The Usefulness of Flow Cytometric CD10 Detection in the Differential ...

Hematopathology / FLOW CYTOMETRIC CD10 DETECTION IN PTCL

The Usefulness of Flow Cytometric CD10 Detection in the Differential Diagnosis of Peripheral T-Cell Lymphomas Alessandra Stacchini, MSc,1 Anna Demurtas, MSc,1 Sabrina Aliberti, MSc,1 Paola Francia di Celle, MSc,3 Laura Godio, MD,2 Giorgio Palestro, MD,2 and Domenico Novero, MD2 Key Words: Angioimmunoblastic; Peripheral T-cell lymphoma; CD10; Flow cytometry DOI: 10.1309/MC7QRGPTV0LRR98X

Abstract We studied the histologic and multiparameter flow cytometry (MFC) features of 12 cases of angioimmunoblastic T-cell lymphoma (AITL), 13 of mature T-cell lymphoma, and 25 control cases of reactive lymphoid hyperplasia to evaluate the role of CD10 in the differential diagnosis of peripheral T-cell lymphomas (PTCLs). A characteristic immunophenotypic profile (CD2+/CD4+) with recurrent phenotypic aberrancies (eg, CD3 and CD7 loss) was identified in most AITL cases; MFC documented CD10 coexpression on T cells in 10 (83%). Mature T-cell lymphoma showed a more heterogeneous altered immunophenotypic pattern, and 2 cases of PTCL, unspecified, had clear evidence of aberrant CD10 expression on T cells. A small physiologic CD3+/CD4+/CD10+ T-cell population was detected by MFC in all control cases tested (range, 0.28%-4.71%), suggesting that a normal subset of peripheral CD10+ T cells exists. CD10 was a highly sensitive but incompletely specific phenotypic marker for diagnosing AITL; the differential diagnosis of PTCL, unspecified, must be related with traditional histologic features. A small number of CD10+ T cells in reactive lymph nodes suggests that this subpopulation may be the normal counterpart of neoplastic T cells in AITL. The biologic role of CD10+ T cells should be studied further.

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Am J Clin Pathol 2007;128:854-864 DOI: 10.1309/MC7QRGPTV0LRR98X

Angioimmunoblastic T-cell lymphoma (AITL) is a distinct clinicopathologic form of peripheral T-cell lymphoma (PTCL), recognized in the World Health Organization classification1 and reinserted into the entity first described as angioimmunoblastic lymphadenopathy with dysproteinemia by Frizzera et al.2 It commonly manifests with lymphadenopathy, hepatosplenomegaly, bone marrow involvement, and systemic symptoms, such as fever and hypergammaglobulinemia.3,4 The lymph nodes involved show partial or total obliteration of the normal architecture by a polymorphic infiltrate of lymphocytes, plasma cells, eosinophils, and immunoblasts accompanied by a prominent proliferation of venules and an expansion of follicular dendritic cell meshwork.1 Even in the presence of histologic criteria indicative of AITL, neoplastic T cells may be scarce and difficult to recognize, leading to diagnostic errors in more than 50% of cases.5 The differential diagnosis includes atypical reactive processes, Hodgkin lymphomas, B-cell lymphomas, and other forms of mature T-cell lymphoma. Therefore, a definitive diagnosis requires additional immunologic, cytogenetic, and molecular data. It has recently been demonstrated that, in most AITL cases, neoplastic T cells aberrantly express the CD10 molecule,5 an antigen absent in reactive and neoplastic T-cell processes. Multiparameter flow cytometry (MFC) is a rapid, highly sensitive, and reproducible method to assess the immunophenotype of T lymphocytes,6 allowing for the precise identification, characterization, and distinction of the altered antigenic profile of the neoplastic population from normal T cells. We report a retrospective examination of 12 cases of AITL, 13 cases of mature T-cell lymphoma, and 25 cases of reactive lymphoid hyperplasia used as control cases, using flow cytometric analysis. The results provide evidence that neoplastic © American Society for Clinical Pathology

Hematopathology / ORIGINAL ARTICLE

T cells in AITL are easily distinguishable from normal counterparts by specific and recurrent phenotypic aberrancies, CD10 is a useful but not exclusive marker able to differentiate this T-cell lymphoma form from other types of mature T-cell lymphoma, and a small number of normal CD10-expressing T cells exist in reactive hyperplasia. MFC proved to be a powerful tool, able to identify normal and neoplastic CD10+ T cells, even when present in small quantities. Moreover, if associated with traditional histologic features, it can also be used in the differential diagnosis of mature T-cell lymphomas.

