Immunophenotypic Analysis of Benign and Malignant Cutaneous Lymphoid Infiltrates ELLEN B. REST, MD, MS THOMAS D. HORN, MD
he distinction between benign and malignant lymphoid infiltrates in the skin may be difficult. Helpful histologic criteria exist,’ but in many cases differentiation of benign from malignant infiltrates is aided by special techniques. Recently, many advances have been made in the characterization of lymphoid infiltrates by the use of cellular immunophenotyping. An expanding knowledge of the normal sequence of hematopoetic cell maturation and accompanying antigen expression has considerably aided the understanding of benign and malignant infiltrates. The concept that a lymphoid malignancy is phenotypically related to a normal leukocyte counterpart allows the identification of aberrant antigen expression. In this chapter, we will discuss technical considerations of the immunoperoxidase method and review the immunophenotypes of certain benign and malignant cutaneous infiltrates. What makes identification of a particular antigen, or combination of antigens, useful in classifying infiltrates as benign or malignant? The antigenic alteration, whether related to inappropriate expression or absence of expression, should rarely be found in benign lesions and should be found in some significant percentage of malignant ones.* Many of the antigens to be discussed are found in benign infiltrates and absent in malignant ones, making appropriate positive and negative controls very important. In certain cases, routine histology coupled with immunophenotypic analysis may not be sufficient to establish a diagnosis. Each case must be evaluated with careful
T
From the Department of Dermatology, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Address correspondence to: Thomas D. Horn, M.D., Department ofDermatology, The ]ohns Hopkins Medical Institutions, Blalock 913 F, 600 N. Wolfe Street, Baltimore, MD 22205. This work was supported, in part, by the Clinician-Scientist Award of The Johns Hopkins University (TDH).
0 2991 by Elsevier Science Publishing
Co., Inc. 0738-081x/91/$3.50 l
consideration given to clinical information and findings of lymph node biopsy, when available. The antigenic characterization of hematopoietic cell lines is an evolving field. The literature of the past 10 - 15 years may be confusing because several antibodies have been used to detect one antigen and antigens may be referred to by multiple names. We will use the cluster designation (CD) numbers for antigens, followed by other relevant names when appropriate.
Technique The basic reaction in immunohistochemical techniques is recognition of and binding to a tissue antigen by a known antibody. The antigen-antibody combination may be detected in a number of ways. In the case of direct immunoperoxidase, an enzyme is conjugated to the antibody, and tissue binding is detected via a color change in a chromogen substrate of the enzyme (Figure 1). More elaborate methods, such as secondary and tertiary antibodies or bridging systems such as avidin-biotin, are now commonly used and allow conservation of antibodies, along with improved sensitivity and specificity. Biopsy specimens may be fresh frozen or paraffin embedded. Frozen tissue may be snap frozen in isopentane or stored in Michel’s transport medium for 3 - 5 days before being embedded in Optimal Temperature Cutting (OCT) compound, followed by freezing at - 70°C. Many antibodies are suitable only for frozen tissue, as fixation and paraffin embedding significantly alter some antigens. Recent work has focused on antibodies reliable in paraffin-embedded tissues .3 Nonetheless, if a lymphoid infiltrate is anticipated clinically one should obtain a portion of tissue in transport medium to assure the best evaluation. The antibodies used may be either polyclonal or monoclonal. Polyclonal antiserum is produced by injection of a purified antigenic molecule, a polypeptide, for example, into an animal and, therefore, is a mixture of antibodies to
261
262
REST AND
Clinics in Dermatology 2991;9:261-272
HORN
Immunology
Direct Method
Indirect Method-Two
Slep
AndIn Euo+m Complex
Figure 1. lmmunoperoxidase staining methods. Direct method: enzyme-conjugated antibody binds to a tissue antigen. indirect method: enzyme-conjugated secondary antibody binds to antigen-specific prima y antibody. Avidin Biotin Complex: prima y antibody is antigen specific. Seconda y antibody is conjugated to biotin, which binds to avidin biotin enzyme complex.
