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Supplementary Figures S1-S2 and Supplementary Tables S1-S5.

Purpose: Chromosome 13q14 deletion occurs in a substantial number of chronic lymphocytic leukemia (CLL) patients and it is believed to play a pathogenetic role. The exact mechanisms involved in this lesion have not yet been fully elucidated because of its heterogeneity and the imprecise knowledge of the implicated genes. This study was addressed to further contribute to the molecular definition of this lesion in CLL.Experimental Design: We applied single-nucleotide polymorphism (SNP)-array technology and gene expression profiling data to investigate the 13q14 deletion occurring in a panel of 100 untreated, early-stage (Binet A) patients representative of the major genetics, molecular, and biological features of the disease.Results: Concordantly with FISH analysis, SNP arrays identified 44 patients with del(13)(q14) including 11 cases with a biallelic deletion. The shorter monoallelic deletion was 635-kb long. The loss of the miR-15a/16-1 cluster occurred in all del(13)(q14) cases except in 2 patients with a monoallelic deletion, who retained both copies. MiR-15a/16 expression was significantly downregulated only in patients with the biallelic loss of the miRNA cluster compared to 13q normal cases. Finally, the natural grouping of SNP profiles by nonnegative matrix factorization algorithm showed that patients could be classified into 2 separate clusters, mainly characterized by short/biallelic versus wide/monoallelic 13q14 deletions. Supervised analyses of expression data showed that specific transcriptional profiles are correlated with these 2 genomic subgroups.Conclusions: Overall, our data highlight the presence of 2 distinct molecular types of 13q14 deletions, which may be of clinical relevance in CLL. Clin Cancer Res; 16(23); 5641–53. ©2010 AACR.

Background: Multidrug-resistant (MDR) or extensively-drug-resistant (XDR) Pseudomonas aeruginosa (PSA) strains infections are a major concern in nosocomial environments being associated both with worse outcomes and with high mortality rates especially in oncologic and haematogic settings. In the Trieste University hospital, we experienced an outbreak of XDR-PSA bacteremia in the haematology unit probably caused by a reservoir in the toilets faucets. Materials/methods: The haematology unit in Trieste hospital has twelve double-bed rooms and a protected area with three single-bed rooms reserved to patients undergoing marrow transplants and severe neutropenia (neutrophils

The hypomethylating agent Decitabine (DAC) is a valuable treatment option in acute myeloid leukemia (AML), particularly in elderly patients (pts) not suitable for intensive chemotherapy (CHT). However, limited data are available about efficacy and safety of DAC in clinical practice.We retrospectively reviewed data of 104 AML pts treated with DAC in eight Italian Hematological Centers from 2015 to 2017. The objective of this study was to evaluate the efficacy and safety of DAC in older AML pts outside of clinical trial. Seventy-five (75%) pts received DAC as first line treatment (Cohort 1) and 29 pts as salvage therapy (Cohort 2). All pts received a DAC schedule of 20 mg/sqm IV for 5-days, every 28 days. The median age was 72.5 years (74 in cohort 1 and 66 in cohort 2) and 16% of pts had an ECOG performance status2 at the start of DAC treatment (with non-significant difference in the two cohorts). The cumulative illness rating scale (CIRS) was6 in 27% of pts. Forty-five pts (43%) had secondary AML. Bone marrow blast count was30% in 64% of patients (67/104). In the relapsed cohort 17/29 (59%) patients were treated with DAC after conventional CHT, 5/29 (17%) after allo-SCT and 7/29 (24%) after azacitidine therapy.A total of 469 DAC cycles were given to the 104 pts with a median of 3 cycles (range 1-21) and 45/104 (43%) pts received4 cycles. The Overall Response Rate (ORR = Complete Remission-CR plus Partial Remission-PR) was 33%, significantly higher in Cohort 1 (42%) compared to Cohort 2 (14%) (p = 0.009). The median duration of response was 6 months (range 1-20). In Cohort 1 the best response (CR or PR) was obtained between 3th and 6th cycle. In multivariate Cox regression analysis, achievement of CR or PR (HR = 0.78; p = 0.0004), CIRS 6 (HR = 0.9; p = 0.04) and complex karyotype (HR = 0.8; p = 0.03) were significant predictors of better overall survival (OS). Median OS from the start of DAC therapy was 11 months for the whole population with a significant OS advantage in Cohort 1 (median OS 12.7 mths vs 6.3 mths; p = 0.003); median OS was significantly longer in responders compared to non-responders (22.6 mths vs 5.7 mths; p 0.0001). At the last follow-up, 56 patients (54%) are still alive and 48 (46%) are dead (71% due to disease progression). The most common toxicities were myelosuppression and documented infectious complications that occurred mainly during the first 4 cycles.These data confirm the efficacy (ORR 33%) and the acceptable safety profile of DAC in the real life management of AML in elderly pts unsuitable for intensive CHT, with a significant better performance in first line therapy (ORR 42%, median OS 12.7 mths). The efficacy of DAC, both in first line and as salvage therapy, may probably be improved with combined treatment strategies and/or with different DAC schedules that could increase its anti-leukemic effect.