Materials and Methods Cases A retrospective examination of 12 lymph nodes, from 12 patients, histologically identified as AITL between January 2000 and November 2006, was performed. The diagnoses were made according to the World Health Organization criteria1 and integrated by the results of T-cell receptor (TCR) gene rearrangement analysis and immunohistochemical analysis. The study also included 12 cases of peripheral T-cell lymphoma, unspecified (PTCLu), diagnosed by lymph node biopsy and 1 case of PTCL diagnosed in the spleen (case 14). Moreover, 25 cases of reactive lymph node hyperplasia were assayed to evaluate the presence of normal CD10-expressing T cells. Histologic and Immunohistochemical Studies Histopathologic examination was performed on diagnostic lymph node and staging bone-marrow biopsy specimens. All tissues were formalin (lymph nodes) or Bouin (bone marrow) fixed, paraffin-embedded for sectioning, and stained with H&E. Immunostaining with an avidin-biotin complex immunoperoxidase method was carried out using an automated immunostainer (Benchmark XT, Ventana Medical Systems, Tucson, AZ). The panel of antibodies included CD3 (clone PS1, Novocastra, Newcastle upon Tyne, England), CD5 (clone 4C7, Novocastra), CD20 (clone L26, DAKO, Carpinteria, CA), CD10 (clone 5GC6, Novocastra) for T and B, CD21 (clone 1F8, DAKO), and CD35 (clone BerMACDCR, DAKO) to highlight the follicular dendritic cells. All cases were also evaluated for CXCL13 expression (clone 53610, R&D Systems, Minneapolis, MN). Staining performed by the Bond Polymer Refine Detection System in a Vision Biosystem instrument (Vision Biosystem, Mount Waverley, Australia) was preceded by antigen retrieval by heat plate at 99°C for 20 minutes in acid buffer (pH 6). In situ hybridization for Epstein-Barr virus (EBV)-encoded RNA (EBER) was performed using the DAKO EBV probe kit according to the manufacturer’s protocol.

Multiparameter Flow Cytometry Fresh lymph node tissue processing and staining with 3or 4- color combinations of monoclonal antibodies, directly conjugated to fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, and allophycocyanin, was performed as previously described.7,8 The MFC panel included various combinations of antibodies against CD2, CD3, CD4, CD5, CD7, CD8, and CD10 (all purchased from Caltag, Burlingame, CA); CD19 and CD20 (Becton Dickinson, San Jose, CA); CD30 (Caltag); CD45RA (Immunotech, Marseille, France); CD45RO, CD56, TCRαβ, TCRγδ, and immunoglobulin κ and λ light chains (Becton Dickinson). Samples were subject to permeabilization and partial fixation by applying the Fix and Perm kit (Caltag), according to the manufacturer’s protocol, for CD3 intracellular staining. Data acquisition and analysis were performed on a FACSCalibur flow cytometer (Becton Dickinson) using CellQuest and Paint-a-Gate software programs (Becton Dickinson). Nonviable cells and debris were excluded, based on forward and side light scatter properties, and cells were analyzed selecting the most appropriate lymphocyte gate based on the combination of forward and side scatter. Positivity for an antigen was defined as at least 10% of lymphoma events beyond a 2% threshold based on an isotypic control tube. Percentages of cell types were determined based on total viable cellular events. Immunophenotypic aberrancies of T lymphocytes were defined as discrete immunophenotypic clusters exhibiting loss or increased or diminished expression of T-cell antigens relative to the internal immunophenotypically normal T-cell population. CD10+ T cells were first identified as CD19–/CD10+ events (using a κ/λ/CD19/CD10 antibody combination) and successively confirmed by using a CD4/CD8/CD3/CD10 antibody combination. Molecular Studies Gene rearrangement studies for T- and B-cell clonality were analyzed by polymerase chain reaction (PCR). DNA was extracted by the Proteinase K and phenol-chloroform method. Immunoglobulin heavy chain (IgH) gene rearrangements were amplified with consensus primers derived from the framework 2 (FR2) of the VH regions and from the JH1-6 regions using a seminested PCR method.9 TCRγ gene rearrangements were amplified with primers that target Vγ1-9 and Jγ gene segments.10 The PCR products were denaturized at 95°C for 5 minutes and then cooled at 4°C for 15 minutes to induce homoduplex and heteroduplex formation on a 12% nondenaturing polyacrylamide gel in case of monoclonal and polyclonal lymph proliferations, respectively. Statistical Analysis The proportion of cases expressing CD10 in the AITL group and in the other PTCLs was compared by using the Am J Clin Pathol 2007;128:854-864

