Blood and lymph node T lymphocytes in cutaneous T cell lymphoma: evaluation by light microscopy.

Cytology of peripheral blood and lymph node lymphocytes from a group of unselected patients with cutaneous T cell lymphoma (CTCL) was studied by light microscopy. Twenty of 45 patients had circulating lymphocytes with convoluted nuclei recognized in routine Wright-Giemsa-stained peripheral blood smears. Cytocentrifuge preparations of E-rosetted lymphocytes showed that greater than 10% of the T cells had convoluted nuclei in each of 16 patients with positive blood smears and in six of 17 whose blood smears were negative or inconclusive. Peripheral blood involvement with greater than 10% convoluted T cells was most frequent in patients with erythroderma (100%) including those with normal of decreased lymphocyte counts, and was not uncommon in patients with mycosis fungoides in the plaque or tumor phase (42%). The light-microscopic morphology of the abnormal cells found in the patients with the plaque or tumor phase of mycosis fungoides was not distinguishable from that of the erythrodermic patients. Increased percentages (less than 15%) of T cells having convoluted nuclei were also found in the lymph node cell suspensions from CTCL patients with adenopathy (18 of 25 patients). These results suggest that a high frequency of extracutaneous involvement occurs in patient with CTCL, the clinical significance of which remains to be determined.     

HLA-G protein up-regulation in primary cutaneous lymphomas is associated with interleukin-10 expression in large cell T-cell lymphomas and indolent B-cell lymphomas.

Primary cutaneous lymphomas (CLs) constitute a spectrum of diseases characterized by a clonal accumulation of lymphocytes in the skin. Most CLs display a T(h)2 cytokine profile, including expression of interleukin-10 (IL-10). Because the up-regulation of HLA-G, a nonclassical class Ib molecule inducible by IL-10, might account for the immunescape of the malignant clone, HLA-G and IL-10 expression has been investigated in 45 cases of primary CL (10 of B-cell and 35 of T-cell origin) with quantitative polymerase chain reaction (PCR) and immunohistochemistry. HLA-G message was present in all cutaneous B-cell (CBCL) and T-cell (CTCL) lymphomas evaluated. Immunohistochemistry revealed HLA-G protein expression in 23 (51%) of 45 cases (7 of 10 CBCL, 16 of 35 CTCL). While in CBCL mostly indolent types displayed HLA-G positivity, in CTCL HLA-G expression was associated with high-grade histology and advanced stage of the disease. Except for neoplastic and infiltrating lymphocytes, other cells such as macrophages and dendritic cells showed HLA-G immunoreactivity. Furthermore, IL-10 protein expression was demonstrated in 16 (73%) of 22 HLA-G(+) cases, which correlated with HLA-G protein presence (P <.001). HLA-G up-regulation together with IL-10 expression in CL might additionally contribute to the evasion of immunosurveillance and facilitate the transition from low- to high-grade lymphomas.     

Prognostic significance of tumor burden in the blood of patients with erythrodermic primary cutaneous T-cell lymphoma

Erythrodermic cutaneous T-cell lymphoma (CTCL) includes patients with erythrodermic mycosis fungoides who may or may not exhibit blood involvement and Sézary syndrome and in whom hematological involvement is, by definition, present at diagnosis. These patients were stratified into 5 hematologic stages (H0-H4) by measuring blood tumor burden, and these data were correlated with survival. The study identified 57 patients: 3 had no evidence of hematologic involvement (H0), 8 had a peripheral blood T-cell clone detected by polymerase chain reaction (PCR) analysis of the T-cell receptor gene and less than 5% Sézary cells on peripheral blood smear (H1), and 14 had either a T-cell clone detected by Southern blot analysis or PCR positivity with more than 5% circulating Sézary cells (H2). Twenty-four patients had absolute Sézary counts of more than 1 x 10(9) cells per liter (H3), and 8 patients had counts in excess of 10 x 10(9) cells per liter (H4). The disease-specific death rate was higher with increasing hematologic stage, after correcting for age at diagnosis. A univariate analysis of 30 patients with defined lymph node stage found hematologic stage (P =.045) and lymph node stage (P =.013) but not age (P =.136) to be poor prognostic indicators of survival. Multivariate analysis identified only lymph node stage to be prognostically important, although likelihood ratio tests indicated that hematologic stage provides additional information (P =.035). Increasing tumor burden in blood and lymph nodes of patients with erythrodermic CTCL was associated with a worse prognosis. The data imply that a hematologic staging system could complement existing tumor-node-metastasis staging criteria in erythrodermic CTCL.     

