Search

Supplementary Table 4 from Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays

Supplementary Figure 1 and Table 1 from CXCL13 Is Highly Produced by Sézary Cells and Enhances Their Migratory Ability via a Synergistic Mechanism Involving CCL19 and CCL21 Chemokines

Genome Data Link from Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays

Supplementary Table 1 from Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays

Supplementary Figure 2 from Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays

In this study, we used single nucleotide polymorphism and comparative genomic hybridization array to study DNA copy number changes and loss of heterozygosity for 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71% and 68% of cases, respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in >30% of tumors: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis; however, when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Integrating mapping and transcriptional data, we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer-related genes such as members of the NF-κB pathway (BAG4, BTRC, NKIRAS2, PSMD3, and TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times, we identify several common candidates that might exert critical roles in SS, such as BUB3 and PIP5K1B. Altogether, our study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggests a novel set of genes of potential interest in SS. [Cancer Res 2009;69(21):8438–46]

Supplementary Table 3 from Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays

Supplementary Table 2 from Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays

BACKGROUND: Mycosis fungoides (MF) is an indolent primary cutaneous T-cell lymphoma. To the authors' knowledge, no data currently are available regarding the evolution over time of the risk of developing specific pathways of disease progression. METHODS: This retrospective study analyzed 1422 patients with MF who were diagnosed and followed from 1975 through 2010 in 27 Italian Study Group for Cutaneous Lymphoma centers. The primary objectives were to ascertain the time course, pathways, and hazards risk trends of cutaneous/extracutaneous disease progression; to evaluate whether different tumor-lymph node-metastasis-blood (TNMB) stages have different pathways of disease progression; and to analyze differences between tumor-stage and erythrodermic MF with regard to clinical onset, disease evolution, and prognosis. The secondary objective was to provide a further validation for the revised International Society for Cutaneous Lymphomas and the Cutaneous Lymphoma Task Force of the European Organization of Research and Treatment of Cancer (ISCL/EORTC) classification. RESULTS: The median follow-up was 14.5 years; stage progression occurred in 29.7% of patients and blood involvement was the most frequent extracutaneous site of disease progression. Patients with stage IA to stage IB disease demonstrated a steady low annual incidence of disease progression to tumor-stage (1%-2%); patients with stage IIA disease had a higher risk within the first years (up to 9.4%). Erythroderma evolved with a significantly higher frequency from patches/plaques (13.9%/28.2%) than tumors (P = .028 and P = .013, respectively). Hazards rates of extracutaneous involvement were low (< 1%). The T-score was found to be associated with extracutaneous involvement site, tumor-stage disease with lymph node/visceral lesions, and erythroderma with blood involvement. TNMB classification and stage progression resulted as independent prognostic variables being detected on multivariate analysis; the type of extracutaneous involvement was found to affect survival . CONCLUSIONS: The data from the current study support the need for a stage-tailored follow-up, suggest that the classification of tumor-stage disease at a stage below erythroderma could be modified, and offer a further validation for the revised TNMB classification. Cancer 2012. © 2012 American Cancer Society.

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.

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.We used flow activated cell sorting analysis to compare the distribution of the T-cell receptor-Vβ 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.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; P0.02). In addition, a skewed usage of T-cell receptor-Vβ subfamilies was observed in the circulating T-cell clone for 61.9% of all patients with Sézary syndrome, T-cell receptor-Vβ 2 and 5.1 subfamilies being the most frequently represented (42.8%), followed by T-cell receptor-Vβ 12 and 13.1.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.

Summary Background Accurate assessment of the somatic mutational status of clonal immunoglobulin variable region (IgV) genes is relevant in elucidating tumour cell origin in B-cell lymphoma; virgin B cells bear unmutated IgV genes, while germinal centre and postfollicular B cells carry mutated IgV genes. Furthermore, biases in the IgV repertoire and distribution pattern of somatic mutations indicate a possible antigen role in the pathogenesis of B-cell malignancies. Objectives This work investigates the cellular origin and antigenic selection in primary cutaneous B-cell lymphoma (PCBCL). Methods We analysed the nucleotide sequence of clonal IgV heavy-chain gene (IgVH) rearrangements in 51 cases of PCBCL (25 follicle centre, 19 marginal zone and seven diffuse large B-cell lymphoma, leg-type) and compared IgVH sequences with their closest germline segment in the GenBank database. Molecular data were then correlated with histopathological features. Results We showed that all but one of the 51 IgVH sequences analysed exhibited extensive somatic hypermutations. The detected mutation rate ranged from 1·6% to 21%, with a median rate of 9·8% and was independent of PCBCL histotype. Calculation of antigen-selection pressure showed that 39% of the mutated IgVH genes displayed a number of replacement mutations and silent mutations in a pattern consistent with antigenic selection. Furthermore, two segments, VH1-69 (12%) and VH4-59 (14%), were preferentially used in our case series. Conclusions Data indicate that neoplastic B cells of PBCBL have experienced germinal centre reaction and also suggest that the involvement of IgVH genes is not entirely random in PCBCL and that common antigen epitopes could be pathologically relevant in cutaneous lymphomagenesis.

