Malignant Cutaneous T-cell Lymphoma Research Articles

Cutaneous lymphoproliferative disorders: Back to the future.

In the 1980s, immunohistochemistry and clonality analyses became instrumental in the recognition and definition of new types of cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma (CBCL) and the development of new classifications. By accepting loss of pan-T-cell antigens and clonal T-cell receptor gene rearrangements as important criteria to differentiate between benign and malignant T-cell proliferations, and monotypic immunoglobulin light-chain expression and clonal immunoglobulin gene rearrangements as crucial criteria to distinguish between benign and malignant B-cell proliferations, many cases, until then diagnosed as cutaneous lymphoid hyperplasia or pseudolymphoma, were reclassified as primary cutaneous CD4+ small/medium T-cell lymphoma (PCSM-TCL) or primary cutaneous marginal zone lymphoma (PCMZL), respectively. However, in recent years there is growing awareness that neither these immunohistochemical criteria nor demonstration of T-cell or B-cell clonality is specific for malignant lymphomas. In addition, many studies have reported that these low-grade malignant CTCL and CBCL have an indolent clinical behavior and an excellent prognosis with disease-specific survival rates of or close to 100%. As a result, recent classifications have downgraded several low-grade malignant cutaneous lymphomas to lymphoproliferative disorder (LPD). Both the 5th edition of the WHO classification (2022) and the 2022 International Consensus Classification (ICC) of mature lymphoid neoplasms reclassified PCSM-TCL as primary cutaneous CD4+ small/medium T-cell LPD and primary cutaneous acral CD8+ T-cell lymphoma as primary cutaneous acral CD8+ T cell LPD. While the 2022 ICC introduced the term "primary cutaneous marginal zone LPD," in the 5th edition of the WHO classification PCMZL is maintained. In this review we describe the background and rationale of the continually changing terminology of these conditions and discuss the clinical consequences of downgrading malignant lymphomas to LPDs.

Integrated transcriptome and trajectory analysis of cutaneous T-cell lymphoma identifies putative precancer populations.

The incidence of cutaneous T-cell lymphoma (CTCL) increases with age, and blood involvement portends a worse prognosis. To advance our understanding of the development of CTCL and identify potential therapeutic targets, we performed integrative analyses of paired single-cell RNA and T-cell receptor (TCR) sequencing of peripheral blood CD4+ T cells from patients with CTCL to reveal disease-unifying features. The malignant CD4+ T cells of CTCL showed highly diverse transcriptomic profiles across patients, with most displaying a mature Th2 differentiation and T-cell exhaustion phenotype. TCR-CDR3 peptide prediction analysis suggested limited diversity between CTCL samples, consistent with a role for a common antigenic stimulus. Potential of heat diffusion for affinity-based trajectory embedding transition analysis identified putative precancerous circulating populations characterized by an intermediate stage of gene expression and mutation level between the normal CD4+ T cells and malignant CTCL cells. We further revealed the therapeutic potential of targeting CD82 and JAK that endow the malignant CTCL cells with survival and proliferation advantages.

Single-cell RNA sequencing unveils the communications between malignant T and myeloid cells contributing to tumor growth and immunosuppression in cutaneous T-cell lymphoma

Cutaneous T cell lymphoma (CTCL) is characterized by the accumulation of malignant T cells in the skin. However, advanced CTCL pathophysiology remains elusive and therapeutic options are limited due to the high intratumoral heterogeneity and complicated tumor microenvironment (TME). By comparing the single-cell RNA-seq (scRNA-seq) data from advanced CTCL patients and healthy controls (HCs), we showed that CTCL had a higher enrichment of T/NK and myeloid cells. Subpopulations of T cells (CXCR3+, GNLY+, CREM+, and MKI67+ T cells), with high proliferation, stemness, and copy number variation (CNV) levels, contribute to the malignancy of CTCL. Besides, CCL13+ monocytes/macrophages and LAMP3+ cDC cells were enriched and mediated the immunosuppression via inhibitory interactions with malignant T cells, such as CD47-SIRPA, MIF-CD74, and CCR1-CCL18. Notably, elevated expressions of S100A9 and its receptor TLR4, as well as the activation of downstream toll-like receptor and NF-κB pathway were observed in both malignant cells and myeloid cells in CTCL. Cell co-culture experiments further confirmed that the interaction between malignant CTCL cells and macrophages contributed to tumor growth via S100A9 upregulation and NF-kb activation. Our results showed that blocking the S100A9-TLR4 interaction using tasquinimod could inactivate the NF-κB pathway and inhibit the growth of CTCL tumor cells, and trigger cell apoptosis. Collectively, our study revealed a landscape of immunosuppressive TME mediated by interactions between malignant T cells and myeloid cells, and provided novel targets and potential treatment strategies for advanced CTCL patients.