Evaluation of circulating malignant cells provides prognostic information in cutaneous T cell lymphoma

Peripheral blood lymphocyte morphology was evaluated prospectively by light microscopy of blood smears and E rosette preparations in 160 patients with cutaneous T cell lymphoma (CTCL). Blood involvement was related to the type of cutaneous T-stage, being present in 90% of patients with erythroderma (T4), 27% of those with cutaneous tumors (T3), 9% of those with generalized (T2), and 0% of those with limited skin plaques (T1). Untreated patients with blood involvement (38 of 105) had a higher frequency of CTCL in lymph nodes and viscera and survival inferior to that of patients with normal or nondiagnostic lymphocyte morphology (P less than .001). Multivariate analysis showed skin stage and age to be the most important pretreatment risk factors for survival. Although blood involvement was not an independent risk factor for the entire group, it appeared to have some adverse influence in the T2/T3 subsets (P = .051). Both lymphocytosis and size distribution of the circulating CTCL cells at initial diagnosis influenced survival. Patients with "mixed cell" cytology (greater than 20% large [greater than 11 microns] CTCL cells), had a worse survival than those with predominantly small circulating CTCL cells (P = .009). The former were more likely to have aggressive features, including lymph node effacement by tumor (P less than .001) and visceral disease (P = .074), than were "small cell" patients. Our data indicate that detailed review of the blood lymphocyte morphology in patients with diagnosed or suspected CTCL is helpful in predicting extent of disease and prognosis.     

Diagnostic value of dominant T-cell clones in peripheral blood in 363 patients presenting consecutively with a clinical suspicion of cutaneous lymphoma

It is now widely accepted that polymerase chain reaction (PCR) analysis of cutaneous T-cell clonality is of diagnostic value in cutaneous T-cell lymphomas (CTCLs) and most helpful in the diagnosis of mycosis fungoides (MF). However, the diagnostic and prognostic value of circulating clonal T cells remains unclear. We studied T-cell clonality in the peripheral blood (PB) and the cutaneous lesion, sampled at the same time, in 363 consecutively seen patients with a clinical suspicion of cutaneous lymphoma. Using a PCR technique providing a specific imprint of T-cell clones (PCRgamma-denaturing gradient gel electrophoresis), we found that detection of identical circulating and cutaneous T-cell clones was associated with the diagnosis of CTCL (P <.001). Detection of circulating tumor cells in patients with MF was infrequent (12.5%), except in those with erythrodermic MF (42%; P =.003). Moreover, among the 46 patients who had identical circulating and cutaneous T-cell clones, 25 (56%) had erythroderma. The finding of a dominant clone in the PB but not in the skin was frequent, regardless of the clinicohistologic classification; it occurred in 30% of patients with CTCL, 41% with non-CTCL malignant infiltrates, and 34% with benign infiltrates. This pattern was significantly more frequent in patients over 60 years of age (P <.002), even in the CTCL group (P <. 01). In conclusion, dominant T-cell clones detected in the PB of patients with MF by using a routine PCR technique are rarely tumoral and are more often related to age. A multicenter prospective study is under way to establish the prognostic value of circulating tumor cells.     