A progression-risk score to predict treatment-free survival for early stage chronic lymphocytic leukemia patients

Second-generation tyrosine kinase inhibitors (2G-TKIs) dasatinib and nilotinib produced historical rates of about 50% complete cytogenetic response (CCyR) and about 40% major molecular response (MMR) in chronic myeloid leukaemia (CML) patients failing imatinib. Direct comparisons between dasatinib and nilotinib are lacking, and few studies addressed the dynamics of deep molecular response (DMR) in a “real-life” setting. We retrospectively analyzed 163 patients receiving dasatinib (n = 95) or nilotinib (n = 68) as second-line therapy after imatinib. The two cohorts were comparable for disease's characteristics, although there was a higher rate of dasatinib use in imatinib-resistant and of nilotinib in intolerant patients. Overall, 75% patients not in CCyR and 60% patients not in MMR at 2G-TKI start attained this response. DMR was achieved by 61 patients (37.4%), with estimated rate of stable DMR at 5 years of 24%. After a median follow-up of 48 months, 60% of patients persisted on their second-line treatment. Rates and kinetics of cytogenetic and molecular responses, progression-free and overall survival were similar for dasatinib and nilotinib. In a “real-life” setting, dasatinib and nilotinib resulted equally effective and safe after imatinib failure, determining high rates of CCyR and MMR, and a significant chance of stable DMR, a prerequisite for treatment discontinuation.

Objective: We performed an external and multicentric validation of the predictive value of abdominal computed tomography (aCT) on time to first treatment (TTFT) in early stage chronic lymphocytic leukemia (CLL) patients. Methods: aCT was performed at diagnosis in 181 Rai 0 patients enrolled in the O-CLL1GISL trial (clinicaltrial.gov ID:NCT00917549). Results: Fifty-five patients showed an abnormal aCT. Patients with an abnormal aCT showed a significantly shorter TTFT than those with normal aCT (P < 0.0001). At multivariate analysis, aCT (P = 0.011), b-2 microglobulin (P = 0.019), and CD38 expression (P = 0.047) correlated with TTFT. Following IWCLL 2008 criteria, 112 (61.9%) cases remained at Rai 0, while 69 (38.1%) satisfied the criteria of clinical monoclonal B-cell lymphocytosis (cMBL). Reclassified Rai 0 patients with an abnormal aCT showed a significantly shorter TTFT than those with a normal aCT (P < 0.0001). At multivariate analysis, only aCT (P = 0.011) correlated with TTFT. Eleven cMBL cases (15.9%) showed an abnormal aCT and were reclassified as small lymphocytic lymphomas (SLL); nonetheless, TTFT was similar for cMBLs and SLLs. Conclusion: Our results confirm the ability of the abnormal aCT to predict progression in early stage cases.

Recent studies have described chromosome 2p gain as a recurrent lesion in chronic lymphocytic leukemia (CLL). We investigated the 2p gain and its relationship with common prognostic biomarkers in a prospective series of 69 clinical monoclonal B-cell lymphocytosis (cMBL) and 218 early stage (Binet A) CLL patients. The 2p gain was detected by FISH in 17 patients (6%, 16 CLL, and 1 cMBL) and further characterized by single nucleotide polymorphism-array. Overall, unfavorable cytogenetic deletions, i.e., del(11)(q23) and del(17)(p13) (P = 0.002), were significantly more frequent in 2p gain cases, as well as unmutated status of IGHV (P < 1 × 10(-4) ) and CD38 (P < 1 × 10(-4) ) and ZAP-70 positive expression (P = 0.003). Furthermore, 2p gain patients had significantly higher utilization of stereotyped B-cell receptors compared with 2p negative patients (P = 0.009), and the incidence of stereotyped subset #1 in 2p gain patients was significantly higher than that found in the remaining CLLs (P = 0.031). Transcriptional profiling analysis identified several genes significantly upregulated in 2p gain CLLs, most of which mapped to 2p. Among these, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Thus, 2p gain can be present since the early stages of the disease, particularly in those cases characterized by other poor prognosis markers. The finding of genes upregulated in the cells with 2p gain provides new insights to define the pathogenic role of this lesion.