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Fisher exact test. The mean fluorescence intensity (MFI) of CD10+ vs CD10– T cells in normal samples was compared by using the paired t test.

Results Clinical Features The clinical features of the cases are summarized in ❚Table 1❚. Data for 25 patients (15 men and 10 women; age range, 4884 years) were studied after obtaining informed consent. Most patients had generalized lymphadenopathy. Other clinical features included fever, skin rash, anemia, hepatomegaly, and splenomegaly. A histologic diagnosis of AITL had been made in 12 cases and of PTCLu in the other 13 cases. Angioimmunoblastic T-Cell Lymphoma Histologic Features All cases demonstrated effacement of the nodal architecture with diffuse proliferation of small lymphocytes, eosinophils,

plasma cells, immunoblasts, and clusters of medium-sized lymphocytes showing abundant pale cytoplasm; florid proliferation of high endothelial venules also was observed in all cases ❚Image 1❚. Immunohistochemical analysis documented the T-cell nature of the medium-sized clear lymphocytes in all AITL cases, as evidenced by the expression of CD3 and CD5 antigens. In 10 of 12 cases, expression of CD10 was observed by immunohistochemical analysis, evidencing a population of CD10+ “bona fide” T lymphocytes when comparing on serial sections the localization of CD3+, CD10+, and CD20+ cells (Image 1D-1F). Positivity for CD21 and CD35 by immunohistochemical analysis revealed expanded follicular dendritic meshwork (Image 1G). EBER was performed in 4 cases, revealing positivity in scattered immunoblasts (Image 1I). Clear-cut cytoplasmic CXCL13 staining with dot-like reinforcement was observed in medium-sized cells clustered in irregular aggregates and in sheets surrounding small venules (Image 1H) in all 11 cases evaluated ❚Table 2❚. Bone marrow staging after the diagnosis of T-cell lymphoma on lymph nodes was performed in 10 of 12 cases. Four

❚Table 1❚ Clinicopathologic Features of AITL and Mature T-Cell Lymphoma Cases Initial Diagnosis/ Case No./ Sex/Age (y)

AITL 1/F/64 2/F/73 3/M/70 4/F/73 5/M/71 6/M/48 7/F/63 8/F/69 9/M/67 10/F/75 11/M/75 12/M/76 PTCLu 13/F/67 14/M/72 15/F/62 16/M/84 17/M/69 18/M/65 19/F/72 20/M/50 21/M/70 22/F/67 23/M/76 24/M/61 25/M/62

Clinical Manifestations

Biopsy Site

BM

MFC Findings*

Rearrangement CD10 by Immunohistochemical Analysis TCR IgH

Fever; lymphadenopathy Lymphadenopathy; skin rash Lymphadenopathy Lymphadenopathy NA Generalized lymphadenopathy Lymphadenopathy Fever; elevated IgG level; skin rash; cervical lymphadenopathy Elevated IgG level; hemolytic anemia; lymphadenopathy Lymphadenopathy Fever; weight loss; itching; lymphadenopathy Generalized lymphadenopathy; splenomegaly

Cervical LN Cervical LN Cervical LN Axillary LN Cervical LN Inguinal LN Cervical LN Cervical LN