Summary Background Cutaneous lymphomas may have a profound impact on patients’ health-related quality of life (HRQoL) and psychological well-being. Objectives To evaluate HRQoL and psychological distress in patients with cutaneous lymphoma, and to evaluate them in relation to personal and clinical characteristics. Methods Patients with cutaneous T-cell lymphoma or cutaneous B-cell lymphoma (CBCL) were consecutively recruited in a dermatological hospital. Data on HRQoL were collected using a dermatology-specific questionnaire, the Skindex-29, and an oncology-specific questionnaire, the EORTC QLQ-C30. Results Of 95 patients, there were 24 with CBCL, 59 with mycosis fungoides (MF) and 12 with Sezary syndrome (SS). The most frequent items reported in Skindex-29 were itching and sensitive skin, being annoyed by the disease, worry that it could get worse, affected interactions, and impairment in sexual life. The most frequent problems appearing from the EORTC QLQ-C30 analysis were fatigue, pain and insomnia. A worse HRQoL was observed for all the scales in patients with SS, followed by MF, and CBCL. HRQoL impairment in all histotypes was higher in women than in men, in patients with probable anxiety or depression, and when the disease worsened. The highest prevalence of probable anxiety or depression was observed in patients treated with systemic steroids (60%) and interferon (50%). Conclusions The detailed evaluation of HRQoL and psychological problems in patients with cutaneous lymphomas, and their relationship with clinical variables, may give important information on the burden of the disease for patients, and thus improve communication and satisfaction with care.

Follicular lesions can either associate with typical patch-/plaque-type mycosis fungoides or, more rarely, be the only clinical manifestation of the disease. We describe 2 adult patients who presented with alopecia and disseminated follicular erythematous papules, and comedones and cysts, respectively. In both patients, histology showed a folliculotropic infiltrate of atypical lymphocytes that spared the epidermis, in the absence of follicular mucinosis. Molecular genetic analysis confirmed the oligo/monoclonal nature of the T-cell infiltrate. Reported cases of purely follicular mycosis fungoides without mucinosis are reviewed.

Introduction Sézary Syndrome (SS) is a rare and aggressive variant of Cutaneous-T-Cell Lymphoma (CTCL) characterized by the presence of malignant lymphocytes named Sezary (SS) cells in the skin, lymph nodes, and peripheral blood. With a poor prognosis, SS has not a specific therapy still available. As a role of skin in SS pathogenesis is not elucidated yet, here we study the contribution of this microenvironment by comparing matched skin and blood derived SS cells for tumor cell proliferation and activation levels of PI3k/AKT pathway. Using our previous SNP array data we also verified the genomic status of members belonging to this pathway in a large cohort of SS patients. Methods Expression of Ki67 proliferation marker was evaluated in perfect-paired blood and skin-paraffin biopsies obtained from eleven SS patients by flow cytometry analysis and immunohistochemistry respectively. KI67+ neoplastic cells were calculated as percentage within neoplastic CD4+ cells recognized by a co-staining with the specific TCRVb rearrangement. Phosphorylation levels of members of PI3k/AKT pathway were compared between matched circulating and skin resident SS cells proteins derived from 2 SS patients using an AKT kinase array (Cell Signaling). Affymetrix SNP6.0 arrays was used to investigate the copy number (CN) status of members of mTORC 1 pathway in a cohort in 37 SS samples derived from 23 SS patients and 3 cell lines. Results Skin derived SS cells showed a significant higher proliferation index (PI) respect to SS cells obtained from blood (12%±11 vs 1,24%+1,18; P = 0,00025). In order to identify the signals responsible for SS proliferation, we used a PI3K/AKT kinase array that revealed an enhanced phosphorylation levels of many components of this cascade, particularly of PRAS40 with a Fold change (Fc) of 6,15; GSK3a (Fc = 4.83), mTOR (Fc = 4.61) mP70S6K (Fc = 4.64) BAD (Fc = 7.09) and 4EBP1 (Fc = 5.40)in skin-SS cells respect to circulating ones. We next deepen our observations in mTORC1 signaling because of this and earlier observations made in CTCL cell lines. Using our SNP6 array data we have verified the genomic status of members of this pathway. Results obtained showed that P70S6K, the kinase downstream TORC1 that plays a crucial role in protein synthesis, showed a mono-allelic gain in 9 out 23 patients (39%) whereas PDCD4, a protein that inhibits protein translation displayed a mono-allelic loss in 10 of 23 individuals (43%). 4 of these patients showed a concomitant P70S6K gain and PDCD4 loss. Overall these genetic defects suggest that an abnormal protein synthesis can occur in these patients.. Conclusion: Our data demonstrate that skin microenvironment enhances SS cell proliferation index and activates mTORC1 signaling, an unbalanced pathway that reveals novel potential therapeutic targets for Sezary Syndrome. Citation Format: Cristina Cristofoletti, Mario Picozza, Antonella Bresin, Maria Cristina Picchio, Enrico Scala, Giuseppe A. Lombardo, Francesca Passarelli, Elisabetta Caprini, Giandomenico Russo, Maria Grazia Narducci. Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 936.