MicroRNA Regulation of T-Cell Exhaustion in Cutaneous T Cell Lymphoma

Cutaneous T cell lymphoma (CTCL) is characterized by a background of chronic inflammation, where malignant CTCL cells escape immune surveillance. To study how microRNAs (miRs) regulate T-cell exhaustion, we performed miR sequencing analysis, qRT-PCR, and in situ hybridization on 45 primary CTCL samples, three healthy skin samples, and CTCL cell lines, identifying miR-155-5p, miR-130b-3p, and miR-21-3p. Moreover, miR-155-5p, miR-130b-3p, and miR-21-3p positively correlated with immune checkpoint gene expression in lesional skin samples and were enriched in the IL-6/Jak/signal transducer and activator of transcription signaling pathway by gene set enrichment analysis. Further gene sequencing analysis showed decreased mRNA expression of the major negative regulators of Jak/signal transducer and activator of transcription signaling: SOCS, PIAS, and PTPN. Transfection of MyLa and HuT78 cells with anti-miR-155-5p, anti‒miR-21-3p, and anti‒miR-130b revealed a considerable increase in SOCS proteins along with a significant decrease in the levels of activated signal transducer and activator of transcription 3 and immune checkpoint surface protein expression as well as decreased cell proliferation. Downregulation of miR-155, miR-130, and miR-21 in CTCL cell lines decreased CTCL cell growth and facilitated CD8+ T-cell-mediated cytotoxic activity, with concordant production of IFN-γ and CD107a expression. Our results describe the mechanisms of miR-induced T-cell exhaustion, which provide a foundation for developing synthetic anti-miRs to therapeutically target the tumor microenvironment in CTCL.

Tre-O2-03 - Mogamulizumab-associated rash frequently mimics cutaneous T-cell lymphoma recurrence but heralds a positive response to therapy: an updated single-center case series

Mogamulizumab is a humanized antibody that targets CCR4, a transmembrane chemokine receptor predominantly expressed on effector T helper type 2 and malignant cutaneous T-cell lymphoma (CTCL) cells. This biologic was FDA approved for relapsed/refractory mycosis fungoides (MF)/Sézary syndrome (SS) in 2018. MARs are the most common adverse events reported; however, the clinicopathologic nature of these eruptions remains unclear. To inform future management and recognition of MAR, we report an update on our retrospective case series of MF/SS patients treated with mogamulizumab since its approval as standard of care. Among 24 CTCL (16 SS, 8 MF) patients, 17 developed MAR (68%). MAR development was associated with complete (11/17) or partial (4/17) responses, for an overall response rate (ORR) of 88% to mogamulizumab among patients with MAR. A lower ORR of 28% (2/7) was observed in patients that did not develop MAR. Median MAR time to onset (TTO) was 2.1 mo. (0.7–9.0). Median time to resolution (TTR) was 6.9 mo. (3.5–9.2) in 8/17 patients with MAR resolution. Five distinct but frequently concurrent clinical patterns were identified: lichenoid papules and plaques (9/17), MF-like lesions (9/17), photodistribution (7/17), psoriasiform plaques (7/17), and SS-like erythroderma (3/17). Histopathologic patterns included spongiotic (n=21), lichenoid (n=14), psoriasiform (n=10), and interface (n=8) features across 27 skin biopsies. Eosinophils (n=15), epithelioid granulomas (n=4), and bizarre bi- and multinucleated cells (n=6) that may represent highly activated macrophages were also noted. Although mild, features associated with CTCL were noted in all suspected MAR tissue samples [epidermotropism (n=10), exocytosis (n=12), and lymphoid atypia (n=16)]. When compared to baseline studies, flow cytometry analysis for circulating Sézary cells and TCR rearrangement studies in skin and blood were most often negative during suspected MAR despite the presence of CTCL-associated histological features, emphasizing the importance of ancillary studies in distinguishing MAR from CTCL. In conclusion, MAR may be characterized by distinct clinical, histopathologic, and molecular patterns. Notably, MAR can clinically mimic CTCL lesions and may occur more often in patients with clinical responses, highlighting the need for clinicians to rule out progressive disease vs. drug eruption in treating a condition that historically suffers from poor outcomes and treatment response.