the varied antigenic determinants present. This antiserum may cross react with a number of antigens, but the cross reactivity can be removed by absorption using the undesired antigenic determinants.2 Polyclonal antisera are available in limited quantities. By contrast, monoclonal antibodies (MAbs) are produced by hybridoma technique and are available in large quantities, They detect only one antigenic determinant, but cross reactivity may occur if that determinant is expressed on more than one antigen. Cross reactivity cannot be removed from MAbs.2 In detecting the bound antibody, the most commonly used enzyme is peroxidase .4 Alkaline phosphatase and glucose oxidase may also be used. Substrates include diaminobenzidine, which gives a brown color, and aminoethylcarbazole, which gives a red color. The slides are then counterstained using hematoxylin or Giemsa stain. In addition to fresh versus fixed tissue, there are a number of technical factors that may affect correct immunophenotyping. Although commercial kits used to detect an antigen or group of antigens are convenient, there may be considerable variation in staining between assays and kits, making appropriate positive and negative controls very important. With some tissues or antibodies, nonspecific staining (background) may be a problem. Variations in fixative, length of fixation, dehydration, embedding materials, drying time, and storage may affect the survival of a particular antigen in paraffin-embedded tissues.2 For example, B5 fixative has been shown to improve staining with two B-cell markers, LNl and LN2.3 Assuming adequate mastery of the immunoperoxidase technique, interpretation of results remains subjective. Crucial estimates must be made of percentages of stained cells (eg, T helper/inducer versus T cytotoxic/suppressor) and loss of staining. Furthermore, the pattern of staining on cells is important; membrane-bound antigens display an annular or ring configuration, whereas cytoplasmic antigens provide a more diffuse pattern.
B and T lymphocytes are derived from bone marrow precursors. Each line undergoes a maturation process that is accompanied by sequential expression of various antigens. Antigens expressed normally only by T cells or B cells are termed lineage-specific or restricted. We will briefly examine the development of each of these cell lines. T lymphocyte precursors migrate to the thymus, where maturation and differentiation occur (Figure 2). The first T lineage-restricted antigen is CD7 (Leu 9, 3A1),5 which is a pan T antigen and is expressed on 90% of peripheral T cells. After maturation, T cells enter the extra-thymic sites and populate lymphoid tissues. Mature T cells express CD3, the T-cell receptor-related antigen, CD5 (Leu l), and CD2. Most mature T cells also express a subset marker. The majority of T cells in the periphery are of the helper/inducer subset, expressing CD4 (Leu3). CD8 (Leu 2)-positive cells are cytotoxic/suppressor cells. Most cutaneous infiltrates preserve the T helper (CD4+) subset majority. B-cell maturation, at least in the early phases, appears to take place in the bone marrow in the adult. There are antigen-independent types of pre B cells and resting B cells, and antigen-dependent/activated B cells, differentiated B cells, and plasma cells (Figure 3).‘j Some antigens are present at each phase, such as the pan B antigens CD19, CD20, and CD22, and some are stage specific,
Figure 2. MAbs can be used to detect sequential stages in Tcell development. Malignancies can be phenotypically related to stages of normal T-cell maturation. Abbreviations: T-ALL, Tcell acute lymphoblastic leukemia; LBL, T-cell lymphoblastic lymphoma; T-CLL, T-cell chronic lymphocytic leukemia; MF/ SS, mycosis fungoides Seza y syndrome; PTL, peripheral T-cell lymphoma; ATL, adult T-cell leukemia/lymphoma; AIL, angiocentric immunoproliferative lesions.
CD7
CD1 CD0 CD4 Transfsrrh receptor Tdt Neoplasias
I
I
Clinics in Dermatology 1991;9:261- 272
Figure 3. MAbs can be used to detect sequential stages in Bcell maturation. The malignant lympkomas and leukemias reflect these stages of normal B-cell development. Abbreviations: HCR, heavy-chain gene rearrangement; KRD/ J..RD,kappa or lambda light-chain rearrangement or deletion; TdT, terminal deoxynucleotidyl transferase; CLL, chronic lympkocytic leukemia. such as surface IgD expression. B cells are not divided according to subsets as are T cells, but eventually are committed to antibody production of only one heavy and light-chain type. With new understanding of normal T- and B-cell ontogeny and the development of MAbs, the characterization of neoplastic lymphoid processes has advanced beyond morphology alone. Neoplasms can be related both functionally and phenotypically to their normal counterparts (Figures 2,3).5 Examples include lymphoblastic lymphomas, which are neoplasms of early precursor T and B cells; the majority expressing an immature T-cell phenotype. Tumors arising from mature B cells are characterized by monoclonal surface immunoglobulin and include small lymphocytic lymphomas, most chronic lymphocytic leukemias, follicular lymphomas, and mantle zone lymphoma. Tumors that secrete immunoglobulin are of fully differentiated B cells or plasma cells and include multiple myeloma and Waldenstroms macroglobulinemia . T-cell neoplasms are a heterogeneous group and include T-cell chronic lymphocytic leukemia, T-cell prolymphocytic leukemia, mycosis fungoides/Sezary syndrome, peripheral T-cell lymphoma, adult T-cell leukemia (HTLV-1 positive), and angiocentric lymphomas.