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma and its pathogenesis is still unknown. Diagnosis/prognosis may strongly ameliorate the management of SS individuals. Here, we profiled the expression of 470 microRNAs (miRNAs) in a cohort of 22 SS patients, and we identified 45 miRNAs differentially expressed between SS and controls. Using predictive analysis, a list of 19 miRNAs, including miR-21, miR-214, miR-486, miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs including again miR-21, potentially able to discriminate patients with unfavorable and favorable outcome. We validated our data for miR-21, miR-214 and miR-486 by qRT-PCR, including an additional set of array-independent SS cases. In addition, we also provide an in vitro evidence for a contribution of miR-214, miR-486 and miR-21 to apoptotic resistance of CTCL cell line.

In this study, we used single nucleotide polymorphism and comparative genomic hybridization array to study DNA copy number changes and loss of heterozygosity for 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71% and 68% of cases, respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in >30% of tumors: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis; however, when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Integrating mapping and transcriptional data, we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer-related genes such as members of the NF-κB pathway (BAG4, BTRC, NKIRAS2, PSMD3, and TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times, we identify several common candidates that might exert critical roles in SS, such as BUB3 and PIP5K1B. Altogether, our study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggests a novel set of genes of potential interest in SS. [Cancer Res 2009;69(21):8438–46]

Abstract 3717 Poster Board III-653 Introduction Primary cutaneous lymphomas (PCL) present in the majority of histologic variants an indolent behaviour and a good prognosis with a prolonged survival. In a very small number of patients (pts), PCL is aggressive at the onset while in pts who are resistant or have relapsed after repeated topic or systemic therapies an advanced disease is more frequently observed. In these patients no therapy is capable of inducing a stable remission of the disease. The efficacy and low toxicity of Pegylated Liposomal Doxorubicin (PLD) as a single agent in second-line therapy of PCL was recently demonstrated. In our study we tested the safety and efficacy of PLD (Caelyx®) (C) in association with three drugs of proven effectiveness in nodal lymphoprolipherative and other primary cutaneous neoplastic disorders: Bleomycin (B), Vinblastine (B) and Dacarbazine (D) (CBVD). Patients and Methods From February 2003 to December 2008 we observed 37 consecutive pts with advanced PCL: 19 Cutaneous T-Cell Lymphomas (CTCL) and 18 cutaneous B-Cell Lymphomas (CBCL). The CTCL pts were: 15 males and 4 females, with median age 59 (27-86) years, of which 7 pts with Anaplastic Large Cell Lymphoma (ALCL) CD30 positive, 7 pts with transformed Mycosis Fungoides (tMF), 3 pts with CD30 negative ALCL, 1pt with Panniculitis-like (Pl) Lymphoma and 1 pt with transformed Sezary Syndrome (tSS), according to the WHO-EORTC consensus classification. Nine pts presented with a nodal involvement and 10 pts were resistant or relapsed after 1 or more systemic treatments. Among the CBCL pts there were: 8 males and 10 females, with median age 61 (42-84) years, 12 pts had a Follicular Centre Lymphoma (FCL), 3 a Marginal Zone Lymphoma (MZL) and 3 a Diffuse Large B-Cell Lymphoma Leg Type (LT). Five pts presented a nodal involvement and 11 pts had received 1 or more previous systemic therapy. All 37 pts received CBVD therapy at following dosage: C: 20 mg/m2, B: 10 mg/m2, V: 6 mg/m2, D: 325 mg/m2 at days 1 and 15, administered intravenously every 4 weeks for a maximum of 6 cycles. Rituximab (R) at dosage of 375 mg/m2 was administered to CBCL pts at 1st day of each cycle. Before the treatment, pts were submitted to a complete staging of disease including TC-scan, bone marrow biopsy, and immunophenotyping of peripheral blood cells. Results In the CTCL group 4 pts received 4 CBVD cycles, 14 pts received 6 CBVD cycles and 1 pt presented a progressive disease after the 1st cycle. Overall Response Rate (ORR) was 94.7% (18/19 pts). Sixteen out of 18 pts (88.8%) had a Complete Remission (CR) with disappearance of nodal involvement and cutaneous lesions, 2/18 pts obtained a Partial Remission (PR) with disappearance of nodal involvement and 75% of cutaneous lesions. In the CBCL group 3 pts received 2 R-CBVD cycles, 3 pts 4 cycles and 11 pts 6 cycles: ORR was 100% and all pts obtained a CR. The occurrence of palmoplantar erytrodisesthesia in 5 pts and grade 2-3 granulocytopenia in 12 pts did not modify the therapy program. Two CTCL pts (1 tSS, 1Pl) and 1 CBCL (LT) pt in CR after therapy received an allogeneic Hemopoietic Stem Cells (HCS) transplant and 1 CBCL(FCL) pt a syngeneic HCS transplant. In CTCL group 11 (61%) pts maintained their response after a median follow up of 15.5 (9-71) months. Among CBCL pts 14 (77.7%) pts are still in CR after a median follow up of 15 (4-36) months. Conclusions Our experience demonstrates that the CBVD association is an effective and safe therapy for advanced PCL in inducing an important tumour burden reduction with a high CR rate. The assessment of response duration requires a longer observation. A larger number of patients in a multicentric trial are needed to confirm our promising results. Disclosures: Off Label Use: liposomial doxorubicin has a peculiar cutaneous tropism and low cardiac toxicity compared with other anthracyclines.

