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Am J Clin Pathol 2009;132:699-706 699699

DOI: 10.1309/AJCPV8LN5ENMZOVY 699© American Society for Clinical Pathology Hematopathology / Immunophenotypic Variatiaons in MCL

CME/SAM

Immunophenotypic Variations in Mantle Cell Lymphoma Juehua Gao, MD, PhD, LoAnn Peterson, MD, Beverly Nelson, MD, Charles Goo lsby, PhD, and Yi-Hua Chen, MD

Key Words:

Mantle cell lymphoma; Immunophenotype; Flow cytometry; Ki-67

DOI: 10.1309/AJCPV8LN5ENMZOVY

Abstract

Mantle cell lymphoma (MCL) expresses pan-B-

cell antigens and is usually CD5+/CD10-/CD23-/ FMC7+. In this study, we evaluated 52 patients with confirmed diagnoses of MCL and identified variant immunophenotypes in 21 patients (19/48 classical and 2/4 variant MCLs), including CD5- in 6 (12%) of 52, CD10+ in 4 (8%) of 50, CD23+ in 10 (21%) of

48, and FMC7- in 4 (11%) of 37 cases. Three cases

showed variations in 2 antigens, including CD5-/

CD23+, CD10+/FMC7-, and CD23+/FMC7-; they

were all classical MCLs. One blastoid variant MCL was CD23+, and one was FMC7-. Evaluation for proliferation index by immunohistochemical analysis for Ki-67 demonstrated no significant difference between MCLs with variant immunophenotypes and

MCLs with typical immunophenotypes. The high

proliferation index (>60%) was exclusively seen in the blastoid and pleomorphic variants. Our results indicate that immunophenotypic variations are common in MCL, and recognizing the variability is important for accurate subclassification of B-cell lymphoma.Mantle cell lymphoma (MCL) accounts for approximately

3% to 10% of non-Hodgkin lymphomas and predominantly

occurs in men of advanced age. Morphologically, classical MCL is characterized by a monomorphic proliferation of small to medium-sized lymphocytes with irregular nuclei and incon- spicuous nucleoli. Several morphologic variants have been recognized, such as blastoid and pleomorphic variants, which are associated with more aggressive clinical behavior.1 Virtually all MCLs harbor t(11;14)(q13;q32) transloca- tion, which juxtaposes the CCND1 gene encoding cyclin D1 to an enhancer of the immunoglobulin heavy chain (IgH) gene, leading to an overexpression of cyclin D1. 1

Therefore,

cyclin D1 has been widely used as an immunohistochemical marker for MCL. Flow cytometric immunophenotyping also serves an important role in the diagnosis of MCL. Typically, MCLs are positive for pan-B-cell antigens (CD19, CD20, and CD22) and usually are CD5+, CD10-, CD23-, and FMC7+. 1 However, variations from the typical immunophenotype have been observed, 2-7 some of which could be misleading and cause diagnostic difficulties, particularly when the mor- phologic features are not classical or the biopsy specimen is small. These cases could be misdiagnosed as other types of B-cell lymphomas such as follicular lymphoma or chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/ SLL). Therefore, recognizing the variability of immunophe- notype in MCL is important for diagnostic workup of patients with lymphoma. Clinically, MCL is considered an aggressive neoplasm with a median survival of 3 to 4 years. However, in a sub- set of patients, the disease may follow an indolent course, and patients may survive more than 10 years.1,8

It has been

recognized in earlier studies that proliferation of the tumor, as

Upon completion of this activity you will be able to:• describe the typical immunophenotype of mantle cell lymphoma.• recognize immunophenotypic variations of mantle cell lymphoma.• discuss the importance of evaluating cyclin D1 or CCND1/IgH translocation in the proper morphologic context to rule out mantle cell lymphoma, particularly in small biopsy and "fluid" samples.The ASCP is accredited by the Accreditation Council for Continuing

Medical Education to provide continuing medical education for physicians

The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. This activity qualifies as an American Board

of Pathology Maintenance of Certification Part II Self-Assessment Module The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to di sclose.