Immunoarchitecture Cutaneous lymphoid infiltrates, whether benign or malignant, occur in several patterns recognizable at low magnifications. The array of cells may be perivascular, bandlike (lichenoid), nodular, or diffuse. Evidence exists that
REST AND HORN
263
CUTANEOUS LYMPHOID INFILTRATES
the immunophenotypic composition of an infiltrate correlates closely with the general architectural features.’ In general, a perivascular architecture suggests an inflammatory, ie, benign, process. One exception to this statement is leukemia cutis, in which early lesions may display a perivascular pattern. Band-like infiltrates, while characteristic of many benign conditions such as lichen planus or lupus erythematosus, are also observed in the patch and plaque stages of mycosis fungoides. The cells in perivascular and band-like infiltrates are typically composed of a variable mixture of T helper/inducer (CD4 +) and T cytotoxic/suppressor (CD8 +) cells. Nodular patterns may represent benign or malignant infiltrates. Lymphocytoma cutis often displays nodular architecture with the lymphoid aggregates typically, but not invariably more concentrated at upper dermal levels. Nodular lymphoma, often of B-cell origin, is described as “bottom heavy” within the dermis. Nodular patterning suggests the presence of B lymphocytes, with a peripheral mantle of T cells of variable number. Germinal center formation, ie, organization paralleling normal lymph node architecture, may be observed in benign and malignant infiltrates, but is more characteristic of the former. Diffuse infiltrates of polymorphous composition are often benign, eg, an arthropod bite reaction. A diffuse infiltrate composed of a monomorphous cell population is commonly malignant. Both T- and B-cell neoplasms may assume this pattern. In this setting, distinction of lymphoid malignancy from a poorly differentiated carcinoma is crucial. Architectural patterns of the infiltrates, observed at low magnifications, give the first clues to the nature of a lymphoid infiltrate. Careful attention to cellular details and mitotic rates are additional crucial factors. The following sections review the immunophenotypic patterns of several benign and malignant cutaneous lymphoid processes. A summary of selected antigens and antibodies commonly used to study these disorders is presented in Table 1.
T-Cell Lymphoma The cutaneous T-cell lymphomas (CTCLs) are a diverse group of disorders. Dermatologists traditionally equate CTCL with mycosis fungoides and Sezary syndrome, but it is clear that there are primary cutaneous T-cell lymphomas that are distinct from the epidermotropic CTCLs, mycosis fungoides, and Sezary syndrome.*-10 A classification scheme for peripheral T-cell lymphomas has been proposed by Suchi et al” based on histopathologic criteria. Mycosis fungoides and Sezary syndrome are placed in a low-grade small cerebriform cell category, whereas a
264
REST
AND
Clinics in Dermatology
HORN
1991;9:261-272
Table 1. Selected Antigens and Antibodies CD Antigen Name
Specificity
Comment
T-Cell -Associated CD1 (Leu 6) CD2 (Leu 5) CD3 (Leu 4)
Thymocytes T cells, natural killer cells T cells
CD4 CD5 CD7 CD8
T cells T cells, subset of B cells 90% of T cells, natural killer cells T cell subset, natural killer cells
(Leu (Leu (Leu (Leu