Background. Use of 2nd generation tyrosine kinase inhibitors (2G-TKIs) dasatinib (DAS) and nilotinib (NIL) in chronic phase (CP) chronic myeloid leukemia (CML) patients failing imatinib (IM) results in around 50% of sustained cytogenetic response, and around 40% major molecular response (MMR). However, these are historical data and it's unclear if there's a significant difference in efficacy of the two 2G-TKIs, especially in the long-term. Aims and methods. We retrospectively analysed 163 CP-CML patients resistant or intolerant to IM that received either DAS (n=95) or NIL (n=68) as second-line therapy. We compared the characteristics of the two groups at the time of CML diagnosis and at the time of IM failure, including the cause of switch to 2G-TKI, duration of IM therapy, IM dose escalation and Hammersmith score to predict the probability of response to 2G-TKIs. Cytogenetic and molecular responses were evaluated according to the ELN recommendations. Sustained deep molecular response (DMR) was defined as MR4 or better lasting ≥ 2 years, ongoing at the last contact, and with at least a Q-PCR test every 6 months. Time to treatment failure (TTF) was calculated from the start of 2G-TKI to any of the followings: progression to accelerated or blast phase (ABP), death for any cause at any time, treatment discontinuation for primary or secondary resistance or intolerance. Progression free survival (PFS) was calculated from the start of 2G-TKI to ABP or death. Overall survival (OS) was calculated from the start of 2G-TKI to death. Results. DAS and NIL cohorts were comparable for age, sex and risk score (Sokal and EUTOS) at diagnosis. Median duration of IM therapy was similar (DAS 19 months, NIL 14 months), but 27/95 patients (28%) had IM dose escalation before DAS compared to only 9/68 (13%) before NIL (p=0.03). There was a higher rate of switch to DAS than to NIL for secondary resistance (26/95, 27% vs 7/68, 10%; p=0.01) while more patients changed from IM to NIL due to intolerance (31/68, 46%, vs 21/95, 22% for DAS; p=0.002). Rates of primary resistance did not differ (47/95, 49% for DAS vs 28/68, 41% for NIL; p=0.37), as well as other causes of switch (1/95, 1% for DAS vs 2/68, 3% for NIL; p=0.77). Hammersmith score was almost identical in the two groups. Complete cytogenetic response (CCyR) was attained in 53/73 (73%) patients not in CCyR at the time of DAS start, and in 31/48 (65%) patients not in CCyR at the time of NIL start (p=0.46). Mean time to CCyR was similar (7.1 months for DAS and 5.3 months for NIL; p=0.30). MMR was achieved in 55/89 (65%) patients not in MMR at the time of DAS start and in 39/61 (65%) patients not in MMR at the time of NIL start (p=0.82). Again, mean time to MMR was not different in the DAS e NIL cohorts (12.4 vs. 8.5 months; p=0.14). DMR was obtained in 39/88 (44%) patients not in DMR at the time of DAS start and in 30/65 (46%) patients not in DMR at the time of NIL start (p=0.95). Sustained DMR was evaluable in 127 patients: 37 patients (29%) achieved sustained DMR, without difference between DAS (24/82, 29%) and NIL (13/45, 29%; p=1.00). With a median follow-up of 44 months (range 1-124), 5-year TTF was similar for DAS (65%, 95%CI 52-75%) and NIL (61%, 95%CI 43-74%; p=0.40) [Figure 1a]. Thirty-two of 95 patients (34%) stopped DAS due to toxicity (19/32, 59%), resistance (11/32, 31%) or other causes (3/32, 10%); 22/68 patients (32%) interrupted NIL for toxicity (11/22, 50%), resistance (8/22, 36%) or other causes (3/22, 14%). Probability of survival and progression were almost identical, with a 5-year PFS of 84% (95%CI 68-89%) for DAS and 92% (95%CI 79-97%) for NIL (p=0.27) [Figure 1b] and a 5-year OS of 89% (95%CI 78-95%) and 96% (95%CI 85-99%) (p=0.31), respectively. Conclusions. With the limits of a retrospective analysis, our data suggest similar efficacy of DAS and NIL after IM failure in CP-CML, with rates of cytogenetic and molecular responses higher than those previously reported and excellent long-term survival. Around 30% achieved sustained DMR with second-line therapy, thus being potentially candidate for TKI discontinuation. Disclosures Tiribelli: Bristol-Myers Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau. Bonifacio:Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy. Fanin:Novartis: Speakers Bureau.

Abstract 2423 Chronic lymphocytic leukemia (CLL) is characterized by an extremely variable clinical course. Mutational status of the immunoglobulin heavy-chain variable (IGHV) region defines two disease subsets with different prognosis. A fraction of CLL cases carries highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3). We performed sequence analysis to characterize IGHV regions in a panel of 1133 CLL patients investigated by a multicenter Italian study group. A total of 1148 rearrangements were identified; the analysis of stereotyped subsets was performed based on previously reported criteria (Messmer et al, J Exp Med 2004; Stamatopuolos et al, Blood 2007). Specifically, we compared all our sequences with those found in three different publicly available data sets (Stamatopoulos et al, Blood 2007; Murray et al, Blood 2008 and Rossi et al, 2009 Clin Cancer Res). In addition, a pairwise alignment within all sequences was performed in order to discover novel potential subsets (HCDR3 identity > 60%). Based on the 2% cut-off used to discriminate between Mutated (M) and Unmutated (UM) cases, 777 sequences (67.59%) were classified as M, while 371 sequences (32.3%) as UM. The most represented IGHV genes within mutated cases were IGHV4-34 (104/118) and IGHV3-23 (85/96), whereas IGHV1-69 (97/112) was the most frequently used in the UM group. Interestingly, the IGHV3-21 gene, reported to be frequently expressed in CLL patients from Northern Europe, was present in only a small fraction of cases (24; 2.07%), confirming a previous finding reported by Ghia et al (Blood 2005) in a smaller panel. In our series, stereotyped HCDR3 sequences were found in 407/1148 (35.45%) patients, 177 of whom were M and 230 were UM cases. Overall, we observed that stereotyped sequences were significantly associated with UM IGHV status (Fisher's exact test, P In our series we identified 407/1148 (35.45%) stereotyped HCDR3 sequences. The percentage was higher than that reported by Stamatopoulos et al and Murray et al. This discrepancy may partially be due to the different approach used in our analysis, namely the matching to a general data set including all published stereotyped subsets instead of the auto-matching performed by those Authors. We demonstrated a significant association between IGHV status and stereotyped sequences and confirmed the finding that #1 is the most frequent subset identified so far. Finally, we were able to identify a series of 23 novel putative subsets that will require further confirmation. Disclosures: No relevant conflicts of interest to declare.