Prec-P-01 - Preclinical investigation of new targeted combination therapies in cutaneous T-cell lymphoma

Prec-P-01 - Preclinical investigation of new targeted combination therapies in cutaneous T-cell lymphoma

Bio-P-08 - Mode-of-action of HDAC inhibitor resminostat in CTCL

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma characterized by malignant skin-homing T cells with an increasing bias towards the T helper cell type 2 (Th2) during disease progression. Patients with advance CTCL have a high disease burden and the majority of CTCL patients suffer from severe itching (pruritus). A key challenge in the treatment of advanced CTCL is to maintain and to stabilize initial therapeutic responses after systemic treatment. Addressing this clinical need, the oral class I, IIb, IV HDAC inhibitor resminostat is currently under clinical evaluation for disease control after systemic therapy (RESMAIN, NCT02953301). Here, the mode-of-action of resminostat was explored. Several CTCL cell lines representing mycosis fungoides (MF) and Sézary syndrome (SS) were used in cell biological and molecular assays. HDAC inhibitors are epigenetic drugs that alter the chromatin landscape and the expression of genes. Indicating an interference with epigenetic processes, resminostat induced hyperacetylation on specific histone residues linked with transcriptional regulation. Whole transcriptome analysis upon resminostat treatment uncovered changes in oncogenic signaling pathways and in the expression of genes associated with the pathogenesis of CTCL. The expression of several skin-homing receptors, which mediate the infiltration of malignant T cells into the skin, was reduced by resminostat. Furthermore, resminostat up-regulated a gene expression signature representative of Th1 cell type and down-regulated genes of the Th2 cell type, thus favoring the beneficial Th1 T cell phenotype. In agreement with this observation, resminostat decreased the mRNA level and protein secretion of the Th2 and itch-mediating cytokine IL-31 suggesting that resminostat might improve pruritus. Moreover, resminostat treatment resulted in anti-proliferative and pro-apoptotic effects in both MF and SzS cells. In addition to counteracting intrinsic pathogenic pathways in malignant CTCL cells, we found that resminostat enhanced natural killer cell-mediated lysis, thus promoting the innate immune response towards malignant cells. In contrast to reports for other HDAC inhibitors, resminostat did not reduce the viability or function of NK cells but directly positively affected their cytolytic function. Moreover, resminostat up-regulated the expression NKG2D-ligands on cancer cells and, thereby, facilitates the recognition of cancer cells by NK cells. Interestingly, the combination of resminostat with opsonizing antibodies (e.g. Mogamulizumab, IPH4102) improved the lysis of CTCL cells in NK cell assays, suggesting a potential of resminostat's combinations with opsonizing antibodies. Taken together, these data corroborate that resminostat has the potential to counteract the malignant T cell population in CTCL. In conclusion, our preclinical data support the hypothesis that resminostat treatment will improve or stabilize CTCL and its symptoms.