+ – – – + – – +

45% CD3+/CD4+/CD10+ 50% CD3–/CD4+/CD10+ 12% CD3–/CD4+ 40% CD3–/CD4+/CD10+ 10% CD3+/CD4+/CD5+dim 23% CD3+/CD4+/CD10+ 13% CD3+/CD4+/CD10+ 9% CD3–/CD4+/CD10+

+ (partial) + (partial) – + – + (dim) + (partial) + (partial)

M M M M M M M M

M P P P P ND P P

Supraclavicular LN

–

8% CD3–/CD4+/CD10+

+

M

P

Supraclavicular LN Cervical LN

ND ND

13% CD3+/CD4+/CD10+ 30% CD3+/CD4+/CD10+

+ +

M M

ND M

Supraclavicular LN

+

25% CD3–/CD4+/CD10+

+

M

ND

Cervical LN Spleen Laterocervical LN Axillary LN LN under jaw Supraclavicular LN Inguinal LN Cervical LN Supraclavicular LN Cervical LN Inguinal LN Inguinal LN Cervical LN

+ + – ND ND – ND + + + – – +

22% CD3–/CD4+ 15% CD3+/CD2– 24% CD3+bright/CD4+ 30% CD3+/CD5– 32% CD3–/CD5+ 86% CD3+/CD30+ 17% CD3+/CD5– 12% CD3–/CD5+ 35% CD3–/CD4+/CD10+ 20% CD3+/CD4+/CD10+ 24% CD3+dim/CD4+ 27% CD3–/CD8+ 19% CD3+dim/CD5+bright

– ND – ND ND ND ND – + + – – –

M M M M M M M M M M M M M

ND ND P ND P P ND ND ND ND ND ND ND

Lymphadenopathy Splenomegaly Lymphadenopathy Lymphadenopathy; fever Parotid swelling NA NA Lymphadenopathy Lymphadenopathy Lymphadenopathy Lymphadenopathy Fever; lymphadenopathy Lymphadenopathy

AITL, angioimmunoblastic T-cell lymphoma; BM, bone marrow; IgH, immunoglobulin heavy chain; LN, lymph node; M, monoclonal; MFC, multiparameter flow cytometry; NA, not available; ND, not done; P, polyclonal; PTCLu, peripheral T-cell lymphoma, unspecified; TCR, T-cell receptor. * Expressed as a percentage of total viable cellular events.

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A

B

C

D

E

F

❚Image 1❚❚ Histologic examination and immunostaining of representative cases of angioimmunoblastic T-cell lymphoma. A, B, and C, Nodal effacement by an atypical lymphoid infiltrate extending into the pericapsular adipose tissue (A, H&E, ×34; B, H&E, ×170) and prominent vascular arborization (C, H&E, ×650). D, CD3 immunostaining of lymphoma T cells (×34). E, CD10 immunostaining of the same T-cell area (×34). F, CD20 immunostaining of nonneoplastic B cells (×34). (continued next page)

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G

H

❚Image 1❚❚ (continued) G, CD21 and CD35 immunostaining showing an expanded follicular dendritic meshwork (×34). H, CXCL13 expression (×340). I, Epstein-Barr virus–encoded RNA positivity in small to intermediate-sized B cells (×340).

I

cases were positive for lymphoma with infiltration varying from a focal aggregate (case 8) to a massive infiltrate (20%30% in cases 1, 5, and 12). Flow Cytometric Analysis The light scatter features and immunophenotypic profile of the AITL cases were evaluated and are detailed in ❚Table 3❚. Neoplastic T cells had the same light scatter features as those of normal T cells in 6 cases, while distinctly larger cells were identifiable ❚Image 2❚ in the other 6 cases. All cases expressed CD2 and CD5 antigens. CD3 was expressed at the surface in 6 cases (50%) and at the cytoplasmic level in 4 cases (33%). Eleven cases expressed CD4, 1 at a dim level; 1 case was CD4–. CD7 was absent or partially expressed in 7 (58%) of 12 cases. A varying percentage of CD10 expression was documented in 10 (83%) of 12 cases, ranging from 21% to 100%, forming a distinct population, as 858 858


