In vitro effects of wound-dressings on key wound healing properties of dermal fibroblasts.

Healing of complex wounds requires dressings that must, at least, not hinder and should ideally promote the activity of key healing cells, in particular fibroblasts. This in vitro study assessed the effects of three wound-dressings (a pure Ca2+ alginate: Algostéril®, a Ca2+ alginate + carboxymethylcellulose: Biatain alginate® and a polyacrylate impregnated with lipido-colloid matrix: UrgoClean®) on dermal fibroblast activity. The results showed the pure calcium alginate to be non-cytotoxic, whereas the other wound-dressings showed moderate to strong cytotoxicity. The two alginates stimulated fibroblast migration and proliferation, whereas the polyacrylate altered migration and had no effect on proliferation. The pure Ca2+ alginate significantly increased the TGF-ß-induced fibroblast activation, which is essential to healing. This activation was confirmed by a significant increase in Vascular endothelial growth factor (VEGF) secretion and a higher collagen production. The other dressings reduced these fibroblast activities. The pure Ca2+ alginate was also able to counteract the inhibitory effect of NK cell supernatants on fibroblast migration. These in vitro results demonstrate that tested wound-dressings are not equivalent for fibroblast activation. Only Algostéril was found to promote all the fibroblast activities tested, which could contribute to its healing efficacy demonstrated in the clinic.

The value of five blood markers in differentiating mycosis fungoides and Sézary syndrome: a validation cohort.

BACKGROUND: Clinical and histological diagnosis of Sézary syndrome (SS) and mycosis fungoides (MF) is challenging in clinical routine. OBJECTIVES: We investigated five blood markers previously described for SS (T-plastin, Twist, KIR3DL2, NKp46 and Tox) in a prospective validation cohort of patients. METHODS: We included 447 patients in this study and 107 patients were followed up for prognosis. The markers were analysed by reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) on peripheral blood leucocytes and CD4+ T cells in a cohort of consecutive patients with early MF, erythrodermic MF and SS and compared with patients presenting with benign inflammatory dermatoses (BID) and erythrodermic BID. The markers were assessed in parallel to gold standard values such as CD4/CD8 ratio, loss of CD7 and CD26 membrane expression and CD4 absolute values. Sensitivity and specificity were analysed by receiver operator characteristic curves. The prognostic value of selected markers was analysed on a subset of patients. This study was conducted in one centre. RESULTS: We defined cut-off values for each marker. T-plastin, Twist and KIR3DL2 had the best validity. SS may be overrepresented. The combination of T-plastin and Twist was able to differentiate between erythrodermic MF or BID and SS. The additional analysis of KIR3DL2 may be useful to predict the prognosis. CONCLUSIONS: We propose T-plastin, Twist and KIR3DL2 measured by RT-qPCR as new diagnostic markers for Sézary syndrome.

Revisiting the initial diagnosis and blood staging of mycosis fungoides and Sézary syndrome with the KIR3DL2 marker.