High-scatter T cells: a reliable biomarker for malignant T cells in cutaneous T-cell lymphoma

In early-stage cutaneous T-cell lymphoma (CTCL), malignant T cells are confined to skin and are difficult to isolate and discriminate from benign reactive cells. We found that T cells from CTCL skin lesions contained a population of large, high-scatter, activated skin homing T cells not observed in other inflammatory skin diseases. High-scatter T (T(HS)) cells were CD4(+) in CD4(+) mycosis fungoides (MF), CD8(+) in CD8(+) MF, and contained only clonal T cells in patients with identifiable malignant Vβ clones. T(HS) cells were present in the blood of patients with leukemic CTCL, absent in patients without blood involvement, and contained only clonal malignant T cells. The presence of clonal T(HS) cells correlated with skin disease in patients followed longitudinally. Clonal T(HS) cells underwent apoptosis in patients clearing on extracorporeal photopheresis but persisted in nonresponsive patients. Benign clonal T-cell proliferations mapped to the normal low-scatter T-cell population. Thus, the malignant T cells in both MF and leukemic CTCL can be conclusively identified by a unique scatter profile. This observation will allow selective study of malignant T cells, can be used to discriminate patients with MF from patients with other inflammatory skin diseases, to detect peripheral blood involvement, and to monitor responses to therapy.     

Expression of three cell proliferation-associated antigens, DNA polymerase alpha, Ki-67 antigen and transferrin receptor in nodal and cutaneous T-cell lymphomas.

We studied the expression of three cell proliferation-associated antigens: DNA polymerase alpha, Ki-67 antigen, and transferrin receptor, in 35 T-cell lymphomas of nodal origin (T-NL) and 40 cutaneous T-cell lymphomas (CTCL). Immunohistochemical staining was carried out on frozen tissue sections of these specimens using three monoclonal antibodies, DAKO-PC, CL22-2-42B (DNA polymerase alpha), and OKT9. The proportion of cells positive for CL22-2-42B, DAKO-PC, or OKT9 among all tumor cells was correlated with four histologic subtypes: malignant lymphoma (ML), diffuse, small; mixed; large; and large cell immunoblastic in both T-NL and CTCL. A strong correlation was noted between positivity for CL22-2-42B and that for DAKO-PC or OKT9. On the other hand, no difference in the expression of these three antigens was noted between T-NL and CTCL in the high, intermediate or low grade-malignancy group. In CTCL as well as in T-NL, cells positive for CL22-2-42B, DAKO-PC or OKT9 were significantly more numerous in the high-grade group than the intermediate-grade group, and in the intermediate-grade group than the low-grade group. Furthermore, a significant correlation between survival period and the number of CL22-2-42B-positive cells was noted when the T cell malignancies, CTCL and T-NL were considered (t value = 2.015, p less than 0.05). Thus, the expression of DNA-polymerase alpha, Ki-67 antigen or OKT9 seems to well reflect the biological behavior and/or clinical prognosis of T-cell lymphoma.     

Quantitative PCR on 5 genes reliably identifies CTCL patients with 5% to 99% circulating tumor cells with 90% accuracy

We previously identified a small number of genes using cDNA arrays that accurately diagnosed patients with Sézary Syndrome (SS), the erythrodermic and leukemic form of cutaneous T-cell lymphoma (CTCL). We now report the development of a quantitative real-time polymerase chain reaction (qRT-PCR) assay that uses expression values for just 5 of those genes: STAT4, GATA-3, PLS3, CD1D, and TRAIL. qRT-PCR data from peripheral blood mononuclear cells (PBMCs) accurately classified 88% of 17 patients with high blood tumor burden and 100% of 12 healthy controls in the training set using Fisher linear discriminant analysis (FLDA). The same 5 genes were then assayed on 56 new samples from 49 SS patients with blood tumor burdens of 5% to 99% and 69 samples from 65 new healthy controls. The average accuracy over 1000 resamplings was 90% using FLDA and 88% using support vector machine (SVM). We also tested the classifier on 14 samples from patients with CTCL with no detectable peripheral involvement and 3 patients with atopic dermatitis with severe erythroderma. The accuracy was 100% in identifying these samples as non-SS patients. These results are the first to demonstrate that gene expression profiling by quantitative PCR on a selected number of critical genes can be employed to molecularly diagnosis SS.     