Purpose: Chromosome 13q14 deletion occurs in a substantial number of chronic lymphocytic leukemia (CLL) patients and it is believed to play a pathogenetic role. The exact mechanisms involved in this lesion have not yet been fully elucidated because of its heterogeneity and the imprecise knowledge of the implicated genes. This study was addressed to further contribute to the molecular definition of this lesion in CLL. Experimental Design: We applied single-nucleotide polymorphism (SNP)-array technology and gene expression profiling data to investigate the 13q14 deletion occurring in a panel of 100 untreated, early-stage (Binet A) patients representative of the major genetics, molecular, and biological features of the disease. Results: Concordantly with FISH analysis, SNP arrays identified 44 patients with del(13)(q14) including 11 cases with a biallelic deletion. The shorter monoallelic deletion was 635-kb long. The loss of the miR-15a/16-1 cluster occurred in all del(13)(q14) cases except in 2 patients with a monoallelic deletion, who retained both copies. MiR-15a/16 expression was significantly downregulated only in patients with the biallelic loss of the miRNA cluster compared to 13q normal cases. Finally, the natural grouping of SNP profiles by nonnegative matrix factorization algorithm showed that patients could be classified into 2 separate clusters, mainly characterized by short/biallelic versus wide/monoallelic 13q14 deletions. Supervised analyses of expression data showed that specific transcriptional profiles are correlated with these 2 genomic subgroups. Conclusions: Overall, our data highlight the presence of 2 distinct molecular types of 13q14 deletions, which may be of clinical relevance in CLL. Clin Cancer Res; 16(23); 5641–53. ©2010 AACR.

Abstract 2375 Poster Board II-352 A prognostic index based on widely available clinical and laboratory features was recently proposed to predict survival in patients with previously untreated patients with chronic lymphocytic leukemia (CLL) by MD Anderson investigators. However, whether proposed clinical risk categories may surrogate new biological variables of prognostic relevance (i.e., mutational status of the IgVH gene regions, ZAP-70 or CD-38 expression, cytogenetic abnormalities) is unclear thus far. In a series of 160 asymptomatic Binet stage A patients enrolled in a Gruppo Italiano Studio Linfomi (GISL) multicentre trial designed to validate prospectively biological parameters in early CLL as well as to assess the impact on clinical outcome of an early versus delayed policy of treatment with subcutaneous alemtuzumab in the high biological risk, we evaluated whether clinical categories derived from newly proposed prognostic index reflected biological risk. Since the original prognostic index was derived from a database including cases with more advanced disease we used an optimal cutoff search to determine how to best split Binet stage A patients in different prognostic groups. To this purpose an independent patient cohort consisting of 310 Binet stage A patients included in a GIMEMA (Gruppo Italiano Malattie EMatologiche Maligne dell'Adulto) database was used. According to recursive partitioning (RPART) model, a classification tree was built that identified two subsets of patients who scored respectively: 0-3 (low risk) and 4-7 (high risk). Therefore, by prognostic index, 48.7% and 51.2% of 160 asymptomatic stage A patients, respectively, met criteria of low risk and high risk disease. In our prospective series high- risk score was more frequently associated with both unmutated IgVH status (P=0.009) and higher CD38-expression (P=0.002); in contrast only a trend towards an increased ZAP-70 expression could be found (P=0.06). As far as cytogenetic abnormalities are concerned, we observed that 11q deletion occurred more frequently among patients belonging to high-risk score (P=0.005), while cases with 13q deletion or trisomy 12 were homogeneously distributed among low- and high-risk patient category(P=0.151 and P=0.452, respectively). We did not consider suitable for correlation analysis 17p deletion since observed only in 2 out of 160 Binet stage A patients. In conclusion, our results demonstrate in a prospective cohort of patients with early CLL that clinical categories of a revised score index may surrogate biological parameters of prognostic relevance. The observation reinforces the revised IWCLL guidelines recommendations to assess the risk of CLL patients on clinical basis and to deserve biological studies to patients eligible for clinical trials. Disclosures: No relevant conflicts of interest to declare.

SummaryIGHV mutational status and ZAP-70 or CD38 expression correlate withclinical course in B-cell chronic lymphocytic leukaemia (CLL). The threemarkers may be discordant in the single case and there is no consensus ontheir combined use in clinical practise. This multicenter study investigatedthis issue. Two-hundred and sixty-two Binet stage A patients were studied forthe three markers. Sixty patients were profiled with HG-U133A geneexpression chips. Disease progression was determined by time from diagnosisto treatment (TTT). The probability of being treatment-free at 3 years wassignificantly shorter in patients with unmutated IGHV genes (IGHVunmut66% vs. 93%, chi square of log-rank = 30, P

This study confirms previous observations of the efficacy of antiviral treatment in the HCV-related B-NHL. A complete remission of the haematological as well as the infectious disease was observed in a large fraction of patients. This approach is not cytotoxic, and therefore, additional conventional chemotherapies are not precluded in non-responders. The haematological response, as well as immunological and clinical responses, was related to the disappearance of HCV-RNA. Unlike Vallisa et al (2005), we used the standard dose of PEG-IFN (1·5 μg/kg), but this did not determine a significantly higher SVR. The resistance of these patients to antiviral therapy is also indicated by the absence of ‘early virological responses’ (HCV-RNA undetectable at the 4th week of therapy) even among the genotype 2 carriers. Finally, this study shows that, as well as in HCV-positive chronic hepatitis, PEG-IFN + RIBA seems more powerful than IFN + RIBA, but the low number of cases prevents definitive conclusions.

