ALBERT

Description: Background. The prognostic role of cell of origin profile (COO) assessed by immunohistochemistry (IHC) is controversial in Rituximab era. FIL conducted a phase III randomized trial aimed at investigating the benefit of intensification with high dose therapy plus autotransplant compared to R-dose-dense therapy as first line in young DLBCL at poor risk (aa-IPI 2-3). Clinical results were reported (Vitolo, ASH 2012). The aim of BIO-DLCL04 was to correlate the biological markers with PFS. Patients and Methods. From 2005 to 2010, 412 untreated DLBCL at aa-IPI 2-3 were enrolled. Central histology revision was mandatory and 13 patients were excluded due to different histologies. Biological markers were analyzed on DLBCL NAS; COO analysis was performed by IHC and cases were classified in germinal center (GC) and non-GC according to Hans' algorithm; COO determined by gene expression profile using the NanoString® nCounter® Analysis System based on 20-gene assay (Lymph2Cx) using formalin fixed paraffin embedded tissue is ongoing; BCL2, BCL6 and MYC anomalies were tested by IHC; final analysis by fluorescent in situ hybridization (FISH) is ongoing. Cases were deemed positive if at least 30% of lymphoma cells were stained with each antibody (with the exception of at least 40% for MYC). Results. At the time of this analysis, 223 DLBCL NAS were analyzed: 131 non-GC and 92 GC; BCL2, BCL6 and MYC anomalies were tested in 196, 74 and 107 cases respectively. Clinical characteristics for non-GC vs GC were: median age 51 years for both, male 49% vs 45%, aa-IPI 3 15% vs 25%, bone marrow involvement (BM) 16% vs 24%. R-HDC was performed in 45% of non-GC patients and in 49% of GC. Complete response was recorded in 105 (80%) non-GC patients and in 62 (67%) GC. At a median follow-up of 49 months, the 3-year PFS for non-GC vs GC was 75% (95% CI: 67-82) vs 57% (95% CI: 46-67) with crude hazard ratio, HR 0.55 (0.35-0.87), p.01 and adjusted (for age, gender, aa-IPI, BM) aHR 0.56 (0.35-0.88), p.013. No significant differences by treatment were reported. Overexpression of MYC by IHC had a relevant prognostic impact, with aHR 1.84 (0.99-3.44), p.054. By IHC, 3-years PFS for double negative vs single BCL2 or MYC overexpression vs double positive, was 85% vs 68% vs 51% respectively, with an aHR for double expressors compared to double negative of 3.91 (1.13-13.53), p.031. At the time of the present report, FISH analysis was conducted in 88 cases: 43 were triple negative, 37 single hit and 8 double/triple hit. By FISH, 3-years PFS for triple negative vs single hit vs double/triple hit was 74% vs 84% vs 25% respectively, with an aHR for double/triple hit compared to triple negative of 5.73 (2.05 to 16.02), p.001. Conclusions. In conclusion, with the limit of the analysis performed by IHC based on Hans' algorithm, BIO-DLCL04 showed an unexpected better outcome for non-GC compared to GC, irrespective of treatment arm. The ongoing analysis conducted by Nanostring will be more informative. The overexpression of MYC was an unfavourable risk factor, mainly if associated with BCL2 overexpression, irrespective of type of treatment. Moreover, double/triple hit patients represent a subgroup with extremely poor prognosis. High dose therapy plus autotransplant was not able to reverse the inferior outcome of neither double expressors nor double hit patients and new strategies are deemed for these poor prognosis patients. Disclosures No relevant conflicts of interest to declare.

Description: Background: The heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL patients (pts) that doesn't have the most recurrent adult rearrangements (BCR-ABL1 t(9;22); TCF3-PBX1 t(1;19); MLL-AF4 t(4;11)) are collectively referred to as "triple negative" (Ph-/-/-) ALL. CRLF2 is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it. Aims: Clustering and biological characterization of Ph-/-/- ALL (that represents 61% of adult B-ALL; Roberts KG, J Clin Oncol. 2016), considering CRLF2 overexpression event, in order to define and assess biomarkers in this subgroup to test new drugs. Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) were performed on 55 Ph-/-/- ALL, 29 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized with the Expression Console Software. Successively we cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in the top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (TruSight Pancancer-Illumina; MLPA and/or dMLPA-MRC-Holland; SNP Array-Affymetrix; 454 Junior-Roche and PCR). Results: Clustering our Ph-/-/- gene expression data using the impact of the 10 single genes in our cohort, we could identify a defined 2-clusters-subdivision (Gr1 and Gr2; Fig 1A). The Gr2 is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) overexpression and it represents 14.1% of all B-ALL. The Gr2 GEP is similar to Ph+ one. Fusion copy number alteration and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mutations or deletion), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. Molecular characterization shed light on a very heterogeneous scenario especially in the group 1, suggesting the need of a more discerning clustering for this group. In spite of the small number of cases is required, preliminary Gr1 subclustering discerns MLLr and ZNF384 gene expression subgroups. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upregulated in the group1 and CDK6 in the Gr2. CRLF2 and CD200 immunoblotting and CD200 immunohistochemistry preliminary analyses suggest that protein expression of CRFL2 and CD200 are higher in Gr2 in comparison to Gr1. Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B-based on 10 genes. 3C-up represents 14.1% of all B-ALL and it is characterized by a) high co-expression of three main genes: CRLF2, CTGF and CD200; b) IKZF1 deletion; c) JAK-STAT pathway mutations/fusions/deletions. Gr1 represents 46.9% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identify new potential therapeutic targets with available drug (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; tyrosine kinase inhibitors already effective on Ph+ and Ph-like) to test. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Figure. Figure. Disclosures Cavo: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Martinelli:Novartis: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Ariad/Incyte: Consultancy; Amgen: Consultancy.