C-reactive protein and multiple complex coronary artery plaques in patients with primary unstable angina

The aim of this study was to investigate the possible association of plasma C-reactive protein (CRP) levels with the presence of angiographically multiple complex lesions (CLs) in patients with primary unstable angina (PUA). For the purpose of this study, 228 consecutive patients with PUA who underwent in-hospital catheterization were evaluated. Plasma CRP levels were measured upon patients' admission. Coronary plaques were classified as CL or non-CL according to Ambrose's criteria. There were 100 (43.9%) patients with no or one CL (/=2). Tertiles of plasma CRP levels upon admission were significantly associated with the number of CLs on angiographic studies. In particular there was a significant gradual increase in either the number of CLs, or the presence of apparently thrombus-containing CLs with increasing of CRP tertiles. By multivariate analysis CRP was independently associated with the presence of either multiple CLs (R.R.=1.8, 95%CI=1.5-2.2, P<0.001), or angiographically apparent thrombus-containing CLs (R.R.=1.4, 95%CI=1.2-1.7, P=0.03).High plasma levels of CRP may reflect a multifocal activation of the coronary tree in patients with PUA. This finding suggests a generalized inflammatory reaction throughout the coronary tree in these patients.     

High frequency of primary cutaneous lymphomas associated with lymphoproliferative disorders of different lineage

In patients suffering from primary cutaneous lymphomas, secondary malignancies of various origin may develop. However, the frequency of a second neoplasm deriving from another lymphoid lineage is still unclear and may be underestimated. We screened all our patients with primary cutaneous lymphomas from a 4-year recruitment period for a coexisting secondary lymphoproliferative disorder. The cohort comprised of a total of 82 patients with primary cutaneous lymphomas, 62 with primary cutaneous T-cell lymphoma (CTCL), 18 with primary cutaneous B-cell lymphomas, and two with CD4+/CD56+ hematodermic neoplasm/blastic lymphomas. Seven patients (8.5%) were identified with a coexisting lymphoma of a different lymphoid lineage. Four patients with Sézary syndrome (SS) suffered from systemic B-cell lymphoma. Two of these developed SS after chemotherapy of their B-cell lymphoma. The other three patients with various types of skin lymphomas (SS, Mycosis fungoides [MF], primary cutaneous marginal zone lymphoma) developed Hodgkin’s disease (hairy cell leukemia). Our data indicate that patients with primary cutaneous lymphomas have an elevated risk for the development of a secondary lymphoproliferative disorder even without previous chemotherapy. Possible explanations for this association include a genetic predisposition, alterations in early progenitor cells, underlying viral infections, and/or stimulation of a B-cell clone by the malignant helper T cells of the primary CTCL and vice versa.     

Photopheresis — Extracorporeal irradiation of 8-MOP containing blood — A new therapeutic modality

Summary In the course of the last four years our group has had experience with the development of an exciting new concept for the treatment of the exfoliative erythrodermic form of cutaneous T cell lymphoma (CTCL), leukemic form, as well as other T cell mediated diseases: extracorporeal photopheresis (EP). In our first experience with this new therapeutic approach, clinical response was observed in 6 of the 7 patients studied. Skin pathology improved significantly so that 5 of 7 patients were able to resume their normal daily activities. Laboratory response was significant in all 7 as measured by a decrease in abnormal T lymphocytes. In spite of an individually adjusted maintenance therapy no significant side effects have been observed. Our initial observations with this new and apparently safe approach for treatment of CTCL and other T cell mediated diseases (e. g. pemphigus vulgaris) suggest that further more extensive clinical trials as well as research into the basic underlying mechanisms involved are warranted.Presented at the 32nd Annual Meeting of the German Society for Hematology and Oncology, October 1986R. M. Knobler was a recipient of the Max Kade Foundation Fellowship     

