ALBERT

Description: Introduction. Chronic lymphocytic leukemia (CLL) with 11q deletion has been associated to a poor prognosis, but the clinical course of patients carrying this lesion is variable. This aberration, most often monoallelic, is present in 10-17% of newly diagnosed CLL and in 20-30% of patients with progressive or chemorefractory disease. The minimal deleted region (MDR) (2-3 Mbp) is located on the 11q22.3-q23.1 region and includes ATM. Moreover, 30-40% of 11q- CLL have also an inactivating ATM mutation on the other allele. The deleted region on 11q can also include BIRC3, a gene that is often deleted or mutated in advanced/chemorefractory stages of the disease. Although BIRC3 disruption has been associated to a poor prognosis, its prognostic implications in addition to ATM deletion are not well defined. The aim of this study was to perform a copy number aberration (CNA) and gene sequencing analyses on a cohort of CLL patients with 11q- in order to identify subgroups with potential prognostic relevance based on: i) the inclusion of BIRC3 in the deleted region; ii) the presence of BIRC3 mutation; iii) the presence of other CNAs. Methods. The study has included 55 untreated CLL patients followed at our Institution or enrolled in GIMEMA clinical trials (2003-2013). Genomic DNA was extracted from peripheral blood samples. CNA analysis was performed by genomic hybridization on the CytoScan HD array (Affymetrix), which contains more than 2.6 x 106 markers for copy number analysis and 750.000 SNPs. Data were analyzed using both Partek Genomics Suite and ChAS (Chromosome Analysis Suite, Affymetrix) software. The resulting CNAs were verified by visual examination of the plotted copy number profiles. BIRC3 mutations (exons 6-9) were evaluated by Sanger sequencing. Time to first treatment (TFT) was calculated from the date of diagnosis to the date of first therapy or last follow-up; progression-free survival (PFS) from the date of first therapy to the date of progression, death or last follow-up. Results. Baseline characteristics of the 55 cases were as follows: median age at diagnosis 59 years (range 39-84), male gender in 81.8% of patients, progressive disease in 62%. All patients showed 11q- by FISH (median 80%, range 25-99% of nuclei); germline IGHV were present in 96.4% of cases; TP53 deletion in 1 case and TP53 mutation in none; NOTCH1 mutation in 4/40 cases; SF3B1 mutation in 5/40 (all mutually exclusive with only 1 case having both SF3B1 and BIRC3 mutations). By CytoScan HD array, the size of 11q- was very variable, ranging from 0.36 Mbp to 65.14 Mbp; the MDR was located on 11q22.3 region, encompassing 4 genes (ACAT1, ATM, CUL5, NPAT). BIRC3 was included in the deleted region in 45/55 cases (81.8%) and was mutated in 4/54 (7.5%), being always deleted on the other allele. Beside 11q-, 51 cases (92.7%) showed several additional CNAs (average 4.9, range 1-14 per patient), with 5 recurrent lesions: 2p gain in 11 cases, del4(p15.2) in 6, del19(p13.3) in 6, 8q gain in 5 and del4(q22.1) in 4. BIRC3 deletion was not associated to the number of additional CNAs nor to specific CNA. After a follow-up of 59.6 months (range 7.4-229.7), 40 of 47 evaluable patients have received treatment (median TFT 15.8 months, range 0-167). BIRC3 deleted cases (n=37) showed a TFT not significantly different from WT cases (n=10). Conversely, BIRC3 mutation was associated to a shorter TFT (p 3 CNAs larger than 5 Mb (n=14) or 〉10 CNAs (n=5), or the presence of 2p gain, del4(p15.2), del19(p13.3) or 8q gain. So far, 22 patients have been evaluated for PFS after first-line therapy (median 28.7 months). BIRC3 deleted cases (n=17) were not associated to a shorter PFS compared to WT cases (n=5), in line with the results from Rose-Zerilli et al (Haematologica 2014). Conclusions. Among CLL with 11q-: 1) BIRC3 deletion involves more than 80% of cases, whilst the mutation is rare (7.5%); 2) BIRC3 deletion is not associated to a higher genomic complexity; 3) BIRC3 deletion does not seem to influence TFT or PFS of 11q- CLL; 4) BIRC3 mutation is strongly associated to a short TFT; 5) BIRC3 biallelic lesions can be associated to a marked hyperleucocytosis at diagnosis and immediate need of treatment. