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The GIMEMA phase II LLC1518 VERITAS trial investigated the efficacy and safety of frontline, fixed-duration venetoclax and rituximab (VenR) combination in young (≤65 years) and fit patients with chronic lymphocytic leukemia (CLL) and unmutated IGHV and/or TP53 disruption. Treatment consisted of the Ven ramp-up, six-monthly courses of the VenR combination, followed by six monthly courses of Ven single agent. A centralized assessment of measurable minimal residual disease (MRD) was performed on the peripheral blood (PB) and bone marrow (BM) by ASO-PCR at the end of treatment (EOT) and during the follow-up. The primary endpoint was the complete remission (CR) rate at the EOT. Seventy-five patients were enrolled; the median age was 54 years (range 38-65), 96% had unmutated IGHV, 9 (12%) had TP53 disruption, and 4% were IGHV mutated with TP53 disruption. The overall response rate (ORR) at the EOT was 94.7%, with a CR rate of 76%. An undetectable (u) MRD was recorded in 69.3% of patients in the PB and 58.7% in the BM. The 12-month MRD-free survival in the 52 patients with uMRD in the PB at the EOT was 73.1%. After a median follow-up of 20.8 months, no disease progressions were observed. Three patients have died, two due to Covid-19 and 1 to tumor lysis syndrome. The first report of the VERITAS study shows that frontline VenR was associated with a high rate of CRs and durable responses with uMRD in young patients with CLL and unfavorable genetic characteristics.

Simple Summary IDH1/2 mutations are a common event in acute myeloid leukemia (AML) and represent a therapeutic target. We designed the GIMEMA AML1516 observational protocol to examine the prevalence of IDH1/2 mutations and the associations between IDH mutations and clinico-biological parameters in a cohort of Italian patients affected by AML. By analyzing 284 consecutive adult AML patients, we confirmed that IDH1 and IDH2 mutations are frequently detected-14% and 18%, respectively-at diagnosis. IDH1/2 mutations were significantly associated with an inferior performance status and non-complex karyotype when compared to IDH1/2-WT. With regards to the outcome, in the subset of IDH1/2-mutated patients the rate of complete remission achievement was 60.5% and overall survival at 2 years was 44.5%: these percentages did not significantly differ from IDH1/2-WT patients. However, given the availability of IDH1/2 inhibitors, it is important to recognize IDH1/2-mutated cases up-front to offer patients the most appropriate therapeutic strategy. IDH1/2 mutations are common in acute myeloid leukemia (AML) and represent a therapeutic target. The GIMEMA AML1516 observational protocol was designed to study the prevalence of IDH1/2 mutations and associations with clinico-biological parameters in a cohort of Italian AML patients. We analyzed a cohort of 284 AML consecutive patients at diagnosis, 139 females and 145 males, of a median age of 65 years (range: 19-86). Of these, 38 (14%) harbored IDH1 and 51 (18%) IDH2 mutations. IDH1/2 mutations were significantly associated with WHO PS >2 (p < 0.001) and non-complex karyotype (p = 0.021) when compared to IDH1/2-WT. Furthermore, patients with IDH1 mutations were more frequently NPM1-mutated (p = 0.007) and had a higher platelet count (p = 0.036). At relapse, IDH1/2 mutations were detected in 6 (25%) patients. As per the outcome, 60.5% of IDH1/2-mutated patients achieved complete remission; overall survival and event-free survival at 2 years were 44.5% and 36.1%, respectively: these rates were similar to IDH1/2-WT. In IDH1/2-mutated patients, high WBC proved to be an independent prognostic factor for survival. In conclusion, the GIMEMA AML1516 confirms that IDH1/2 mutations are frequently detected at diagnosis and underlines the importance of recognizing IDH1/2-mutated cases up-front to offer the most appropriate therapeutic strategy, given the availability of IDH1/2 inhibitors.