CD4(+) cutaneous T-cell lymphoma cells express the p140-killer cell immunoglobulin-like receptor

Tumor cells of patients with cutaneous T-cell lymphoma (CTCL) have the cell surface phenotype of mature T-helper lymphocytes, and it may be impossible to differentiate them from nonmalignant lymphocytes in skin and blood. Until now, no specific cell membrane marker of CTCL has been reported. In the current study, it is reported for the first time that CTCL cells express the major histocompatibility complex class I binding p140-killer cell immunoglobulin-like receptor, which has been described on a minor subset of natural killer lymphocytes and on a marginal circulating CD8(+) T lymphocyte subset. Interestingly, the molecular characterization of this KIR expressed by CTCL allowed us to isolate a novel allelic form of p140-KIR3DL, resulting in 4 amino acid substitutions, 3 in the extracellular immunoglobulin-like domain of the protein and one in the cytoplasmic region. This finding is likely to be important both for the pathophysiology and for the clinical treatment of patients with CTCL.     

Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients. The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth-promoting effect depends on direct cell-cell contact and soluble factors such as interleukin-2. In conclusion, we demonstrate that SE triggers a bidirectional cross talk between nonmalignant T cells and malignant CTCL cells that promotes growth of the malignant cells. This represents a novel mechanism by which infections with SE-producing bacteria may contribute to pathogenesis of CTCL.     

Mycosis fungoides

Mycosis fungoides (MF) constitutes the most frequent cutaneous T-cell lymphoma. Sezary syndrome is considered by some authors to be an erythrodermic leukemic variant of MF, but is classified separately in the new WHO-EORT classification of cutaneous lymphomas. MF usually occurs in old adults with a 2:1 male to female ratio. Its prognosis is variable and strongly conditioned by the extent and type of skin involvement and presence of extracutaneous disease. Patients with stage IA-disease have an excellent prognosis with an overall long-term life expectancy that is similar to an age-, sex-, and race-matched control population. Almost all patients with stage IA MF will die from causes other than MF, with a median survival >33 years. Only 9% of these patients will progress to more extended disease. Patients with stage IB or IIA have a median survival greater than 11 years. These patients with T2 disease have a likelihood of disease progression of 24% and nearly 20% die of MF. Subgroups with stage IB or IIA have similar prognosis. Patients with cutaneous tumors or generalized erythroderma have a median survival of 3 and 4.5 years, respectively. The majority of these patients will die of MF. Extracutaneous dissemination is observed in less than 10% of patients with patch or plaque disease and in 30-40% of patients with tumors or generalized erythrodermatous involvement. Extracutaneous involvement is directly correlated to the extent of cutaneous disease. The most commonly involved organs are lung, spleen, liver, and gastrointestinal tract. Patients with extracutaneous disease at presentation involving either lymph nodes or viscera have a median survival of <1.5 years. Patients with plaque-type or erythrodermic MF may develop cutaneous tumors with large cell histology, often expressing CD30, which share a common clonal origin as observed in their preexisting MF and are associated with a less favourable outcome.     

In vitro growth of cutaneous T-cell lymphomas.

It is relatively easy to propagate EB virus-transformed normal and malignant B lymphocytes in vitro. Normal T lymphocytes divide for short periods of time after exposure to many mitogens. Exposure of normal T lymphocytes to lymphocyte-conditioned medium (LCM) permits them to divide for longer periods of time, but permanent cell lines cannot be established. Intial attempts to grow malignant T cells from patients with cutaneous T-cell lymphomas (CTCL) in unsupplemented growth medium resulted in the establishment of four EB virus-transformed B-lymphoblastoid lines. The responses of CTCL cells to mitogens and LCM varied from unresponsive to near normal. Subsequent attempts to grow CTCL cells in medium supplemented with mitogens or LCM resulted in the establishment of two cell lines lacking B-cell markers or EB virus. The cells of both lines initially had the ability to form E rosettes, although one of the lines has lost this ability with passage. The two lines can be distinguished from normal T cells by having some or all of the following properties: long-term proliferation, tumorigenecity in nude mice, gradual loss of dependence on mitogen or LCM, and convoluted nuclear morphology.     