BACKGROUND: The early diagnosis of Sézary syndrome (SS) is challenging. Loss of CD7 and CD26 expression on CD4+ T cells is the currently used criterion in the initial diagnosis and staging of patients with SS. OBJECTIVES: Our aim was to evaluate the respective value of CD26, CD7 and KIR3DL2 expression on CD4+ T cells and total lymphocytes at initial diagnosis of SS. METHODS: This prospective study included 254 patients with clinical features consistent with cutaneous T-cell lymphoma seen at our institution between March 2014 and February 2019. Peripheral blood analysis by flow cytometry was performed for each patient at the time of diagnosis and during follow-up. The diagnosis of SS was based on ISCL/EORTC criteria. RESULTS: The presence of KIR3DL2+ Sézary cells (SCs) ≥ 200 µL-1 correlated with the diagnosis of SS, with sensitivity of 88·6% and specificity of 96·3%. All 154 patients with either inflammatory skin disease or other haematological disease had KIR3DL2+ cells < 200 µL-1 , while eight of them had CD4+ CD26- T cells ≥ 1000 µL-1 . Of five patients with SS and lymphopenia, four had CD4+ CD7- T cells < 1000 µL-1 and three had CD4+ CD26- T cells < 1000 µL-1 . However, all of them had KIR3DL2+ CD4+ T cells ≥ 200 µL-1 . Among patients with available samples during evolution, all B1-staged patients with ≥ 200 µL-1 KIR3DL2+ SCs at diagnosis evolved to B2 stage within 7 months. CONCLUSIONS: KIR3DL2 expression on T cells is highly specific and helps the early diagnosis of SS, especially in those patients with lymphopenia. What's already known about this topic? In the ISCL/EORTC cutaneous T-cell lymphoma (CTCL) categorization of blood involvement (B0-B2), B2 is defined as a T-cell receptor clonal rearrangement in blood, associated with high blood-smear Sézary cell (SC) count. Flow cytometry was developed to circumvent interobserver variability of SC manual counts; however, it mostly relies on detection of cells lacking CD7 and/or CD26 expression. We previously reported the reliability of KIR3DL2 as the first positive SC marker. What does this study add? Based on our analysis of 254 patients, we propose that KIR3DL2 be added to the ISCL/EORTC criteria for initial diagnosis of Sézary syndrome (SS) and B2 staging. This marker improved sensitivity of SS B2-stage CTCL diagnosis with a specificity > 95%, especially for patients with lymphopenia. We found KIR3DL2 helped early diagnosis of SS and was more reliable than CD26 in assessing blood tumour burden during therapy. What is the translational message? SC quantification is the major means of staging at initial diagnosis and monitoring blood tumour burden in a clinical trials setting. We recommend using a threshold value of KIR3DL2+ SCs ≥ 200 µL-1 or KIR3DL2+ SCs/lymphocytes ≥ 10% in the diagnostic criteria of SS and propose a novel algorithm for CTCL B2 blood staging.

Cutaneous presentation of adult T-cell leukemia/lymphoma (ATLL). Single-center study on 37 patients in metropolitan France between 1996 and 2016.

INTRODUCTION: Adult T-cell leukemia/lymphoma (ATLL) is a hematological malignancy associated with chronic HTLV-1 infection. AIM: To describe skin lesions in ATLL. METHODS: A descriptive, retrospective study between 1996 and 2016, including all patients diagnosed with ATLL at Saint-Louis Hospital (Paris, France). RESULTS: Thirty-seven ATLL patients were included. Fifteen patients (41%) had a cutaneous localization of the disease, which was present from the beginning of the disease for two thirds of them. ATLL types in patients with cutaneous localization of the disease were as follows: lymphoma, n=5, chronic, n=4, smoldering, n=4, acute, n=2. Half the patients had 2 or more cutaneous manifestations. The cutaneous localizations observed were as follows: nodulotumoral (n=8), plaques (n=7), multipapular (n=6), macular (n=4), purpuric (n=2). Among the 15 patients with cutaneous localization, median overall survival was significantly shorter in the acute and lymphoma types compared to the smoldering and chronic types (8.7 months vs. 79 months, P=0.003). DISCUSSION: ATLL is a hematologic malignancy with variable expression that is diagnosed only very rarely in metropolitan France, but that should be sought in patients from countries with high HTLV-1 prevalence in the event of a chronic eruption with patches, papules, plaques and/or tumors. The chronic and smoldering types are relatively indolent, whereas the acute and lymphoma forms have a poor prognosis.

PDE4D promotes FAK-mediated cell invasion in BRAF-mutated melanoma.

The cyclic AMP (cAMP) signaling pathway is critical in melanocyte biology for regulating differentiation. It is downregulated by phosphodiesterase (PDE) enzymes, which degrade cAMP itself. In melanoma evidence suggests that inhibition of the cAMP pathway by PDE type 4 (PDE4) favors tumor progression. For example, in melanomas harboring RAS mutations, the overexpression of PDE4 is crucial for MAPK pathway activation and proliferation induced by oncogenic RAS. Here we showed that PDE4D is overexpressed in BRAF-mutated melanoma cell lines, constitutively disrupting the cAMP pathway activation. PDE4D promoted melanoma invasion by interacting with focal adhesion kinase (FAK) through the scaffolding protein RACK1. Inhibition of PDE4 activity or inhibition of PDE4D interaction with FAK reduced invasion. PDE4D expression is increased in patients with advanced melanoma and PDE4D-FAK interaction is detectable in situ in metastatic melanoma. Our study establishes the role of PDE4D in BRAF-mutated melanoma as regulator of cell invasion, and suggests its potential as a target for preventing metastatic dissemination.