Summary Multicomponent reactions (MCRs) are ideal platforms for the generation of a wide variety of organic scaffolds in a convergent and atom-economical manner. Many strategies for the generation of highly substituted and diverse structures have been developed and among these, the Petasis reaction represents a viable reaction manifold for the synthesis of substituted amines via coupling of an amine, an aldehyde and a boronic acid (BA). Despite its synthetic utility, the inherent drawbacks associated with the traditional two-electron Petasis reaction have stimulated continuous research towards more facile and tolerant methodologies. In this regard, we present the use of free alkyl BAs as effective radical precursors in this MCR through a single-electron transfer mechanism under mild reaction conditions. We have further demonstrated its applicability to photo-flow reactors, facilitating the reaction scale-up for the rapid assembly of complex molecular structures., Graphical abstract, Highlights • Multicomponent photocatalyzed Petasis reaction • Boronic acids as alkyl radical precursors under mild reaction conditions • Photo-flow chemistry application for Petasis reaction, Catalysis; Chemistry; Engineering; Green chemistry

To shed light onto the molecular basis of Philadelphia chromosome-positive acute lymphoblastic leukemia and to investigate the prognostic role of additional genomic lesions, we analyzed copy number aberrations using the Cytoscan HD Array in 116 newly diagnosed adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia enrolled in four different GIMEMA protocols, all based on a chemotherapy-free induction strategy. This analysis showed that patients with Philadelphia chromosome-positive acute lymphoblastic leukemia carry an average of 7.8 lesions/case, with deletions outnumbering gains (88% versus 12%). The most common deletions were those targeting IKZF1, PAX5 and CDKN2A/B, which were detected in 84%, 36% and 32% of cases, respectively. Patients carrying simultaneous deletions of IKZF1 plus CDKN2A/B and/or PAX5 had a significantly lower disease-free survival rate (24.9% versus 43.3%; P=0.026). The only IKZF1 isoform affecting prognosis was the dominant negative one (P=0.003). Analysis of copy number aberrations showed that 18% of patients harbored MEF2C deletions, which were of two types, differing in size: the longer deletions were associated with the achievement of a complete molecular remission (P=0.05) and had a favorable impact on disease-free survival (64.3% versus 32.1% at 36 months; P=0.031). These findings retained statistical significance also in multivariate analysis (P=0.057). KRAS deletions, detected in 6% of cases, were associated with the achievement of a complete molecular remission (P=0.009). These results indicate that in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia a detailed evaluation of additional deletions - including CDKN2A/B, PAX5, IKZF1, MEF2C and KRAS - has prognostic implications and should be incorporated in the design of more personalized treatment strategies.

In Ph+ acute lymphoblastic leukaemia (Ph+ ALL), minimal residual disease (MRD) is the most relevant prognostic factor. Currently, its evaluation is based on quantitative real-time polymerase chain reaction (Q-RT-PCR). Digital droplet PCR (ddPCR) was successfully applied to several haematological malignancies. We analyzed 98 samples from 40 Ph+ ALL cases, the majority enrolled in the GIMEMA LAL2116 trial: 10 diagnostic samples and 88 follow-up samples, mostly focusing on positive non-quantifiable (PNQ) or negative samples by Q-RT-PCR to investigate the value of ddPCR for MRD monitoring. DdPCR BCR/ABL1 assay showed good sensitivity and accuracy to detect low levels of transcripts, with a high rate of reproducibility. The analysis of PNQ or negative cases by Q-RT-PCR revealed that ddPCR increased the proportion of quantifiable samples (p

Adolescents and young adults (AYA) with acute lymphoblastic leukemia (ALL) represent a unique patient population with specific characteristics and needs. Growing evidences suggest that pediatric-inspired approaches improve the outcome in AYA. These results prompted the design of a pediatric AIEOP-BFM ALL 2000-based regimen - the GIMEMA LAL-1308 protocol - for newly diagnosed AYA (range 18-35 years) with Philadelphia negative (Ph-) ALL. The protocol included minimal residual disease (MRD) analysis at two different time-points (TP), that is, at the end of induction IA and consolidation IB, and a modulation in post-consolidation intensity according to MRD. Seventy-six patients were eligible between September 2010 and October 2014. The regimen was well tolerated, with 2.7% induction deaths and no deaths in the post-consolidation phase. The complete response (CR) rate was 92%; the 48-month overall survival (OS) and disease-free survival (DFS) were 60.3% and 60.4%. Both OS and DFS were significantly better in T-ALL than B-ALL. A molecular MRD

With the outbreak of COVID‐19 pandemic (Wang et al, 2020), Italy ‐ and in particular the largest Northern regions ‐ became disease epicenter with a dramatic raise in infections and deaths in the month of March.