Distinct genetic abnormalities, such as TP53 deletion at 17p13.1, have been identified as having adverse prognostic relevance in B-cell chronic lymphocytic leukemia (B-CLL), and conventional cytogenetic studies have shown that TP53 deletion in B-CLL is mainly associated with the loss of 17p due to complex chromosomal rearrangements. We used an integrative genomic approach to investigate the significance of 17p loss in 18 B-CLLs in Binet stage A, carrying a TP53 monoallelic deletion detected by means of fluorescence in situ hybridization (FISH). Genome-wide DNA analysis using single nucleotide polymorphism (SNP) arrays of 12 of 18 samples showed 17p loss in 11 cases, with breakpoints scattered along the 17p11.2 region. FISH analysis confirmed these findings and revealed 17p loss in a small fraction of leukemic cells in the remaining TP53-deleted case, and it also indicated 17p loss in the six cases not investigated by means of SNP arrays. Mutations in exons 2–11 of the remaining TP53 allele were found in 9 of 12 deleted samples. Gene-expression profiling of 60 B-CLLs, including seven patients with 17p loss, identified 40 differentially expressed genes in 17p- versus 17p normal samples, 35 of which were downregulated in 17p-tumors. The majority (30 of 35) of these transcripts, including putative tumor suppressor genes, mapped to 17p, thus indicating a remarkable gene-dosage effect. Our data provide evidence that 17p loss may play an additional pathogenetic role in B-CLL and suggest that the concomitant loss of multiple tumor suppressor genes could be responsible for the highly adverse prognostic relevance associated with TP53 loss. © 2008 Wiley-Liss, Inc.

Background: Over the last two decades several phenotypic, molecular, and chromosomal markers have been identified that are significantly associated with the prognosis of CLL patients. Therefore, clinicians managing CLL patients would benefit from a simplified prognostic index. Methods: We analyzed prospectively collected data from 337 Binet A CLL patients enrolled in Italian the O-CLL1-GISL protocol with the aim of developing scores capable of predicting treatment free survival (TFS). Factors independently associated with TFS were included in the prognostic indexes. To account for differences in the magnitude of the association between the individual independent factors and TFS, we assigned a weighted risk score to each factor based on ranges of their corresponding hazard ratios (HRs) (i.e., 1 point for HR 1.1-1.9; 2 points for HR 2.0-2.9, etc.). The total risk score was then calculated by the sum of the ratings of each factor on individual basis. Risk groups were identified combining risk categories with a non-statistically different TFS. Results: We developed two scores based on weighted multivariable models: the first included clinical and laboratory parameters [clinical score (c-score)], while the second was based on biological markers [biological score (b-score)] (Table 1). The c-score allowed to predict the TFS of patients through the combination of Rai stage, b2-microglobulin and absolute lymphocyte count (ALC), while the b-score predicted TFS by IGHV mutational status and CD38 expression. The c-score showed a C-statistic of 0.72, while the b-score was 0.67, although cases stratified according to the b-score showed a more specific mRNA/microRNA profile. When the two scores were forced in a multivariate analysis, both showed an independent predictive value on TFS with a similar HR, demonstrating their complementarity. Thus, we attempted to integrate the two scores performing a further multivariate analysis in which all parameters, significantly associated with TFS at univariate analysis, were tested (Table 1). ALC, Rai stage, b2-microglobulin together with IGHV mutational status, resulted independently associated with TFS. We constructed a weighted score [comprehensive score (co-score)], including all the above 4 variables, which allowed the identification of 3 different risk groups with significantly different TFS (Figure 1). The C-statistic of the g-score was 0.75, showing a better concordance than the other two scores. Moreover, its validity was externally validated in a series of 297 newly diagnosed Binet A CLL patients from the Mayo Clinic, USA. Conclusions: Using this multistep process and external validation, we developed a score with high discriminatory power and predictive significance on the individual patient level. Table 1. Univariate and multivariate Cox proportional Hazards Models Variable Univariate analysis Multivariate analysis Clinical model Biological model Comprehensive model HR (95% CI) P HR (95% CI) P score HR (95% CI) P score HR (95% CI) P score Age (years) 60 1.12 (0.73-1.74) 0.59 - - - - - - - - - Sex Male/Female 0.93 (0.6-1.44) 0.93 - - - - - - - - - Rai stage 0/I-II 2.30 (1.47-3.50) 10 3.43 (1.99-5.92) Figure 1. TFS according to comprehensive progression risk score. Figure 1. TFS according to comprehensive progression risk score. Disclosures Shanafelt: Cephalon: Research Funding; Glaxo-Smith-Kline: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Jannsen: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding. Kay:Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding; Tolero Pharma: Research Funding; Pharmacyclics: Research Funding.

