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Because the efficacy of new drugs reported in trials may not translate into similar results when used in the real‐life, we analyzed the efficacy of idelalisib and rituximab (IR) in 149 patients with relapsed/refractory chronic lymphocytic leukemia treated at 34 GIMEMA centers. Median progression‐free survival (PFS) and overall survival were 22.9 and 44.5 months, respectively; performance status (PS) ≥2 and ≥3 previous lines of therapy were associated with shorter PFS and overall survival (OS). 48% of patients were on treatment at 12 months; the experience of the centers (≥5 treated patients) and PS 0–1 were associated with a significantly longer treatment duration (p = 0.015 and p = 0.002, respectively). TP53 disruption had no prognostic significance. The overall response rate to subsequent treatment was 49.2%, with median OS of 15.5 months and not reached in patients who discontinued, respectively, for progression and for toxicity (p

National audience; Ce travail vise à démontrer les capacités d’un nouveau type de détecteur de particules chargées, bénéficiant des progrès récents en intégration microélectronique 3D, pour des applications médicales avancées telles que l’hadronthérapie (ex. réduction des incertitudes dans la fluence pour délivrer la bonne dose, étiquetage précis d'un temps d'arrivée des ions pour la sélection en temps de vol des rayons gamma prompts) et la radiographie - tomographie proton (ex. filtrage des neutrons et des rayons gamma, amélioration potentielle de la résolution spatiale).Ce détecteur intégré en technologie CMOS est appelé 3D-SiCAD (3D Silicon Coincidence Avalanche Detector).Il met en oeuvre un mode de coïncidence interne innovant, objet d’une forte activité R&D au niveau international, basé sur un empilement vertical de pixels de type SPAD -Single Photon Avalanche Diode (mode Geiger) permettant d’améliorer significativement la discrimination entre les évènements liés au passage de particules (déclenchement simultané de deux pixels) et le bruit intrinsèque. L’originalité de notre démarche consiste en l’application de ce concept innovateur aux applications médicales.Les étapes de conception du premier prototype ainsi que les principaux résultats obtenus seront présentés avec notamment :i) les performances de la diode SPAD seule réalisée en technologie CMOS 0.35μm avec par exemple un taux de comptage dans l’obscurité moyen de 70Hz/μm²,ii) la démonstration expérimentale de la diminution du bruit apparent (taux de comptage de faux événements) grâce à la détection en coïncidence dans une courte fenêtre temporelle. En pratique, le taux de réjection du bruit peut atteindre un facteur x103 dans les conditions optimales,iii) la mise en oeuvre de cette solution technologique pour la mesure de l’activité d’une source de strontium.Finalement les performances actuelles, les pistes d’amélioration possibles ainsi qu’une revue des travaux en cours et futurs seront brièvement discutés.

The mammalian target of rapamycin (mTOR) signalling pathway has emerged as an important therapeutic target for acute myeloid leukaemia (AML). This study assessed the combination of temsirolimus, an mTOR inhibitor, and lower-dose clofarabine as salvage therapy in older patients with AML. Induction consisted of clofarabine 20 mg/m2 on days 15 and temsirolimus 25 mg (flat dose) on days 1, 8 and 15. Patients achieving complete remission with (CR) or without (CRi) full haematological recovery could receive monthly temsirolimus maintenance. In 53 evaluable patients, the overall remission rate (ORR) was 21% (8% CR, 13% CRi). Median disease-free survival was 3.5 months, and median overall survival was 4 months (9.1 months for responders). The most common non-haematological severe adverse events included infection (48%), febrile neutropenia (34%) and transaminitis (11%). The 30-d all-cause induction mortality was 13%. Laboratory data from 25 patients demonstrated that a >50%in vivo inhibition of S6 ribosomal protein phosphorylation was highly correlated with response rate (75% with inhibition versus 0% without inhibition; P = 0.0001), suggesting that targeting the mTOR pathway is clinically relevant. The acceptable safety profile and the predictive value of target inhibition encourage further investigation of this novel regimen.

