Rein Willemze

Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation

Recruitment of activated T‐cells to the skin is a common feature in a wide variety of inflammatory skin diseases. As CXCR3 activating chemokines CXCL10 (IP‐10), CXCL9 (Mig), and CXCL11 (IP‐9/I‐TAC) specifically attract activated T‐cells, this study addressed the question of whether differences in the expression of these chemokines correlate with the site and cellular composition of the skin infiltrates in different types of inflammatory skin disease. Skin biopsies from lichen planus, chronic discoid lupus erythematosus, allergic patch test reactions, psoriasis, and Jessners lymphocytic infiltration of the skin were investigated for chemokine expression using RNA in situ hybridization, and for the expression of CXCR3 using immunohistochemistry. The results showed differential expression of CXCL10, CXCL9, and CXCL11, which correlated with differences in the localization and cellular composition of the infiltrates. Whereas CXCL10 and CXCL11 were mainly expressed by basal keratinoctyes, CXCL9 mRNA expression was located predominantly in the dermal infiltrates. Correlation with immunohistochemical data suggested that macrophages and activated keratinocytes were the main producers of these chemokines. CXCR3 was expressed by a majority of both CD4+ and CD8+ infiltrating T‐cells, suggesting a functional interaction between locally produced chemokines and CXCR3‐expressing T‐cells. In conclusion, these findings indicate that these CXCR3 activating chemokines play a significant role in the recruitment and maintenance of T‐cell infiltrates in the inflammatory skin diseases studied. Copyright

bcl-2 protein expression in primary cutaneous large B-cell lymphoma is site-related.

PURPOSE Primary cutaneous large B-cell lymphoma (PCLBCL) that presents on the leg has recently been recognized as a distinct disease entity. These lymphomas have a reduced disease-free survival and a worse prognosis as compared with the more common, morphologically similar PCLBCL that present on the head or trunk. Studies in noncutaneous diffuse large B-cell lymphomas suggest a relationship between the expression of bcl-2 protein and clinical behavior. In the present study, we investigated whether these two groups of PCLBCL differ in the expression of bcl-2 protein and the presence of t(4;18), known as one of the causes of bcl-2 overexpression. PATIENTS AND METHODS Paraffin sections from pretreatment biopsies of 14 PCLBCLs of the head or trunk and nine PCLBCLs of the legs were investigated for expression of bcl-2 protein using immunohistochemistry, and for the presence of the 14;18 translocation using polymerase chain reaction (PCR) amplification with primers against both the major breakpoint region (mbr) and the minor cluster region (mcr) of bcl-2. For reasons of comparison, nine secondary cutaneous large B-cell lymphomas (SCLBCLs) were also studied. RESULTS Expression of bcl-2 protein was found in all nine PCLBCLs of the leg and in all nine SCLBCLs, but not in any of the 14 PCLBCLs on the head and trunk. The t(14;18) was only detected in two of seven SCLBCLs, but not in the five PCLBCLs of the leg or the eight PCLBCLs on the head or trunk studied. CONCLUSION The striking differences in bcl-2 expression between PCLBCL of the head or trunk and PCLBCL on the leg suggest that bcl-2 expression is site-related and may contribute to the different clinical behavior between these two groups of lymphomas. In addition, they underscore that PCLBCL on the head and trunk and PCLBCL on the leg are distinct disease entities, as recently recognized in the European Organization for Research and Treatment of Cancer (EORTC) classification for primary cutaneous lymphomas.

Expression of Fas and Fas-ligand in primary cutaneous T-cell lymphoma (CTCL): association between lack of Fas expression and aggressive types of CTCL

Background Fas (CD95; APO‐1) is a transmembrane protein that mediates apoptosis upon cross‐linking with Fas‐ligand (Fas‐L). Interaction of Fas‐L expressed by cytotoxic T cells with Fas‐expressing tumour cells plays an important part in antitumour immune responses.

Differential Expression of Thymus and Activation Regulated Chemokine and Its Receptor CCR4 in Nodal and Cutaneous Anaplastic Large-Cell Lymphomas and Hodgkin's Disease

Recent studies demonstrated that Hodgkin/Reed-Sternberg (H/RS) cells in Hodgkins disease (HD) express thymus and activation-regulated chemokine (TARC), whereas reactive lymphocytes surrounding H/RS cells express its ligand, CC-chemokine receptor 4 (CCR4). Because in vitro studies showed that CCR4 expression is a marker for lymphocytes bearing a T-helper 2 (Th2) phenotype, it was suggested that expression of TARC is a new immune escape mechanism in HD. To find out whether this mechanism might also be operative in CD30+ malignant lymphomas other than HD, TARC and CCR4 expression was investigated by immunohistochemistry on paraffin and frozen-tissue sections of 39 nodal CD30+ anaplastic large cell lymphomas (ALCL), including 27 ALK-negative and 12 ALK-positive ALCL, 25 primary cutaneous CD30+ ALCL, including 11 patients with lymphomatoid papulosis, and 31 cases of HD. TARC was expressed by the neoplastic cells in 12/27 (44%) nodal ALK-negative ALCL and all cases of classic HD, but not in nodal ALK-positive ALCL (0/12) and only rarely in primary cutaneous CD30+ ALCL (3/25). In contrast, CCR4 was expressed by the neoplastic cells in 9/9 cutaneous CD30+ ALCL, and in 9/15 (60%) nodal ALK-negative ALCL, but only in 1/4 (25%) nodal ALK-positive ALCL and not by the H/RS cells in HD (0/8). Apart from three cases of HD showing 10 to 15% CCR4-positive lymphocytes surrounding TARC-positive H/RS cells, CCR4-positive reactive T cells were few (<5%) in all other cases studied. Our results demonstrate a differential expression of TARC and CCR4 in different types of CD30+ malignant lymphomas. The small number of CCR4-positive reactive T cells in most cases studied argues against an important role of TARC expression in the evasion of antitumor responses.