Chemokine and chemokine receptors expressed by normal and neoplastic lymphocytes play a key role in cell recruitment into skin and lymph nodes. The aim of this study was to get further insights into the role of chemokines in pathogenesis and progression of cutaneous T-cell lymphoma (CTCL) with particular regard to Sézary Syndrome (SS), a CTCL variant with blood involvement. Here, we show that functional CXCL13 homeostatic chemokine is strongly up-regulated in SS cells, well-detectable in skin lesions and lymph nodes, and measurable at high concentration in plasma of SS patients, at different levels during disease progression. Furthermore, we show that the addition of CXCL13 to CCL19 or to CCL21, the selective CCR7 agonists responsible for lymph node homing, strongly enhances the migration of CCR7+ SS cells. We also show that neutralization of the CCR7 receptor strongly impairs CCL19/21-induced chemotaxis of SS cells both in the absence or presence of CXCL13. Additional experiments performed to investigate the survival, adhesion, and metalloproteases secretion indicate that CXCL13 combined with CCL19 and CCL21 mainly affects the chemotaxis of SS cells. Our findings suggest that this newly described CXCL13 expression in SS represents a new pathogenetic mechanism of diagnostic significance. [Cancer Res 2008;68(17):7137–46]

A descriptive study in a Dermatological Research Institution in Rome, Italy, was conducted to test the feasibility and acceptability of health related quality of life (HRQoL) assessment in the daily routine. Quality of life, and psychosocial distress evaluations were obtained for each patient. Patients were invited to complete the Skindex-29, GHQ-12, and SF-36. Results were returned to the clinical staff using standardised feed-back forms with: a) "categories" of QoL scores to help interpreting Skindex-29 scores; b) "warnings" pointing out problems that patients experienced "all the time"; c) categories of GHQ-12 scores for minor psychiatric problems; d) the classical SF-36 graph depicting the patient's "QoL profile" with normative references. The clinical staff were trained, and then their attitudes and behaviours were surveyed using a standardised questionnaire. For all 170 participants enrolled (63% males, 35% age64 years), feed-back forms were provided within three hours from data collection. For patients with repeated hospitalisations time-trends in HRQoL measurements were also provided. The acceptability, both for patients and the clinical staff, was high but the actual impact on clinical activities was limited. The routine assessment of HRQoL in dermatology is feasible and well accepted both by patients and by the clinical staff. The application of these widely used questionnaires should be implemented in a larger scale and evaluated in different settings.

Sézary syndrome (SS) is a rare form of cutaneous T-cell lymphoma (CTCL) characterized by a distinct metastatic pattern mainly involving blood and skin. Chemokines and their receptors play a critical role in cellular recruitment and homing to tissues and in the metastatic process of several tumors including non-Hodgkin T-cell lymphomas (NHLs). Here we report that SS cells express a functionally active CXCR4 and that its ligand SDF-1 is abundantly produced in the skin, which represents the main destination of SS cell spreading. SDF-1 is normally inactivated by proteolytic cleavage by the CD26/dipeptidylpeptidase IV (DPPIV). The lack of CD26 from the cell surface is a hallmark of circulating SS cells. We also show that the CD26- phenotype is maintained also in skin-infiltrating neoplastic T lymphocytes and that SS-affected individuals exhibit a reduced activity of plasma soluble CD26. Finally, we observe that the addition of soluble CD26 reduces the migratory response of SS cells to SDF-1 whereas the inhibition of the CD26 peptidase activity in Hut78, a CD26+ CTCL cell line, enhances the SDF-1-induced migration of these cells. Our findings suggest that the SDF-1-CXCR4 axis could play an important role in skin homing of SS through the regulatory activity of CD26.

Introduction. Rituximab has been demonstrated to be effective either as intralesional or as systemic therapy in PCBCL, We report our experience in the treatment of PCBCL with intravenous Rituximab. P atients and Methods From February 1999 to February 2014 we treated 75 patients: 47 Follicle Center Lymphoma, 23 Marginal Zone Lymphoma, 5 Diffuse Large Lymphoma Leg type. The stage was T1 in 38 pts, T2 in 22 pts and T3 in 15 pts. In 24 patient prior treatment included: CHT (11), Radiotherapy (4), Surgery (4) or alpha2Interferon (IFN) (5). Rituximab at dosage of 375 mg/m2 for a minimum of 4 cycles, was administered alone (51 patients) or in association with CHT (13). RT (2) or IFN (3). Results. No patient presented adverse effects during the Rituximab infusion. A reduction of circulating B lymphocytes was observed for 11 months, on the average, without an increased risk of infections. No added toxicity was observed in patients treated with Rituximab plus CHT. Overall response rate was 97,3% (CR 82,6 %, PR 14,6 %). Five–years Overall Survival (OS) was 86,9% with Disease Free Survival of 57%. According to stage OS was in T1 94,3%, in T2 90,5%, in T3 73,6%. (T1+T2 vs T3: p Conclusions. Rituximab is effective and safe in the treatment of PCBCL even in heavily-treated or elderly patients. In our patients only the stage of disease was significant for the prognosis. A higher number of patients are necessary to indicate Rituximab in biological and clinical subsets of patients as a front-line therapy. Disclosures No relevant conflicts of interest to declare.