❚Table 2❚ Proportion of Cases of AITL and PTCLu Expressing CD10 and CXCL13 by Immunohistochemical Analysis* Lymphoma Subtype

CD10+

CXCL13+

AITL (n = 12) PTCLu (n = 13)

10/12 (83) 2/13 (15)

11/11 (100) 3/12 (25)

AITL, angioimmunoblastic T-cell lymphoma; PTCLu, peripheral T-cell lymphoma, unspecified. * Data are given as number positive/total tested (percentage).

shown in Image 2. All cases lacked CD56. CD30 was partially expressed in 4 cases; this finding was confirmed by immunohistochemical analysis. The cases tested for naive vs memory phenotype manifested CD45RO positivity (not shown). All AITL cases demonstrated at least 1 phenotypic aberrancy (eg, loss of CD3 or CD7; CD3/CD10 or CD4/CD10 coexpression; © American Society for Clinical Pathology


❚Table 3❚ Light Scatter and Immunophenotypic Characteristics of Angioimmunoblastic T-Cell Lymphoma Cases Case No.

Aberrant Light Scatter of % of Total T Cells (%) Aberrant T Cells T Cells CD2

CD3

CD4

CD5

CD7

CD8

CD10

CD30

αβ CD56 TCRα

TCRγγδ

1 2 3 4 5 6 7 8 9 10 11 12

45 50 12 40 26 23 13 9 8 13 30 25

+ – (ND) – (cy+) – (ND) + (d) + + – (cy+) – (cy+) + + (d) – (cy+)

+ + + – + + + + + + + (d) +

+ + + + + + + + + + + +

– + – + + + (pt) + + – – – –

– – – – – – – – – – – –

+ (pt) + (pt) – + – + + + (pt) + + + +

– – – – – – + (pt) – + (pt) – + (pt) + (pt)

– – – – – – – – – – – –

– – – – – – – – – – – –

N N N N N N L L L L L L

73 81 68 78 73 75 35 52 55 39 68 63

+ + + + + + + + + + + +

+ (d) – – – + + + – – + + –

cy, cytoplasmic; d, dim; L, large cells; N, normal; ND, not done; pt, partial.

or dim expression of CD4 or CD3). ❚Image 3❚ shows examples of the immunophenotypic features seen in AITL. Molecular Analysis PCR analysis of TCRγ-chain gene rearrangements was performed in all cases (Table 1). All cases were positive. A monoclonal IgH gene rearrangement was also identified in 2 (17%) of 12 AITL cases.

104

800

103

103

600 400

102 101

0 0

200 400 600 800 1,000 FSC-Height

100 100

102 101

101

102 103 CD4 PE

100 100

104

104

104

800

103

103

600 400

CD10 APC

1,000

CD10 APC

SSC-Height

CD10 APC

104

200

B

Histologic Features All mature T-cell lymphoma cases studied showed effacement of nodal architecture, with a relatively monomorphic infiltrate of medium to large atypical lymphoid cells ❚Image 4A❚ and ❚Image 4B❚. A moderate degree of vascular proliferation was present without prominent arborizing networks. No evident proliferation of follicular dendritic cells

1,000

CD10 APC

SSC-Height

A

Mature T-Cell Lymphoma

102 101

200 0 0

200 400 600 800 1,000 FSC-Height

100 100

101 102 103 CD3 PerCP

104

101 102 103 CD3 PerCP

104

102 101

101

102 103 CD4 FITC

104

100 100

❚Image 2❚❚ Flow cytometric features of 2 representative cases of angioimmunoblastic T-cell lymphoma. A, Normal (blue) and neoplastic (red) T cells with the same light scatter distribution; CD3 is not expressed in the CD4/CD10 double-positive neoplastic cells. B, Neoplastic cells have greater forward light scatter than normal T cells and express CD3 at the surface level. APC, allophycocyanin; FITC, fluorescein isothiocyanate; FSC, forward scatter; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; SSC, side scatter.