The Involvement of STAT/SOCS Signaling in miRNA-Induced T Cell Exhaustion in Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell lymphoma (CTCL), collectively known as mycosis fungoides (MF) and Sézary syndrome (SS) arises from CD4+ T cells in a background of chronic inflammation. The chronic inflammation fosters the growth of CTCL cells and may facilitate T-cells exhaustion that is characterized by deprived effector function and sustained expression of inhibitory receptors. As a result, malignant CTCL cells escape immune surveillance and are not eliminated. At present, the roles of miRNAs or signaling pathways involved in the expression of immune checkpoints in CTCL has yet to be elucidated. Therefore, this project is aimed to elucidate how immune checkpoints are regulated by miRNAs, and how this regulation contributes to T-cell exhaustion and the development of CTCL. Methods: We first conducted miRNAseq analysis to assess the miRNA profile of 50 CTCL patient tumor samples. The sequencing data analysis was performed at the City of Hope Integrative Genomics Core. Next, we verified the expression of 3 highly upregulated miRNAs (miR-155, -21 and -130) from the miRNAseq analysis in 5 CTCL cell lines and tumor samples using qRT-PCR and in situ hybridization (ISH). Finally, we transfected the CTCL cell line Myla 2059 and HuT 78 with anti-miR-155, -21, -130 or Scramble (Scr) control using the Lonza nucleofection kit and nucleofector machine. Cell lysates were prepared 72 hours after transfection and then subjected to Western Blot analysis. We probed the blot with antibodies against SOCS, PTEN, pSTAT3 and GAPDH as a loading control. Results: The data analysis revealed that miR-155 had the highest correlation with CTLA-4 (r = 0.59, P < 0.0001), PD1 (r = 0.42, P = 0.0021), PD-L1 (r = 0.42, P = 0.0025), TIM3 (r = 0.63, P < 0.0001), LAG3 (r = 0.57, P < 0.0001), and ICOS (r = 0.74, P < 0.0001) mRNA; -21 had the highest correlation with PD-L1 (r = 0.44, P = 0.0012), TIM3 (r = 0.52, P < 0.0001), and ICOS (r = 0.37, P < 0.0073) mRNA; and -130 had the highest correlation with CTLA-4 (r = 0.5, P = 0.0002), PD-L1 (r = 0.54, P < 0.0001), TIM3 (r = 0.69, P < 0.0001), LAG3 (r = 0.49, P = 0.0003), and ICOS (r = 0.68, P < 0.0001) mRNA. qRT-PCR and ISH revealed that miRs-155, -21 and -130 were upregulated in all 5 CTCL cell lines and primary tumor samples. There was a dramatic increase in SOCS proteins and significant decrease in pSTAT3 expression in Myla 2059 and HuT 78 cells transfected with anti-miRs-155, -21 or -130, compared to cells transfected with Scr. Conclusions: Immune checkpoints and ligands like PD-L1 expression in CTCL are regulated by miRs-155, -21 and -130. The SOCS family of proteins, negative regulators of STAT signaling, are involved in miRs-155, -21 and -130-induced immune checkpoints expression in CTCL. Taken together, these results demonstrate the mechanisms of miRNA-induced T cell exhaustion and pave the way for the development of miRNA therapeutics in CTCL. Disclosures Rosen: Seattle Genetics: Consultancy; Celgene: Speakers Bureau; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau; NeoGenomics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; Pebromene: Consultancy. Querfeld:Celgene: Research Funding; Stemline: Consultancy; Trillium: Consultancy; MiRagen: Consultancy; Kyowa Kirin: Consultancy; Bioniz: Consultancy; Helsinn: Consultancy.

The Thioredoxin-Interacting Protein TXNIP Is a Putative Tumour Suppressor in Cutaneous T-Cell Lymphoma

Background: The thioredoxin-interacting protein (TXNIP) is involved in cellular metabolism and cell proliferation, and recently, deficient expression of TXNIP has been associated with progression and poor outcome for cancer patients. Objectives: To assess TXNIP expression and function in malignant T cells from cutaneous T-cell lymphoma (CTCL). Methods: CTCL-derived malignant (MyLa2059, PB2B) and non-malignant (MyLa1850) cell lines were analysed by Western blotting and qPCR for TXNIP expression. Subsequently, the malignant CTCL cell lines were treated with GSK126 – an inhibitor of enhancer of zeste homolog 2 (EZH2) methyltransferase activity or assessed by bisulphite sequencing for TXNIP promoter methylation. Methylation was also assessed with the demethylating agent 5-azacytidine (5AZA). Finally, TXNIP was overexpressed in the malignant PB2B cell line via plasmid transduction, and the effect of TXNIP was further analysed by flow cytometry. Results: We report on low expression of TXNIP protein in all cell lines representing different subtypes and stages of CTCL when compared to non-malignant T cells. Epigenetic silencing and other mechanisms were involved in the repression of TXNIP whereas forced expression of TXNIP strongly inhibited proliferation of malignant T cells. Conclusions: Epigenetic silencing and other as yet unknown mechanisms repress TXNIP expression in malignant T cells. As forced expression of TXNIP inhibits malignant proliferation, we propose that TXNIP is a putative tumour suppressor in CTCL.

