Role of Neutrophil-to-Lymphocyte Ratio (NLR) in Patients with Mycosis Fungoides.

BACKGROUND: The neutrophil/lymphocyte ratio (NLR) at baseline has been demonstrated to correlate with higher stages of disease and to be a prognostic factor in numerous cancers. However, its function as a prognostic factor for mycosis fungoides (MF) has not been yet clarified. OBJECTIVE: Our work aimed to assess the association of the NLR with different stages of MF and to outline whether higher values of this marker are related to a more aggressive MF. METHODS: We retrospectively calculated the NLRs in 302 MF patients at the moment of diagnosis. The NLR was obtained using the complete blood count values. RESULTS: The median NLR among patients with early stage disease (low-grade IA-IB-IIA) was 1.88, while the median NLR for patients with high-grade MF (IIB-IIIA-IIIB) was 2.64. Statistical analysis showed positive associations of advanced MF stages with NLRs higher than 2.3. CONCLUSIONS: Our analysis demonstrates that the NLR represents a cheap and easily available parameter functioning as a marker for advanced MF. This might guide physicians in recognizing patients with advanced stages of disease requiring a strict follow-up or an early treatment.

Circulating miR-1246 and miR-485-3p as Promising Biomarkers of Clinical Response and Outcome in Melanoma Patients Treated with Targeted Therapy.

Despite the significant improvements in advanced melanoma therapy, there is still a pressing need for biomarkers that can predict patient response and prognosis, and therefore support rational treatment decisions. Here, we investigated whether circulating miRNAs could be biomarkers of clinical outcomes in patients treated with targeted therapy. Using next-generation sequencing, we profiled plasma miRNAs at baseline and at progression in patients treated with BRAF inhibitors (BRAFi) or BRAFi + MEKi. Selected miRNAs associated with response to therapy were subjected to validation by real-time quantitative RT-PCR. Receiver Operating Characteristics (ROC), Kaplan-Meier and univariate and multivariate Cox regression analyses were performed on the validated miR-1246 and miR-485-3p baseline levels. The median baseline levels of miR-1246 and miR-485-3p were significantly higher and lower, respectively, in the group of patients not responding to therapy (NRs) as compared with the group of responding patients (Rs). In Rs, a trend toward an increase in miR-1246 and a decrease in miR-485-3p was observed at progression. Baseline miR-1246 level and the miR-1246/miR-485-3p ratio showed a good ability to discriminate between Rs and NRs. Poorer PFS and OS were observed in patients with unfavorable levels of at least one miRNA. In multivariate analysis, a low level of miR-485-3p and a high miR-1246/miR-485-3p ratio remained independent negative prognostic factors for PFS, while a high miR-1246/miR-485-3p ratio was associated with an increased risk of mortality, although statistical significance was not reached. Evaluation of miR-1246 and miR-485-3p baseline plasma levels might help clinicians to identify melanoma patients most likely to be unresponsive to targeted therapy or at higher risk for short-term PFS and mortality, thus improving their management.

Combined High-Throughput Approaches Reveal the Signals Driven by Skin and Blood Environments and Define the Tumor Heterogeneity in Sézary Syndrome.

Sézary syndrome (SS) is an aggressive variant of cutaneous t-cell lymphoma characterized by the accumulation of neoplastic CD4+ lymphocytes-the SS cells-mainly in blood, lymph nodes, and skin. The tumor spread pattern of SS makes this lymphoma a unique model of disease that allows a concurrent blood and skin sampling for analysis. This review summarizes the recent studies highlighting the transcriptional programs triggered by the crosstalk between SS cells and blood-skin microenvironments. Emerging data proved that skin-derived SS cells show consistently higher activation/proliferation rates, mainly driven by T-cell receptor signaling with respect to matched blood SS cells that instead appear quiescent. Biochemical analyses also demonstrated an hyperactivation of PI3K/AKT/mTOR, a targetable pathway by multiple inhibitors currently in clinical trials, in skin SS cells compared with a paired blood counterpart. These results indicated that active and quiescent SS cells coexist in this lymphoma, and that they could be respectively treated with different therapeutics. Finally, this review underlines the more recent discoveries into the heterogeneity of circulating SS cells, highlighting a series of novel markers that could improve the diagnosis and that represent novel therapeutic targets (GPR15, PTPN13, KLRB1, and ITGB1) as well as new genetic markers (PD-1 and CD39) able to stratify SS patients for disease aggressiveness.

