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Abstract 2713 Primary cutaneous T-cell lymphomas (CTCL) are usually characterized by a T-helper CD4+ immunophenotype and show an indolent clinical course; on the contrary, cases harbouring a CD8+ cytotoxic immunophenotype exhibiting epidermotropism (AECTCL) or pleomorphic morphology (PTL-NOS), as well as cases with gamma-delta + (CGDL) immunophenotype, display a very different course. Clinically, these tumours are very aggressive, with poor outcome in adults. Chemotherapy, preferably based on intensive CHOP-like regimens, represents the mainstay of treatment, when possible followed by stem cell transplantation. Histologically, tumour cells constitute a population of pleomorphic medium-large size elements. In these rare CTCL subtypes few reports have been published dealing with oncogenomic investigations. With this study, by array-based comparative genomic hybridization (a-CGH) and gene expression profiling (a-GEP), we aimed to explore genomic alterations possibly involved in tumorigenesis of aggressive CTCL referred to our department. We focused on 9 cases of CD8+ AECTCL, 3 cases of CD8+ PTL-NOS and 4 cases of CGDL, who were extensively investigated by a-CGH; a-GEP was applied in 5 cases of AECTCL to further expose genomic profile of neoplastic cells. Among AECTCL patients, immunophenotype was always typical and EBV was demonstrated only in one case. Results were assessed by statistical analysis to reveal more significant common chromosomal aberrations. By a-CGH investigations, we found the presence of extensive gains and losses of both large and small chromosomal regions; copy number gains were more frequent than losses. In CD8+ cases, we observed gains of 3p21.33-p21.2 in 10 out of 12 cases, 6p21.2-p21.1 in 8/12, 7q11.23 in 9/12, 7q21.2-q22.1 in 10/12, 7q36.1-q36.3 in 7/12, 8q24.3 in 8/12, 11pter in 11/12, 11q12.3-q13.2 in 10/12, 16p13.3 in 10/12, 17q in 10/12, trisomy 19 (as a mosaic aberration) in 12/12, and trisomy 22 in 8/12; losses involved 4q12-q22.2 in 8/12, 9p21.3 in 10/12 (homozygous in 7) and 14q11.2 in 9/12. In CGDL, recurrent copy number alterations (observed in 50% to 75% of cases) were gains of 2q13-qter, 17q21.3, 19 (trisomy, as mosaic) and 22q11.21, along with losses of 4q12-q22, 6q23.3 and 9p21.3. Summarizing, most of our genomic results have been previously described in other cutaneous lymphomas: gains of 3p21 in diffuse large B-cell lymphomas (DLBCL), of 7q21 in mycosis fungoides (MF), of 8q24 in PTL-NOS and in Sézary syndrome (SS), of 11q22-q13 in DLBCL, of 16p in DLBCL and in angioimmunoblastic T-cell lymphoma (AITL), of 19p in AITL and natural killer lymphomas, trisomy 22 in AITL, PTL-NOS and SS, gain of 17q in SS and in ALCL, MF and PTL-NOS. However, we underline the loss of p21.3, determining CDKN2A deletion, as a common alteration in these three CTCL entities. We also found other common anomalies in AECTCL and PTL-NOS, with particular interest with regard to gain of 17q, 19p13 and 19q13.11-q32, which associate with the JAK/STAT signaling pathway activation. Other alterations in AECTCL cases involve c-MYC (8q24), CCND1/CDK4–6 (11q13) and IL21R (16p13.3). Worth mentioning, GEP analysis in AECTCL confirmed altered expression of CDKN2A, JAK3 and STAT6 genes. Although most genetic aberrations detected in our study have already been described in other lymphoproliferative disorders, we conclude that the combination of aberrations appears characteristic in these aggressive disorders. Disclosures: No relevant conflicts of interest to declare.