Early recognition of Philadelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) cases could impact on the management and outcome of this subset of B-lineage ALL. In order to assess the prognostic value of the Ph-like status in a pediatric-inspired, minimal residual disease (MRD)- driven trial, we screened 88 B-lineage ALL cases negative for major fusion genes (BCR-ABL1, ETV6-RUNX1, TCF3-PBX1 and KTM2Ar) enrolled in the GIMEMA LAL1913 front-line protocol for adult BCR/ABL1-negative ALL. The screening - performed using the “BCR/ABL1-like predictor” - identified 28 Ph-like cases (31.8%), characterized by CRLF2 overexpression (35.7%), JAK/STAT pathway mutations (33.3%), IKZF1 (63.6%), BTG1 (50%) and EBF1 (27.3%) deletions, and rearrangements targeting tyrosine kinases or CRLF2 (40%). The correlation with outcome highlighted that: i) the complete remission rate was significantly lower in Ph-like compared to non-Phlike cases (74.1% vs. 91.5%, P=0.044); ii) at time point 2, decisional for transplant allocation, 52.9% of Ph-like cases versus 20% of non-Ph-like were MRD-positive (P=0.025); iii) the Ph-like profile was the only parameter associated with a higher risk of being MRD-positive at time point 2 (P=0.014); iv) at 24 months, Ph-like patients had a significantly inferior event-free and disease-free survival compared to non-Ph-like patients (33.5% vs. 66.2%, P=0.005 and 45.5% vs. 72.3%, P=0.062, respectively). This study documents that Ph-like patients have a lower complete remission rate, event-free survival and disease-free survival, as well as a greater MRD persistence also in a pediatric-oriented and MRD-driven adult ALL protocol, thus reinforcing that the early recognition of Ph-like ALL patients at diagnosis is crucial to refine risk-stratification and to optimize therapeutic strategies. Clinicaltrials gov. Identifier: 02067143.

BCR/ABL1-like acute lymphoblastic leukemia (ALL) accounts for 15% to 30% of B-lineage ALL, with a peak of incidence occurring in adolescence. This subgroup of patients is characterized by a peculiar transcriptional profile that resembles that of true BCR/ABL1-positive cases, and have a heterogeneous genetic background and a poor outcome. Next-generation sequencing studies have demonstrated that the majority of patients carry rearrangements of tyrosine kinases or cytokine receptors and mutations of janus kinase (JAK)/signal transducer and activator of transcription (STAT), thus opening the way to the possible use of targeted therapeutic approaches. However, several issues remain unresolved at both the diagnostic and therapeutic level, such as the definition of a standardized method to identify BCR/ABL1-like ALL and the design of ad hoc clinical trials examining tyrosine kinase inhibitors or other tailored treatments. These aspects are discussed in this review.

Background Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile. Patients and methods Patients with absence of disease progression for over 10 years (10–34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultra-deep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis. Results WES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients. Conclusions The genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.

Summary Copy number aberrations (CNAs) represent cooperating events in B-lineage acute lymphoblastic leukaemia (B-ALL); however, their clinical relevance across different age cohorts is unclear. We analysed the recurrent CNAs in 157 age-stratified B-ALL negative cases for recurrent rearrangements (B-NEG ALL), and their association with patients’ clinico-biological features. We found that: (i) CDKN2A/RB1-deleted and EBF1-deleted adults had a shorter disease-free survival than those with wild-type, (ii) among the unfavourable markers, CDKN2A/RB1 deletions and K/NRAS mutations retained their impact in multivariate analysis, encouraging the evaluation of CDKN2A/RB1 deletions and RAS mutations in the diagnostic/prognostic workflow to refine ALL risk assessment.

TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response to ibrutinib. We hereby report that ibrutinib-induced in vitro apoptosis and proliferation inhibition were significantly lower in TP53-mutated (TP53-M) CLL cells compared to TP53 wild-type cells. Contrariwise, venetoclax effectively killed TP53-M cells. Gene expression profile analysis of TP53-M cells revealed a downmodulation of B-cell receptor (BCR)-related genes and an upmodulation of genes with anti-apoptotic/pro-survival activity, suggesting that the survival and proliferation of TP53-M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53-M CLL patients.