Genetically Driven CD39 Expression Affects Sezary Cell Viability and IL-2 Production and Detects Two Patient Subsets with Distinct Prognosis.

Sézary syndrome (SS) is a rare and aggressive variant of cutaneous T-cell lymphoma. It is characterized by the copresence of CD4+ neoplastic lymphocytes, named Sezary cells, mainly in the blood, lymph nodes, and skin where they induce chronic inflammation that in turn impairs the patient's QOL and fuels neoplastic cells. SS is not readily cured, but immunotherapy is becoming an effective option for this lymphoma. In this study, we investigated, in a large cohort of patients with SS, the expression and function of the immune checkpoint molecule CD39, which degrades proinflammatory extracellular adenosine triphosphate. We showed that the SNP rs10748643 A/G within the ENTPD1 gene coding for the CD39 protein controls its expression level. Patients carrying the A/G‒G/G genotype showed a significantly higher frequency of clonal CD4+CD39+ SS cells than those carrying the A/A genotype. Different from other cancers, high CD39 expression correlates with a better prognosis. Comparing primary G/G with A/A lymphoma cells, we observed that G/G SS cells have a higher ability to degrade adenosine triphosphate, increased apoptotic susceptibility, and upon activation, reduced IL-2 production. Accordingly, CD39 enzymatic inhibition enhances SS cell viability and IL-2 production on activation. These results strongly suggest a special caution for SS treatment with therapeutic inhibitors of CD39.

Continuous low-dose gemcitabine in primary cutaneous T cell lymphoma: A retrospective study.

The aim of our retrospective study was to evaluate the efficacy of a continuous therapy with a lower dosage of gemcitabine compared to those usually administered in patients with cutaneous T cell lymphomas (CTCL). Twenty-two patients received different dosages of gemcitabine. Dosage and schedule of the drug were chosen on the basis of clinical features. Gemcitabine was given at 1000 mg every 15 days in 13 patients (four mycosis fungoides [MF], nine Sezary syndrome [SS]); at 1000 mg at days +1, +8, +15 in six cases (three MF, three SS). All patients had been previously treated: four patients had received both skin directed and systemic treatments. Eighteen patients had received photopheresis, IFN, chemotherapy and immunotherapy. The objective response rate (CR + PR) among all patients was 54.5% (12 of 22 patients) with a CR of 4.5% (one of 22 patients) and a PR of 50% (11 of 22 patients). Patients with SS had an ORR of 61.5% (eight of 13 patients) with one CR (7%) and seven PR (53.8%); patients with MF showed an ORR of 55.6% (five of nine patients) but no patients experienced CR (0%). The schedule with the highest efficacy and the lowest toxicity profile was 1000 mg every 15 days. Median progression free survival and overall survival in all patients were 17 and 45 months respectively. Gemcitabine was generally well tolerated. We have demonstrated that a much lower dose of gemcitabine (1000 mg once every 15 days) in patients with advanced-stage and refractory CTCL can lead to a durable response, with tolerable and manageable adverse effects.

Challenging Cutaneous T-Cell Lymphoma: What Animal Models Tell us So Far.

Cutaneous T-cell lymphomas are characterized by heterogeneity of clinical variants, further complicated by genomic and microenvironmental variables. Furthermore, in vitro experiments are hampered by the low culture efficiency of these malignant cells. Animal models are essential for understanding the pathogenetic mechanisms underlying malignancy and for discovering new anticancer treatments. They are divided into two main categories: those in which tumors arise in the host owing to genetic modifications and those that use tumor cell transplantation. In this review, we summarize the attempts to decipher the complexity of the pathogenesis of cutaneous T-cell lymphoma by exploiting genetically modified and xenograft models.

Reduction of T Lymphoma Cells and Immunological Invigoration in a Patient Concurrently Affected by Melanoma and Sezary Syndrome Treated With Nivolumab.