Abstract 3889 Inflammatory cytokines play a biological role in the pathogenesis of Chronic lymphocytic leukemia (CLL). IL23 is a pro-inflammatory cytokine involved in T-cell responses and in tissue remodeling. It has been shown that the IL23 receptor (IL23R) is up-regulated in primary acute lymphoblastic leukemia (ALL) cells, and that IL23 inhibits ALL cell growth. Nevertheless, the anti-tumor function of IL23 still remains controversial. The role of the IL23R/IL23 axis in CLL has not been investigated so far. Herein we evaluated the expression pattern of IL23R/IL23 axis and its correlation with progression free survival (PFS) in CLL patients. A total of 233 newly diagnosed Binet stage A CLL cases from Italian institutions (clinicaltrials.gov NCT00917540) were studied for IL23R expression by flow-cytometry (FC) (median percentage IL23R expression=22.7, range 1.2–91.1). The median follow-up was 23 months (range 1–47). PFS information was obtained in 203 patients. Using the median value of 23% of IL23R as threshold, 8/102 IL23Rneg and 23/101 IL23Rpos CLL cases progressed with therapy requirement. The 2-year PFS probability of IL23Rneg patients was 89.7% as compared to 80.7% of IL23Rpos cases [χ2 7.7, P=.006; HR=3.0, 95%CI (1.3–6.6)]. Cases were then stratified according to IL23R positivity [IL23Rneg (102 cases) versus IL23Rpos (101 cases)]. No significant difference in terms of CD38 and ZAP-70 positive cases was observed, however, the IGVH mutational status could distinguish the two groups: IGHV-mutated in 92 (78.6%) of IL23Rneg vs 70 (61.9%) IL23Rpos and IGHV-unmutated in 25 (21.4%) vs 43 (38.1%), p=.006]. FISH analysis showed that IL23Rneg and IL23Rpos cases carrying 13q14.3 were respectively 53 (51.4%) and 44 (42.7%), while the number of patients with trisomy 12 were 8 and 10 respectively in cases with low and high IL23R expression. Deletion of 11q was detected in 3.9% (4/103) of IL23Rneg and in 8.7% (9/103) of IL23Rpos cases. Only 3 cases with 17p deletion were seen in this cohort of early CLL patients and all belonged to the IL23Rpos group. Overall, no significant differences in the incidence of the major genetic lesions were observed between the two groups. Il23R expression still remained independently associated with PFS also in multivariate analysis. In situ expression analysis of IL23R and of its ligand IL23 was then performed by immunohistochemistry (IHC) in 16 CLL samples [10 lymph node (LN) and 6 bone marrow (BM) biopsies] collected on diagnosis and in 8 control biopsies (4 lymph nodes with reactive follicular hyperplasia and 4 normal BM biopsies). IL23R was variably expressed in CLL and significantly expressed in the neoplastic clones of 9 (6 lymph nodes and 3 BM biopsies) of the 16 cases tested; IL23R was diffusely present along the membrane and cytoplasm of neoplastic cells effacing the lymph node or BM architecture (Fig. 1, upper-left). In CLL cases with low IL23R expression, IL23R was detected in few scattered lymphoid cells intermingling with neoplastic lymphocytes (Fig. 1, upper-right). IL23 was also detected, with a variable staining intensity (Fig. 1, middle-left), paralleling in part that of IL23R. Double-marker analysis confirmed the concomitant expression of IL23 and IL23R in CLL neoplastic infiltrates highlighting the co-localization of the two markers (Fig.1 middle-right) and suggesting the possibility of an autocrine IL23/IL23R loop in CLL clones. We speculated that the microenvironment of CLL cases rich in IL23R and IL23 could be enriched in IL17-producing cells. The IHC expression of IL17 in CLL cases with low or high IL23R and IL23 expression showed that CLL cases rich in IL23Rpos cells, also characterized by high IL23 expression, displayed significantly higher numbers of IL17pos infiltrating cells (Fig. 1 bottom-left), as compared with CLL cases with no or low expression of IL23R or IL23 (Fig. 1 bottom-right). In conclusion, our study shows that high IL23R expression predicts a worse PFS. Furthermore, we linked this picture with, the in situ engendering of a clone-related microenvironment characterized by the preponderancy of pro-inflammatory signals such as those of the IL23/IL23R/IL17 axis, and its correlates in the peripheral blood (i.e. IL23R expression on circulating CLL cells), may endorse its strong prognostic significance. This analysis prompts further investigation into the specific function of the IL23/IL23R/IL17 axis and its targets in the context of CLL. Figure 1. Figure 1. Disclosures: No relevant conflicts of interest to declare.