Abstract 2714 Sézary Syndrome (SS) is an aggressive type of cutaneous CD4+ T-cell lymphoma characterized by erythroderma, generalized lymphadenopathy and presence of malignant T cells in peripheral blood. Patients with SS have a generally poor median survival (2–4 years), with allogeneic stem cell transplantation as the only curative treatment option. Genome-wide analysis of chromosomal alterations represents a current powerful tool to investigate pathophysiology in hematological malignancies, possibly leading to development of new therapeutic agents. A recent important study reported gain of 17q22–25 and 8q22–24, as well as loss of 17p13 and 10q25 as characteristic genomic aberrations in SS. In this study, by array-based comparative genomic hybridization (a-CGH), we aimed to further explore genomic alterations in 25 patients with Sézary Syndrome (SS) referred to our Institution. The patient series included 11 males and 14 females, with a median age of 65 years (range 29 to 85). At diagnosis, 3 patients were in stage IIIB, 18 in stage IVA1 and 4 in stage IVA2. Flow cytometry analysis unveiled typical CD4+/CD7±/CD26- lymphocytic immunophenotype, while molecular analysis showed clonal rearrangement of T-cell receptor beta and/or gamma chains in all patients. At the time of blood samples collection, 21% of patients were untreated. Among treated ones, photopheresis alone was used in 37% whereas all the others received also chemotherapy-based therapies. Lymphocyte count was higher than 3000/mcL in 75% of patients, higher than 6000/mcL in 46% and higher than 9000/mcL in 25%. Elevated LDH levels were observed in 29%. Genomic DNA was isolated from peripheral blood mononuclear cells of 10 patients and from CD4+/CD14− cells of 15 patients, selected by an immunomagnetic method. Quantity and quality of all gDNA samples were assessed using UV- VIS spectrophotometry and agarose gel electrophoresis. Genome-wide array-based comparative genomic hybridization (aCGH) was performed using the Agilent Human Genome CGH Microarray Kit 4×44K. Copy number profiles from CGH arrays were compared using Integrative Genome Viewer. Most frequently observed recurrent copy number alterations involved gains in chromosomes 7, 8 and 17 and losses in chromosome 10, 17 and 19. In particular, chromosomal gains involved 7q11.21-7q11.23 in 32% of patients, 7q21.3-7q22.1 in 36%, 8q24.2-8q24.3 in 44% (with amplification of the MYC oncogene in 36%), while chromosomal losses involved 10p11.22 in 44% of patients, 10q11.22–21.1 in 48%, 10q23.3 (harbouring the PTEN tumor suppressor gene) in 40%, 10q24 (involving NFkB2 gene) in 56%, 10q25.1-q26.3 in 56%, and 19p13.3 (involving the cell growth/apoptosis regulating GADD45B gene) in 32%. With regard to chromosome 17, we observed loss of region 17p13.1 (containing the TP53 gene) in 60% of cases, whereas a gain in 17q21 (harbouring genes coding for STAT3, STAT5A e STAT5B) was documented in 64% of patients. Worth mentioning, 52% of cases showed both losses in the p arm and gains in the q arm within chromosome 17. In summary, our results partially confirmed those previously reported with regard to alterations in chromosomes 7, 8, 10 and 17, resulting in amplification of oncogenes and deletion of tumor suppressor genes. We also observed genome alterations associated with activation of the signal transduction JAK/STAT pathway, possibly involved in SS malignant phenotype. Further genome alterations emerged in this study, such as those in chromosome 7 and 19, are also worth investigating for their possible pathophysiological meanings. Disclosures: No relevant conflicts of interest to declare.

Abstract 3565 Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, haematologic malignancy. The disease may occur at any age, with strong male predominance. Skin presentation is very common. Histologically, tumour cells constitute a uniform population of round or more pleomorphic medium size elements. Clinically this tumour is very aggressive, with poor outcome in adults (median survival 13 months). Chemotherapy (CT), preferably with acute leukemia-type regimens, is the mainstay of treatment, when possible associated with stem cell transplantation (SCT). Few studies investigated copy number changes by array-based comparative genomic hybridization (array-CGH) and gene expression profiling, highlighting a peculiar set of chromosomal losses involving regulatory genes. The present study focused on 23 cases of BPDCN, that were extensively investigated cytogenetically (array-CGH and SNP6 array) and immunohistochemically. Clinico-pathologic findings and molecular data were analyzed in order to identify prognostic markers and possible clinical subsets. At onset 12 cases had diffuse, 7 multiple non contiguous and 4 localized skin involvement. Cases 16 and 18 were father and his daughter. Specific therapy was administered in 20 cases: 10 ALL-type CT; 4 NHL-type CT; 2 mono-CT with 1 radiotherapy; 2 radiotherapy alone; 2 SCT. Ten patients relapsed, in a median time of 10 months: 9 cases received further CT and 2 underwent allo-SCT. At last follow-up, 11 patients had died of disease and 2 of therapy-related causes. Median overall survival (OS) was 21 months. At last follow-up 10 patients were alive, 6 disease-free (8 to 28 months) and 4 with disease. Immunophenotype was typical (CD4, CD56, BDCA2, Tcl-1, and CD123) in all but three patients: 1 negative both for CD4 and CD56, 1 for CD56 and 1 for CD4. All cases expressed CD45RA, 19 CD68/KP1, 10 CD2 and/or CD7 and, when tested, for BDCA2/CD303. T-cell and B-cell receptor genes were germline with no evidence of EBV infection. We observed chromosomal imbalances in all patients. Loss of CDKN2A locus (9p21.3) occurred in 15/23 patients, and in 5 cases a biallelic loss was found. FISH confirmed 9p21.3 loss in all 5 cases. Of note, patients with biallelic loss of locus 9p21.3, in respect to those with hemizygous loss, had reduced survival probability (hazard ratio 11.98; confidence interval [CI] 1.21–118.96; log-rank test, p=0.0349). Median OS was 20 and 35 months and median time to relapse 11 and 15 months, respectively. Furthermore, loss of RB1 (13q13.1-q14.3) and CDKN1B (12p13.2-p13.1) locus were observed in 13 cases. An additional, yet unreported, cytogenetic alteration was the loss of 7p12 in 5 of our patients. This is the locus of IKZF1 gene, which encodes the transcription factor Ikaros. Of note, biallelic and monoallelic deletions of IKZF1 has been shown to be associated with chemoresistance and very poor outcome in a subset of BCR-ABL1 positive acute lymphoblastic leukemias. Array-CGH and SNP6 in our series confirmed that BPDCN is characterized by a complex pattern of genomic losses, predominantly affecting chromosomes 9 (71%), 13 (61%), 12 (57%), 5 (19%), 7 (19%), 14 (14%) and 15 (14%). In conclusion, this is the largest reported series of BPDCN patients, confirming high aggressiveness of the disease. We found negative correlation between age and time to relapse (p=0.0053) and a lower rate of bone marrow involvement in the group of patients with localized skin disease (2 of whom in CR after local skin radiotherapy). Furthermore, we observed absence of CD4 and/or CD56 expression in 3/23 cases. We are the first to report reduced OS probability among patients with homozygous loss of locus 9p21.3 in comparison to those with hemizygous loss, and the possible loss of 7p12-IKZF1 gene in BPDCN. Disclosures: No relevant conflicts of interest to declare.