Despite the recent availability of several new drugs in hemato-oncology, T-cell lymphomas are still incurable and PD-1 blockade could represent a therapeutic chance for selected patients affected by these malignancies, although further studies are required to understand the biological effects of anti-PD-1 mAbs on neoplastic T-cells and to identify biomarkers for predicting and/or monitoring patients' response to therapy. Sezary Syndrome (SS) represents a rare and aggressive variant of cutaneous T cell lymphoma (CTCL) with a life expectancy of less than 5 years, characterized by the co-presence of neoplastic lymphocytes mainly in the blood, lymph nodes and skin. In this study we analyzed longitudinal blood samples and lesional skin biopsies of a patient concurrently affected by SS and melanoma who underwent 22 nivolumab administrations. In blood, we observed a progressive reduction of SS cell number and a raise in the percentage of normal CD4+ and CD8+ T cells and NK cells over total leukocytes. Eight weeks from the start of nivolumab, these immune cell subsets showed an increase of Ki67 proliferation index that positively correlated with their PD-1 expression. Conversely, SS cells displayed a strong reduction of Ki67 positivity despite their high PD-1 expression. On skin biopsies we observed a marked reduction of SS cells which were no more detectable at the end of therapy. We also found an increase in the percentage of normal CD4+ T cells with a concomitant decrease of that of CD8+ and CD4+ CD8+ T cells, two cell subsets that, however, acquired a cytotoxic phenotype. In summary, our study demonstrated that nivolumab marked reduced SS tumor burden and invigorated immune responses in our patient. Our data also suggest, for the first time, that Ki67 expression in circulating neoplastic and immune cell subsets, as well as an enrichment in T cells with a cytotoxic phenotype in lesional skin could be valuable markers to assess early on treatment SS patients' response to PD-1 blockade, a therapeutic strategy under clinical investigation in CTCL (ClinicalTrials.gov NCT03385226, NCT04118868).

Preclinical Evidence for Targeting PI3K/mTOR Signaling with Dual-Inhibitors as a Therapeutic Strategy against Cutaneous T-Cell Lymphoma.

The phosphoinositide 3-kinase(PI3K)/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway is hyperactivated in many tumors, as well as in cutaneous T-cell lymphoma (CTCL), which includes the mycosis fungoides and the aggressive variant known as Sezary syndrome (SS). TORC1 signaling is activated in SS cells by cytokines and chemokines, which are overexpressed in SS tissues. Furthermore, the recurrent copy number variation of genes belonging to this cascade, such as PTEN, LKB1, and P70S6K, contributes to the hyperactivation of the pathway. The aim of this study was to investigate the therapeutic potential of mTOR inhibitors in CTCL. We compared the efficacy of three rapalogs (rapamycin, temsirolimus, and everolimus) and the dual-mTOR/PI3K inhibitor PF-04691502 (hereinafter PF-502) in four CTCL cell lines. PF-502 was revealed to be the most effective inhibitor of cell growth. Interestingly, PF-502 also exerted its antitumor activity in patient-derived CTCL cells and in a xenograft mouse model, where it induced significant apoptosis and increased survival of treated mice. Furthermore, we found an inverse correlation between PTEN gene expression and the ability of PF-502 to induce apoptosis in SS cells. Our data strongly support the therapeutic potential of dual PI3K/mTOR inhibitors in CTCL.

Protein kinase Akt2/PKBß is involved in blastomere proliferation of preimplantation mouse embryos.

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.

Loss of ß-arrestin-2 gene and possible functional consequences on Sezary Syndrome.

Sezary Syndrome is an aggressive T-cell Lymphoma involving blood, skin and lymphonodes Involvement of the CXCR4-SDF1 has been previously shown. We here present evidence also of the involvement of B-arrestin a downstream regulator of CXCR4, that is depleted and downregulated as well as a potential functional role for this depletion.

Blood and skin-derived Sezary cells: differences in proliferation-index, activation of PI3K/AKT/mTORC1 pathway and its prognostic relevance.