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Ibrunitib (IBR) is active in chronic lymphocytic leukemia (CLL) patients (pts) with TP53 aberrations. Few data describing the dynamics of TP53 mutated clones under IBR are available. We analyzed a cohort of 40 treatment-naïve and relapsed CLL pts treated with IBR to investigate the dynamics of clonal and subclonal TP53 mutations (TP53-mut). Methods. Forty pts (Table) underwent a longitudinal TP53 monitoring (117 samples) by ultra-deep sequencing (UDS): 26 received IBR + rituximab (IBR+RTX) in first line as part of the GIMEMA LLC 1114 protocol (IBR exposition: 8 months in 7 pts and 14 months in 19 pts) (cohort 1), while 14 received IBR single agent after a median of 1.5 (range: 1-4) chemo-immunotherapy lines (IBR exposition: 2.1 to 4 years in 12 pts) (cohort 2). Samples were analyzed by UDS on a MiSeq sequencer (Illumina, Inc.) to obtain a 5000X coverage/base. For variant calling, the MiSeq Reporter software and an in-house bioinformatics pipeline were applied. All mutations were checked on the IARC TP53 database and those with a variant allele frequency (VAF)

Description: Introduction. The fludarabine, cyclophosphamide, rituximab (FCR) regimen is associated with high complete response (CR) rates and a negative residual disease status in a significant proportion of cases and is considered the optimal front-line treatment for fit patients with chronic lymphocytic leukemia (CLL). In addition, long-term follow-up of patients treated with FCR at the MD Anderson Cancer Center, in the multicenter German CLL8 study and at Italian institutions indicate that a sizable fraction of patients characterized by a favorable biologic profile remains free from progression in excess of 10 years. FC combined with ofatumumab (FC-O), a human monoclonal antibody which targets an epitope of the CD20 molecule, has also been associated with a high CR rate. The aim of this study was to evaluate whether a double dose of ofatumumab (O2) combined with FC could improve the CR rate in young (≤65 yrs) and fit patients with CLL. Methods. Sixty-one fit CLL patients from 15 Italian institutions were enrolled in this front-line study and treated with the FC-O2 regimen based on the FC schedule (F 25 mg/sqm i.v. d1-3, C 250 mg/sqm i.v. d1-3) combined with 13 doses of O (300 mg i.v. d14; 1000 mg d21 at the first cycle; 1000 mg d1 and d15 at cycles 2-6 and d28 at cycle 6). As infection prophylaxis, patients received bactrim and peg-filgrastim in order to prevent granulocytopenia. CLL diagnosis, treatment requirement and response were assessed according to the 2008 iwCLL guidelines. Minimal residual disease (MRD) was evaluated by flow cytometry in the peripheral blood (PB) and bone marrow (BM), and also by RQ-PCR in flow negative cases. CT scan evaluation was included in the response assessment. Adverse events (AEs) were graded according to the NCI-CTCAE. Results. The median age of patients was 60 years (range 36-65), Binet stages B and C were recorded in 86% of cases, B-symptoms in 21%, increased β2M values in 74% and bulky nodes (≥5 cm) in 10%. An IGVH unmutated status was recorded in 60% of cases, deletion 13q in 37%, no aberrations in 33%, deletion 11q in 14%, trisomy 12 in 12%, 17p deletion and/or TP53 mutation were found in 10% of cases. At present, the median follow-up of patients is 7 months (range 1-20). Response to treatment has been assessed in 29 patients after a median number of 6 courses of treatment (range 2-6). The overall response rate is 90%, with a CR rate of 69% (20 patients). No evidence of MRD was observed by flow cytometry in both PB and BM in 15/20 CR patients (75%). To date, 11 patients with cytometric MRD negative CR have been evaluated by RQ-PCR and no residual disease was detected in 3. Grade 3-4 granulocytopenia was recorded in 4 patients (7%), a severe infection in 4 (7%) and 5 patients (8%) experienced a severe infusion-related reaction during