Description: Abstract 4631 Background Peripheral T cell lymphomas (PTCLs) are a heterogeneous group of tumors representing approximately 12% of lymphoid neoplasms, basically subdivided into specified and not specified (NOS) forms. PTCL/NOS, corresponding to about 60%–70% of PTCLs, cannot be further classified on the basis of morphology, phenotype, or conventional molecular studies. Clinically, PTCLs/NOS are highly aggressive lymphomas, with a poor response to therapy, and dismal overall survival (20-30%). Their pathobiology is poorly known, though recent gene expression profiling (GEP) studies have provided some hints for better understanding their pathogenesis. In particular, GEP and immunohistochemical studies on tissue-microarrays (TMAs) demonstrated PDGFRA to be systematically activated in almost all PTCLs/NOS, by nominating it as potential therapeutic target. Aims In this study, we aimed to identify the determinants of PDGFRA activation in PTCL/NOS. Specifically, we studied PDGFRA locus in order to identify possible mutations, translocations, or copy number variations and we explored the possible existence of an autocrine/paracrine loop sustaining an otherwise integer kinase. Methods The PDGFRA locus (4q1.1-4q1.3) was studied by FISH and wide-genome SNPs analysis (Affymetrix 500K Array). Direct sequencing of all PDGFRA exons and introns as well as of the promoter region was also performed in 90 cases. IHC and ELISA were adopted in order to study the expression of PDGF-A, PDGF-B and PDGF-C on tissue sections and in supernatants from PTCL/NOS cell cultures, respectively. Finally, the expression of PDGFRA and its activated (phosphorilated) form, p-PDGFRA, was assessed by IHC on TMAs, and by flow-citometry in PTCL/NOS cultured cells as well as in a FIP1L1-PDGFRApos chronic eosinophilic leukemia cell line (EOL-1) before and after the exposure to an anti-PDGF ligand neutralizing antibody (R&D System), given at various concentrations (20-40-60-80 ug/mL). Vitality assessments, proliferation/cell cycle assay (by In Situ Cell Proliferation kit, FLUOS – Roche) and evaluation of PDGFRA and p-PDGFRA were performed at 24, 48, 96 hours. A human PDGF peptide (R&D Sytems) was added to cultured cells for 6 hours to evaluate whether PDGFRA de-phosphorilation was really due to PDGF ligand remotion. Results First, FISH, SNPs analysis and direct sequencing showed preserved integrity of PDGRA locus. Thus we tested the hypothesis of an autocrine/paracrine stimulation. PDGF-A, PDGF-B and PDGF-C were found to be expressed by neoplastic cells at IHC in 93-95% of cases. In addition, PDGF-AA was found to be secreted by cultured neoplastic cells by ELISA. Notheworthy, PTCL cells secreted much more ligand than any other cell taken as control. We then tested whether PDGFRA phosphorylation was actually due to the presence of a PDGF ligand. Indeed, PTCL cells treated with anti-PDGF ligand neutralizing antibody at various concentrations showed PDGFRA dephosphorilation ranging from 30% up to 90% in a time dependent manner. Notably, the effect was specific as in EOL-1 PDGFRA phosphorylation was not modified at all. In addition, PTCL cells treated with a minimum of 20ug/mL of anti-PDGF ligand neutralizing antibody for 48h showed a 70% blockade of proliferation in comparison to untreated cells (BrdU assay). A further addition of 20 ug/ml of inhibitory antibody at 48 hours, increased the proliferation arrest up to 80% at 96 hours. Finally, the addition of a natural human PDGF peptide to cells previously treated with the anti-PDGF antibody, could restore PDGFRA phosphorylation confirming that PDGFRA de-phosphorilation was due to ligand remotion. Conclusions Taken together, our data demonstrate that PDGFRA activity is sustained by an autocrine loop in PTCL/NOS. In fact, though, in vivo, a possible additive paracrine effects mediated by reactive components cannot be excluded, we provide evidence that the phenomenon is largely due to neoplastic cells. Importantly, as PDGFRA signaling abrogation was associated to proliferation arrest, PDGFRA was confirmed as potential therapeutic target. Acknowledgments: this work was supported by Centro Interdipartimentale per la Ricerca sul Cancro “G. Prodi”, BolognAIL, AIRC, FIRB, RFO, Fondazione Cassa di Risparmio in Bologna, Fondazione della Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006, and Vanini-Cavagnino grant. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2942 Poster Board II-918 Introduction: LMO2 is a transcription factor with a central role in haematopoietic development and angiogenesis. High levels of LMO2 transcript were reported in CD34+ stem cells and strong protein expression was observed in endothelia, germinal centre B cells (GCB) and some B cell lymphomas. Activation of LMO2 has been demonstrated in T-ALL as a consequence of translocations or deletions and its expression in diffuse large B-cell lymphomas is associated with favourable outcome. Furthermore, correlation between LMO2 expression and tumour progression has been reported in prostatic carcinoma. Aims: The expression pattern of a newly developed rabbit monoclonal antibody raised against a fixation-resistant epitope of the LMO2 protein was studied in human normal and pathologic samples. Material and Methods: Normal haematopoietic and non-haematopoietic tissue sections, tissue micro-arrays including 1452 neoplastic samples, 11 lymphoma-derived cell lines and CD34+/CD38− stem cells, purified from human umbilical cord blood, were investigated by single and double immunostaining. Results and discussion: Strong LMO2 expression was found in CD34+/CD38− stem cells and in contrast to previous studies, the anti-LMO2 rabbit monoclonal distinctively labelled normal mantle, monocytoid and splenic marginal zone B cells. A few LMO2+/CD34+ cells were detected in the thymic cortex, most likely representing T cell precursors. Tables 1 and 2 summarise the results obtained in tumours and lymphoma-derived cell lines. Among B cell neoplasms, LMO2 was highly expressed in B-lymphoblastic lymphoma/leukaemia (87%) and heterogeneously in MCL (14%), FL (57%) and BL (37%). In DLBCL, LMO2 positivity was closely related to those cases with a “germinal centre” phenotype. LMO2 nuclear positivity was observed in all cases of LPHL; interestingly Reed-Sternberg cells in CHL showed LMO2 cytoplasmic staining in 10% of cases. The constant expression in T-ALL highlights the pathogenetic role and diagnostic significance of LMO2 in this setting as opposed to PTCLs (usually negative). Detection of LMO2 was observed in the majority of AML and CML cases investigated. Different types of carcinoma expressed LMO2 that was found to be mainly cytoplasmic. Conclusion: The newly developed anti-LMO2 rabbit monoclonal antibody represents a powerful reagent for detection of LMO2 protein in routine samples as compared to the previously published monoclonal anti-LMO2. Its frequent expression among haematological malignancies (e.g. T-ALL, B-ALL and AML) indicates LMO2 as putative therapeutic target as evidenced by RNA-silencing studies in experimental models. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1890 Poster Board I-913 Background. Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterised by a proliferation of predominantly megakaryocytes and granulocytes in bone marrow that in fully developed disease is replaced by fibrous tissue. At molecular level, no specific defect has been identified yet. Cytogenetic abnormalities occur in up to 30% of patients, the commonest including del(13)(q12-22), der(6)t(1;6)(q21-23;p21.3), del (20q), and partial trisomy 1q. In addition, approximately 50% of patients with PMF exhibit a single, recurrent, somatic mutation in the gene encoding the cytoplasmic tyrosine kinase Janus kinase 2 (JAK2). However, such mutation is not specific, also occurring in other MPN. Recently a couple of reports dealt with single-nucleotide polymorphism (SNP) array karyotyping of MPD, including some PMF. Importantly, such studies could identify previously uncovered genetic lesions, highlighting the importance of novel high resolution technologies for the detection of formerly unknown, cryptic aberrations. In this study we performed high resolution karyotyping by SNP oligonucleotide microarray by using the most updated Affymetrix array (Genome-Wide Human SNP Array 6.0) in 20 cases of myelofibrosis (MF) in order to identify novel cryptic genomic aberrations. Methods. DNA (500 ng) was extracted from peripheral blood cells (PBMNC) of 14 primary and 6 secondary MF patients. PBMNC were depleted from lymphocytes by magnetic beads. Briefly, CD3+ cells were labeled with anti-CD3 MoAb directly coupled to magnetic microbeads (Miltenyi Biotech), washed and subsequently purified using Mini-MACS technology. After selection, cell present in the positive (CD3) and negative (PBMNC) fractions were counted and submitted to flow cytometry analysis. DNA was processed and hybridized to the Affymetrix SNP arrays 6.0 as for manufacturer instruction. A whole-genome copy