Questions appear on p 798. Exam is located at www.ascp.org/ajcpcme.Downloaded from https://academic.oup.com/ajcp/article/132/5/699/1766006 by guest on 04 July 2023

700 Am J Clin Pathol 2009;132:699-706700

DOI: 10.1309/AJCPV8LN5ENMZOVY © American Society for Clinical PathologyGao et al / Immunophenotypic Variatiaons in MCL

evaluated by the mitotic index or the proliferation-associated antigen Ki-67, was the best predictor of survival in patients with MCL. 9-11 Recent studies have demonstrated that it remains an important prognostic marker in the era of anti-CD20 therapy and is independent of other clinical factors. 12,13

Previous reports on unusual immunophenotypes in

MCL are about patients with single antigen variation, 2-7 and the data on correlation of variant immunophenotypes with morphologic features, proliferation of the tumor, or clinical outcome are rare. 6,7

In the present study, flow cytometric

analysis was performed to investigate the types and frequen- cies of the variant immunophenotypes in a cohort of 52 patients with confirmed diagnoses of MCLs. The findings were correlated with morphologic features, immunohis- tochemical results, and proliferation index of the tumor to investigate whether MCLs with variant immunophenotypes differ in morphologic features or biologic behavior from

MCLs with typical immunophenotypes.

Material and Methods

Case Selection

This study was approved by the institutional review board of Northwestern Memorial Hospital, Chicago, IL. Cases diag- nosed as MCL between January 1994 and September 2008 that had been evaluated by morphologic examination of the tissue biopsy specimens and flow cytometric immunophenotyping were obtained by searching the database of the Department of Pathology, Northwestern Memorial Hospital. A total of 52 cases in which the diagnosis of MCL was confirmed by overex- pression of cyclin D1 and/or the presence of t(11;14)-CCND1/ IgH translocation by fluorescence in situ hybridization (FISH) or cytogenetic studies were included in the study. Some cases included in this study have been previously reported. 7

Flow Cytometric Immunophenotyping

Specimens collected after September 2005 (25 speci- mens) were analyzed on the BD Biosciences LSR II 6-color flow cytometer, and data analysis was performed using FACSDiva software (BD Biosciences, San Jose, CA). The following monoclonal antibodies (BD Biosciences) were used: CD20-allophycocyanin (APC)-Cy7 (L27), CD19- peridinin chlorophyll protein-Cy5.5 (SJ25C1), κ-fluorescein isothiocyanate (FITC), and λ-phycoerythrin (PE). Additional monoclonal antibodies used were from Beckman Coulter (Miami, FL): CD5-PE-Cy7 (BL1a), CD10-APC (ALB1), CD79b-PE (CB3-1), FMC7-FITC (FMC7), and CD23 (also called B6)-PE (HD50). Samples collected before September 2005 (27 specimens) were analyzed on a standard 4-color Beckman Coulter EPICS

ALTRA flow cytometer, and data analysis was performed using EXPO32 software (Beckman Coulter). The following mono-clonal antibodies were used: CD20-PE-Cy5 (B9E9), CD19-energy-coupled dye (PE-Texas red) (J4.119), CD5-FITC (SFCI24T6612), CD10-PE-Cy5(ALB1), CD23-PE(9P25), FMC7-FITC (FMC7), κ-FITC, λ-PE (Beckman Coulter), and

CD79b-FITC (SN8, DAKO, Carpinteria, CA).

Fresh cell suspensions were prepared and analyzed as described previously. 14

Briefly, a cursor was established

based on the negative background staining observed on the granulocyte or T-cell populations. The B cells exhibit- ing staining characteristics with virtual complete overlap with the internal negative reference cell populations were considered negative. Positive staining was defined when no or limited overlap was seen with the negative reference cell populations. Dim staining was defined as greater intensity of the relevant antigen but with significant overlap with the negative reference populations. In our laboratory, nega- tive staining for CD5 was always confirmed by repeated staining with 2 additional independent anti-CD5 antibodies against the different epitopes of CD5 molecule (LI7F12, BD Biosciences; SFCI24T6G12 [T1], Beckman Coulter).