ofatumumab administration. Treatment was discontinued in 8 patients as a result of toxicity (infection, 2 cases; FUO, 1; infusion-related toxicity, 1; autoimmune hemolytic anemia, 1; recurrent granulocytopenia, 1; tachyarrhythmia, 1; non-specified toxicity,1). A non-treatment-related death (traumatic aortic transaction due to a dislocated aortic endoprostheses) has been recorded in a patient after 2 months from treatment discontinuation and 1 showed a disease progression after 4 courses of FC-O2. Conclusions. Taken together, the first analysis of this ongoing front-line study suggests that the combination of FC with an increased dose of ofatumumab is well tolerated with acceptable and no unexpected toxicity. Our preliminary results show that the FC-O2 treatment is associated with a high rate of cytometric MRD-negative CR in young and fit patients with previously untreated CLL. Disclosures Carella: Seattle Genetics Inc.: Research Funding. Foà:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Conventional cytogenetics and molecular analyses allow to stratify acute myeloid leukemia (AML) patients into subgroups with different clinical and prognostic relevance. AMLs with unsuccessful cytogenetics and no known recurrent mutations are a subgroup of cases for which information on possible underlying genetic lesions of the leukemic cells is lacking and with a poorly defined outcome. Previous studies have quantified the rate of unsuccessful karyotyping in approximately 10% of the analyzed AML samples and it is conceivable that if cases with molecular rearrangements were to be included this figure could be lower. With the aim of investigating the prevalence and impact of cases with an undefined genetic profile (UGP), we studied 437 AML patients - 228 males and 209 females, with a median age of 50 years (range 1-81) - treated on successive intensive chemotherapy protocols at our Institution. Conventional cytogenetic and molecular analyses - RUNX1-RUNX1T1, CBFB-MY11, DEK-NUP214, FLT3-ITD, NPM1, BCR-ABL, MLL-PTD - were performed at diagnosis. According to the genetic alterations, patients were comprehensively stratified into three subgroups: a favorable risk group - t(8;21) RUNX1-RUNX1T1, inv(16) CBFB-MY11, normal karyotype with mutated NPM1 without FLT3-ITD - with a 5-year overall survival (OS) of 65%, an intermediate risk group - normal karyotype with mutated or wild type NPM1 and FLT3 ITD or wild-type NPM1 without FLT3-ITD, t(9;11)(p22;q23), cytogenetic abnormalities not classified as favorable or adverse - with a 5-year OS of 27% and an unfavorable risk group - inv(3)(q21q26) or t(3;3)(q21;q26), t(6;9)(p23;q34), DEK-NUP214, t(v;11)(v;q23), -5 or del(5q), -7, complex karyotype - with a 5-year OS of 11%. Thirty-three patients (7.5%) were identified as having an UGP and their baseline characteristics, as well as clinical outcome, were compared to those of patients with defined molecular and cytogenetic features. Patients with an UGP were older at the onset of the disease than those with a delineated genetic profile (median 55 vs 49 years). In addition, the proportion of UGP cases increased with age, being 3% in patients 50 years. The complete remission (CR) rate for UGP patients (69.6%) was similar to that of intermediate risk patients (71.1%), but inferior to that of patients with a favorable risk profile (90.5%) (p=0.0046) and better than that of unfavorable genetic risk patients (63.7%). After adjusting for age, gender, WBC and platelet count, Hb, marrow blast percentage at diagnosis and treatment, UGP remained an independent factor for lower CR rate with respect to patients with a favorable genetic risk profile. The frequency of relapse was significantly higher in patients with UGP compared to the favorable risk group (60.8% vs 32%) (p=0.011). In multivariate analysis, the 5-year OS of patients with UGP was significantly worse than that of patients with a favorable genetic risk profile (p