number variation (CNV), genotyping, loss of heterozygosity (LOH) and uniparental disomy (UPD) analyses were performed using the Partek Suite 6.0. Ten lab-specific as well as 90 HapMap samples relative to Caucasian healthy donor were used as control reference. Genomic abnormalities were defined as recurrent when occurring in at least 25% of cases. JAK2 mutational status was assessed as reported, by alle-specific PCR. Clinical information and complete follow up were retrieved for all cases. Direct sequencing, FISH, qPCR and immunohistochemistry (IHC) has been chosen for validation. Results. In all patients we could detect several CNV. The median number of CNV was 60 (range, 34-72), including 46 amplifications (A) and 14 deletions (D). All commonest previously described abnormalities were detected. In addition, several formerly uncovered recurrent lesions were identified, mainly involving 1p, 1q, 2p, 4p, 4q, 5q, 6p, 6q, 7q, 8p, 9q 10q, 11p 11q, 12p, 14q, 15q, 16p, 16q, 17q, 18q, 19q, 20p, 22q. The median size of such CNV was 424,582 Kbp (1,379 Kbp-71,277 Mbp). We then compared JAK2+ vs. JAK2− cases. Of note, we found numerous definite aberrations (A or D) distinguishing the two groups and specifically affecting 16q23.1, 1p36.13, 3q26, 14q13.2, 5q33.2, 6q14.1, 7q33, 8p23.1, and 9p11.2. Grippingly, several genes of potential interest for PMF pathogenesis were identified within the involved loci, including RET, SCAPER, WWOX and SIRPB1. Among others, the product of such genes has been selected for validation by IHC. Similarly, many miRNA were recognized, which may deserve further investigation. Conclusions. By using a newly developed highly sensitive array we identified novel cryptic lesions in patients affected by MF. Future studies on larger series, as well as functional analyses will definitely assess their role in the pathogenesis of the disease. Of note, consistent differences were recorded in JAK2+ vs. JAK2−, supporting the hypothesis of different genetic mechanisms occurring in the two sub-groups. Acknowledgments: this work was supported by AIL Pesaro Onlus, Centro Interdipartimentale per la Ricerca sul Cancro “G. Prodi”, BolognAIL, AIRC, FIRB, RFO, Fondazione Cassa di Risparmio in Bologna, Fondazione della Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006.*GV and MRS equally contributed to this work. Disclosures: No relevant conflicts of interest to declare.

Description: Background. Peripheral T-cell lymphoma not otherwise specified (PTCL/NOS) is the most common T-cell lymphoma; however, it remains a complex entity showing great variety regarding morphology, immunophenotype and clinical behaviour. Recently, gene expression profiling (GEP) revealed PDGFRA as possible PTCL-associated molecule, by nominating it as potential therapeutic target. Aims and Methods. We investigated the possible determinants of PDGFRA activity in PTCL/NOS. We performed GEP of 28 PTCLs/NOS and 20 samples of normal T-cell subpopulations, by using the Affymetrix HG-U133 2.0 plus microarray. Moreover, we built tissue-microarrays (TMAs) including 145 PTCLs/NOS for protein analyses. The PDGFRA locus (4q1.1–4q1.3) was studied by FISH on those TMAs, while direct sequencing of all PDGFRA exon and introns as well as of the promoter region was performed in 90 cases. Immunohistochemistry (IHC) and ELISA were adopted in order to study the expression of PDGF-A, PDGF-B and PDGF-C on tissue sections and in supernatants from PTCL/NOS cell cultures, respectively. Finally, the expression of PDGFRA and its activated (phosphorilated) form, p-PDGFRA, was assessed by IHC on TMAs, and by flow-citometry in PTCL/NOS cultured cells before and after the exposure to an anti-PDGF ligand neutralizing antibody (R&B System). Results. First, GEP showed over-expression of PDGFRA in all tested PTCLs/NOS, while IHC confirmed the expression of PDGFRA and p-PDGFRA. FISH, SNPs analysis and direct sequencing showed preserved integrity of PDGRA locus. We then studied PDGF-A, PDGF-B and PDGF-C, which turned out to be expressed and secreted by PTCL/NOS cells. In order to verify the hypothesis of an autocrine loop, we tested whether the remotion of PDGF ligands from the supernatants of cultured PTCL/NOS cells determined PDGFRA de-phosphorilation. Notably, 48h after the exposure to 20 mg of anti-PDGF antibody, we already appreciated a significant effect with reduction of PDGFRA phosphorilation up to 75%. Conclusions. Taken together, our data demonstrate that PDGFRA activity is sustained by an autocrine loop in PTCL/NOS.