Enhancement of the host immune responses in cutaneous T-cell lymphoma by CpG oligodeoxynucleotides and IL-15

Patients with advanced cutaneous T-cell lymphoma (CTCL) exhibit profound defects in cell-mediated immunity. Host immune functions appear to play an integral role in mediating disease-controlling responses in CTCL, therefore we investigated the effects of synthetic oligode-oxynucleotides with CpG motifs (CpG ODN), which have been recognized as immune stimulatory by virtue of activation of dendritic cells (DCs) following binding to Toll-like receptor (TLR) 9. Peripheral blood mononuclear cells (PBMCs) from patients with advanced CTCL (erythroderma with circulating malignant T cells) and healthy volunteers were cultured with either CpG-A or CpG-B ODN. Patients' PBMCs exhibited marked induction of interferon-alpha (IFN-alpha) release following culture with CpG-A. Similarly significant activation of NK cells and CD8 T cells occurred as assessed by up-modulation of CD69 expression and by natural killer lytic activity. Nevertheless, the PBMCs of patients exhibited blunted responses to CpG-A compared to healthy volunteers. In such cases, IL-15 was capable of producing levels of NK activation that were superior to CpG-A, while the combined effects of CpG-A plus IL-15 induced maximal activation of NK cells and further enhanced activation of CD8 T cells. These findings have important implications for the potential enhancement of antitumor immunity among patients with advanced CTCL.     

Experimental treatment of chronic lymphocytic leukemia with extracorporeal photochemotherapy

Summary Based on the encouraging results obtained with extracorporeal photochemotherapy (EP) in the treatment of the exfoliative erythrodermic form of cutaneous T-cell lymphoma (CTCL), leukemic form, as well as other T-cell-mediated diseases we evaluated the therapeutic potential of EP in patients with chronic lymphocytic leukemia (B-CLL). Three patients with B-CLL were treated for a period of 1 year. Two patients showed stabilization of disease, as demonstrated by reduction in their peripheral white blood cell count, with one patient showing lymph-node resolution. A third patient with significant intolerance to previous chemotherapy did not respond within the observed period. No significant side effects of EP were observed. Our observations suggest that EP may have a positive effect on the course of B-CLL in selected patients. Additional clinical trials are warranted to further define the role of EP alone or in combination therapy in the management of B-CLL.     

The growth of cutaneous T-cell lymphoma is stimulated by immature dendritic cells

In the initial stage of cutaneous T-cell lymphoma (CTCL), proliferating CTCL cells are concentrated in the epidermis in close association with an immature dendritic cell (DC), the Langerhans cell. Because long-term in vitro culture of CTCL cells has proven difficult, the in vivo association with the major antigen-presenting cell (APC) of the epidermis has been postulated to play a role in directly stimulating the clonal T-cell proliferation. We report that CTCL cells can be reproducibly grown in culture for 3 months when cocultured with immature DCs. CTCL cells retain the phenotype and genotype of the initial malignant clone, whereas the APCs are a mixture of immature and mature DCs. CTCL cell and DC survival was dependent on direct membrane contact. Growth was inhibited by antibodies that bound to the T-cell receptor (TCR) or interfered with the interaction of CD40 with its ligand on the CTCL cell. Addition of antibody to CD3 or the clonotypic TCR caused rapid CTCL cell apoptosis followed by engulfment by avidly phagocytic immature DCs and subsequent DC maturation. The opportunity to study CTCL cells and immature DCs for prolonged periods will facilitate studies of tumor cell biology and will allow investigation of the intriguing hypothesis that CTCL cell growth is driven through TCR recognition of class II-presented self-peptides. In addition, the culture of CTCL cells will permit evaluation of therapies in vitro before clinical intervention, thereby improving safety and efficacy.