Description: The management of Ph+ acute lymphoblastic leukemia (ALL) patients has profoundly changed during the last decade. Virtually all adult Ph+ ALL patients, including the elderly where the abnormality accounts for over 50% of cases, can obtain a complete remission (CR) with the use of TK inhibitors (and steroids) without systemic chemotherapy (Vignetti M et al. Blood 2007;109:3676; Foà R et al. Blood 2011;118:6521). Most patients remain, however, minimal residual disease (MRD)-positive. The possibility of targeting MRD via an immune-mediated control is particularly appealing in these patients. Previous studies have shown that natural killer (NK) cells with killing activity against autologous blasts may be expanded from ALL patients in CR (Torelli GF et al. Haematologica 2005;90:785). NK cell recognition of malignant targets is regulated by activating and inhibitory receptors. The major receptors with activating functions are NKG2D, DNAM-1 and the natural cytotoxicity receptors (NCRs) (NKp30, NKp44 and NKp46). MIC-A/B and ULBPs are ligands for NKG2D, PVR and Nec-2 for DNAM-1, while NCRs are orphan receptors. The pathways of NK-ALL recognition are unclear. Possible differences in NK cell killing susceptibility and in the expression of NK cell activating ligands among subgroups of ALL patients have been suggested. The aims of this study were: 1) to analyze the pathways of NK-ALL recognition, with particular attention to Ph+ samples; and 2) to verify whether differences in NK cell activating receptor ligand expression among molecularly-defined subgroups of patients correlate with the susceptibility to recognition and killing by NK cells activated and expanded under GMP conditions. PBMCs were collected from 23 healthy donors and 3 adult Ph+ ALL patients in CR. NK cells were enriched and cultured for 14 days in the presence of irradiated autologous feeder cells, autologous plasma, IL-2 and IL-15. The expression of the activating receptors NKG2D, DNAM-1 and NCRs was then analyzed. Samples from 46 newly diagnosed adult ALLs, median age 34 years (18-74), were also investigated: 39 patients had B-ALL - 15 BCR-ABL+, 7 MLL-AF4+, 2 E2A-PBX1+, 15 negative - and 7 had T-ALL. The expression of the NKG2D and DNAM-1 ligands on ALL blasts was analyzed. The cytotoxic activity of ex vivo expanded NK cells against primary ALL blasts was determined in a 51Cr release assay. For blocking experiments, NK cells were pre-treated with the anti-NKG2D or anti-DNAM-1 neutralizing mAbs. NK cells from healthy donors and from Ph+ ALL patients could be expanded respectively 33.2±15.2 and 39.1±19.3 fold. Expanded NK cells were represented by a homogenous population displaying a high expression of CD56 and CD16, in the absence of CD3. DNAM-1, NKG2D, NKp30 and NKp44 activating receptors presented a significantly increased expression after expansion from healthy donors (DNAM-1 p=.0007; NKG2D p=.0004; NKp30 p=.05; NKp44 p=.001). DNAM-1 and NKG2D showed a significantly increased expression after expansion also from Ph+ ALL patients (DNAM-1 p=.0012; NKG2D p=.045), while the expression of NCRs was not tested on these samples. The phenotypic analysis performed within molecularly-defined subgroups of ALL revealed that Ph+ cases presented an overall high surface expression of NKG2D and DNAM1 ligands. In particular, when compared to ALLs carrying no known molecular markers, Ph+ samples showed significantly higher levels of ULBP-1, ULBP-3 and MIC-B (p=.008, p=.026 and p=.033, respectively). In line with the phenotypic results, primary blasts from Ph+ ALL (n=5) appeared significantly more susceptible to NK-dependent lysis than B-ALL without molecular aberrations (n=6) (p=.007). When cytotoxic assays were performed in the presence of neutralizing mAbs, the NK cell killing potential was significantly inhibited by anti-DNAM-1 (p=.006), suggesting a pathway of recognition of ALL blast cells in the setting of the Nec-2/DNAM-1 interaction. The high expression of ligands for activating receptors in Ph+ ALL cases, together with the highest levels of susceptibility to NK cell-mediated lysis by this subgroup of ALL, point to a possible therapeutic use of autologous NK cells activated and expanded ex vivo. Management of Ph+ ALL patients - particularly the elderly - with TK inhibitors plus an immune-based strategy aimed at controlling/eradicating MRD without the use of systemic chemotherapy and/or transplant program is worthy of being investigated. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4373 In chronic lymphocytic leukemia (CLL), the distribution and prognostic impact of genetic and molecular markers have been mostly investigated in retrospective studies including patients in different phases of the disease. From November 2002 to May 2009, we prospectively assessed the clinical and biologic features of 219 young CLL patients ('65 years), distributed in three different cohorts: 124 cases at diagnosis, of which 99 in Binet stage A (group 1), 68 at first progression (group 2), 27 at progression after one or more therapies (group 3). Median age was 52 years for group 1, 57 years for group 2 and 61 years for group 3. Advanced disease defined as Rai stage III/IV was present in 6%, 19% and 48% patients and as Binet stages B/C in 19%, 68% and 78% patients in the 3 cohorts, respectively. The proportion of unmutated IgVH cases progressively and significantly increased, being 36% in group 1, 48.5% in group 2 and 75% in group 3 (p 0.001). The same trend was found for CD38 (p 0.01). The incidence of ZAP-70 expression and p53 mutations showed a trend towards a progressive increase among the three groups (41%, 55% and 56.5% for ZAP-70 and 4%, 7% and 12.5% for p53 mutations), without however reaching significance. The incidence of del(17p) ≥20% raised progressively from group 1, to groups 2 and 3 (2%, 4.5% and 18.5%, respectively; p 0.001). The incidence of del(11q) increased from group 1 to 2 (10% and 16%; p 0.18), though it was not higher in group 3 (7.5%). Focusing the comparison to groups 1 and 2 only, i.e. patients with CLL at diagnosis in all stages and patients at first progression of the disease requiring therapy, the distribution of prognostic markers did not differ significantly, except for a lower proportion of cases expressing CD38, although the proportion of del(17p) 〉20% and 〉10% and of +12 doubled. When the analysis was restricted to stage A CLL at diagnosis, there was a highly significant difference in the lower proportion of unmutated IgVH (0.003), CD38 expression (p 0.009), ZAP-70 expression (p 0.004), del(17p) (p 0.034) and in the higher proportion of del(13q) (p 0.028) in comparison with CLL at first progression. No patient with stage A CLL at diagnosis showed del(17p) ≥20%, whilst 2% showed del(17p) 〉10% and 3% harbored p53 mutations. Of the 40 patients evaluated by FISH at multiple time points, 35% showed clonal evolution. Only previous treatment was significantly associated with the development of clonal evolution, whilst no correlation with the IgVH mutational status, ZAP-70 or CD38 expression was found. Patients of group 1 required treatment after a median time of 49.6 months from diagnosis. Treatment-free interval (TFI) was significantly shorter in cases with IgVH unmutated vs. mutated (at 48 months, 16.5% and 68.3%, respectively p

Description: The CD15 antigen is an adhesion molecule normally expressed on neutrophils that mediates phagocytosis and chemiotaxis: it is also expressed on blasts of patients with acute myeloid leukemia (AML). Its prognostic role has been tested in different studies, including or not acute promyelocytic leukemia (APL), with conflicting results and its significance still remains unclear. To address this issue, a cohort of 460 AML patients of all ages with, the exclusion of APL, [M/F 243/217, median age 50.6 years (range 0.9 – 81.2)] intensively treated at our Institute between 1/1999 and 12/2010 was retrospectively evaluated. Overall, 61 patients (13.3%) evolved from a documented myelodysplastic phase (MDS): AML-ETO, CBFβ-MYH11, FLT3-ITD and NPM were positive in 35/438 (8.2%), 30/427 (7.0%), 55/409 (13.4%) and 67/200 (14.6%) evaluable patients, respectively. A favorable karyotype was found in 90/436 patients (20.6%) while an unfavorable profile was documented in 60/436 cases (13.8%). CD15 