BCR/ABL1-like acute lymphoblastic leukaemia (ALL) is a subgroup of B-lineage acute lymphoblastic leukaemia that occurs within cases without recurrent molecular rearrangements. Gene expression profiling (GEP) can identify these cases but it is expensive and not widely available. Using GEP, we identified 10 genes specifically overexpressed by BCR/ABL1-like ALL cases and used their expression values - assessed by quantitative real time-polymerase chain reaction (Q-RT-PCR) in 26 BCR/ABL1-like and 26 non-BCR/ABL1-like cases to build a statistical "BCR/ABL1-like predictor", for the identification of BCR/ABL1-like cases. By screening 142 B-lineage ALL patients with the "BCR/ABL1-like predictor", we identified 28/142 BCR/ABL1-like patients (19·7%). Overall, BCR/ABL1-like cases were enriched in JAK/STAT mutations (P < 0·001), IKZF1 deletions (P < 0·001) and rearrangements involving cytokine receptors and tyrosine kinases (P = 0·001), thus corroborating the validity of the prediction. Clinically, the BCR/ABL1-like cases identified by the BCR/ABL1-like predictor achieved a lower rate of complete remission (P = 0·014) and a worse event-free survival (P = 0·0009) compared to non-BCR/ABL1-like ALL. Consistently, primary cells from BCR/ABL1-like cases responded in vitro to ponatinib. We propose a simple tool based on Q-RT-PCR and a statistical model that is capable of easily, quickly and reliably identifying BCR/ABL1-like ALL cases at diagnosis.

SummaryFollicular lymphoma (FL) is an indolent disease, but 30%–40% of cases undergo histologic transformation to an aggressive malignancy, typically represented by diffuse large B cell lymphoma (DLBCL). The pathogenesis of this process remains largely unknown. Using whole-exome sequencing and copy-number analysis, we show here that the dominant clone of FL and transformed FL (tFL) arise by divergent evolution from a common mutated precursor through the acquisition of distinct genetic events. Mutations in epigenetic modifiers and antiapoptotic genes are introduced early in the common precursor, whereas tFL is specifically associated with alterations deregulating cell-cycle progression and DNA damage responses (CDKN2A/B, MYC, and TP53) as well as aberrant somatic hypermutation. The genomic profile of tFL shares similarities with that of germinal center B cell-type de novo DLBCL but also displays unique combinations of altered genes with diagnostic and therapeutic implications.

Background and aims. B-lineage acute lymphoblastic leukemia (B-ALL) carrying t(4;11)(q21;q23)/KMT2A-AFF1 accounts for roughly 10% of adult B-ALL, it is associated to pro-B immunophenotype and aggressive clinical features. However, the role of KMT2A-AFF1 in pediatric-like, minimal residual disease (MRD)-driven clinical trials and the impact of transplant in this poor prognostic subgroup are still debated. To address these issues we investigated the incidence, clinical features and outcome of KMT2A-AFF1-positive ALL treated in 6 consecutive GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) clinical trials. Materials and methods. Between November 1996 and September 2016, 926 BCR-ABL1-negative B-ALL patients were enrolled in the GIMEMA clinical trials LAL0496, LAL2000, LAL0904, LAL1104, LAL1308, LAL1913. The median follow-up is 25.4 months (range: 0.1-146.9). Starting from LAL2000, KMT2A-AFF1-positive patients were classified as high-risk and managed more intensively. LAL1308 and LAL1913 adopted a similar pediatric-like backbone, at variance from LAL0496, LAL2000 and LAL0904. Overall, median age was 34.3 (range, 14.1 - 81.9), there were 502 males (54.2%) and 424 females (45.8%) and the median WBC count was 8.80 x 109/L (range, 0.4- 872.3). Incidence, clinical features and outcome of KMT2A-AFF1-positive were analyzed in comparison with KMT2A-AFF1-negative B-ALL. MRD evaluation after induction treatment was available in 197 patients enrolled in the more recent protocols. Results. Overall, 97/926 (10.5%) samples harbored KMT2A-AFF1 fusion gene. The comparison between KMT2A-AFF1-positive and the 829 KMT2A-AFF1-negative B-ALL patients revealed that KMT2A-AFF1-positive patients had a higher median age (42 vs 32.5 years old, p Conclusions. The present study examined a cohort of 926 BCR-ABL1-negative B-ALL treated in Italian multicenter clinical trials. Overall, though not differing in the achievement of CR and MRD negativity, the analysis of survival curves confirms that KMT2A-AFF1-positive patients have a significantly shorter survival than KMT2A-AFF1-negative patients. This result holds true also when censoring for transplant, thus suggesting that allograft does not confer a survival advantage to KMT2A-AFF1-positive patients. Moreover, KMT2A-AFF1-positive patients perform poorly also in more intensive regimen, as emerged from the analysis of the GIMEMA LAL1913 clinical trial. These findings encourage the adoption of alternative strategies: among the available targeted therapies, preclinical models of KMT2A-leukemia show sensitivity to BCL2-inhibitors, thus providing the rationale for testing this approach in upcoming clinical trials for KMT2A-AFF1-positive patients. Figure Disclosures Chiaretti: Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Krampera:Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy. Bassan:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: 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; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Vignetti:Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Educational Training; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Educational Training.

