Kiel classification: distinction of centroblastic and immunoblastic lymphomas is a significant prognostic risk factor. Blood.1997;89:2291-2297. 4. Non-Hodgkin’s Lymphoma Pathologic Classification Project. National Cancer Institute sponsored study of classification of non-Hodgkin’s lymphomas: summary and description of a Working Formation for clinical usage. Cancer. 1982;49:21122135. 5. Brittinger G, Bartels H, Common H, et al. Clinical and prognostic relevance of the Kiel classification of non-Hodgkin lymphomas: results of a prospective multicenter study by the Kiel Lymphoma Study Group. Hematol Oncol. 1984;2:269306. 6. Nathani B, Dixon D, Jones S, et al. The clinical significance of the morphological subdivision of diffuse “histocytic” lymphoma: a study of 162 patients treated by the Southwest Oncology Group. Blood. 1982;60:1068-1074. 7. Kwak L, Wilson M, Weiss L, et al. Clinical significance of morphologic subdivision in diffuse large cell lymphoma. Cancer. 1991;68:1988-1993. 8. Salar A, Fernandez de Servilla A, Romagosa V, et al. Diffuse large B-cell lymphoma: is morphologic subdivision useful in clinical management? Eur J Haematol. 1998;60:202-208.


9. Letarte M, Vera S, Tran R, et al. Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. J Exp Med. 1998;168:1247-1253. 10. Shipp MA, Look AT. Hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key! Blood. 1993;82:1052-1070. 11. LeBien TW, McCormack RT. The common acute lymphoblastic leukemia antigen (CD-10): emancipation from a functional enigma. Blood. 1989;73:625-635. 12. Almasri NM, Ituraspe JA, Braylan RC. CD10 expression in follicular lymphoma and large cell lymphoma is different from that of reactive lymph node follicles. Arch Pathol Lab Med. 1998;122:539-544. 13. Gregory CD, Tursz T, Edwards CF, et al. Identification of a subset of normal B cells with a Burkitt’s lymphoma (BL)-like phenotype. J Immunol. 1987;139:313-318. 14. Hsu SM, Jaffe ES. Phenotypic expression of B-lymphocytes, 1: identification with monoclonal antibodies in normal lymphoid tissues. Am J Pathol. 1984;114:387-395. 15. Hoffmann FG, Knapp W, Thioerfelder S. Anatomical distribution of call antigen expressing cells in normal lymphatic tissue and in lymphomas. Leuk Res. 1982;6:761-767. 16. Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome 18. Proc Natl Acad Sci U S A. 1985;82:7439-7443. 17. Johnson A, Brun A, Dictor M, et al. Incidence and prognostic significance of t(14;18) translocation in follicular center cell lymphoma of low and high grade. Ann Oncol. 1995;6:789794. 18. Garcia CF, Weiss LM, Warnke RA. Small noncleaved cell lymphoma: an immunophenotypic study of 18 cases and comparison with large cell lymphoma. Hum Pathol. 1986;17:451-461. 19. Ritz J, Pesando JM, McConarty N, et al. A monoclonal antibody to human acute lymphoblastic leukaemia antigen. Nature. 1980;283:583-585. 20. Fang JM, Finn WG, Hussong JW, et al. CD10 antigen expression correlates with the t(14;18)(q32;q21) major breakpoint region in diffuse large B-cell lymphoma. Mod Pathol. 1999;12:295-300. 21. Gascoyne RD, Adomat A, Krajewski S, et al. Prognostic significance of bcl-2 protein expression and bcl-2 gene rearrangement in diffuse aggressive non-Hodgkin’s lymphoma. Blood. 1997;90:244-251. 22. Hill ME, MacLennan KA, Cunningham DC, et al. Prognostic significance of bcl-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin’s lymphoma: a British National Lymphoma Investigation study. Blood. 1996;88:1046-1051. 23. Skinnider BF, Horsman DE, Dupuis B, et al. Bcl-6 and bcl-2 protein expression in diffuse large B-cell lymphoma and follicular lymphoma: correlation with 3q27 and 18q21 chromosomal abnormalities. Hum Pathol. 1999;30:803-808. 24. Kramer MHH, Hermans J, Wijburg E, et al. Clinical relevance of bcl2, bcl6, and MYC rearrangements in diffuse large B-cell lymphoma. Blood. 1998;92:3152-3162. 25. Hermine O, Haioun C, Lepage E, et al. Prognostic significance of bcl-2 protein expression in aggressive nonHodgkin’s lymphoma. Blood. 1996;87:265-272. 26. Korsmeyer SJ. Bcl-2 initiates a new category of oncogenes: regulator of cell death. Blood. 1992;80:879-886.