positivity was found in 171/406 evaluable patients (42.1%): in particular, CD15 was positive in 13/42 evaluable patients evolved from MDS (31.0%) compared with 158/364 evaluable patients without previous MDS (43.4%) (p=0.123). Induction treatments consisted of anthracycline (ACY) + cytarabine (Ara-C) +/- etoposide in 448 patients and of a fludarabine-based regimen in 12 patients. A complete remission (CR) was achieved by 334 patients (72.6%), while 82 (17.8%) were resistant and 44 (9.6%) died during induction: the median CR duration was 15.5 months (range 0.6 – 176.0), with a 2-year disease-free survival (DFS) rate of 45.1% (95% CI 39.6 – 50.6). The median overall survival (OS) was 14.4 months (range 0.3 – 177.0), with a 2-year OS rate of 42.2% (95% CI 37.5 – 46.9). Among the several variables tested at univariate analysis for CR achievement, age

Description: Conflicting results have been reported regarding the correlation between CD133, a surface marker of immature progenitors, and outcome in acute myeloid leukemia (AML). The expression of this antigen has also been reported in myelodysplastic syndromes (MDS), in particular in high-risk diseases, but always in small cohorts of patients and without a focus on the prognostic role of this antigen. Aim of our study was to establish a clinico-biologic correlation between CD133 expression and disease features at baseline in a large series of AML patients of different ages, with particular regard to the older age.Seven hundred AML patients consecutively diagnosed at a single Institution were retrospectively analyzed and enrolled in this study. There were 395 males and 305 females, with a median age of 54 years (range 1.1-90.4). A previous MDS phase was recognized in 124 patients. Several clinical and biologic features were recorded at baseline and retrospectively collected, such as age, gender, FAB and WHO morphologic classification, cytogenetic analysis, molecular alterations, hematologic parameters (Hb, platelet and WBC count), response to treatment and outcome. Overall, 157 patients expressed CD133. This first analysis was carried out on the older patient population (≥65 years) on the basis of the CD117 positivity. Seventy-three older patients expressed CD133 at baseline, whereas 36 patients were CD117+CD133-. Comparison between the two groups showed a significant prevalence of a previously recognized MDS phase in CD133+ patients (27% vs 13%, p=0.01), higher incidence of a complex karyotype or typical MDS cytogenetic aberrations (trisomy 8, del20q, del5q) (30% vs 8%, p=0.001) and of dysplastic morphologic features detectable in patients without a previous dysplastic identification (63% vs 27%, p=0.002). Forty-three patients in the CD133+ group and 21 patients in the CD133- group received intensive chemotherapy: the remission rate was 52% and 64%, respectively (p=0.06). The relapse rate was 25.5% in the CD133+ and 19% in the CD133- group, respectively (p=0.08). No differences were observed with regard to the hematologic parameters at baseline or in overall survival between the two groups. We then assessed the characteristics of cases negative for CD117, but CD133+ (13 patients) that were compared to the entire cohort of cases that were CD117+CD133+ (144 patients): again we found that, independently from the positivity for CD117, CD133 identified patients with a previously reported MDS phase (61% of patients CD117-CD133+), with a higher median age (69 years) and with dysplastic morphologic changes (100% CD117-CD133+). Taken together, our findings strongly suggest that CD133 can identify at diagnosis a previous MDS phase. In particular, the presence of this antigen in the setting of older de novo AML patients should be used to recognize early a subset of patients who, for the associated biologic features, could benefit from the use of hypomethylating agents as first line treatment. Further analyses, aimed at identifying the prognostic role of this antigen in a large cohort of patients treated with azacitidine, are warranted. Disclosures No relevant conflicts of interest to declare.