Expression of killer cell inhibitory receptors is restricted to true NK cell lymphomas and a subset of intestinal enteropathy-type T cell lymphomas with a cytotoxic phenotype.

Background/Aims—Killer inhibitory receptors (KIR) have a modulating effect on the cytotoxic functions of natural killer (NK) cells and T cells. Because lymphoma cells often have the same receptors as their non-neoplastic counterparts, this study investigated the expression of KIR on well defined groups of NK and T cell lymphomas, with and without a cytotoxic phenotype, from different sites of origin. Methods—Nine CD56+/CD3− NK cell lymphomas, 29 CD3+/CD56− T cell lymphomas with a cytotoxic phenotype, and 19 T cell lymphomas without a cytotoxic phenotype were stained for KIR using monoclonal antibodies specific for CD94, CD158a, and CD158b. In addition, the expression of KIR was studied on normal lymphoid tissues. Results—KIR expression was seen in five of nine true NK cell lymphomas including three of four nasal, one of four cutaneous, and one of one intestinal lymphoma nasal type. Double staining for CD56 and CD94 in normal lymphoid tissues revealed that KIR was predominantly expressed by CD56+ NK cells and sporadically on CD8+ T cells. Moreover, enteropathy-type T cell lymphomas with a cytotoxic phenotype showed KIR expression (three cases expressing CD94 and one case expressing CD158a). All nodal and extranodal non-intestinal T cell lymphomas with or without a cytotoxic phenotype lacked expression of KIR. Conclusions—These results show that KIR expression is restricted to CD56+/CD3− true NK cell lymphomas originating from the nose, gut, and skin, as well as in a subset of extranodal T cell lymphomas originating from the small intestine, which possessed a cytotoxic phenotype. Thus, the presence of KIR on NK/T cell lymphomas seems to mimic the distribution of KIR found on NK and T cells in normal lymphoid tissue.

A cytotoxic phenotype does not predict clinical outcome in anaplastic large cell lymphomas

AIM: To investigate whether anaplastic large cell lymphomas (ALCL) expressing cytotoxic proteins have a relatively worse clinical outcome compared with ALCL lacking a cytotoxic phenotype. METHODS: 59 primary cases of ALCL originating from different sites were investigated by immunohistochemistry for the presence of the cytotoxic proteins T cell intracytoplasmic antigen (TIA-1) and granzyme B in the neoplastic cells. Since site of origin and expression of anaplastic lymphoma kinase (ALK) strongly influence prognosis, the presence of a cytotoxic phenotype was also investigated in relation to the primary site of origin (lymph node, gut, or skin) and ALK expression. The prognostic value was investigated by analysis of overall and relapse-free survival time, including Cox regression analysis. RESULTS: 39 of 59 ALCL (66%) appeared to have a cytotoxic phenotype as shown by expression of TIA-1 or granzyme B or both in the neoplastic cells. The presence of a cytotoxic phenotype did not have any influence on prognosis. Even when the survival data were corrected for site of origin and stage at presentation or were analysed separately for ALK positive and negative cases, no prognostic influence of a cytotoxic phenotype was observed. CONCLUSIONS: In primary biopsies of patients with ALCL, the presence of a cytotoxic phenotype is not related to clinical outcome of the disease.

Differentiation between actinic reticuloid and cutaneous T cell lymphoma by T cell receptor gamma gene rearrangement analysis and immunophenotyping.