Background BCR/ABL1-like (alias Ph-like) acute lymphoblastic leukemia (ALL) is a clinically challenging subgroup. Its incidence varies from 10% in children to 30% in adult ALL. The underlying genomic background, usually characterized by the presence of kinase activating lesions, opens the way to targeted treatment. However, one of the major issues of this subset is represented by the identification of a standardized assay for their recognition at diagnosis. Indeed, while gene expression profile (GEP) and next-generation sequencing (NGS) are efficient techniques to identify these cases, they are difficult to be implemented in the diagnostic routine. Our group previously developed a rapid, simple and cost-effective algorithm based on a quantitative real time-polymerase chain reaction (Q-RT-PCR) named "BCR/ABL1-like predictor". Briefly, the expression levels of 10 genes - NUDT4, SEMA6A, ADGRE5, SOCS2, JCHAIN, CRLF2, TP53INP1, CD99, IFITM1 and IFITM2 - are used to generate a score: cases with a score ≥-0.3 are classified as BCR/ABL1-like. Aims After developing the BCR/ABL1-like predictor, we sought to: further refine the ability of this model to correctly predict BCR/ABL1-like ALL by evaluating NGS and RNA-sequencing of BCR/ABL1-like and non-BCR/ABL1-like cases, and to define the incidence of specific lesions;evaluate the reproducibility of the predictor when performed in external laboratories or when samples with a known genetic background were analyzed in house by the predictor. Results NGS and RNA-sequencing were carried out in 28 BCR/ABL1-like cases: CRLF2 overexpression, was found 35.7% of cases, with 3 harboring a CRLF2 rearrangement and 1 with a concomitant CRLF2 mutation. Furthermore, 13 JAK/STAT pathway mutations - JAK1, n=5; JAK2, n=3; IL7R and CRLF2, n=2 each, JAK3, n=1 - were identified in 33.3% of cases. Finally, RNA-sequencing and/or FISH experiments of the BCR/ABL1-like ALL cases revealed 11 lesions: 5 ABL-class fusion genes (3 NUP214/ABL1, 1 ZC3HAV1/ABL2 and 1 EBF1/PDGFRB), 2 BCR/JAK2, 3 CRLF2-r and 1 DDX3X/USP9X. In order to verify the reproducibility of the predictor, a collaboration was started across Europe with the Institute of Hematology and Blood Transfusion (ÚHKT, Prague), Josep Carreras Leukaemia Research Institute (Barcelona) and Munich Leukemia Laboratory (Munich), through the exchange of material and/or data. The first collaboration was carried out with the ÚHKT and Josep Carreras Leukaemia Research Institute laboratories: 11 cDNA samples (from 1 μg of total RNA), previously studied by our laboratory and classified as BCR/ABL1-like (n=5) and non-BCR/ABL1-like (n=6) ALL were shipped blindly and evaluated for the model in these laboratories. The technical set-up was sent to each laboratory, consisting of experimental procedures, PCR protocol and the threshold for Q-RT-PCR analysis. We received the raw data (i.e. 2^(-ΔCt) values) and uploaded them in the on-line BCR/ABL1-like predictor tool. We had 100% concordance with the ÚHKT and 81.8% with the Josep Carreras Leukaemia Research Institute. The main discrepancies were with 2/5 BCR/ABL1-like cases resulted as non-BCR/ABL1-like in the external laboratory: 1 was a p210 BCR/ABL1-positive case, used as control, which proved borderline, and the second was a BCR/ABL1-like case that, on the other hand, was not studied for the genetic features. A further collaboration was carried out with the MLL laboratory: we received 12 RNA samples - already analyzed by NGS and FISH - for evaluation by the BCR/ABL1-like predictor. The BCR-ABL1-like predictor classified 5 cases as BCR/ABL1-like and 7 as non-BCR-ABL1-like: all 5 BCR/ABL1-like carried a typical signature, consisting of a CRLF2 rearrangement plus JAK/STAT pathway mutations. Discordant cases were represented by 2 non-BCR-ABL1-like cases: one had a CRLF2 rearrangement plus JAK/STAT mutations and the other an ABL1 rearrangement. It is important to underline that the latter case had a borderline score (-0.482). Thus, the concordance rate was 83.3%. Conclusions This study shows that the BCR/ABL1-like predictor is a valid and reproducible tool across laboratories to identify rapidly these cases, with the goal of introducing genetic-driven therapeutic approaches upfront. One critical aspect is represented by borderline cases: in these patients, NGS experiments are required to define the underlying genomic lesion. Further investigations are currently underway. Disclosures Chiaretti: Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees. Machova:Novartis: Consultancy; Incyte: Consultancy; BMS: Consultancy, Research Funding. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Foà:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau.

Chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor (BCR) belonging to subset #1 (IGHV1-5-7/ IGKV1-39) display a poor outcome. To characterize their genetic and genomic features and BCR function, we selected 20 subset #1 CLL from a series of 579 cases. Subset #1 CLL, all showing unmutated IGHV, were associated with the presence of del(11q) (50%) in comparison with unmutated CLL, unmutated stereotyped CLL other than subset #1 and with cases using the same IGHV genes but a heterogeneous VH CDR3 (non-subset #1 CLL). There were no distinctive features regarding CD38, ZAP-70, and TP53 disruption. NOTCH1, SF3B1, and BIRC3 were mutated in 15%, 0%, and 5% of cases, respectively, while BIRC3 was deleted in 22% of cases. Microarray unsupervised analysis on 80 unmutated/mutated/stereotyped/non-stereotyped CLL showed a tight clustering of subset #1 cases. Their genomic signature exhibited several differentially expressed transcripts involved in BCR signal transduction, apoptosis regulation, cell proliferation, and oxidative processes, regardless of del(11q). Accordingly, BCR ligation with anti-IgM revealed a significant higher proliferation of subset #1 versus unmutated non-subset #1 CLL, both at baseline and after 24–48 hr stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function, and patients prognosis.

Gene profile and functional changes upon IgD cross-linking were evaluated in chronic lymphocytic leukemia (CLL). Microarrays highlighted responsiveness to IgD in all cases, independently of clinico-biological characteristics. Stimulated samples exhibited the down-regulation of transcripts of B-cell receptor signaling and cell-adhesion at 24h and the up-modulation of differentiation and apoptosis genes at 48 h. A significant increase in apoptosis upon ligation was also documented. Furthermore, comparison between IgD and IgM stimulation displayed a differential transcriptional/functional response. In conclusion, CLL respond to IgD displaying expression changes and cell-death enhancement, indicating the apoptosis induction via-IgD as an alternative approach for CLL management.

Next-generation whole-exome sequencing has revealed two novel genes, namely NOTCH1 and SF3B1, whose mutations predict poor outcome and preferentially associate with chemorefractory chronic lymphocytic leukemia (CLL). Analysis of 539 CLL cases documents that NOTCH1 mutations i) represent one of the most frequent cancer gene mutations involved at presentation; ii) cluster with cases harboring trisomy 12 and tend to be mutually exclusive with TP53 disruption among genetic subgroups; iii) identify high-risk patients showing poor survival similar to that associated with TP53 abnormalities; and iv) exert a prognostic role independent of widely accepted clinical and genetic risk factors. Mutations of SF3B1, a splicing factor that is a critical component of the spliceosome, recurrently associate with fludarabine-refractory CLL, occur at a low rate at CLL presentation and have a minor role in Richter transformation, corroborating the notion that CLL histological shift is molecularly distinct from chemorefractory progression without the Richter transformation.