Description: The development of agents directed specifically against the BCR-ABL tyrosine kinase has opened therapeutic avenues that have profoundly modified the management of diseases which harbor the genetic abnormality that leads to the production of the BCRABL transcript. While the role of tyrosine kinase inhibitors of 1st and 2nd generation in chronic myeloid leukemia (CML) patients is well established, important clinical responses have been documented also in BCR-ABL+ acute lymphoblastic leukemia (ALL), the most relevant genetic ALL subgroup which accounts for about 25% of adult cases and is associated with a dismal prognosis. Following the administration of tyrosine kinase inhibitors alone as induction treatment, complete hematologic remissions have in fact been recorded in virtually all cases. Thus, the possibility of identifying the presence of the BCR-ABL transcript in the shortest possible time and with a simple and broadly utilizable technique would translate into the timely implementation of a targeted therapy. The availability of a method capable of highlighting the presence of the BCR-ABL protein has important implications because it can document the effective transduction of the molecular transcript; since tyrosine kinase inhibitors are effective on the protein, this would unequivocally support the use of such specific agents in these cases. For this purpose, we have utilized the BCR-ABL Protein Kit* (BD Biosciences, San José, CA) which consists of an immunoassay that identifies qualitatively the presence of the BCR-ABL protein in the lysates of leukemic cell populations. The BCR-ABL fusion protein is captured and detected on Cytometric Bead Array (CBA) and analyzed on a FACSCanto (BD Biosciences) flow cytometer. Lysates from normal mononuclear cells and from the K562 cell line were used as negative and positive controls, respectively. The presence of the BCR-ABL protein was investigated on fresh bone marrow or peripheral blood samples from 56 consecutive cases of acute leukemia aged between 11 and 81 years referred to our Institution. The BCR-ABL protein could be documented in 11 of the 56 cases analyzed. By conventional immunophenotypic analysis they proved to be a B-lineage ALL in 9 cases and a B lymphoid blast crisis of CML in 2. In all 11 cases, the presence of the BCR-ABL transcript was subsequently documented by molecular biology. In the other 45 cases the diagnosis was: B-lineage ALL in 26 (2 of which were ALL/AF4+), T-ALL in 2, acute myeloid leukemia in 22 (5 of which were PML-RARα+) and acute biphenotypic leukemia in 1. None of these latter 45 cases harbored the BCRABL transcript. The assay utilized was successfully completed within a maximum of 4 hours from the marrow or blood collection on 20 × 106 cells containing at least 20% of leukemic cells. The correlation between the presence/absence of the BCR-ABL protein evaluated using the BCR-ABL Protein Kit* and the positivity/negativity of the transcript assessed according to conventional genetic techniques has been absolute. In our laboratory, the assay has shown to be reliable, reproducible and of simple execution. It allows in a very short time to identify the presence of the BCR-ABL protein – both p190 and p210 – through a flow cytometric analysis and thus to reliably offer proteomic information to investigators researching targeted anti-tyrosine kinase treatment.

Description: Fixed-duration treatment with venetoclax (Ven), a highly selective Bcl-2 inhibitor combined with an anti-CD20 monoclonal antibody, showed high efficacy inducing high rates of deep responses with undetectable minimal residual disease (uMRD) in patients with previously treated and untreated chronic lymphocytic leukemia (CLL). The efficacy and safety of the Ven and rituximab (VenR) combination have been investigated in a multicenter, prospective study of the GIMEMA group that included young patients with previously untreated CLL (LLC 1518, VERITAS, NCT03455517). The primary endpoint of this study was the CR rate assessed according to the iwCLL criteria. Inclusion criteria were: treatment requirement per iwCLL criteria, age ≤65 years, cumulative Illness rating scale score ≤6, creatinine clearance ≥30 mL/min, and an unfavorable biologic profile with IGHV unmutated and or TP53 disruption. Treatment consisted of the Ven dose ramp-up (from 20 to 400 mg daily, during 5-weeks) followed by Ven 400 mg daily, combined with R for six 28-day courses (375 mg/m2, course 1; 500 mg/m2, courses 2-6). Patients continued with Ven single agent, 400 mg daily, until month 13. Tumor lysis syndrome (TLS) prophylaxis measures included hydration, allopurinol, or rasburicase. All patients received PneumocystisJirovecii prophylaxis. G-CSF was given in patients with recurrent and severe granulocytopenia. Adverse events (AEs) were graded according to the CTCAE criteria v.5, TLS events were classified according to Howard's criteria. Response was assessed at months 7 and 15 and included clinical examination, PB evaluation, BM aspirate, BM biopsy, and CT scan. MRD was checked centrally in the PB and BM by a 6/4-color flow-cytometry assay with a sensitivity of at least 10-4 according to the internationally standardized European Research Initiative on CLL. Quantitative MRD results assessed by flow-cytometry were categorized as uMRD (uMRD4;