Immunohistochemical Analysis

Immunohistochemical stains for cyclin D1 and Ki-67 were performed on all 52 cases on formalin-fixed, paraffin- embedded whole tissue sections. Immunohistochemical stains for CD20, CD5, CD10, and BCL-6 were performed on cases with variant immunophenotypes. All immunostains were per- formed semiautomatically on the BenchMark XT (Ventana Medical Systems, Tucson, AZ) using the Advanced HRP detection system (Ventana Medical Systems). The primary antibodies used were polyclonal anti-cyclin D1 at a dilution of

1:25 (NeoMarkers, Fremont, CA), prediluted monoclonal anti-

Ki-67 (Ventana), prediluted monoclonal anti-CD20 (Ventana), monoclonal anti-CD5 at a dilution of 1:50 (Novocastra, Newcastle upon Tyne, England), monoclonal anti-CD10 at a dilution of 1:20 (Vector, Novi, MI), and monoclonal anti- BCL-6 at a dilution of 1:10 (DAKO). Cyclin D1+ MCL was used as a positive control for cyclin D1 staining, and normal tonsil tissue was used as a control for the rest of the antigens examined in this study. The proliferation index was assessed by counting Ki-67+ neoplastic lymphocytes in 10 random high-power fields (HPF; ×600) in biopsy specimens with adequate size and 5 HPF in small biopsy specimens. In each HPF, 500 cells were counted, and the average percentage of Ki-67+ neoplastic lymphocytes of all neoplastic lymphocytes counted was used as the proliferation index.

Fluorescence In Situ Hybridization

Interphase FISH for

CCND1 /IgH fusion was performed on

touch imprints or paraffin-embedded tissues at Mayo Medical Downloaded from https://academic.oup.com/ajcp/article/132/5/699/1766006 by guest on 04 July 2023

Am J Clin Pathol 2009;132:699-706 701701

DOI: 10.1309/AJCPV8LN5ENMZOVY 701© American Society for Clinical PathologyHematopathology / Original Article

Laboratories (Rochester, MN) using the dual-color and dual- fusion probes for

CCND1 (11q13) and IgH (14q32) (Abbott

Laboratories, Des Plaines, IL) as previously described. 15,16

Results

Case Information

Among the 52 patients, 45 were men and 7 were women, and ages ranged from 29 to 83 years (median, 61 years). Of the cases, 48 were diagnosed as classical MCL, and 4 were variant MCLs, including 3 blastoid variants and 1 pleomorphic variant. The 52 specimens included 35 lymph node, 2 nasopharyngeal,

11 gastrointestinal, 2 tonsillar, and 2 soft tissue biopsies; 32

were the initial diagnostic biopsy specimens, and 20 were biop- sy specimens in recurrent disease. Immunohistochemical analy- sis for cyclin D1 was performed and showed positive nuclear staining in all cases. FISH for CCND1/IgH translocation was performed on touch imprints or paraffin-embedded tissues in 18 cases and was positive in 15. An additional 2 cases were posi- tive for t(11;14)(q13;q32) by conventional cytogenetic studies. We did not identify cyclin D1- MCLs in our series.

Flow Cytometric Immunophenotyping

All 52 cases of MCL showed surface immunoglobulin

light chain restriction (30 λ and 22 κ) and were positive for pan-B-cell antigens CD20 and CD19. The variant immunophe- notypes were defined in this study as CD5-, CD10+, CD23+, or FMC7-. These variant immunophenotypes were identified

in 21 patients, including CD5- in 6 (12%) of 52, CD10+ in 4 (8%) of 50, CD23+ in 10 (21%) of 48, and FMC7- in 4 (11%) of 37 cases

Table 1

individual cases are listed in

Table 2

3 showed variations in 2 antigens, including CD5-/CD23+,

CD10+/FMC7-, and CD23+/FMC7- (Table 2). The lack of CD5 staining was confirmed by repeating the flow cytometric analysis with 2 additional independent monoclonal antibodies recognizing different epitopes of CD5. By comparison with the immunophenotyping results of the diagnostic samples, 16 of the 20 recurrent MCLs showed the same immunopheno- types as previously identified, but 4 previously CD23- cases were dim CD23+ (cases 13, 17, and 18) or CD23+ (case 15) in the recurrent disease.