Sézary syndrome (SS) is a rare and aggressive variant of Cutaneous T-Cell Lymphoma characterized by neoplastic distribution mainly involving blood, skin, and lymph-node. Although a role of the skin microenvironment in SS pathogenesis has long been hypothesized, its function in vivo is poorly characterized. To deepen this aspect, here we compared skin to blood-derived SS cells concurrently obtained from SS patients highlighting a greater proliferation-index and a PI3K/AKT/mTORC1 pathway activation level, particularly of mTOR protein, in skin-derived-SS cells. We proved that SDF-1 and CCL21 chemokines, both overexpressed in SS tissues, induce mTORC1 signaling activation, cell proliferation and Ki67 up-regulation in a SS-derived cell line and primary-SS cells. In a cohort of 43 SS cases, we observed recurrent copy number variations (CNV) of members belonging to this cascade, namely: loss of LKB1 (48%), PTEN (39%) and PDCD4 (35%) and gains of P70S6K (30%). These alterations represent druggable targets unraveling new therapeutic treatments as metformin here evaluated in vitro. Moreover, CNV of PTEN, PDCD4, and P70S6K, evaluated individually or in combination, are associated with reduced survival of SS patients. These data shed light on effects in vivo of skin-SS cells interaction underlying the prognostic and therapeutic relevance of mTORC1 pathway in SS.

Sézary Syndrome, recent biomarkers and new drugs.

Sezary syndrome (SS) is a primary cutaneous T-cell lymphoma (CTCL) characterized by erythroderma, lymphadenopathy and leukemic involvement of the peripheral blood. The high relapse rates and a poor prognosis complicate its clinical course and treatment. The phenotypic characterization and genomic/transcriptomic approaches revealed high heterogeneity of Sezary cells, identifying a wide spectrum of biomarkers implicated in the development of this lymphoma. In this context, we discuss the major malignancy-related biomarkers reported in the literature for the diagnosis, prognosis and staging of SS. The hope for a single reliable diagnostic marker appears increasingly unrealistic, but the discovery of multiple potential biomarkers, with pathogenetic implications, paves the road to promising personalized therapies in SS.

Loss of the candidate tumor suppressor ZEB1 (TCF8, ZFHX1A) in Sézary syndrome.

Cutaneous T-cell lymphoma is a group of incurable extranodal non-Hodgkin lymphomas that develop from the skin-homing CD4+ T cell. Mycosis fungoides and Sézary syndrome are the most common histological subtypes. Although next-generation sequencing data provided significant advances in the comprehension of the genetic basis of this lymphoma, there is not uniform consensus on the identity and prevalence of putative driver genes for this heterogeneous group of tumors. Additional studies may increase the knowledge about the complex genetic etiology characterizing this lymphoma. We used SNP6 arrays and GISTIC algorithm to prioritize a list of focal somatic copy-number alterations in a dataset of multiple sequential samples from 21 Sézary syndrome patients. Our results confirmed a prevalence of significant focal deletions over amplifications: single well-known tumor suppressors, such as TP53, PTEN, and RB1, are targeted by these aberrations. In our cohort, ZEB1 (TCF8, ZFHX1A) spans a deletion having the highest level of significance. In a larger group of 43 patients, we found that ZEB1 is affected by deletions and somatic inactivating mutations in 46.5% of cases; also, we found potentially relevant ZEB1 germline variants. The survival analysis shows a worse clinical course for patients with ZEB1 biallelic inactivation. Multiple abnormal expression signatures were found associated with ZEB1 depletion in Sézary patients we verified that ZEB1 exerts a role in oxidative response of Sézary cells. Our data confirm the importance of deletions in the pathogenesis of cutaneous T-cell lymphoma. The characterization of ZEB1 abnormalities in Sézary syndrome fulfils the criteria of a canonical tumor suppressor gene. Although additional confirmations are needed, our findings suggest, for the first time, that ZEB1 germline variants might contribute to the risk of developing this disease. Also, we provide evidence that ZEB1 activity in Sézary cells, influencing the reactive oxygen species production, affects cell viability and apoptosis.

T Cell Leukemia/Lymphoma 1A is essential for mouse epidermal keratinocytes proliferation promoted by insulin-like growth factor 1.