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Molecular Analysis PCR analysis of TCRγ-chain gene rearrangements was performed in all cases (Table 1). As expected all cases were positive. All cases tested for monoclonal IgH gene rearrangement were polyclonal.

was observed. An evident number of CD10+ lymphoid cells was observed in 2 cases of PTCLu, focally clustered in perifollicular T cell–rich areas largely outnumbering B cells ❚Image 4C❚ and ❚Image 4D❚. EBV latent membrane protein was performed in the 2 CD10+ cases, revealing positivity in B immunoblasts in 1 case (case 21). In 3 cases, a significant amount of CXCL13 positivity was observed, with distribution similar to AITL in 2 cases (cases 14 and 15); the third case (case 21) showed numerous scattered positive cells without aggregation; this last case was 1 of 2 CD10+ cases. Bone marrow involvement was evaluated in 10 of 13 cases. A positive infiltrate was detected in 6 cases, ranging from 5% to 30%.

Reactive Lymph Node Hyperplasia All reactive lymph nodes showed preservation of the architecture and hyperplastic lymphoid follicles with moderate paracortical expansion. The use of multicolor flow cytometry allowed for the identification of a discrete population of normal T cells positive for CD10 in all cases, ranging from 0.28% to 4.71% (average ± SD, 1.47 ± 0.1) of the total lymphoid T-cell population. All CD10+ T cells were CD4+/CD8– with a slightly lower intensity of CD3 expression (MFI ± SD, 75 ± 26.87) than in the CD3+/CD10– population (MFI ± SD = 90.58 ± 26.16); the difference in the MFI was statistically significant (P = .048; paired t test) ❚Image 5❚. The immunophenotypic profile of this subpopulation was further studied ❚Table 5❚ in 4 cases. In all 4 cases, the CD3+/CD4+/CD10+ T cells expressed CD2, CD5, CD7, and TCRαβ and displayed a memory phenotype (CD45RO+/CD27+). The activation marker HLA-DR was

Flow Cytometric Analysis The immunophenotypic profiles of mature T-cell lymphoma are summarized in ❚Table 4❚. Although combined light scatter and immunophenotypic aberrancies were identifiable in all cases, unlike AITL, no common immunophenotypic profile was found. CD10 expression was evident in 2 cases, at 66% and 33% of the total T population. The difference between the AITL group and the PTCL group in CD10 expression was statistically significant (χ2 = 7.58; P = .005).

A

B

C

D

❚Image 3❚❚ Immunophenotypic characteristics in various cases of angioimmunoblastic T-cell lymphoma. All neoplastic T cells are depicted in red and normal T cells in blue. A, CD7– neoplastic T cells. B, CD30 dim expression. C, Expansion of a CD10+/CD19– population. D, CD45RO expression. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP-Cy5.5, peridinin chlorophyll protein–cyanin 5.5 tandem conjugate.

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A

B

C

D

❚Image 4❚❚ (Case 22) Histologic examination and immunostaining of a case of CD10+ peripheral T-cell lymphoma, unspecified. A, Low-power view showing interfollicular colonization of lymphomatous cells (H&E, ×34). B, High magnification shows blastic morphologic features of interfollicular cells (H&E, ×340). C, CD3 immunostaining of lymphoma T cells (×70). D, CD10 immunostaining of the same T-cell area (×70). ❚Table 4❚ Light Scatter and Immunophenotypic Characteristics of Cases of Peripheral T-Cell Lymphoma, Unspecified Case No.

Aberrant Light Scatter of T Cells (%) Aberrant T Cells

% of Total T Cells CD2

CD3

CD4

CD5

CD7

CD8

CD10 CD30

CD56

αβ TCRγγδ TCRα

13 14 15 16 17 18 19 20 21 22 23 24 25

22 15 24 30 32 86 17 12 35 20 24 27 19

81 58 71 62 64 91 93 56 46 47 66 50 64

– (cy+) + + (b) + – (cy+) + + – (cy+) – (cy+) + + (d) – (cy+) + (d)

+ – + – – + – + + + + – +

+ + + – + + + + + + + + + (b)

– – + – – – + – – + – – –

– – – + – – + – – – – + –

– – – – – – – – + + – – –

ND – – – – – – – – – – – ND

– + + + – + + – – + – ND –

L L N N N N N L L L L L N

+ – + + + + + + (d) + + + + +

ND ND ND – – + – – + – ND – ND

– – – – – – – – – – – ND –

b, bright; cy, cytoplasmic; d, dim; L, large cells; N, normal; ND, not done.