Loss of CDKN2A, encoding p16/INK4A and p14/ARF tumor suppressors, has been associated with poorer survival and higher leukemia-initiating- cell-frequency in xenograft models (Notta F et al. Nature 2011). Recently, using genome-wide single nucleotide polymorphisms arrays and gene candidate deep exon sequencing, we identified CDKN2A deletions in 29% (29/101) adult newly diagnosed Ph+ ALL patients and in 47% relapsed cases. Accordingly, here we aimed to investigate the functional consequences of genomic deletions and their prognostic value. By quantitative real-time PCR a significant difference in the expression of CDKN2A was observed among cases with heterozygous (p = 0.04) and homozygous (p = 0.01) loss compared to normal cases, suggesting that deletions lead to CDKN2A haploinsufficiency. In order to investigate their clinical implications, data were collected from 81 patients (median follow-up: 25.2 months- range 2.1-148.1; median age at diagnosis: 54 years- range, 18-76; CDKN2A loss in 29, 36%, cases). 72 patients (89%) were enrolled in the GIMEMA clinical trials (12 patients in GIMEMA LAL0201-B protocol, 13 in LAL2000 and 47 in LAL1205 protocols), while 9 patients (11%) were enrolled into institutional protocols. By univariate analysis a shorter overall survival (OS) and disease-free survival (DFS) were found in patients with CDKN2A deletion compared with those wild-type (OS: 27.7 v 38.2 months, respectively, p = 0.0206; DFS: 10.1 vs 56.1 months, respectively, p = 0.0010). Moreover, a higher cumulative incidence of relapse for patients with CDKN2A deletion (73.3 vs 38.1; p = 0.0014) was also recognized. The multivariate analysis confirmed the negative prognostic impact of CDKN2A deletion on DFS (p = 0051). These results show that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation, cell growth and tumor formation by the action of MDM2 and CDK4/6, attractive drugs to prevent disease recurrence could be represented by the inhibitors of MDM2 and CDK4/ CDK6. These drugs are currently under investigation in in vitro studies by our group. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program, Programma di Ricerca Regione – Università 2007 – 2009.

Introduction. Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed. Methods. 23 SNPs spanning the MTAP, CDKN2A/Band CDKN2BAS loci, as well as relative intergenic regions were genotyped in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples, and 183 healthy controls. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Results. A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with MAF 20% missing call rates, were instead excluded from the association analysis and potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20 to 3.33; P=7.1¥103). Conclusions. Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione - Università 2007-2009.

Summary Metaphase (M-) and array (A-) Comparative Genomic Hybridization (CGH) were used to investigate 40 cases of T- and 32 of B-cell acute lymphoblastic leukaemia (ALL) with normal/failed cytogenetics. M-CGH was performed in all cases and A‐CGH in 10/12 T-ALL cases with uncertain/normal M-CGH results. M-CGH was abnormal in 38/72 cases, with a total of 110 imbalances (60 gains, 50 losses). 25/40 patients with T-ALL (62AE5%) showed 77 imbalances, with at least 1 genomic imbalance and a mean of 3 aberrations/ patient (range 1‐12). 13/32 patients with B-ALL (40AE6%) presented 34 imbalances, with a mean of 2AE6 imbalances (range 1‐8). A-CGH detected 4 more T-ALL cases with genomic imbalances. A-CGH identified NF1/17q11AE2 deletion and interphase fluorescence in situ hybridization provided a 10AE8% estimated overall incidence of NF1/17q11AE2 deletion in T-ALL. In all but one case (6/7) with NF1 deletion, denaturing high-performance liquid chromatography and direct sequencing detected NOTCH1 gene mutations. Three or more imbalances in CGH-positive cases were significantly associated with resistance to treatment and death during or after induction therapy. We suggest that the work-up for ALL at diagnosis should include CGH investigations, particularly when cytogenetics is uninformative, because they may provide potentially valuable information with prognostic and therapeutic implications.

Deletions of the 9p21 chromosomal region are frequent events for the development of a variety of cancers, including solid tumors and hematological malignancies, such as childhood ALL. This region contains the 40-kb region encoding the p16INK4a/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15INK4b/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. In order to explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate the influence on the prognosis, 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18–76) and the median blast percentage was 90% (range, 18–99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21. PCR amplification and mutation screening of each exon by cloning and subsequent sequencing were also performed. Moreover, gene-expression profiling was performed (Affymetrix Human Exon 1ST Array) to draw a specific signature associated with deletions. At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8–31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm eliminating a large number of genes. In contrast, cases with bi-allelic inactivation were 28% with the majority of deletions (75%) limited to CDKN2A/ARF/CDKN2B genes. FISH analysis was performed using three different BAC clones, but since they overlooked microdeletions we only appreciated a mild fluorescent signal reduction. In order to assess whether 9p21 loss is responsible for progression, the genomic status of this locus was assessed at the time of relapse and an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06) was found. Functionally, deletions led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005), as demonstrated by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. Finally, the mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by point mutations with only the identification of the missense D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5′UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)] and a variation, known as SNP was found in the exon 2 (rs3731249). Finally, 2 SNPs were found in the 3′ UTR region of CDKN2A/ARF (rs11515 and rs3088440). The role of the mutations identified in the UTR is not yet defined, but the comparison with the germline material from saliva samples showed that these mutations were inherited. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort, finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.003) and cumulative incidence of relapse (p= 0.004). In conclusion, the inactivation of the tumor suppressor genes CDKN2A/ARF is a frequent event in Ph+ ALL. The main mechanism of inactivation is represented by genomic deletions, whereas missense mutations are rare. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis.