Nodal marginal zone lymphoma (NMZL) is a rare, indolent B-cell tumor that is distinguished from splenic marginal zone lymphoma (SMZL) by the different pattern of dissemination. NMZL still lacks distinct markers and remains orphan of specific cancer gene lesions. By combining whole-exome sequencing, targeted sequencing of tumor-related genes, whole-transcriptome sequencing, and high-resolution single nucleotide polymorphism array analysis, we aimed at disclosing the pathways that are molecularly deregulated in NMZL and we compare the molecular profile of NMZL with that of SMZL. These analyses identified a distinctive pattern of nonsilent somatic lesions in NMZL. In 35 NMZL patients, 41 genes were found recurrently affected in ≥3 (9%) cases, including highly prevalent molecular lesions of MLL2 (also known as KMT2D; 34%), PTPRD (20%), NOTCH2 (20%), and KLF2 (17%). Mutations of PTPRD, a receptor-type protein tyrosine phosphatase regulating cell growth, were enriched in NMZL across mature B-cell tumors, functionally caused the loss of the phosphatase activity of PTPRD, and were associated with cell-cycle transcriptional program deregulation and increased proliferation index in NMZL. Although NMZL shared with SMZL a common mutation profile, NMZL harbored PTPRD lesions that were otherwise absent in SMZL. Collectively, these findings provide new insights into the genetics of NMZL, identify PTPRD lesions as a novel marker for this lymphoma across mature B-cell tumors, and support the distinction of NMZL as an independent clinicopathologic entity within the current lymphoma classification.

// Monica Messina 1,* , Sabina Chiaretti 1,* , Jiguang Wang 2,* , Anna Lucia Fedullo 1 , Nadia Peragine 1 , Valentina Gianfelici 1 , Alfonso Piciocchi 3 , Fulvia Brugnoletti 1 , Filomena Di Giacomo 4 , Simona Pauselli 1 , Antony B. Holmes 5 , Maria Cristina Puzzolo 1 , Giulia Ceglie 1 , Valerio Apicella 1 , Marco Mancini 1 , Geertruy te Kronnie 6 , Anna Maria Testi 1 , Antonella Vitale 1 , Marco Vignetti 3 , Anna Guarini 1 , Raul Rabadan 2 and Robin Foa 1 1 Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome, Italy 2 Department of Systems Biology, Biomedical Informatics and Center for Computational Biology and Bioinformatics, Columbia University, New York, NY, USA 3 GIMEMA Data Center, Rome, Italy 4 Department of Molecular Biotechnology and Health Science, and Center for Experimental Research and Medical Studies (CeRMS), University of Torino, Torino, Italy 5 Institute for Cancer Genetics and The Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA 6 Department of Women’s and Children’s Health, University of Padova, Padova, Italy * These authors have contributed equally to this work Correspondence to: Robin Foa, email: // Keywords : acute lymphoblastic leukemia, next generation sequencing, copy number aberrations, novel prognostic markers, genetic-driven targeted therapy Received : October 23, 2015 Accepted : January 28, 2016 Published : February 12, 2016 Abstract To shed light into the molecular bases of B-lineage acute lymphoblastic leukemia lacking known fusion transcripts, i.e. BCR-ABL1 , ETV6-RUNX1 , E2A-PBX1 , and MLL rearrangements (B-NEG ALL) and the differences between children, adolescents/young adults (AYA) and adults, we analyzed 168 B-NEG ALLs by genome-wide technologies. This approach showed that B-NEG cases carry 10.5 mutations and 9.1 copy-number aberrations/sample. The most frequently mutated druggable pathways were those pertaining to RAS/RTK (26.8%) and JAK/STAT (12.5%) signaling. In particular, FLT3 and JAK/STAT mutations were detected mainly in AYA and adults, while KRAS and NRAS mutations were more frequent in children. RAS/RTK mutations negatively affected the outcome of AYA and adults, but not that of children. Furthermore, adult B-NEG ALL carrying JAK/STAT mutations had a shorter survival. In vitro experiments showed that FLT3 inhibitors reduced significantly the proliferation of FLT3 -mutated primary B-NEG ALL cells. Likewise, PI3K/mTOR inhibitors reduced the proliferation of primary cells harboring RAS and IL7R mutations. These results refine the genetic landscape of B-NEG ALL and suggest that the different distribution of lesions and their prognostic impact might sustain the diverse outcome between children, adults and partly AYA - whose genomic scenario is similar to adults - and open the way to targeted therapeutic strategies.