Identification of CD245 as myosin 18A, a receptor for surfactant A: A novel pathway for activating human NK lymphocytes.

CD245 is a human surface antigen expressed on peripheral blood lymphocytes, initially delineated by two monoclonal antibodies DY12 and DY35. Until now, CD245 molecular and functional characteristics remained largely unknown. We combined immunological and proteomic approaches and identified CD245 as the unconventional myosin 18A, a highly conserved motor enzyme reported as a receptor for the surfactant protein A (SP-A), that plays a critical role in cytoskeleton organization and Golgi budding. We report that the recruitment of CD245 strongly enhanced NK cell cytotoxicity. Further, we show that the enhancement of the NK lymphocytes killing ability toward CD137-ligand expressing target cells could result from the induction of CD137 expression following CD245 engagement. The SP-A receptor could therefore represent a novel and promising target in cancer immunotherapy.

KIR3DL2 (CD158k) is a potential therapeutic target in primary cutaneous anaplastic large-cell lymphoma.

BACKGROUND: KIR3DL2, an inhibitory receptor expressed by natural killer cells and a subset of normal CD8(+) T cells, is aberrantly expressed in neoplastic cells in transformed mycosis fungoides and Sézary syndrome. Anti-KIR3DL2 targeted antibody therapy has shown potent activity in preclinical models for these diseases. OBJECTIVES: To examine the expression of KIR3DL2 and its potential use as a therapeutic target in patients with primary cutaneous anaplastic large-cell lymphoma (pcALCL), the most aggressive cutaneous CD30(+) lymphoproliferative disease. METHODS: Samples from 11 patients with pcALCL and three CD30(+) lymphoproliferative disease cell lines - Mac1, Mac2a and Mac2b - were used in KIR3DL2 expression studies using immunohistochemistry, flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. The effect of IPH4102, a monoclonal humanized IgG1 targeting KIR3DL2, was assessed by in vitro cytotoxicity assays against Mac1, Mac2a and Mac2b using allogeneic peripheral blood mononuclear cells as effectors. RESULTS: KIR3DL2 mRNA and protein were found in all human samples of pcALCL, and in the Mac2a and Mac2b cell lines. KIR3DL2 protein expression was present on 85·8 ± 14·0% of CD30(+) skin-infiltrating tumour cells. In vitro functional studies showed that KIR3DL2(+) Mac2a and Mac2b pcALCL lines are sensitive to antibody-derived cytotoxicity mediated by IPH4102, through activation of natural killer cells, in a concentration-dependent manner. CONCLUSIONS: pcALCL tumour cells express KIR3DL2, and we provide preclinical proof of concept for the use of IPH4102, a humanized anti-KIR3DL2 antibody, to treat patients with primary cutaneous CD30(+) ALCL.

Hypoxia and MITF regulate KIT oncogenic properties in melanocytes.

KIT mutations are frequent in acral, mucosal and chronic sun-damage (CSD) melanoma, but little is known about the mechanisms driving the transformation of KIT-mutated melanocytes into melanoma cells. We showed that exposition of melanocytes harboring the (L576P)KIT mutation to a hypoxic environment induced their transformation into malignant cells. Transformed (L576P)KIT melanocytes showed downregulation of MITF expression characteristic of melanoma initiating cells (MICs). In agreement, these cells were able to form spheres in neural crest cell medium and low-adherence conditions, also a characteristic of MICs. Downregulation of MITF by RNA interference induced transformation of KIT-mutated melanocytes in normoxia and acquisition of a MIC phenotype by these cells. Hence, low level of MITF cooperates with oncogenic KIT to transform melanocytes. Activation of the cAMP pathway in transformed (L576P)KIT melanocytes stimulated MITF expression, and reduced cellular proliferation and sphere formation. These findings highlight the essential role of MITF in revealing the oncogenic activity of KIT in melanocytes and suggest that the cAMP pathway is a therapeutic target in KIT-mutated melanoma.

Long-term efficacy and safety of alemtuzumab in advanced primary cutaneous T-cell lymphomas.