3A) 1) 9) 2)
T-cell receptor-related antigen Helper/inducer subset
Cytotoxic/suppressor
subset
B-Cell -Associated CD10 CD19 CD20 CD21 CD22
(Leu 12) (Leu 16) (B2) (Leu 14)
(I-eu 8)
Pre B cells, polymorphonuclear cells B cells, follicular dendritic cells B cells B cells, follicular dendritic cells B cells Mature B cells T cells
Activation CD15 (Leu Ml) CD25 CD30 (Ki-1) (Ber-H2) CD45 (Leu 18) CD71 Ki-67 HLA-DR, DQ, DP
EMA
CALLA
C3d receptor
and Other Antigens
Granulocytes, monocytes macrophages Activated T and nonlymphoid cells Activated T & B cells Lymphoid cells except plasma cells, Reed Stemberg cells Activated T & B cells Proliferating cells B lymphocytes and activated cells of many types, including T lymphocytes Epithelial cells, plasma cells, some Reed-Stemberg cells, some large cell lymphomas
number of the other entities to be discussed, such as Ki1+ lymphoma and adult T cell leukemia/lymphoma (ATLL), are in the high-grade category. Immunophenotypic studies of CTCL show differences from benign infiltrates in two areas. The malignant infiltrating cells often fail to express certain pan T antigens such as CD7, CD3, and/or CD5. Abnormalities of subset antigen expression occur, such as the simultaneous expression of both helper (CD4) and cytotoxic (CD8) markers on a subset of infiltrating cells, leading to estimates of CD4 + plus CD8 + cells totaling greater than 100% of the infiltrate. For example, a normal estimate would be 80% CD4 -I- cells and 20% CD8 + cells, whereas in CTCL the count may be 80% CD4 i- and 40% CD8 +. Subset markers may not be expressed at all. Evidence of monoclonal origin in these neoplasms may be obtained by studying rearrangements of the T-cell receptor gene, a technique discussed elsewhere in this volume. We will consider the findings in mycosis fungoides, followed by
IL-2 receptor Leukocyte common antigen Transferrin receptor Nuclear antigen expressed by cells in mitosis Class II major histocompatability antigens Epithelial membrane antigen
other T-cell lymphomas. features exist.
However,
many overlapping
Mycosis Fungoides Mycosis fungoides (MF) often begins as persistent localized or disseminated patches that may progress to plaques and nodules. Preceding, nonspecific, chronic, eczematous disorders may not show the histologic features of MF. There is no specific test to aid in the diagnosis of early MF. Nonspecific findings on routine staining often correlate with nonspecific immunophenotype studies. Early studies indicated that the infiltrating T cells had a mature helper/inducer phenotype and were not significantly different from T cells in benign lichenoid dermatoses.12 Further work indicates that most of the infiltrating cells in CTCL are of the T helper/inducer phenotype, but that a large proportion of these cells have an aberrant immunophenotype, with loss of certain pan T-cell anti-
Clinics in Dermatology 7991:9:261- 272
CUTANEOUS
REST AND HORN LYMPHOID INFILTRATES
265
Figure 4. Mycosis fungoides. Top: Staining for CD4 shows that most of the infiltrating cells are T helper/inducer subset (hematoxylin counterstain X 200). Left: Epidermotropic T cells are CD4 positive (hematoxylin counterstain X320). Right: Staining with pan T-cell antigen CD7 shows loss of antigen expression (hematoxylin counterstain X320).