Abstract 4814 Sezary Syndrome (SS) is characterized by specific chromosomal abnormalities, however is not completely clarified yet which are the genetics hits associated to the initial phase or associated to disease progression. In this study, employing both high density Comparative Genomic Hybridization array (aCGH) and Single Nucleotide Polymorphism (SNP) array technologies, we elucidated the most frequent and significant chromosomal gain and loss regions, and new potentially relevant aberration for the disease progression onset. A total of 30 samples derived from 18 SS patients were analyzed on the Gene Chip Human Mapping 10K Array (Affymetrix). Genotype call and signal information performed by dChip SNP Software (http://www.dchip.org) provided us normalization and simultaneous measurement of Loss of Heterozygosity (LOH) and DNA Copy Number (CN) changes. We have further refined our analysis with a systematic method, Genomic Identification of Significant Targets in Cancer (GISTIC), able to identify regions that were significantly amplified o deleted, assigning a G-score that considers the amplitude of the aberration as well as the frequency of its occurrence across samples (http://genepattern.broadinstitute.org). Our data, generating by the integration of this two methods (see Table 1), revealed 19 significant focal event, including 10 amplification (10p, 17q, 8q, 6p, 4p, 1q, 18q, 21q, 3q, 1p) and 9 deletions (14q, 17p, 10p, 9p, 8p, 13q, 12p, 6q, 2p). In addition, new significant aberrant regions were identified, such as losses of 9p21.3, locus of an important tumor-suppressor gene CDKN2A (p16INK4a/p14ARF) and CDKN2B (p15INK4b). Recently, several study have reported the great influence of alteration in p16INK4a/p14ARF for prognosis of Diffuse large B-cell lymphoma (DLBL), primary cutaneous DLBCL leg-type, Blastic plasmacytoid dendritic cell neoplasm (BPDCN). Across our set of samples, 9p21.3 deletion occur in 8/30 samples from 6 patient, prevalently in heterozygosity state (Log2 ratio from −0.3484 to –0.7691). Two cases presented homozygous deletion (Log2 ratio from −1.2157 to –1.5342), and in four patients losses appeared only in a following phase of observation, suggesting a tumor progression event. Table 1 Significant regions of chromosomal gain Genes in Region Significant regions of chromosomal loss Genes in Region Cytoband Q value GISTIC peak Cytoband Q value GISTIC peak 10p12.33 8.07E-13 17765313-19264851 8 14q11.2 1.85E-40 21395737-22130391 1 17q11.1 3.54E-09 20147238-62034035 563 17p13.1 2.15E-10 1-15053486 234 8q22.3 2.93E-07 70887466-146274826 314 10p11.22 1.98E-07 30280444-32664641 8 6p25.3 0.0020062 1-1263549 6 9p21.3 0.00074825 21283632-22089567 11 4p15.32 0.0071803 17395812-20340589 4 8p23.3 0.00076308 1-32177352 179 1q23.3 0.039674 158849705-159641626 8 13q21.31 0.0028295 63681448-65120691 0 18q22.3 0.1913 1-76117153 251 12p13.1 0.025412 9304151-17414118 85 21q22.3 0.1913 1-46944323 223 6q24.1 0.029124 139319577-141635965 4 3q29 0.2191 169050880-199505740 163 2p23.3 0.043403 24636437-43910832 126 1p36.31 0.11633 1-20338260 239 Disclosures: No relevant conflicts of interest to declare.

Sezary syndrome (SS) represents a very aggressive form of Cutaneous T Cell Lymphomas with a median overall survival of 5.1 years (range, 0.4-18.6 years). As it appears evident there are in this disease either short survivors than long survivors. Previous studies of prognostic indicators in SS showed that circulating Sézary cell count, high CD4/CD8 ratio, advanced age, high lactate dehydrogenase serum level and a high white blood cell count were associated with an unfavorable outcome. Up to now, few data have been provided concerning possible associations between immunological and genetic markers, who might provide also clues on tumor progression in this disease. In the last 10 years we have observed more than 50 Sezary Syndromes in our Institute and phenotyped them with new immunologic markers. We have also performed genetic analysis with Single Nucleotide polymorphysms (SNPs) for evaluation of genomic imbalances, mRNA expression and lately microRNA(miRNA) expression profiling. In this study we have analyzed the expression profile of 470 miRNAs using Agilent platform array in 22 SS patients. We investigated the relationship between the expression level of miRNAs and the clinical outcome of SS patients by Kaplan-Meier method and risk assessment by multivariate analysis. We also functionally investigated the role of miR-21, mapping in one of the region more frequently amplified in SS and some of its targets. We identified 45 miRNAs differentially expressed between SS and healthy controls. Using predictive analysis, a list of 19 miRNAs, including miR-21 and miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs able to discriminate patients with unfavorable and favorable outcome. We show that miR-21 knockdown increases apoptosis and modulates TGF beta receptor 2 expression in vitro. The antipoptotic effect appears to be regulated through PTEN. Conversely, we were not able to observe a direct miR-21 regulation on PDCD4 gene mapping to chromosome 10q24, a frequently imbalanced region in SS. In conclusion we have identified a new prognostic miRNAs signature in SS and characterized the role of miR-21, one of the most involved in cancer, recognized as a disease and prognostic classifier in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 150. doi:10.1158/1538-7445.AM2011-150