Rupture of the urinary bladder secondary to abdominal trauma is uncommon and is usually associated with visceral or bone injuries; less than 10% of all cases occur without associated injuries. We present two cases of bladder rupture in intoxicated patients after minor abdominal trauma and without evidence of associated injuries. Given the absence of any symptoms in either case, we did not initially suspect bladder rupture. One of the two patients also presented with an intravesical herniation of the rectosigmoidal junction.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease; some patients have a rapidly progressing disease and others exhibit an indolent course and survive for many years without treatment. Mutation status of IgVH genes utilized by CLL cells represents a very reliable predictor of clinical outcome in B-CLL, but its analysis is expensive and beyond the capacities of most diagnostic laboratories. To identify surrogate markers we performed a gene expression profiling analysis of CD19+ purified cells from 80 B-CLL untreated patients in Binet stage A, by means of Affymetrix GeneChip® HGU133A arrays. The comparison of 46 IgVH-unmutated versus 34 mutated samples using the Prediction Analysis of Microarrays software identified 78 differentially expressed probes, specific for 59 well-characterized genes. Specifically, 43 genes had a higher and 16 genes a lower average expression in the IgVH unmutated group. These genes are involved in cellular functions, including cell cycle regulation (SEPT7, SEPT10, CDK2AP1), cell proliferation (SLAMF1, LDOC1), apoptosis (CD63, IFT57, P2RX1, RNF130, TNFRSF1B), cell adhesion (CNTNAP2, C1orf38, PCDH9), immune response (ZAP70, IFI44), signal transduction (AKAP13, RASGRP1, USP6NL, TGFBR3, AKAP12), lipid metabolism and fatty-acid degradation (FADS3, LPL, LASS6), cell-cell signalling (FCRL2), phospholipid biosynthetic process (AYTL2), regulation of circadian rhythm (EGR3, CRY1, OPN3), DNA-dependent regulation of transcription (MYBL1, NR4A2, NRIP1, ZBTB20), muscle development (VAMP5, SRI, DMD). The expression signature identified in the proprietary database was then validated by means of a meta-analysis of a publicly available gene expression dataset of 100 B-CLL (Haslinger et al., 2005), showing classification accuracy measures leading to a global classification rate of 82.93% of the test set and thus suggesting the strength of the identified expression signature. The expression levels of 11 genes (LPL, ZBTB20, ZAP70, CRY1, COBLL1, SEPT10, LDOC1, TNFRSF1B, DMD, SRI, NRIP1) were confirmed by means of quantitative real-time PCR (Q-RT-PCR) in a subset of 40 CLL patients. The prognostic impact for Time To Treatment (TTT) of the 59 candidate genes of our classifier model was investigated in 77 patients. Forty-nine (36.4%) of these received treatment after a median follow up of 4 years. As expected, patients with unmutated IgVH genes had a risk of therapy requirement that was about 3 times higher (HR: 3.1,95% C.I. 1.6–5.8, p

B-cell chronic lymphocytic leukemia (B-CLL) is a genetically heterogeneous disease with a variable clinical course. Chromosomal changes have been identified by FISH in approximately 80% of patients, and the presence of specific lesions, such as trisomy 12 and 13q14, 11q23, 17p13.1 and 6q23 deletions represent prognostic markers for disease progression and survival. In order to characterize further the complexity of B-CLL genomic lesions, we performed high density, single nucleotide polymorphism (SNP) array analysis in highly purified neoplastic cells (>92%) from a panel of 100 untreated, newly diagnosed patients (57 males and 43 females; age, median 63 years, range 30–87) in Binet stage A. All patients were investigated by FISH for the presence of trisomy 12 (21 cases); 13q14 deletion (44 cases, 34 as the sole abnormality); 11q22.3, 17p13.1 and 6q23 (15, 7 and 2 patients, respectively). In addition, ZAP-70 and CD38 expression resulted positive in 42 and 46 patients, whereas IgVH genes were mutated in 45 patients. Genome-wide DNA profiling data were generated on GeneChip® Human Mapping 250K NspI arrays (Affymetrix); copy number alterations (CNA) were calculated using the DNA copy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS) (Olshen et al, 2004). A total of 782 CNAs (ranging from 1 to 31 per sample, mean and median values 7.82 and 7, respectively) were detected; DNA losses (365/782=46.67% loss; 194/782=24.81% biallelic deletion) were found to be more frequent than gains (148/782=18.93% gain; 75/782=9.59% amplification). The most recurrent alterations detected by FISH were all confirmed by SNP array analysis, strengthening further the good reliability of such high-resolution technology. We identified 12 minimally altered regions (MARs) larger than 100 kb with a frequency higher than 5%. Among well known alterations, the largest was represented by chromosome 12 trisomy, followed by 6q, 17p and 11q23 deletions (32.87, 19.09 and 10.43 Mb, respectively) and 13q14 deletion (635 kb). Gain of 2p25.3 involves a common region of 4.39 Mb region in 7 patients, although it was extended to the whole short arm of chromosome 2 in 3 cases. Among those alterations previously described in B-CLL, we found losses at 14q32.33 (12 pts) and 22q11.2 (5 pts) involving the IGH and IGLλ loci, respectively. With regard to novel regions, we identified losses at 4q35.2 (5 pts) and 11q25 (6 pts). In addition we found a high frequency of losses/gains at 14q11.2 (42 pts) and 15q11.2 (33 pts), two genomic regions reported to be affected by DNA copy number variations in normal individuals. As regards correlations between CNAs and biological markers, we found that the number of CNAs is significantly higher in cases with unmutated IgVH (9.4; range 2–31) as compared with mutated IgVH (6; range 1–13) (p=0.002), while neither CD38 nor ZAP-70 expression showed significant correlation. In addition, a significant higher number of either CNAs (p=0.001), total MARs (p