AIMS: Differentiation between actinic reticuloid and cutaneous T cell lymphoma can be extremely difficult. Demonstration of clonal T cell receptor (TCR) gene rearrangements has been suggested as a potential diagnostic criterion, but the results obtained thus far have been conflicting. This study investigated whether TCR gamma gene rearrangement analysis, using polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) and immunohistochemistry, can serve as a diagnostic criterion. METHODS: PCR/DGGE was performed on skin, peripheral blood mononuclear cells, and/or lymph nodes of seven patients with actinic reticuloid, 11 patients with Sézary syndrome, and 15 patients with a benign form of erythroderma. The results of PCR/DGGE and Southern blot analysis of TCR beta gene rearrangements were compared. In addition, CD4:CD8 ratios in skin and peripheral blood samples were investigated. RESULTS: Clonal T cell populations were detected in 19 of 21 samples obtained from patients with Sézary syndrome but were not detected in any of the 12 samples from patients with actinic reticuloid. Clonal T cells were detected in the peripheral blood of only one of 15 patients with a benign form of erythroderma. PCR/DGGE and Southern blot analysis gave concordant results in 28 of 29 samples. Immunophenotypic analysis demonstrated increased proportions of CD8+ T cells in skin (seven of seven cases) and peripheral blood (four of seven cases) of patients with actinic reticuloid. CONCLUSION: The results of this study demonstrate that gene rearrangement analysis, in combination with immunohistochemistry, may be an important adjunct in differentiating between actinic reticuloid and cutaneous T cell lymphoma. In patients suspected of having actinic reticuloid, application of both techniques is recommended.

Epstein–Barr virus is present in neoplastic cytotoxic T cells in extranodal, and predominantly in B cells in nodal T non‐Hodgkin lymphomas

Epstein–Barr virus (EBV)‐positive T non‐Hodgkin lymphomas (T‐NHLs) have been described, but it is at present unknown how EBV infects T lymphocytes. It has been postulated that cytotoxic T cells (CTLs) or natural killer (NK) cells can be infected by EBV during the killing of an EBV‐infected target cell. The objective of this study was therefore to determine whether the neoplastic cells in EBV‐positive T‐NHLs (n=221) of various locations have a cytotoxic phenotype. To identify EBV‐harbouring cells, combinations were used of EBV‐encoded RNA (EBER) in situ hybridization (RISH) and immunohistochemistry for T‐ and B‐cell markers and the cytotoxic proteins TIA‐1 and granzyme B. EBV was detected in the neoplastic cells of all nasal T‐NHLs (n=9), 5/34 gastrointestinal (GI) T‐NHLs, and 2/6 lung T‐NHLs, but not in primary cutaneous T‐NHLs (n=103). Moreover, EBV was found in the neoplastic cells of 2/48 nodal anaplastic large cell lymphomas (ALCLs), but not in neoplastic T cells of other nodal T‐NHLs. However, 5/17 nodal peripheral T‐NHLs not otherwise specified (PTCLs NOS) and 1/4 T‐prolymphocytic leukaemias did contain EBV‐positive non‐T cells. Double staining revealed that in EBV‐positive extranodal T‐NHLs (n=16), the EBER‐positive cells had a cytotoxic phenotype (TIA‐1‐ and/or granzyme B‐positive). In nodal non‐ALCL T‐NHLs, the EBER‐positive cells were not positive for TIA‐1 or granzyme B, nor did they express CD3, CD21 or HECA452. Instead, most of these cells expressed the B‐cell marker CD20. These PTCLs NOS with EBER‐positive cells showed features of AILD‐like T‐NHL. It is concluded that neoplastic cells of EBV‐positive extranodal T‐NHLs always have a cytotoxic phenotype, supporting the view that EBV can infect CTLs. In nodal non‐ALCL T‐NHL, EBV is only found in T‐NHL with AILD‐like features and is present in B cells, where it may contribute to the outgrowth of a malignant B‐cell clone. Copyright

Analysis of helper activity on pokeweed mitogen- and interleukin 2-driven immunoglobulin synthesis by neoplastic T4+ cells.

The neoplastic T cells of a series of seven patients with chronic T-cell neoplasia were tested for helper activity on pokeweed mitogen (PWM)-induced and interleukin 2 (IL-2)-induced Ig synthesis. The neoplastic T cells of all patients had a T3+4+8-11+I1- phenotype but differed in expression of the 3A1 antigen. The neoplastic T cells of three patients had helper activity on both PWM- and IL-2-driven Ig synthesis, and in addition produced IL-2 in response to PWM stimulation. Two of these patients had hypergammaglobulinemia. In contrast, the neoplastic T cells in the remaining four patients did not produce IL-2 and did not support PWM-driven Ig synthesis. The T4+ cells of these four patients, however, provided excellent helper activity on IL-2-driven Ig synthesis. These findings emphasize the role of IL-2 in T cell-dependent Ig synthesis and clearly show that IL-2 production is required for helper activity in the PWM-driven system. It is concluded that the combined use of PWM- and IL-2-driven Ig synthesis systems allows separate analysis of IL-2 production and T-helper activity in health and disease.

Primary Cutaneous CD30 (KI-1) Positive Large Cell Lymphomas: Definition and Differential Diagnostic Aspects

Recent studies have suggested that primary cutaneous CD30 (Ki-1) positive T-large cell lymphomas represent a distinct type of cutaneous lymphoma with a favorable prognosis. Here the results of recent studies aimed to define this new type of cutaneous lymphoma more precisely are presented. The importance of differentiating these lymphomas from other CD30+ lymphoproliferative disorders that may occur in the skin is emphasized. Recent evidence suggesting that these primary cutaneous CD30+ large cell lymphomas are closely related to lymphomatoid papulosis is discussed.