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104

104

103

103

103

102 101 100 0

200 400 600 800 1,000 SSC-Height

CD10 APC

104

CD10 APC

CD3 PerCP


102 101 100 100

10

1

2

10 10 CD3 PerCP

3

10

4

102 101 100 100

101

102 103 CD4 FITC

104

❚Image 5❚❚ CD10+ normal T cells in a representative case of reactive follicular hyperplasia, restricted to the CD4+ subset (gate on CD3+ in red). The mean fluorescence intensity of CD3 displayed by the CD10+ T cells is weaker compared with the CD10– cells (see the text). APC, allophycocyanin; FITC, fluorescein isothiocyanate; PerCP, peridinin chlorophyll protein; SSC, side scatter. ❚Table 5❚ Immunophenotypic Profile of Normal CD3+/CD4+/CD10+ T Cells in Representative Cases of Reactive Lymph Node Hyperplasia Case No.

CD3+/CD4+/CD10+ T Cells (%)* CD2

CD5

CD7

CD8

CD45RO

CD45RA

CD56

CD57

CD25

CD27

αβ HLA-DR TCRα

TCRγγδ

1 2 3 4

1.23 1.58 1.36 1.08

+ + + +

+ + + +

– – – –

+ + + +

– – – –

– – – –

– – – –

– – – –

+ + + +

+ + + –

– – – –

*

+ + + +

+ + + +

Total CD10+ T cells detected in the lymph node.

expressed in 3 of 4 cases, whereas the interleukin-2 receptor, CD25, was expressed at less than 20%. No reactivity for CD56 or CD57 was registered.

Discussion This study was carried out to evaluate CD10 expression in cases of AITL and mature T-cell lymphoma. Our findings support those of previous studies indicating that the CD10 antigen is expressed at least by a fraction of neoplastic T cells in the lymph nodes of most AITL cases. However, CD10 cannot be considered an exclusive hallmark of AITL because a high number of CD10+ T cells were also identified in 2 cases of PTCLu. Recent studies have highlighted the role of CD10 in the diagnosis of AITL.5,11-14 In our AITL cases, the coexpression CD4/CD10 represents the major phenotypic aberrancy (83%), followed by the partial or total absence of CD7 (58%) and loss of surface CD3 (50%). The loss of CD7 was considered with caution because, as reported by Jamal et al,15 a large proportion of CD4+ cells may lack CD7 in blood, bone marrow, and lymph node. In our cases, diminished or lost CD7 expression was always observed along with other phenotypic aberrancies and was, therefore, considered significant. The immunophenotypic profiles of the AITL cases evaluated were consistent with those previously reported in literature.16,17 In all cases, neoplastic 862 862


T cells expressed CD2, CD4 (only 1 negative), and CD5 and were negative for CD8 and CD56. There was, however, a certain degree of variability in the CD3 expression, which was frequently deleted. The CD4/CD10 phenotypic association seems to be the most useful in identifying circulating angioimmunoblastic lymphoma T cells, as evidenced by recent studies in which the leukemic phase of the disease was easily diagnosed by CD4/CD10 double-positive cells.13,18,19 A high fraction of CD10+ T cells was also identified in 2 cases of PTCLu, 35% CD3–/CD4+ and 20% CD3+/CD4+. Both cases lacked the morphologic features typical of AITL, the most significant being the absence of the vascular pattern and the expansion of the follicular dendritic cell meshwork. In particular, case 21 was characterized by medium to large CD30+ lymphocytes and showed a consistent number of large B cells that were positive at immunostaining for EBER. The presence of EBER+ B cells, frequently observed in AITL, is thought to be associated with the immunodeficiency that permits the proliferation of EBV-infected B cells.3 In these cases, the presence of clonal B cells would also explain the rearrangements of JH genes observed along with the TCR rearrangement in a small number of AITL cases. Although case 21 displayed an immunophenotype compatible with that of AITL, it did not display the typical histologic pattern of AITL. It could be hypothesized that the expression of CD10 and CXCL13 and EBER positivity are a heritage in a true angioimmunoblastic lymphoma that has undergone a © American Society for Clinical Pathology