T Cell Leukemia/Lymphoma 1A is expressed during B-cell differentiation and, when over-expressed, acts as an oncogene in mouse (Tcl1a) and human (TCL1A) B-cell chronic lymphocytic leukemia (B-CLL) and T-cell prolymphocytic leukemia (T-PLL). Furthermore, in the murine system Tcl1a is expressed in the ovary, testis and in pre-implantation embryos, where it plays an important role in blastomere proliferation and in embryonic stem cell (ESC) proliferation and self-renewal. We have also observed that Tcl1-/- adult mice exhibit alopecia and deep ulcerations. This finding has led us to investigate the role of TCL1 in mouse skin and hair follicles. We have found that TCL1 is expressed in the proliferative structure (i.e. the secondary hair germ) and in the stem cell niche (i.e. the bulge) of the hair follicle during regeneration phase and it is constitutively expressed in the basal layer of epidermis where it is required for the correct proliferative-differentiation program of the keratinocytes (KCs). Taking advantage of the murine models we have generated, including the Tcl1-/- and the K14-TCL1 transgenic mouse, we have analysed the function of TCL1 in mouse KCs and the molecular pathways involved. We provide evidence that in the epidermal compartment TCL1 has a role in the regulation of KC proliferation, differentiation, and apoptosis. In particular, the colony-forming efficiency (CFE) and the insulin-like growth factor 1 (IGF1)-induced proliferation are dramatically impaired, while apoptosis is increased, in KCs from Tcl1-/- mice when compared to WT. Moreover, the expression of differentiation markers such as cytokeratin 6 (KRT6), filaggrin (FLG) and involucrin (IVL) are profoundly altered in mutant mice (Tcl1-/-). Importantly, by over-expressing TCL1A in basal KCs of the K14-TCL1 transgenic mouse model, we observed a significant rescue of cell proliferation, differentiation and apoptosis of the mutant phenotype. Finally, we found TCL1 to act, at least in part, via increasing phospho-ERK1/2 and decreasing phospho-P38 MAPK. Hence, our data demonstrate that regulated levels of Tcl1a are necessary for the correct proliferation and differentiation of the interfollicular KCs.

Single TCR-Vß2 evaluation discloses the circulating T cell clone in Sezary syndrome: one family fits all!

Sézary Syndrome (SS/L-CTCL) is a rare but aggressive variant of cutaneous T cell lymphoma (CTCL), characterized by erythroderma, lymphadenopathy, and the presence of a circulating memory CD4(+) T cell malignant clone with a skin homing behavior, lacking CD26 and CD49d and over-expressing CD60. The availability of a panel of monoclonal antibodies recognizing distinct TCR-Vß families, allows to typify the clone by flow cytometry in about 70 % of cases. The TCR-Vß repertoire of 533 individuals, comprising 308 patients affected by CTCL, 50 healthy donors, and subjects affected by various non-neoplastic dermatological affections was evaluated by flow cytometry. Statistical analyses were performed using the SPSS statistical software package for Microsoft Windows (SPSS, version 21, Chicago, IL). TCR-Vß2 levels below 5.4 % or above 39.5 %, within total CD4(+) T cells, showed the best balance between sensitivity (98.1 %) and specificity (96 %) to identify the presence of a clone in the peripheral blood of patients affected by SS. Based on this observation, a "two-step" procedure in the detection of the malignant T cell clone in CTCLs is herein suggested. TCR-Vß2 assessment in all cases (first step). In the case of TCR-Vß2 levels above 39.5 %, the presence of a clonal expansion of this family is suggested, deserving further confirmation by means of T cell gene rearrangement evaluation. In patients having a TCR-Vß2 reactivity below 5.4 % (second step), the entire TCR-Vß repertoire should be evaluated to typify the expanded clone. In conclusion, the single TCR-Vß2 expression check, instead of the entire repertoire assessment, represents an easy and cost-effective method for the recognition of CTCL aggressive leukemic variant.

Comprehensive analysis of PTEN status in Sezary syndrome.

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma characterized by recurrent chromosomal alterations, among which, chromosome 10q deletion is very frequent. In this study, we investigated the PTEN status, on locus 10q23, in 44 SS patients; our findings show that PTEN is deleted in 36% of SS cases, whereas PTEN downregulation is observed in almost all of the samples evaluated by quantitative reverse-transcriptase polymerase chain reaction and Western blotting analysis. Neither DNA sequence mutation nor promoter hypermethylation were found at the PTEN locus, but we demonstrate that PTEN level can be also reduced by a group of miRs previously found upregulated and of prognostic relevance in SS; particularly, miR-21, miR-106b, and miR-486 were able to control PTEN abundance either in vitro or in vivo. Finally, because reduced PTEN activates the PI3/AKT-mediated pathway of cell growth and survival, we demonstrate that PTEN deficiency is associated with activated AKT in skin resident but not circulating SS cells, suggesting that the cutaneous milieu may strongly contribute to the SS cell growth. To our knowledge, this is the first study fully exploring the PTEN status in a large cohort of SS patients, unveiling potential elements of clinical utility in this malignancy.