Abstract 2341 Poster Board II-318 Background. The clinical heterogeneity of chronic lymphocytic leukemia (CLL) requires parameters to stratify patients into prognostic subgroups to adapt treatment ranging from ‘watch and wait’ to allogeneic stem cell transplantation. To this end, several parameters such as lymphocyte doubling time, β-2 microglobulin, CD38 and ZAP-70 expression, immunoglobulin variable heavy chain (IgVH) mutation status and genetic abnormalities, as assessed by fluorescence in situ hybridization (FISH), have been integrated in clinical practice. Aims. In the present study, we investigated by FISH the incidence of the known major cytogenetic alterations (+12 and 13q14, 17p13, 11q23 deletions) in a series of Binet A B-CLL patients included in the prospective O-CLL1 GISL study started in April 2007. Methods. Molecular markers characterization and FISH analyses were performed as previously reported (Cutrona et al. Haematologica, 2008; Fabris et al. GCC, 2008). A cut-off value of 2% was used to distinguish mutated and unmutated patients. CD38 and ZAP-70 were determined by flow-cytometry and a 30% cut-off was used to distinguish between positive or negative cases. Results. Up to date, 326 patients have been enrolled in the trial and FISH data concerning trisomy 12 and 13q14, 17p13, 11q23 deletions were available in 305 patients. At least one abnormality was found in 197 (64%) cases. The most frequent was del(13)(q14) (150/305, 49%), followed by +12 (40/303, 13%) (in one and three cases accompanied by 17p13 and 13q14 deletions, respectively), del(17)(p13) (7/305, 2%) and del(11)(q23) (17/305, 5%). 13q14 deletion was found as a sole abnormality in 134 patients; in the remaining cases, it was combined with +12 (3 pts) and 17p13 (3 pts) or 11q23 (10 pts) deletions. Among patients with 13q14 deletions, 99 were monoallelic, 12 biallelic and 39 showed a combination of the two patterns. Biomarkers data were available in all of the patients: 95/305 (31%) cases had unmutated IgVH genes; ZAP-70 and CD38 were positive in 117/305 (38%) and 72/305 (23%) cases, respectively. Concerning the distribution of cytogenetic aberrations, the unmutated IgVH group included 29/150 (19%) 13q14 deleted cases, 23/40 (57%) cases with trisomy 12 and 4/7 (57%) and 16/17 (94%) with 17p13 and 11q23 deletions, respectively. ZAP-70-positive groups included 43/150 (28%) 13q14 deleted cases, 26/40 (65%) cases showing trisomy 12 and 5/7 (71%) and 12/17 (70%) with 17p13 and 11q23 deletions, respectively. Finally, CD38-positive cases included 18/150 (12%) 13q14 deleted cases, 26/40 (65%) cases carrying trisomy 12 and 5/7 (71%) and 7/17 (41%) with 17p13 and 11q23 deletions, respectively. The percentages of IgVH mutations significantly correlated with cytogenetic alterations; namely, 5.8±0.3 for cases with del(13)(q14), 4.6±0.4 for normal karyotype, 2.6±0.5 in +12, 0.3±0.2 in del(11)(q23), and 1.7±0.9 in del(17)(p13) cases (p for trend Conclusions. Our preliminary results indicate that in Binet stage A B-CLL patients at diagnosis cytogenetic abnormalities with an expected negative clinical impact are relatively few (7.2%) but significantly associated with prognostic biomarkers which negatively predict the clinical outcome in B-CLL. Disclosures: No relevant conflicts of interest to declare.

Patients whose cells utilize unmutated Ig VH region genes and/or express ZAP-70 or CD38 have a more aggressive course than patients whose cells have mutated Ig VH genes and/or do not express ZAP-70 or CD38. We have conducted here a study on 500 patients characterized for marker expression. By ROC curve analysis, we found 30% as the best cut-off value of CD38 which discriminates between mutated and unmutated cases in CLLs. CD38 expression, had low sensitivity (66%), but relatively high specificity (80%), with a positive and a negative predictive value respectively of 68% and 78% in anticipating VH mutational status. Moreover, the agreement between CD38 expression and VH mutational status was low although significant (K=0.46, p < 0.001). ZAP-70 showed high sensitivity (76%) and specificity (75%), high negative (89%) but a low positive (54%) predictive value and a low, although significant, K statistic (0.45, p < 0.001). Furthermore, we combined CD38 and Zap-70 expression to evaluate whether both variables provided more precise information in estimating VH mutational status. We found sensitivity, 42%; specificity, 97%; positive predictive value, 90%; negative predictive value, 72%; K statistic 0.43, p < 0.001. In conclusion neither CD38 nor ZAP-70 by themselves or in combination were able to anticipate VH mutational status, meaning that CD38 and/or Zap-70 expression could surrogate the VH mutational status. In the second part of this study we wanted to validate this findings on clinical ground. Clinical information was available for 150/500 CLL cases investigated. After a median follow-up of 38 months, 83 cases remained untreated, while 67 cases received treatment. We show that these markers predict the clinical course by using time to first treatment (TTT) as a measure for disease progression. Each of the three markers was capable of discriminating two distinct groups of patients (p < 0.0001 for CD38, p < 0.00001 for ZAP-70 and Ig VH mutations) with different clinical behavior, although marker combinations provided a more precise definition of prognosis. Although many patients expressed all favorable or all unfavorable markers, there also were patients with different marker combinations. We devised a scoring system that subdivides patients based on the absence (score 0) or presence of 1 (score 1), 2 (score 2), or 3 (score 3) unfavorable prognostic markers. Using this scoring system, we have identified 3 groups with significantly different clinical courses: i.e., low- (score 0), intermediate-risk (score 1) and high-risk (score2–3) patients. This scoring system has potential utilization for prognostic stratification of CLL in designing prospective clinical trials.