Background: Sézary Syndrome (SS) is a rare and aggressive leukemic variant of Cutaneous T-Cell Lymphoma (CTCL) characterised by erythroderma and the presence of a malignant circulating memory CD4+ T cell clone with skin homing characteristics, lack of CD26 and CD49d and over-expression of CD60. The availability of a large panel of monoclonal antibodies to CDR3 Vß epitopes, allows the study of the TCR repertoire by flow cytometry and a more precise identification of the malignant clone phenotypic characteristics. Objective: The aim of this study was to evaluate both the distribution of TCR-Vß family and CD60/CD49d expression within the clonal T cell population in patients affected by SS. Methods: We studied, by five-color flow cytometry analysis, TCR-Vß repertoire in 62 patients affected by SS, in 180 patients with MF, 6 cases of B cell Lymphomas (BCL), 19 with chronic Eczema (CE) recruited and followed from 1998 to 2008. A sequential gating strategy was used in order to examine the CD60 and CR49d expression within the neoplastic clonal population identified through the TCR-Vß repertoire analysis. Results and Conclusion: A skewed usage of only 4 out of 25 TCR-Vß subfamilies was observed in circulating T cell clone in 61.9% of patients affected by SS. TCR-Vß 2 and 5.1 were the most frequently represented (42.8% of TCR-Vß reactive patients), followed by TCR-Vß 12 and 13.1. In 20 cases the TCR-Vß panel was unable to identify the neoplastic clone, recognizing less than 5% of T cells, thus representing an indirect proof for the presence of a clonal population. CD60 was over expressed and CD49d down regulated within clonal T lymphocytes. This phenotype of the dominant T cell clone in the peripheral blood of patients with SS was significantly associated with a lower survival rate (chi-square 5.467, p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1736.

Abstract 3238 Poster Board III-175 We have used single nucleotide polymorphism (SNP) and comparative genomic hybridization array (aCGH) to study DNA copy number (CN) changes and loss of heterozygosity (LOH) in a series of 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous-T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71 and 68% of cases respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in more than 30% of tumours: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis, however when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Crossing genomic data mapping with those obtained from gene expression profiles of matching samples we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer related genes such as members of the NF-kB pathway (NKIRAS2, PSMD3, BAG4, BTRC, TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times we identify several common candidates that might exert critical roles in SS, like PIP5K1B and BUB3. Taken together this study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggest a novel set of genes of potential interest in SS. Disclosures No relevant conflicts of interest to declare.

Primary cutaneous B-cell lymphomas (PCBCL) are a distinct group of primary cutaneous lymphomas with few and controversial reports on their treatment and prognostic factors. The aim of this retrospective study of a large international series of PCBCL patients was to analyze the patient and lymphoma characteristics as well as treatment-related variables associated with clinical outcome. From 1980 to 2006, 507 patients were referred to 19 cancer centers of 6 countries all over the world. The median age was 55 years (range,16–92 years) and the M/F ratio was 1.4. According to the WHO-EORTC classification indolent lymphomas included 341 FCL and 122 MZL, while aggressive NHL were represented by DLBCL, leg type (n=44). Sixty patients (12%) had stage II. The majority of cases was diagnosed in trunk/arms (52%), while in 29% in head/neck and in 13% in the legs; 7% of patients had a generalized disease (>1 site). The maximal lesion diameter was >4 cm in 21% of cases and ≥2 lesions were recorded in 39%. The prevailing type of lesions were nodules (74%), while only a minority of patients (6%) were affected by tumors. Few patients had B symptoms (5%), poor ECOG-PS (9%) or elevated LDH (7%). Two hundred eighty-four out of 446 patients (64%) were treated only with surgery (n=86) or chemotherapy (n=95), mostly consisting of a short course of anthracyclin containing regimens, or radiotherapy (30–50 Gy) (n=103). One hundred sixty two cases (36%) received combined therapy, mostly including surgery or chemotherapy, followed by radiotherapy. A small subgroup of 35 patients were given rituximab alone (n=19) or in combination with other treatments. The remaining 26 patients did not receive any therapy. The response rate of 446 patients was the following: 402 achieved CR (86%), 38 PR and 6 were in SD. Neither histology nor treatment significantly influenced CR rate. Among 402 responders, 128 (32%) eventually relapsed, 86% in the skin, 10% in extracutaneous sites and 4% in both. The relapse rate varied according to histology, ranging from 52% in DLBCL leg-type to 29% in MZL and 28% in FL. Moreover, combined treatments significantly reduced relapse rate (24% vs. 37%; p=0.008). The achievement and maintenance of CR significantly influenced the long-term disease specific survival (at 20 year 99 % vs. 45%; p=0.0001). The CR rate of subgroup of 35 patients treated with rituximab, was 74%, while the relapse rate was 35%. These results were not influenced by the addition of other therapies to rituximab. After a median follow-up of 53 months (range, 2–333 months), 5 and 10-year estimate of OS, disease-specific survival, PFS and DFS were 91%, 92%, 61%, 65% and 82%, 88%, 49% and 56%, respectively. Cox multivariate analysis, stratified for age with a stepwise selection of the significant variables, identified DLBCL, leg-type histology, elevated LDH, type of lesion (nodules and tumors), B symptoms and female gender, as significant predictors of a poor OS. In conclusion this retrospective analysis confirms, on a large series of cases, that patients with PCBCL belong to different risk categories requiring a tailored treatment approach. These data can be usefully taken into account for an adequate management strategy of PCBCL patients.