gens and preservation of others. In a study of 56 patients with MF, 64% of those patients with patch or plaque lesions showed loss of one pan-T antigen. Of patients with nodular lesions or lymph node involvement, 100% showed loss of one or more pan-T antigens.13 Preesman et al” sampled tumor and plaque lesions from patients that had both types of lesions, and found that in 2 of 5 patients there was loss of more pan-T antigens in tumor than in plaque-stage lesions. CD7 (Leu 9) is a pan-T antigen, normally expressed on 90% of T cells, which is often deficient in the infiltrates of mycosis fungoides (Figure 4). Wood et ali5 found that CD7 was deficient in 59% of patients with MF in all types of lesions, versus 3% of controls. The decreased CD7 expression was also noted in patients with poikilodermatous MF, atrophic large plaque parapsoriasis,“j and pagetoid reticulosis (Woringer-Kolopp disease).” The CD7 deficiency was more marked on T cells present in the
epidermis.“j In contrast, CD7 deficiency was noted in the infiltrate of only one of eight patients with erythroderma due to Sezary syndrome. la Moderate CD7 deficiency has been reported in two cases of benign cutaneous lymphoid hyperplasia, but patients with MF had expression of CD7 on
he distinction between benign and malignant lymphoid infiltrates in the skin may be difficult. Helpful histologic criteria exist,’ but in many cases differentiation of benign from malignant infiltrates is aided by special techniques. Recently, many advances have been made in the characterization of lymphoid infiltrates by the use of cellular immunophenotyping. An expanding knowledge of the normal sequence of hematopoetic cell maturation and accompanying antigen expression has considerably aided the understanding of benign and malignant infiltrates. The concept that a lymphoid malignancy is phenotypically related to a normal leukocyte counterpart allows the identification of aberrant antigen expression. In this chapter, we will discuss technical considerations of the immunoperoxidase method and review the immunophenotypes of certain benign and malignant cutaneous infiltrates. What makes identification of a particular antigen, or combination of antigens, useful in classifying infiltrates as benign or malignant? The antigenic alteration, whether related to inappropriate expression or absence of expression, should rarely be found in benign lesions and should be found in some significant percentage of malignant ones.* Many of the antigens to be discussed are found in benign infiltrates and absent in malignant ones, making appropriate positive and negative controls very important. In certain cases, routine histology coupled with immunophenotypic analysis may not be sufficient to establish a diagnosis. Each case must be evaluated with careful
T
From the Department of Dermatology, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Address correspondence to: Thomas D. Horn, M.D., Department ofDermatology, The ]ohns Hopkins Medical Institutions, Blalock 913 F, 600 N. Wolfe Street, Baltimore, MD 22205. This work was supported, in part, by the Clinician-Scientist Award of The Johns Hopkins University (TDH).
0 2991 by Elsevier Science Publishing
Co., Inc. 0738-081x/91/$3.50 l
consideration given to clinical information and findings of lymph node biopsy, when available. The antigenic characterization of hematopoietic cell lines is an evolving field. The literature of the past 10 - 15 years may be confusing because several antibodies have been used to detect one antigen and antigens may be referred to by multiple names. We will use the cluster designation (CD) numbers for antigens, followed by other relevant names when appropriate.
Technique The basic reaction in immunohistochemical techniques is recognition of and binding to a tissue antigen by a known antibody. The antigen-antibody combination may be detected in a number of ways. In the case of direct immunoperoxidase, an enzyme is conjugated to the antibody, and tissue binding is detected via a color change in a chromogen substrate of the enzyme (Figure 1). More elaborate methods, such as secondary and tertiary antibodies or bridging systems such as avidin-biotin, are now commonly used and allow conservation of antibodies, along with improved sensitivity and specificity. Biopsy specimens may be fresh frozen or paraffin embedded. Frozen tissue may be snap frozen in isopentane or stored in Michel’s transport medium for 3 - 5 days before being embedded in Optimal Temperature Cutting (OCT) compound, followed by freezing at - 70°C. Many antibodies are suitable only for frozen tissue, as fixation and paraffin embedding significantly alter some antigens. Recent work has focused on antibodies reliable in paraffin-embedded tissues .3 Nonetheless, if a lymphoid infiltrate is anticipated clinically one should obtain a portion of tissue in transport medium to assure the best evaluation. The antibodies used may be either polyclonal or monoclonal. Polyclonal antiserum is produced by injection of a purified antigenic molecule, a polypeptide, for example, into an animal and, therefore, is a mixture of antibodies to
261
262
REST AND
Clinics in Dermatology 2991;9:261-272
HORN
Immunology
Direct Method
Indirect Method-Two
Slep
AndIn Euo+m Complex
Figure 1. lmmunoperoxidase staining methods. Direct method: enzyme-conjugated antibody binds to a tissue antigen. indirect method: enzyme-conjugated secondary antibody binds to antigen-specific prima y antibody. Avidin Biotin Complex: prima y antibody is antigen specific. Seconda y antibody is conjugated to biotin, which binds to avidin biotin enzyme complex.