Correlation of Variant Immunophenotypes With

Morphologic Features, Immunohistochemical Results, CCND1 /IgH Translocation, and Proliferation Index The lack of CD5 expression in 6 cases as determined by flow cytometry was also confirmed by immunohistochemi- cal analysis. As shown in Table 2, all 6 CD5- MCLs were

Table 1

Frequencies of Variant Immunophenotypes in Mantle Cell

Lymphoma

Variant Immunophenotype Frequency (%)

CD5-

6/52 (12)

CD10+

4/50 (8)

CD23+

10/48 (21)

FMC7-

4/37 (11)

Table 2

Detailed Information on MCLs With Variant Immunophenotypes Case No. Variant Immunophenotype Tissue Diagnosis Cyclin D1 FISH (CCND1/IgH) Ki-67 (%) 1

CD5- Ileum Classical MCL + + 10

2

CD5- LN Classical MCL + + <10

3

CD5- Tonsil Classical MCL + - 20

4

CD5- LN Classical MCL + ND 10

5

CD5- LN Classical MCL + + 40

6

CD5-/CD23+ LN Classical MCL + ND <10

7

CD10+ Colon Classical MCL + + <10

8

CD10+/CD5 dim+ Breast Classical MCL + + 50

9

CD10+/FMC7- LN Classical MCL + + 10

10

CD10 dim+ LN Classical MCL + + <10

11

CD23+/FMC7- Colon Classical MCL + ND 20

12

CD23+ LN Classical MCL + + 10

13

CD23 dim+ Esophagus Classical MCL + ND <10

14

CD23+ Duodenum Classical MCL + ND 50

15

CD23+ LN Classical MCL + + 30

16

CD23+ LN Blastoid MCL + + 80

17

CD23 dim+ Rectum Classical MCL + ND <10

18

CD23 dim+ Colon Classical MCL + ND <10

19

CD23+ LN Classical MCL + ND <10

20

FMC7- LN Classical MCL + ND <10

21

FMC7- LN Blastoid MCL + + 80

FISH, fluorescence in situ hybridization; LN, lymph node; MCL, mantle ce

ll lymphoma; ND, not done.Downloaded from https://academic.oup.com/ajcp/article/132/5/699/1766006 by guest on 04 July 2023

702 Am J Clin Pathol 2009;132:699-706702

DOI: 10.1309/AJCPV8LN5ENMZOVY © American Society for Clinical PathologyGao et al / Immunophenotypic Variatiaons in MCL

positive for cyclin D1, and FISH analysis for CCND1/ IgH translocation was positive in 3 of the 4 cases tested. Morphologically, all 6 CD5- MCLs were classical types. An additional case (case 8) was reported to be CD5-/CD10+ at the time of the initial workup, and the diagnosis of MCL was based on morphologic suspicion and confirmation by positive cyclin D1 and CCND1/IgH translocation. Reevaluation of the flow cytometric data for this case showed dim CD5 expres- sion in the monotypic B cells

Image 1

no convincing CD5 staining seen when assessed by immuno- histochemical analysis

Image 2

Three CD10+ cases

were identified in addition to case

8; 2 were positive (cases 7 and 9), and 1 was dim positive

(case 10). The expression of CD10 was also demonstrated by immunohistochemical analysis. The diagnosis of MCL in the 4 CD10+ cases was supported by positive staining for cyclin D1 and the presence of CCND1/IgH translocation (Table 2). Morphologically, they were all classical MCLs. Immunohistochemical analysis for BCL-6 was performed and demonstrated that 3 of the 4 CD10+ MCLs were also positive for BCL-6 (Image 2). The expression of CD23 was detected in 10 (21%) of 48 cases; 9 were classical MCLs, and 1 was the blastoid vari- ant. All CD23+ MCLs were positive for cyclin D1, and 3 were also confirmed by positive CCND1/IgH translocation, including the blastoid variant (case 16). Negative staining for FMC7 was seen in 4 cases (cases 9, 11, 20, and 21). Two showed additional immunophenotypic variations; 1 was FMC7-/CD10+ (case 9), and 1 was FMC7-/CD23+ (case 11). Morphologically, 3 cases were classical MCLs and 1 was the

blastoid variant (case 21). All 4 FMC7- MCLs were positive for cyclin D1, and the 2 cases analyzed by FISH were also positive for CCND1/IgH translocation (cases 9 and 21).

The proliferation rate of the tumor was evaluated by immunohistochemical analysis for Ki-67 in all cases included in the study, and the percentage of positive cells was used as the proliferation index. The 52 MCLs showed variable proliferation index values: less than 10% in 21 (40%), 10% to 30% in 19 (37%), 31% to 60% in 8 (15%), and more than

Image 1

(Case 8) Mantle cell lymphoma. Selected immuno- phenotypes showing a monotypic (

λ) B-cell population

(purple) that was dim CD5+/CD10+. AB

Image 2

(Case 8) Mantle cell lymphoma. Aquotesdbs_dbs17.pdfusesText_23

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