Cutaneous lymphoma is a visible cutaneous malignancy, which especially in the more severe forms may have a profound effect on patients lives. The aim of this study was to evaluate quality of life (QoL), psychological distress, and alexithymia in patients with cutaneous lymphoma referring to a dermatological hospital, and to correlate these results with clinical parameters. QoL was evaluated with the Skindex-29, that measures QoL in skin conditions on three scale (symptoms, emotions, and functioning), and the EORTC QLQ-C30, that assesses the QoL of cancer patients. Presence of minor non-psychotic psychiatric disorders was assessed using the GHQ-12, defining as case patients scoring 4 or more; alexithymia was evaluated using the TAS-20 questionnaire. In our ongoing study, we analysed data from the first 66 patients. Of them, 67% were men, the mean age was 58 years (range: 26–85y), and there were 15 patients (23%) with cutaneous B-cell lymphoma (CTBL), and 51 with T-cell lymphoma (CTCL), 6 of them with Sézary Syndrome. In our population, 31.3% of patients were GHQ-cases, 18.6% had alexithymia, and another 22% possible alexithymia. Dermatology-specific QoL was particularly impaired in the symptoms and the emotions scales. QoL was always significantly more impaired in patients with CTCL than in those with CBCL. Also, the prevalence of GHQ-cases was higher (35% in CTCL vs 13% in CBCL), as well as the prevalence of people with alexithymia (48% vs 20%). Skindex-29 and TAS-20 scores significantly correlated with the T stage of lymphoma (i.e., the higher the stage, the lower the QoL and the higher the prevalence of alexithymia). The evaluation of quality of life and psychological problems in patients with cutaneous lymphomas may help clinicians to better manage the disease and its burden on patients life. Also, the relation between quality of life impairment and clinical variables may give important information on the course of the disease as well as the possible effect of treatment.

Sézary Syndrome (SS) is a rare and aggressive form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving blood and skin. Our expression analyses performed by microarrays demonstrated that many chemokines resulted up-regulated in this type of lymphoma. Since these chemoattractant molecules play a critical role in cellular recruitment and homing to tissues and in the metastatic process of several tumors, we focused our attention on one of them named CXCL13, a lymphoid chemokine involved in B-cell compartmental homing within secondary lymphoid organs. Peripheral Blood Mononuclear cells (PBMCs) were isolated from blood obtained from SS patients and controls by Ficoll-Hypaque density gradient centrifugation (Sigma Aldrich). SS cells and healthy resting CD4+ lymphocytes were purified by positive selection using an anti-human-CD4 conjugated dynabeads (Oxoid). Total RNA was extracted using the Trizol reagent (Life Technologies). Quantitative-Real Time RT-PCR analysis was performed on CD4+ sorted from 14 SS patients and 3 controls. CXCL13 primers were designed by means of the Primer Express software package (Applied Biosystems). The qRT-PCR were performed with a SYBR Green I dye chemistry and AmpliTaq Gold DNA Polymerase on an ABI PRISM 7000 machine (Applied Biosystems). Immunohistochemistry analyses for CXCL13 were performed on formalin-fixed, paraffin-embedded skin biopsies from 15 SS, 15 MF, 6 MF-B cell rich patients using streptoavidin-biotin peroxidase labeling method (DAKO). Sections were counterstained with hematoxylin. Plasma CXCL13 levels were determined using a CXCL13 ELISA kit (BD Pharmingen). Results can be summarized as follow: qRT-PCR analysis revealed that 6 out 13 of SS patients showed an high mRNA levels of CXCL13; Immunohistochemistry analysis showed that CXCL13 is abundantly expressed by neoplastic skin-infiltrating lymphocytes of 9 out 15 SS skin biopsies. Conversely, CXCL13 is weakly expressed on scattered neoplastic skin-infiltrating lymphocytes of 1 out 15 MF and 1 out 6 MF-B cell rich biopsies. Plasma CXCL13 concentrations in SS patients (n = 10) were 1362 ± 134 pg/mL. Conversely, those in MF patients (n = 10) and healthy donors (n = 5) were 70 ± 43 and 13 ± 10 pg/mL, respectively. Compared with healthy controls, plasma CXCL13 levels were significantly higher in patients with SS (p