the varied antigenic determinants present. This antiserum may cross react with a number of antigens, but the cross reactivity can be removed by absorption using the undesired antigenic determinants.2 Polyclonal antisera are available in limited quantities. By contrast, monoclonal antibodies (MAbs) are produced by hybridoma technique and are available in large quantities, They detect only one antigenic determinant, but cross reactivity may occur if that determinant is expressed on more than one antigen. Cross reactivity cannot be removed from MAbs.2 In detecting the bound antibody, the most commonly used enzyme is peroxidase .4 Alkaline phosphatase and glucose oxidase may also be used. Substrates include diaminobenzidine, which gives a brown color, and aminoethylcarbazole, which gives a red color. The slides are then counterstained using hematoxylin or Giemsa stain. In addition to fresh versus fixed tissue, there are a number of technical factors that may affect correct immunophenotyping. Although commercial kits used to detect an antigen or group of antigens are convenient, there may be considerable variation in staining between assays and kits, making appropriate positive and negative controls very important. With some tissues or antibodies, nonspecific staining (background) may be a problem. Variations in fixative, length of fixation, dehydration, embedding materials, drying time, and storage may affect the survival of a particular antigen in paraffin-embedded tissues.2 For example, B5 fixative has been shown to improve staining with two B-cell markers, LNl and LN2.3 Assuming adequate mastery of the immunoperoxidase technique, interpretation of results remains subjective. Crucial estimates must be made of percentages of stained cells (eg, T helper/inducer versus T cytotoxic/suppressor) and loss of staining. Furthermore, the pattern of staining on cells is important; membrane-bound antigens display an annular or ring configuration, whereas cytoplasmic antigens provide a more diffuse pattern.
B and T lymphocytes are derived from bone marrow precursors. Each line undergoes a maturation process that is accompanied by sequential expression of various antigens. Antigens expressed normally only by T cells or B cells are termed lineage-specific or restricted. We will briefly examine the development of each of these cell lines. T lymphocyte precursors migrate to the thymus, where maturation and differentiation occur (Figure 2). The first T lineage-restricted antigen is CD7 (Leu 9, 3A1),5 which is a pan T antigen and is expressed on 90% of peripheral T cells. After maturation, T cells enter the extra-thymic sites and populate lymphoid tissues. Mature T cells express CD3, the T-cell receptor-related antigen, CD5 (Leu l), and CD2. Most mature T cells also express a subset marker. The majority of T cells in the periphery are of the helper/inducer subset, expressing CD4 (Leu3). CD8 (Leu 2)-positive cells are cytotoxic/suppressor cells. Most cutaneous infiltrates preserve the T helper (CD4+) subset majority. B-cell maturation, at least in the early phases, appears to take place in the bone marrow in the adult. There are antigen-independent types of pre B cells and resting B cells, and antigen-dependent/activated B cells, differentiated B cells, and plasma cells (Figure 3).‘j Some antigens are present at each phase, such as the pan B antigens CD19, CD20, and CD22, and some are stage specific,
Figure 2. MAbs can be used to detect sequential stages in Tcell development. Malignancies can be phenotypically related to stages of normal T-cell maturation. Abbreviations: T-ALL, Tcell acute lymphoblastic leukemia; LBL, T-cell lymphoblastic lymphoma; T-CLL, T-cell chronic lymphocytic leukemia; MF/ SS, mycosis fungoides Seza y syndrome; PTL, peripheral T-cell lymphoma; ATL, adult T-cell leukemia/lymphoma; AIL, angiocentric immunoproliferative lesions.
CD7
CD1 CD0 CD4 Transfsrrh receptor Tdt Neoplasias
I
I
Clinics in Dermatology 1991;9:261- 272
Figure 3. MAbs can be used to detect sequential stages in Bcell maturation. The malignant lympkomas and leukemias reflect these stages of normal B-cell development. Abbreviations: HCR, heavy-chain gene rearrangement; KRD/ J..RD,kappa or lambda light-chain rearrangement or deletion; TdT, terminal deoxynucleotidyl transferase; CLL, chronic lympkocytic leukemia. such as surface IgD expression. B cells are not divided according to subsets as are T cells, but eventually are committed to antibody production of only one heavy and light-chain type. With new understanding of normal T- and B-cell ontogeny and the development of MAbs, the characterization of neoplastic lymphoid processes has advanced beyond morphology alone. Neoplasms can be related both functionally and phenotypically to their normal counterparts (Figures 2,3).5 Examples include lymphoblastic lymphomas, which are neoplasms of early precursor T and B cells; the majority expressing an immature T-cell phenotype. Tumors arising from mature B cells are characterized by monoclonal surface immunoglobulin and include small lymphocytic lymphomas, most chronic lymphocytic leukemias, follicular lymphomas, and mantle zone lymphoma. Tumors that secrete immunoglobulin are of fully differentiated B cells or plasma cells and include multiple myeloma and Waldenstroms macroglobulinemia . T-cell neoplasms are a heterogeneous group and include T-cell chronic lymphocytic leukemia, T-cell prolymphocytic leukemia, mycosis fungoides/Sezary syndrome, peripheral T-cell lymphoma, adult T-cell leukemia (HTLV-1 positive), and angiocentric lymphomas.