Screening Novel Agent Combinations to Expedite CTCL Therapeutic Development

Screening Novel Agent Combinations to Expedite CTCL Therapeutic Development

PD-L1 Expression Is Regulated By microRNAs-21 and -130 in Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell lymphoma (CTCL), collectively known as mycosis fungoides (MF) and Sézary syndrome (SS), is characterized by the accumulation of neoplastic memory T- cells in the skin in a background of chronic inflammation. This observation suggests that chronic inflammation fosters the growth of CTCL cells, and may impair successful anti-tumor responses mediated by the CD8+ tumor-infiltrating T-cells. T-cell exhaustion plays an important role in the pathogenesis of CTCL. PD-1 and other inhibitory immune checkpoints are overexpressed in exhausted T-cells (Cancer Immunol Res 6; 2018). Their binding to corresponding ligands like PD-L1 on dendritic cells (DCs), macrophages and tumor cells in the microenvironment directly reduces the functional and proliferative capabilities of T-cells by repressing T-cell receptor signaling and inducing genes that impair T-cell function. As a result, malignant CTCL cells escape immune surveillance and are not eliminated. The miRNA profile of CTCL correlates with a dysfunctional/exhausted immunophenotype, suggesting that epigenetic regulation is involved in the regulation of immune checkpoints. Signaling pathways such as JAK-STAT are constitutively active in CTCL and may also be involved in immune checkpoint regulation. At present, the exact role of miRNAs in regulating the expression of PD-L1 in CTCL has yet to be elucidated. Therefore, the goal of this project is to understand how PD-L1 is regulated, how this regulation contributes to T-cell exhaustion, and how this regulation is altered to contribute to the development of CTCL. To this end, we set out to test the hypothesis that PD-L1 is regulated by oncogenic miRNAs in CTCL.Methods: We first conducted a high-throughput miRNAseq analysis to assess the miRNA profile of 50 CTCL patient tumor samples. Library preparation, miRNA sequencing, and correlation calculation of miRNAs with PD-L1 mRNA expression was performed by the City of Hope Integrative Genomics Core. Next, we verified the expression of 2 highly upregulated miRs-miRs-21and -130- from the miRNAseq analysis in 5 CTCL cell lines and tumor samples using qRT-PCR and in situ hybridization (ISH). Finally, we transfected the CTCL cell line Hut78 with anti-miR-21, -130 or Scramble (Scr) control using the Lonza nucleofection kit and nucleofector machine. Cell lysates were prepared 72 hours after transfection and then subjected to Western Blot analysis. We probed the blot with antibodies against PD-L1, pSTAT3 and GAPDH as a loading control.Results: The miRNAseq data revealed that miRs-21 and -130 had the highest correlation with PD-L1 mRNA. qRT-PCR and ISH revealed that miRs-21 and -130 were upregulated in all 5 CTCL cell lines and primary tumor samples. There was a drastic decrease in PD-L1 and pSTAT3 expression in Hut78 cells transfected with anti-miRs-21 or -130, compared to cells transfected with Scr.Conclusions: PD-L1 expression in CTCL is regulated by miRs-21 and -130. miRs-21 and -130 may be regulating PD-L1 indirectly by silencing negative regulators of STAT signaling such as the SOCS family of proteins. This would explain the decrease in pSTAT3 levels in Hut78 cells transfected with anti-miRs. Taken together, these results demonstrate the ability of miRNAs to regulate immune checkpoints and ligands like PD-L1, and pave the way for the creation of miRNA therapeutics to reverse T-cell exhaustion and slow CTCL disease progression. DisclosuresQuerfeld:Acelion: Membership on an entity's Board of Directors or advisory committees; Kyowa: Membership on an entity's Board of Directors or advisory committees; Bioniz: Membership on an entity's Board of Directors or advisory committees; Medivir: Membership on an entity's Board of Directors or advisory committees; Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Vitamin D controls apoptosis and proliferation of cutaneous T‐cell lymphoma cells

Vitamin D controls apoptosis and proliferation of cutaneous T‐cell lymphoma cells

Ectopic expression of B-lymphoid kinase in cutaneous T-cell lymphoma

AbstractB-lymphoid kinase (Blk) is exclusively expressed in B cells and thymocytes. Interestingly, transgenic expression of a constitutively active form of Blk in the T-cell lineage of mice results in the development of T-lymphoid lymphomas. Here, we demonstrate nuclear factor–kappa B (NF-κB)–mediated ectopic expression of Blk in malignant T-cell lines established from patients with cutaneous T-cell lymphoma (CTCL). Importantly, Blk is also expressed in situ in lesional tissue specimens from 26 of 31 patients with CTCL. Already in early disease the majority of epidermotropic T cells express Blk, whereas Blk expression is not observed in patients with benign inflammatory skin disorders. In a longitudinal study of an additional 24 patients biopsied for suspected CTCL, Blk expression significantly correlated with a subsequently confirmed diagnosis of CTCL. Blk is constitutively tyrosine phosphorylated in malignant CTCL cell lines and spontaneously active in kinase assays. Furthermore, targeting Blk activity and expression by Src kinase inhibitors and small interfering RNA (siRNA) inhibit the proliferation of the malignant T cells. In conclusion, this is the first report of Blk expression in CTCL, thereby providing new clues to the pathogenesis of the disease.