BACKGROUND: Alemtuzumab has been proposed as salvage therapy for refractory cutaneous T-cell lymphomas (CTCLs). Long-term follow-up data are scarce. OBJECTIVES: To assess the efficacy and safety of alemtuzumab in the treatment of advanced CTCL. METHODS: A multicentre retrospective analysis was carried out of 39 patients with advanced CTCL treated with alemtuzumab between 2003 and 2013. RESULTS: Thirty-nine patients (median age 62 years, range 20-83) with Sézary syndrome (SS, n = 23) or advanced mycosis fungoides (MF, n = 16) received alemtuzumab 30 mg two to three times per week for a median duration of 12 weeks (range 1-35). Fifteen patients received maintenance therapy for a median duration of 24 weeks (range 6-277). Eleven patients (28%) had transformed disease (MF, n = 10; SS, n = 1). After a median follow-up of 24 months (range 0.3-124), eight patients (21%) were still alive. The overall response rate was 51% in the whole study group (partial response, n = 13; complete response, n = 7); 70% in patients with SS and 25% in patients with MF (P = 0.009). The median time to progression was 3.4 months (range 0.4-42). Six patients (15%; SS, n = 5; MF, n = 1) remained progression free for > 2 years (median 56 months, range 28-117). Five patients experienced cutaneous large T-cell transformation during alemtuzumab treatment and one patient developed primary cutaneous large B-cell lymphoma. Twenty-four patients (62%) had a grade three or higher infectious adverse event and 10 (26%) a haematological toxicity, which led to treatment discontinuation in 17 cases (44%) and death in two (5%). CONCLUSIONS: Alemtuzumab may induce long-term remission in SS but seems ineffective in MF and transformed CTCL.

ENTPD1/CD39 is a promising therapeutic target in oncology.

Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells that are essential for maintaining the homeostasis of the immune system, limiting self-reactivity and excessive immune responses against foreign antigens. In cancer, infiltrated Tregs inhibit the effector lymphocytes and create a favorable environment for the growth of the tumor. Although Tregs mediate immunosuppression through multiple, non-redundant, cell-contact dependent and independent mechanisms, a growing body of evidence suggests an important role for the CD39-CD73-adenosine pathway. CD39 ectonucleotidase is the rate-limiting enzyme of a cascade leading to the generation of suppressive adenosine that alters CD4 and CD8 T cell and natural killer cell antitumor activities. Here, we review the recent literature supporting CD39 as a promising therapeutic target in oncology. In vitro and in vivo experiments involving knockout models and surrogate inhibitors of CD39 provide evidence in support of the anticancer activity of CD39 inhibition and predict a favorable safety profile for CD39 inhibitory compounds. In addition, we report the ongoing development of CD39-blocking monoclonal antibodies as potential anticancer drugs. Indeed, CD39 antagonistic antibodies could represent novel therapeutic tools for selectively inhibiting Treg function without depletion, a major limitation of current Treg-targeting strategies.

[New developments in Sézary syndrome]. / Actualités sur le syndrome de Sézary.

Sézary syndrome (SS) represents 3% of cutaneous T-cell lymphomas (CTCL). It is an aggressive epidermotropic CTCL with a 5-year survival rate of 24%. According to EORTC (European organization for research and treatment of cancer), SS is defined by erythroderma, diffuse lymphadenopathy, atypical T lymphocytes (>1000/mm(3)), and the presence of a major blood, cutaneous and nodal T cell clone. A specific marker for atypical tumoral T lymphocytes known as Sézary cells was identified in 2001, namely KIR3DL2 (CD158k) receptor, which allows more specific diagnosis of SS; levels of this marker are highly correlated with the clinical course of the disease. In therapeutic terms, clinical trials are being conducted on new molecules that point towards an improved prognosis for this disease. We propose a review of Sézary syndrome, which is currently the subject of scientific papers concerning both physiopathology and therapeutics, with new prospects of targeted therapy.

c-Kit mutants require hypoxia-inducible factor 1alpha to transform melanocytes.