Sézary Syndrome (SS) is a rare and aggressive form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving blood and skin and whose etiology and molecular pathogenesis are still unclear. Conventional cytogenetics studies have shown that most SS patients have chromosome aberrations; however allelotyping studies and genome-wide surveys for chromosome imbalances in this tumour are still very limited (Mao X. et al. 2003 Genes Chromosome Cancer 36:250–260). High-density single nucleotide polymorphism (SNP) arrays allow high-resolution and genome-wide detection of both loss of heterozygosity (LOH) and copy number (CN) abnormality. Therefore we used Affymetrix 10K SNP mapping array containing 11,560 tiled SNPs to investigate genomic aberrations of 13 individuals affected by SS. Genotype calls and signal information were obtained using GeneChip Operating Software (GCOS 1.4) and GeneChip DNA Analysis Software (GTYPE4.0). SNP calls were exported to be analysed with DNA-chip Analyser (dChip v1.3+) genotyping software which allows the simultaneous measurement of DNA copy number changes and LOH events (Zhao X. et al. 2004 Cancer Res. 64:3060–71; Lin M et al. 2004 Bioinformatics 20:1233–40). Our findings indicate that chromosomes 17p, 10/10q and 9 are most frequently affected by LOH events, while gains of CN were observed more often for chromosome 17q and 8/8q. Among our patients almost all individuals showing loss of the 17p arm have also gain of the 17q, suggesting the presence of the isochromosome 17q, a frequently reported abnormality in SS. In addition to this, we characterised the chromosome LOH pattern identifying seven regions of significant loss shared by multiple tumours. Sample clustering based on significant LOH regions identified two groups of patients: one of them consists of 4 patients with a lower rate of chromosomal losses while the other contains 9 patients mainly characterised by the co-occurrence of LOH at chromosome 17 and chromosome 10. The frequency and pattern of chromosomal changes in our group of 13 SS patients are in substantial agreement with previously described results using more conventional techniques, demonstrating the feasibility of the 10K SNP mapping array system to assess allelic imbalance in this tumour. The genome-wide approach and SNP high density allowed the identification of a larger number of LOH regions, including, however, those already described (chromosome 9p, 10q and 17p). Even though no significant statistical association can be observed due to the low number of cases available, we observed a lower overall survival in the group of 9 patients showing simultaneous LOH events at chromosome 17 and 10.

Sezary Syndrome (SS) is a rare form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving skin and blood. Chemokine and chemokine receptors have been implicated in the spreading process of many cancers including various forms of non-Hodgkin T-cell lymphomas (NHL). In this study we report that chemokine receptor CXCR4 is over-expressed by both circulating and skin-homing neoplastic T-lymphocytes of SS patients and is functionally active as demonstrated by the migration of freshly isolated Sezary (SzS) cells along the chemical gradient of its natural ligand SDF-1. To shed light on the regulation of CXCR4/SDF1 interaction, we also investigated the enzymatic activity of CD26/dipeptidylpeptidase IV (DPPIV) since SDF-1 is efficiently inactivated by CD26, in physiological condition.. This is of particular relevance because one of the hallmark of the circulating SzS cells is the loss of CD26 from the cell surface. We first demonstrated that the CD26 negative phenotype is similarly maintained also in the skin-homing neoplastic T lymphocytes; we then observed that the addition of exogenus soluble CD26 reduces the migratory response of SS cells to SDF-1 whereas the inhibition of the CD26 peptidase activity in Hut78, a CD26-positive CTCL cell line, enhances the SDF-1-induced migration of these cells. We finally showed that SS individuals exhibit a reduced activity of the soluble CD26 as revealed by the measurements performed on the patients derived plasma. Our findings suggest that the SDF-1-CXCR4 axis could play an important role in skin homing of SS through the regulatory activity of CD26.

We created a relational database of patients with cutaneous T-cell lymphoma (CTCL) to collect in a standardised fashion, anagraphic variables, clinical history, clinical, histological, haematological, and immunological information on CTCL patients hospitalised at IDI-IRCCS, Rome, Italy. At present, there are data on 424 patients, hospitalised from 1983 to July 2005. Active follow-up is performed yearly to ensure a standardised ascertainment of survival time. For deceased patients, the actual date of death (for all causes) is recorded, while surviving patients are censored at the date of last contact. The database includes 29 patients with Sezary syndrome (SS). Follow-up times ranged from 0 to 105 months. At first hospitalisation the median values of cells/mL were: white blood cells (WBC) 8750, neutrophils 4250, eosinophils 140, basophils 120, lymphocytes 2760, monocytes 500, CD3+ 2780, CD4+ 2431, CD8+ 192, CD19+ 96. Seventeen patients were deceased. We included in the Kaplan-Meier survival analysis only patients who were diagnosed before July 2004 (n=26). Median survival time from diagnosis was 52 months. No significant differences were observed in mortality for WBC (cutoff 9000 cells/uL), neutrophils (cutoff 4500 cells/uL), basophils (cutoff 200 cells/uL), lymphocytes (cutoff 3000 cells/uL), monocytes (cutoff 500 cells/uL), CD3+ (cutoff 2000 cells/uL), CD4+ (cutoff 2000 cells/uL), CD8+ (cutoff 200 cells/uL), CD19+ (cutoff 70 cells/uL). A lower survival was observed for patients with eosinophils Survival in patients with SS does not seem to be influenced by haemathologic parameters. However, patients with long-term survival (>90 months) are observed, and their characteristics should be further investigated. Survival analysis of 26 patients with Sézary syndrome, Rome, Italy, 1991–2004. Survival analysis of 26 patients with Sézary syndrome, Rome, Italy, 1991–2004.

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.