Introduction. Although treatment with tyrosine kinase inhibitors has revolutionized the management of adult patients with BCR-ABL1-positive ALL and significantly improved response rates, relapse is still an expected and early event in the majority of them. It is usually attributed to the emergence of resistant clones with mutations in BCR-ABL1 kinase domain or to BCR-ABL1-independent pathways but many questions remain unresolved about the genetic abnormalities responsible for relapse. Patients and methods. In an attempt to better understand the genetic mechanisms responsible for this phenomenon, we have analyzed matched diagnosis-relapse samples from 20 adult BCR-ABL1-positive ALL patients using high resolution Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI, n=15 pairs and Genome-Wide Human SNP 6.0, n=5 pairs). Genetic differences were analyzed in terms of copy number changes and loss of heterozygosity (LOH) events. Patients were enrolled in clinical trials of GIMEMA AL Working Party and treated with imatinib alone or in combination with conventional chemotherapy (40%) or dasatinib as frontline therapy (60%). The median age at diagnosis was 54 years (range 23-74) and the median blast cell count was 97% (range 60-99). The median time to relapse was 27 months (range, 9-104). Results. In 2/20 (10%) patients no genomic differences between diagnosis and relapse samples were found (“stable group”), suggesting that only BCR-ABL1 mutations or extraleukemic factors (e.g., drug distribution, drug metabolism, compliance) contributed to the relapse. In the remaining cases (90%), new acquired copy number alterations (CNAs) were detected at relapse (“unstable group”). Acquired macroscopic alterations (>1.5 MB) were detected in 6 (30%) cases and included gain of chromosome 1q, 16q22, 9q and 22q (regions flanking the ABL and BCR genes), 19p13.3 (ABCA7, APC2, ARID3A) and 11q12.1-11q25; macroscopic losses affected the locus 1p36 in 2 cases. The gene most frequently affected by microscopic CNAs (

Recent genome-wide analyses in B-ALL have identified a high frequency of DNA copy-number abnormalities, but the biological and prognostic implications of these abnormalities have not been defined. In order to address this issue, a cohort of 149 adult ALL patients (106 BCR-ABL1-postive, 3 ALL1/AF4, 1 EA/PBX, 39 negative for known molecular rearrangements) were analyzed with the use of singlenucleotide– polymorphism (SNP) microarrays (Affymetrix 250K NspI and SNP6.0), FISH, transcriptional profiling, and resequencing of samples obtained at diagnosis. Lesions varied from loss or gain of complete chromosome arms (trisomy 4, monosomy 7, loss of 9p, gain of 1q) to micro-alterations targeting genomic intervals in which the minimal common region of change involved one or few genes. Lesions in genes involved in early B-cell differentiation (60%) and cell cycle regulation (40%) were observed with relatively high frequency (60%). The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1 (75% in BCR-ABL1 positive and 58% in BCR-ABL1-negative ALL), which encodes the transcription factor Ikaros required for the earliest stages of lymphoid lineage commitment. FISH analysis using a pool of fosmid probes for IKZF1 and genomic quantitative PCR confirmed SNP results. Among the 149 patients, the entire IKZF1 locus was deleted in 19 (13%); in 84 (56%) additional patients, a subgroup of exons or the genomic region immediately upstream of IKZF1 was deleted. In 48 of them, there was a deletion of coding exons 4 through 7, which results in expression of a dominant-negative isoform, Ik6, which cytoplasmatic localization. Using gene-set enrichment analysis to compare the geneexpression signatures of patients with IKZF1 deletion versus not-deleted patients, we identified a unique signatures independent by BCRABL1 and characterized by down-regulation of B-cell lineage genes (e.g. VPREB1, VPREB3, IGLL3, BLK) and up-regulation of genes involved in cell-cycle progression (STK17B, SERPINB9, CDKN1A). We next investigated whether the IKZF1 deletions associated with a poor outcome. Univariate analysis showed that the IKZF1 deletion negatively influenced the cumulative incidence of relapse (p=0.0103) and disease-free survival (p=0.0229). Conclusion. Deletion of IKZF1 is an important event in the development of B-progenitor ALL which significantly influences clinical outcome. European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus.