Specific IgE toward allergenic molecules is a new prognostic marker in patients with Sézary syndrome.

BACKGROUND: Sézary syndrome (SS) is the aggressive leukemic form of cutaneous T cell lymphoma characterized by erythroderma, the presence of a malignant circulating T memory cells with skin homing potential, and the ability to produce a variety of Th2 soluble factors, such as IL-4 and IL-5. We measured total and specific IgE in SS patients as a further parameter of a Th2-skewed immune system, and studied their clinical impact. METHODS: Specific IgE production in a cohort of 55 SS patients was evaluated by the molecule-based ISAC microarray system. We then evaluated survival times and the hazard ratios in this cohort by Kaplan-Meier and Cox methods. RESULTS: Twenty-four (43.6%) SS patients had specific IgE to both environmental and food allergens. For survival analysis, patients found positive to at least one allergen were defined as IgE+. By comparing IgE+ versus IgE- we found a significant difference in the median survival times, 2.9 versus 8.9 years (p < 0.001). Conversely, no survival difference could be observed when total IgE levels were considered. IgE+ patients had higher levels of CD60+CD49-CD4+ T cells, which also represent another worse prognostic index recently identified. CONCLUSION: SS patients had specific IgE to both environmental and food allergens. IgE+ SS patients had a significant lower survival rate. High levels of CD60-CD49+CD4+ T cells associated with an IgE- phenotype allow the identification of a restricted group of long survivor SS patients. Therefore, specific measurement of IgE seems to be useful in discriminating survival.

The role of 9-O-acetylated ganglioside D3 (CD60) and {alpha}4{beta}1 (CD49d) expression in predicting the survival of patients with Sezary syndrome.

BACKGROUND: Sézary syndrome is a rare and very aggressive leukemic variant of cutaneous T-cell lymphoma characterized by extensive skin involvement and a malignant circulating CD4(+) T-cell clone which homes to the skin, over-expresses CD60, and lacks CD7, CD26 and CD49d. So far prognostic markers in this disease are limited to treatment with systemic steroids, age, serum lactate dehydrogenase, and a white blood cell count of 20×10(9)/L or higher: no other biological marker with prognostic value, especially related to malignant cells, has been described. DESIGN AND METHODS: We used flow activated cell sorting analysis to compare the distribution of the T-cell receptor-Vß repertoire and several surface molecules (CD7, CD26, CD49d and CD60) within the circulating CD4(+) T-cell population in 62 patients with Sézary syndrome, 180 with mycosis fungoides, 6 with B-cell lymphomas, and 19 with chronic eczema. We calculated the 5-year overall survival of patients with Sézary syndrome after first hospital admission using Kaplan-Meier product-limit estimates and hazard ratios from the Cox proportional hazards model. RESULTS: We found that both higher number of CD60(+) and lower number of CD49d(+) cells within circulating CD4(+) T cells at disease presentation were significantly associated with a lower probability of survival. An exceedingly high risk of death was observed for patients with a combination of a high proportion of CD4(+)CD60(+) cells (≥ 0.5×10(9)/L) and low proportion of CD4(+)CD49d(+) cells (<0.5×10(9)/L) (hazard ratio = 12.303, 95% confidence interval 1.5-95.9; P<0.02). In addition, a skewed usage of T-cell receptor-Vß subfamilies was observed in the circulating T-cell clone for 61.9% of all patients with Sézary syndrome, T-cell receptor-Vß 2 and 5.1 subfamilies being the most frequently represented (42.8%), followed by T-cell receptor-Vß 12 and 13.1. CONCLUSIONS: In this study we showed that up-regulation of CD60 and down-regulation of CD49d on circulating CD4(+) T cells are two useful markers for predicting a very poor outcome in patients with Sézary syndrome.