Advanced tumor-stage mycosis fungoides (MF) and Sézary syndrome (SS) are cutaneous T-cell lymphomas (CTCL) characterized by very poor prognosis. Allogeneic haematopoietic stem cell transplantation (allo-SCT) represents an experimental treatment which has been shown to be very effective in achieving long lasting complete remission, possibly leading to cure in selected patients. However, high transplant-related mortality (TRM) limit its feasibility in the vast majority of patients with CTCL. Reduced-intensity conditioning regimens have been demonstrated to decrease TRM, allowing gradual establishment of full donor chimerism and possible graft-versus-lymphoma effect. In this setting, evaluation of minimal residual disease (MRD) is particularly useful to guide post-transplant strategies such as donor lymphocyte infusion (DLI). Detection of TCRgamma-chain gene rearrangements, owing to the relatively limited complexity of its genetic elements, is routinely used to detect MRD in T-cell malignancies. Nonetheless, due to the extensive combinatorial repertoire and the large hypervariable regions, TCRbeta represents the best target for MRD monitoring, allowing more sensitive detection of patient-specific rearrangements. With this study, we aimed to identify patient-specific TCRbeta rearrangements to monitor MRD in patients enrolled in a clinical phase II trial of reduced intensity allo-SCT for advanced stage refractory MF/SS. Skin biopsy and peripheral blood samples at diagnosis and at different time points after transplant were obtained from 6 out of 9 evaluable patients, all having achieved clinical complete remission (still enduring in 5). The BIOMED-2 multiplex TCRbeta PCR heteroduplex assay (InVivoScribe Technologies, USA) was used for identification of monoclonal TCRbeta rearrangements, clonal PCR products were directly sequenced in both directions using the complete set of V or J primers, and V, D, J segments identified using the ImMunoGeneTics database (http://imgt.cines.fr). Then, clonespecific PCR assays were performed on samples collected from every single patient and specificity tested by parallel amplification of normal polyclonal DNA samples. In all patients a monoclonal TCRbeta rearrangement has been sequenced allowing to obtain clone-specific primers for PCR assays. In 2 patients we identified the presence of a MRD at the early controls after transplant (+2 and +3 months, respectively) when both were polyclonal by standard TCRgamma rearrangement; clone-specific PCR assays for TCRbeta became negative afterwards, in concomitance with the achievement of full donor chimerism. In 3 patients polyclonality of TCRbeta was observed in all post-transplant controls. One developed chronic cutaneous GvHD and skin biopsies verified the absence of clone-specific T-lymphocytes. In another patient, all post-transplant assays performed by the standard TCRgamma-rearrangement were persistently positive, suggesting the presence of a non disease-specific monoclonality. The last of the 6 patients relapsed 52 months after transplant. Retrospective clone-specific analysis of TCRbeta rearrangements in DNA from skin biopsies unveiled the presence of molecular relapse already 24 months before, whereas standard TCRgamma assays became positive only after clinical relapse. Altogether, we observed good stability of monoclonal TCRbeta rearrangements over time. Our results suggest that the analysis of TCRbeta is more sensitive and more specific than the analysis of standard TCRgamma for detection of MRD in CTCLs, allowing earlier identification of relapses and adopatiention of pre-empatientive treatment such as DLI. With this method, we also demonstrated disappearance of MRD in concomitance with the achievement of full donor chimerism after allo-SCT. Finally, disease clone-specific TCRbeta rearrangement detection might be helpful in distinguishing cutaneous manifestations of acute GvHD from early relapse of MF. TCRbeta analyses of samples from the remaining 3 evaluable patients are ongoing and will be included in the final presentation.