Immunoarchitecture Cutaneous lymphoid infiltrates, whether benign or malignant, occur in several patterns recognizable at low magnifications. The array of cells may be perivascular, bandlike (lichenoid), nodular, or diffuse. Evidence exists that
REST AND HORN
263
CUTANEOUS LYMPHOID INFILTRATES
the immunophenotypic composition of an infiltrate correlates closely with the general architectural features.’ In general, a perivascular architecture suggests an inflammatory, ie, benign, process. One exception to this statement is leukemia cutis, in which early lesions may display a perivascular pattern. Band-like infiltrates, while characteristic of many benign conditions such as lichen planus or lupus erythematosus, are also observed in the patch and plaque stages of mycosis fungoides. The cells in perivascular and band-like infiltrates are typically composed of a variable mixture of T helper/inducer (CD4 +) and T cytotoxic/suppressor (CD8 +) cells. Nodular patterns may represent benign or malignant infiltrates. Lymphocytoma cutis often displays nodular architecture with the lymphoid aggregates typically, but not invariably more concentrated at upper dermal levels. Nodular lymphoma, often of B-cell origin, is described as “bottom heavy” within the dermis. Nodular patterning suggests the presence of B lymphocytes, with a peripheral mantle of T cells of variable number. Germinal center formation, ie, organization paralleling normal lymph node architecture, may be observed in benign and malignant infiltrates, but is more characteristic of the former. Diffuse infiltrates of polymorphous composition are often benign, eg, an arthropod bite reaction. A diffuse infiltrate composed of a monomorphous cell population is commonly malignant. Both T- and B-cell neoplasms may assume this pattern. In this setting, distinction of lymphoid malignancy from a poorly differentiated carcinoma is crucial. Architectural patterns of the infiltrates, observed at low magnifications, give the first clues to the nature of a lymphoid infiltrate. Careful attention to cellular details and mitotic rates are additional crucial factors. The following sections review the immunophenotypic patterns of several benign and malignant cutaneous lymphoid processes. A summary of selected antigens and antibodies commonly used to study these disorders is presented in Table 1.
T-Cell Lymphoma The cutaneous T-cell lymphomas (CTCLs) are a diverse group of disorders. Dermatologists traditionally equate CTCL with mycosis fungoides and Sezary syndrome, but it is clear that there are primary cutaneous T-cell lymphomas that are distinct from the epidermotropic CTCLs, mycosis fungoides, and Sezary syndrome.*-10 A classification scheme for peripheral T-cell lymphomas has been proposed by Suchi et al” based on histopathologic criteria. Mycosis fungoides and Sezary syndrome are placed in a low-grade small cerebriform cell category, whereas a
264
REST
AND
Clinics in Dermatology
HORN
1991;9:261-272
Table 1. Selected Antigens and Antibodies CD Antigen Name
Specificity
Comment
T-Cell -Associated CD1 (Leu 6) CD2 (Leu 5) CD3 (Leu 4)
Thymocytes T cells, natural killer cells T cells
CD4 CD5 CD7 CD8
T cells T cells, subset of B cells 90% of T cells, natural killer cells T cell subset, natural killer cells
(Leu (Leu (Leu (Leu
3A) 1) 9) 2)
T-cell receptor-related antigen Helper/inducer subset
Cytotoxic/suppressor
subset
B-Cell -Associated CD10 CD19 CD20 CD21 CD22
(Leu 12) (Leu 16) (B2) (Leu 14)
(I-eu 8)
Pre B cells, polymorphonuclear cells B cells, follicular dendritic cells B cells B cells, follicular dendritic cells B cells Mature B cells T cells
Activation CD15 (Leu Ml) CD25 CD30 (Ki-1) (Ber-H2) CD45 (Leu 18) CD71 Ki-67 HLA-DR, DQ, DP
EMA
CALLA
C3d receptor
and Other Antigens