Am J Clin Pathol 2001;116:183-190

189


27. Kramer MHH, Hermans J, Parker J, et al. Clinical significance of Bcl2 and p53 protein expression in diffuse large B-cell lymphoma: a population-based study. J Clin Oncol. 1996; 14:2131-2138. 28. Miyashita T, Reed JC. Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood. 1993;81:151-157. 29. Hochenbery DM, Zutter M, Hickey W, et al. Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci U S A. 1991;88:6961-6965. 30. Walton MI, Whysong D, O’Conner PM, et al. Constitutive expression of human bcl-2 modulates nitrogen mustard and camptothecin induced apoptosis. Cancer Res. 1993;53:18531861. 31. McDonnell TJ, Korsmeyer SJ. Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14;18). Nature. 1991;349:254-256. 32. Willemze R, Kerl H, Sterry W, et al. EORTC classification for primary cutaneous lymphomas: a proposal from the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer. Blood. 1997;90:354-371. 33. DeVita VT, Jaffe ES, Mauch P, et al. Lymphocytic lymphomas. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology. Philadelphia, PA: Lippincott; 1990:1741-1798. 34. Kwak LW, Wilson M, Weiss LM, et al. Similar outcome of treatment of B-cell and T-cell diffuse large-cell lymphomas: the Stanford experience. J Clin Oncol. 1991;9:1426-1431. 35. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project: a predictive model for aggressive nonHodgkin’s lymphoma. N Engl J Med. 1993;329:987-994.

190

Am J Clin Pathol 2001;116:183-190

36. Harada S, Suzuki R, Uehira K, et al. Molecular and immunological dissection of diffuse large B cell lymphoma: CD5+, and CD5– with CD10+ groups may constitute clinically relevant subtypes. Leukemia. 1999;13:1441-1447. 37. Alizadeh AA, Eisen MB, Davis RE. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503-511. 38. Uherova P, Singleton TP, Ross CW, et al. The clinical significance of CD10 antigen expression in diffuse large B-cell lymphoma [abstract]. Mod Pathol. 2000;13:165A. 39. Yamaguchi M, Ohno T, Oka K, et al. De novo CD5-positive diffuse large B-cell lymphoma: clinical characteristics and therapeutic outcome. Br J Haematol. 1999;105:1133-1139. 40. Kaufmann O, Flath B, Spath-Schwalbe E, et al. Immunohistochemical detection of CD10 with monoclonal antibody 56C6 on paraffin sections. Am J Clin Pathol. 1999;111:117122. 41. Bavikatty NR, Ross CW, Finn WG, et al. Anti-CD10 immunoperoxidase staining of paraffin-embedded acute leukemias: comparison with flow cytometric immunophenotyping. Hum Pathol. 2000;31:1051-1054. 42. Xu Y, McKenna RW, Kroft SH. Comparison of immunohistochemistry and flow cytometry with cluster analysis for the detection of CD10 [abstract]. Mod Pathol. 2001;14:183A. 43. Monni O, Joensuu H, Franssila K, et al. Bcl2 overexpression associated with chromosomal amplification in diffuse large Bcell lymphoma. Blood. 1997;90:1168-1174. 44. Monni O, Franssila K, Joensuu H, et al. Bcl2 overexpression in diffuse large B-cell lymphoma. Leuk Lymphoma. 1999;34:45-52.