morphologic transformation and was, at the time of observation, histologically unrecognizable. The expression of CD10 in T-cell lymphomas not diagnosed as AITL has occasionally been documented in the literature by sporadic studies, eg, de Leval et al20 described 2 cases of CD10+ PTCL with a follicular growth pattern; Reichard et al21 reported CD10 expression in 2 PTCLu cases complicated by a large B-cell proliferation; and Magro et al22 described a case of primary T-cell lymphoma in the skin with an unusual CD20weak/CD10+/CD8+ phenotype. These findings and our data show that CD10 may be present on T neoplastic cells other than AITL, although in a sporadic manner. The biologic role of CD10 on T neoplastic cells remains unknown. The CD10 antigen is a cell surface protein with endopeptidase activity23 present on a variety of hematopoietic cells, such as granulocytes24 and B-cell23 and T-cell25 precursors but not on resting B and mature T cells. Cutrona et al26 reported that mature T cells subjected to apoptotic stimuli express CD10 and hypothesized some kind of role in the apoptotic process of lymphoid cells. Attygalle et al5 also postulated that CD10 expression in angioimmunoblastic lymphoma may be the result of a disturbance of apoptosis in these cells, similar to what is seen in bcl-2–overexpressing follicular lymphoma cells. However, it must not be overlooked that apoptosis is not a characteristic feature in AITL. The existence of a normal small subset of CD10+ peripheral T cells located primarily within follicular structures was recently demonstrated by Cook et al,27 who affirmed that, if present in small quantities, they should not be considered an indicator of a T-cell neoplasm. This observation was confirmed in our study when MFC was used for the evaluation of a series of reactive lymph nodes, in which the normal amount of CD10+ T cells ranged between 0.28% and 4.71%, thus supporting the existence of a physiologic CD4/CD10 double-positive T subpopulation with a function that is, to date, unknown. Moreover, it is still not clear whether the expansion of the CD10+ T cell might be due to the transformation of a cell normally present in lymphoid tissues or, more simply, a manifestation of an aberrant phenotype that could confer a survival advantage to the neoplastic T cell. The recent demonstration that T cells in AITL express CXCL13, a chemokine highly up-regulated in germinal center T-helper cells, offers an intriguing scenario that supports the hypothesis of a possible derivation of AITL from follicular helper T cells.28,29 Moreover, as suggested by Grogg et al,28 CXCL13 expression as a distinctive feature of AITL may help to redefine this subtype of PTCL through recognition of histologic variants. Our study confirmed the expression of CXCL13 in all AITL cases tested. The 3 PTCLu cases positive for CXCL13 could be included in a minor subset of PTCL having borderline morphologic and phenotypic features with AITL previously described.28,29

The presence of CD10 on normal lymphoid T cells can easily be detected by MFC. The presence of a small number of CD4+/CD10+ T cells, in the absence of other phenotypic aberrancies, should not be considered a sign of the presence of neoplastic cells. Although the expansion of these cell compartments strongly suggests the presence of an AITL, correlation with all available morphologic and immunophenotypic data is necessary to make a definitive diagnosis. Further studies on CD10+ T cells would help in the understanding of its role in normal and neoplastic cells. From the 1Flow Cytometry Unit, 2Laboratory of Pathology, Department of Pathology, Molinette Hospital, Turin, Italy; and 3Center for Experimental Research and Medical Studies, Turin. Address reprint requests to A. Stacchini: II Servizio di Anatomia Patologica, Ospedale Molinette, via Santena 7, 10126 Torino, Italy. Acknowledgments: We thank O. Gaiola, A. Di Tomasso, and D. Corino for their valuable and excellent technical assistance that helped make this article possible. We also thank Barbara Wade for linguistic advice.

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