Many studies have highlighted the critical role of c-Kit in normal melanocyte development but its role in melanoma development remains unclear. Although c-Kit expression is often lost during melanoma progression, a subset of melanoma has been found to overexpress c-Kit and mutations activating c-Kit have recently been identified in some acral and mucosal melanoma. To address the role of these c-Kit mutants in the transformation of melanocytes, we characterized the physiological responses of melanocytes expressing the most frequent c-Kit mutants found in melanoma (K642E and L576P) and a novel mutant we identified in an acral melanoma. We analysed signaling pathways activated downstream of c-Kit and showed that all three mutants led to a strong activation of the phosphatidyl-inositol-3 kinase (PI3K) pathway but only weak activation of the Ras/Raf/Mek/Erk pathway, which was not sufficient to promote uncontrolled melanocyte proliferation and transformation. However, in hypoxic conditions or coexpressed with a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha), c-Kit mutants activate the Ras/Raf/Mek/Erk pathway, stimulate proliferation and transform melanocytes. Proliferation of melanocytes transformed by these mutants was specifically inhibited by imatinib. These results show for the first time that melanocytes require a specific epigenetic environment to be transformed by c-Kit mutants and highlight a distinct molecular mechanism of melanocyte transformation.

Absolute CD3+ CD158k+ lymphocyte count is reliable and more sensitive than cytomorphology to evaluate blood tumour burden in Sézary syndrome.

Background CD158k/KIR3DL2 is a specific marker for Sézary cells which can be used to diagnose Sézary syndrome (SS) in erythrodermic patients with abnormal circulating T cells. Objectives To evaluate the suitability of CD158k/KIR3DL2 for detecting and evaluating blood tumour load during the follow up of patients with SS. Methods The absolute CD3+ CD158k+ lymphocyte count was compared with the absolute count of cytomorphological Sézary cells and was correlated with clinical flares in a cohort of patients with SS. Twenty-five patients were included in the study and 48 blood samples were analysed. Results The absolute count of CD3+ CD158k+ cells strongly correlated with the absolute count of atypical circulating cells (r = 0.97, P < 10(-15)). The CD3+ CD158k+ lymphocyte cell count was in eight cases more sensitive than cytomorphology for detecting atypical circulating cells especially for small-sized tumour cells. The tumour burden evaluated by CD3+ CD158k+ immunostaining was significantly associated with clinical flare (P < 10(-4)). Conclusions CD3+ CD158k+ phenotyping is a reliable and objective test to monitor the blood tumour burden in patients with SS under systemic therapy.

Identification and analysis of the human CD160 promoter: implication of a potential AML-1 binding site in promoter activation.

CD160 is a glycosylphosphatidylinositol-anchored multimer expressed at the cell surface of subsets of cytotoxic T-lymphocytes and natural killer cells. Although CD160 is an important molecule for the control of cell-mediated cytotoxic responses, the mechanisms of regulation of its expression is unknown. We investigated the regulation of CD160 transcription by localizing and analyzing its promoter. The CD160 gene is encoded on chromosome 1, contains 6 exons with the translation initiation codon in exon 3. Bioinformatics analysis pointed to three potential promoter regions, two upstream of exon 1 and one in front of exon 3. RACE-PCR analysis identified a single transcription start site (TSS) and reporter gene transfections localized the active region immediately upstream of exon 1. Sequential deletion analysis led to the identification of a 371 bp sequence, located between -314 and +57 relative to the TSS, as the core promoter sequence driving CD160 gene transcription. Sequence alignment of the mouse and human CD160 genomic promoter region revealed a strong homology in the 371 bp sequence identified and pointed out to three conserved transcription factor binding sites for acute myelogenous leukemia-1 (AML-1), FREAC-4 and Sox17. Site-directed mutagenesis showed that the predicted AML-1 site is essential for the regulation of CD160 gene expression.

Involvement of IFNbeta on IFNgamma and nitric oxide (NO) production by bone marrow (BM) cells in response to lipopolysaccharide.

Bone marrow (BM) cells fractioned in Percoll gradients yield a low-density fraction (Fr3) highly enriched in suppressor activity. Previously, it has been demonstrated that BM associated suppressor activity was mediated by early myeloid cells, through a mechanism dependent on endogenous IFNgamma and nitric oxide production after bacterial stimuli, e.g. lipopolysaccharide (LPS). However, the mechanism(s) through which the IFNgamma is produced in BM has not yet been fully elucidated. Therefore, in the present study we investigated the involvement of IL-12, IL-18 and IFNbeta on the production of IFNgamma and nitric oxide in cultures of BM Fr3 cells, and characterized the IFNgamma-producing cells, in response to LPS. The results show that both IL-12 and IFNbeta, but not IL-18, are involved on IFNgamma production. However, only IFNbeta appears to be critical on nitric oxide production. Furthermore, we found that cells of the Thy1.2+CD3+ phenotype produce IFNgamma and are tightly involved on nitric oxide production by BM Fr3 cells. In conclusion, IFNbeta appears to be critical on IFNgamma- and nitric oxide production by BM cells in response to LPS, through a mechanism that is dependent on Thy1.2+CD3+ IFNgamma-producing cells.