Jak3- and JNK-dependent vascular endothelial growth factor expression in cutaneous T-cell lymphoma

Biopsies from patients with cutaneous T-cell lymphoma (CTCL) exhibit stage-dependent increase in angiogenesis. However, the molecular mechanisms responsible for the increased angiogenesis are unknown. Here we show that malignant CTCL T cells spontaneously produce the potent angiogenic protein, vascular endothelial growth factor (VEGF). Dermal infiltrates of CTCL lesions show frequent and intense staining with anti-VEGF antibody, indicating a steady, high production of VEGF in vivo. Moreover, the VEGF production is associated with constitutive activity of Janus kinase 3 (Jak3) and the c-Jun N-terminal kinases (JNKs). Sp600125, an inhibitor of JNK activity and activator protein-1 (AP-1) binding to the VEGF promoter, downregulates the VEGF production without affecting Jak3 activity. Similarly, inhibitors of Jak3 inhibit the VEGF production without affecting JNK activity. Downregulation of Stat3 with small interfering RNA has no effect, whereas curcumin, an inhibitor of both Jak3 and the JNKs, almost completely blocks the VEGF production. In conclusion, we provide evidence of VEGF production in CTCL, which is promoted by aberrant activation of Jak3 and the JNKs. Inhibition of VEGF-inducing pathways or neutralization of VEGF itself could represent novel therapeutic modalities in CTCL.

Expression of apoptosis regulators in cutaneous T-cell lymphoma (CTCL) cells.

This study examined cutaneous T-cell lymphoma (CTCL) cell lines and cutaneous lesions for the presence of Bcl-2 gene family members and found that the two apoptosis-inhibiting members Bcl-xL and Mcl-1 and the two apoptosis-supporting members Bad and Bax were expressed. However, Bad was at least partially inactivated by phosphorylation. In skin lesions, the translocation of Bad from the nucleus to the cytoplasm may reflect the Bad inactivation by phosphorylation identified in vivo. Bax is also ineffective, as the non-steroidal anti-inflammatory drug sulindac, whose cytotoxic effect is mediated by Bax, could not induce apoptosis in CTCL cell lines. The expression of Bcl-2, Bcl-xL, and Mcl-1 may therefore be sufficient to guarantee the survival of malignant CTCL cells. The Bcl-x, Mcl-1, Bad, and Bax proteins were also expressed in all CTCL skin lesions tested. In two patients from whom two biopsies from two different time points of the disease were available, a significant increase in Mcl-1 expression was found in the later-stage skin lesion. Overexpression of Mcl-1 and synthesis of non-functional Bax may be responsible for the resistance of CTCL cells to the anti-cancer drugs chlorambucil and sulindac.

Inability to culture the dominant T-cell clone from the skin of primary cutaneous T-cell lymphoma as proven by TCR gamma-chain gene sequencing.

Molecular analysis of T-cell receptor (TCR) chain rearrangement has recently become an attractive tool for demonstrating the clonal origin of cutaneous T-cell lymphoma (CTCL) and for identifying the malignant clone at the molecular level. Over the past decade a number of attempts have been made to culture malignant CTCL cells using standard procedures and these attempts have resulted in several cell lines from the peripheral blood of Sézary syndrome, mycosis fungoides and CD30+ lymphoma patients. However, so far it has not been proven by sequence analysis that the cultured T cells truly represent the malignant cells. Aiming to functionally analyze the malignant T cells at a clonal level, we generated a total of 150 T-cell clones (TCC) from lesional skin and peripheral blood of three patients with mycosis fungoides and one patient with a CD30+ lymphoma. Cells were grown either in the presence of autologous irradiated peripheral blood feeder cells using various conditions for T-cell stimulation by direct outgrowth or from skin specimens with various cytokine combinations. In order to identify the malignant TCC we used N-region-specific PCR and compared TCR gamma-chain sequences from clones of lesional skin with in vitro-generated TCC. With the methods employed, none of the 150 established cell lines was found to be identical to the malignant TCC which was readily detected in lesional skin. Our results indicate that standard cell culture methods are not suitable for growing low-grade CTCL cells from the skin but give rise only to benign infiltrating T cells.