Bcr-Abl kinase domain (KD) mutations may cause resistance to tyrosine kinase inhibitor (TKI) therapy in Ph+ acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML). Mutations in rare Ph+ cells have been detected in some imatinib-naïve advanced-phase CML patients (pts), but it is unclear whether low level mutations may already be present at diagnosis in chronic-phase (CP) CML as well as in Ph+ ALL, and whether their detection predicts for subsequent treatment failure. We analyzed cDNA samples from 24 newly diagnosed pts with Ph+ ALL (n=13) or CP-CML (n=11) who subsequently received TKI therapy. Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a. 206-524) in a bacterial vector and sequencing 200 clones for each pt. All pts had evidence of aberrant KD sequences. Three to twelve different mutations were detected in each pt. A total of 115 mutations (41 silent, 5 nonsense, 69 missense mutations) were observed. The majority (107/115, 93%) have never been reported in association with TKI resistance and are likely not to confer any advantage under TKI selective pressure. Interestingly, 103/115 (90%) mutations were transitions: G>A (n=30), A>G (n=25), C>T (n=25), T>C (n=23). One of the eleven CP-CML pt received hydroxyurea for 6 months before starting imatinib therapy. In this pt, high-sensitivity mutation screening was performed again immediately before imatinib start and showed further accumulation of mutations. Eight Ph+ ALL pts and three CML pts subsequently relapsed with mutations, but only two with a mutation (T315I) already detectable at diagnosis. The remaining thirteen pts are in persistent remission (follow-up, 12-52 months), although four of them were harbouring known imatinib-(H396P, D276G, E355G) or dasatinib-(F317L) resistant mutations at low levels. We can conclude that: a) mutations can probably be found at diagnosis in all CP-CML and Ph+ ALL pts; b) mutations seem to arise randomly and most of them are silent/not conferring any growth advantage; c) generation of mutations seems to be linked to Bcr-Abl-driven genetic instability; d) TKI-resistant mutations present at low levels at diagnosis do not always outgrow and lead to relapse, probably because some of them arise in cell clones with limited self-renewal capacity. This warns against high-sensitivity mutation screening of all pts before the start of therapy.

Hematopoietic cell transplantation (HCT) and prolonged chemotherapy are standard postremission strategies for adult acute lymphoblastic leukemia in first complete remission, but the optimal strategy remains controversial. There are no randomized trials of allogeneic HCT. In the present study, updated individual patient data were collected and analyzed from studies with information on availability of matched sibling donor (used to mimic randomization) and from randomized trials of autograft versus chemotherapy. Data from 13 studies including 2962 patients, excluding Philadelphia chromosome–positive patients, showed a survival benefit for having a matched sibling donor for patients < 35 years of age (OR = 0.79; 95% CI, 0.70-0.90, P = .0003) but not for those ≥ 35 years of age (OR = 1.01; 95% CI, 0.85-1.19, P = .9; heterogeneity P = .03) because of the higher absolute risk of nonrelapse mortality for older patients. No differences were seen by risk group. There was a trend toward inferior survival for autograft versus chemotherapy (OR = 1.18; 95% CI, 0.99-1.41; P = .06). No beneficial effect of autografting was seen compared with chemotherapy in this analysis. We conclude that matched sibling donor myeloablative HCT improves survival only for younger patients, with an absolute benefit of approximately 10% at 5 years. Improved chemotherapy outcomes and reduced nonrelapse mortality associated with allogeneic HCT may change the relative effects of these treatments in the future.

The objective of this study was to investigate the health-related quality of life (HRQOL) aspects valued the most by patients with chronic myeloid leukemia (CML) receiving targeted therapies (TT), and to compare their perception with that of health-care professionals' (HCPs). Semi-structured interviews were conducted with 137 CML patients receiving TT from five different countries. An additional sample of 99 CML patients, completing an online interview, was considered for supportive analyses. A sample of 59 HCPs from 12 countries also participated in the study. Patients and HCPs were asked to rate and rank the importance of a predefined list of 74 HRQOL aspects of potential relevance for CML patients. Patients and HCPs agreed that the following five aspects are most important: fatigue, muscle cramps, swelling, worries, and uncertainty about health condition in the future, and importance of social support in coping with the disease. However, the difference in rankings between the two groups was substantial with respect to other HRQOL aspects investigated. Patients valued some issues related to symptoms much higher than HCPs, thus suggesting that a better symptom management could be the crucial aspects to improve HRQOL of CML patients.© 2012 Springer-Verlag.