Protocadherin 15 (PCDH15): a new secreted isoform and a potential marker for NK/T cell lymphomas.

Natural killer cells are well known to play an important role in immune defense against tumor development and viral infections. To further characterize new functionally relevant structures in these cells, we studied a series of monoclonal antibodies that we have raised against the NK cell line YT. One of these antibodies previously described as AY19, recognizes a 85 kD surface glycoprotein. Here we report the identification of a new secreted isoform of protocadherin 15, PCDH15C, which represents a potential associated protein for p85. Importantly, whereas protocadherins are absent from the surface of normal hematopoietic cells, we describe, for the first time, that PCDH15 is expressed in cytotoxic tumor-derived T- and NK-cell lines as well as in biopsies of nasal NK/T-cell lymphomas.

BCL2 and JUNB abnormalities in primary cutaneous lymphomas.

BACKGROUND: BCL2 is upregulated in nodal and extranodal B-cell non-Hodgkin's lymphomas, with a consequent antiapoptotic effect. However, loss of BCL2 has also been noted in some malignancies, suggesting a different molecular pathogenesis. OBJECTIVES: To investigate genomic and protein expression status of BCL2 and to compare the results with that of JUNB in primary cutaneous lymphomas (PCLs). METHODS: We analysed gene copy number of BCL2 and JUNB in 88 DNA samples from 80 patients with PCL consisting of Sézary syndrome/mycosis fungoides (SS/MF), primary cutaneous B-cell lymphoma (PCBCL) and primary cutaneous CD30+ anaplastic large cell lymphoma (C-ALCL) by the use of real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). Real-time PCR and IHC findings were subsequently compared with the results of additional fluorescent in situ hybridization (FISH) analysis of 23 cases of SS and Affymetrix cDNA expression microarray study of two primary cutaneous T-cell lymphoma (CTCL) cell lines. RESULTS: Real-time PCR analysis showed loss of BCL2 gene copy number in 22 of 80 PCL cases (28%), including 17 of 42 SS/MF, three of 13 C-ALCL and two of 33 PCBCL samples, and gain of BCL2 in four PCBCL samples. Gain of JUNB was identified in 18 of 71 PCL cases (25%), including nine of 35 SS/MF, seven of 13 C-ALCL and two of 31 PCBCL samples. IHC analysis revealed absent nuclear expression of BCL2 protein in 47 of 73 PCL cases, comprising 28 of 36 SS/MF, eight of eight C-ALCL and 11 of 29 PCBCL cases. In contrast, BCL2 protein expression was detected in 26 of 73 PCL cases, consisting of 18 of 29 PCBCL and eight of 36 SS/MF cases. JUNB protein expression was present in tumour cells from 30 of 33 of SS/MF and eight of eight C-ALCL, and was absent in tumour cells from 18 of 27 PCBCL cases. A comparison between BCL2 and JUNB revealed loss of BCL2 and gain of JUNB in five of 35 SS/MF samples, and expression of JUNB protein and absent BCL2 expression in 25 SS/MF and eight of eight C-ALCL cases. In contrast, expression of BCL2 and absent JUNB expression were detected in 67% of PCBCL cases. Additional FISH analysis revealed deletion of BCL2 in 19 of 23 SS cases (83%), including eight cases with BCL2 loss shown by real-time PCR. Furthermore, Affymetrix expression microarray demonstrated decreased expression of proapoptotic and antiapoptotic genes involved in BCL2 signalling pathways such as BOK, BIM, HRK, RASA1 and STAT2 in two CTCL cell lines with BCL2 loss and absent BCL2 expression. Increased expression of JUNB was also identified in the MF cell line. CONCLUSIONS: These findings provide a comprehensive assessment of BCL2 and JUNB status in PCL, and suggest that there is a selection pressure in a subset of CTCL cases for tumour cells showing BCL2 loss and upregulation of JUNB primarily through chromosomal deletion and amplification, respectively.