Granulocytes, monocytes macrophages Activated T and nonlymphoid cells Activated T & B cells Lymphoid cells except plasma cells, Reed Stemberg cells Activated T & B cells Proliferating cells B lymphocytes and activated cells of many types, including T lymphocytes Epithelial cells, plasma cells, some Reed-Stemberg cells, some large cell lymphomas
number of the other entities to be discussed, such as Ki1+ lymphoma and adult T cell leukemia/lymphoma (ATLL), are in the high-grade category. Immunophenotypic studies of CTCL show differences from benign infiltrates in two areas. The malignant infiltrating cells often fail to express certain pan T antigens such as CD7, CD3, and/or CD5. Abnormalities of subset antigen expression occur, such as the simultaneous expression of both helper (CD4) and cytotoxic (CD8) markers on a subset of infiltrating cells, leading to estimates of CD4 + plus CD8 + cells totaling greater than 100% of the infiltrate. For example, a normal estimate would be 80% CD4 -I- cells and 20% CD8 + cells, whereas in CTCL the count may be 80% CD4 i- and 40% CD8 +. Subset markers may not be expressed at all. Evidence of monoclonal origin in these neoplasms may be obtained by studying rearrangements of the T-cell receptor gene, a technique discussed elsewhere in this volume. We will consider the findings in mycosis fungoides, followed by
IL-2 receptor Leukocyte common antigen Transferrin receptor Nuclear antigen expressed by cells in mitosis Class II major histocompatability antigens Epithelial membrane antigen
other T-cell lymphomas. features exist.
However,
many overlapping
Mycosis Fungoides Mycosis fungoides (MF) often begins as persistent localized or disseminated patches that may progress to plaques and nodules. Preceding, nonspecific, chronic, eczematous disorders may not show the histologic features of MF. There is no specific test to aid in the diagnosis of early MF. Nonspecific findings on routine staining often correlate with nonspecific immunophenotype studies. Early studies indicated that the infiltrating T cells had a mature helper/inducer phenotype and were not significantly different from T cells in benign lichenoid dermatoses.12 Further work indicates that most of the infiltrating cells in CTCL are of the T helper/inducer phenotype, but that a large proportion of these cells have an aberrant immunophenotype, with loss of certain pan T-cell anti-
Clinics in Dermatology 7991:9:261- 272
CUTANEOUS
REST AND HORN LYMPHOID INFILTRATES
265
Figure 4. Mycosis fungoides. Top: Staining for CD4 shows that most of the infiltrating cells are T helper/inducer subset (hematoxylin counterstain X 200). Left: Epidermotropic T cells are CD4 positive (hematoxylin counterstain X320). Right: Staining with pan T-cell antigen CD7 shows loss of antigen expression (hematoxylin counterstain X320).
gens and preservation of others. In a study of 56 patients with MF, 64% of those patients with patch or plaque lesions showed loss of one pan-T antigen. Of patients with nodular lesions or lymph node involvement, 100% showed loss of one or more pan-T antigens.13 Preesman et al” sampled tumor and plaque lesions from patients that had both types of lesions, and found that in 2 of 5 patients there was loss of more pan-T antigens in tumor than in plaque-stage lesions. CD7 (Leu 9) is a pan-T antigen, normally expressed on 90% of T cells, which is often deficient in the infiltrates of mycosis fungoides (Figure 4). Wood et ali5 found that CD7 was deficient in 59% of patients with MF in all types of lesions, versus 3% of controls. The decreased CD7 expression was also noted in patients with poikilodermatous MF, atrophic large plaque parapsoriasis,“j and pagetoid reticulosis (Woringer-Kolopp disease).” The CD7 deficiency was more marked on T cells present in the
epidermis.“j In contrast, CD7 deficiency was noted in the infiltrate of only one of eight patients with erythroderma due to Sezary syndrome. la Moderate CD7 deficiency has been reported in two cases of benign cutaneous lymphoid hyperplasia, but patients with MF had expression of CD7 on
