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Objectives: Detectable minimal residual disease (MRD) after therapy for acute lymphoblastic leukemia (ALL) is the strongest predictor of hematologic relapse. This study evaluated outcomes of patients with B-cell precursor ALL with MRD of ≥10−4 Methods: Study population was from ALL study groups in Europe managed in national study protocols 2000–2014. MRD was measured by polymerase chain reaction or flow cytometry. Patients were age ≥15 years at initial ALL diagnosis. Patients were excluded if exposed to blinatumomab within 18 months of baseline or prior alloHSCT. Results: Of 272 patients in CR1, baseline MRD was ≥10−1, 10−2 to −1, 10−3 to −2, and 10−4 to −3 in 15 (6%), 71 (26%), 109 (40%), and 77 (28%) patients, respectively. Median duration of complete remission (DoR) was 18.5 months (95% confidence interval [CI], 11.9–27.2), median relapse-free survival (RFS) was 12.4 months (95% CI, 10.0–19.0) and median overall survival (OS) was 32.5 months (95% CI, 23.6–48.0). Lower baseline MRD level (P ≤ .0003) and white blood cell count P ≤ .0053) were strong predictors for better RFS and DoR. Allogeneic hematopoietic stem cell transplantation (alloHSCT) was associated with longer RFS (hazard ratio [HR], 0.59; 95% CI, 0.41–0.84) and DoR (HR, 0.43; 95% CI, 0.29–0.64); the association with OS was not significant (HR, 0.72; 95% CI, 0.50–1.05). Discussion: In conclusion, RFS, DoR, and OS are relatively short in patients with MRD-positive ALL, particularly at higher MRD levels. AlloHSCT may improve survival but has limitations. Alternative approaches are needed to improve outcomes in MRD-positive ALL.

The introduction and the extended clinical use of nilotinib in the first-line treatment of chronic myeloid leukemia have been based on company-sponsored trials. Independent confirmations are extremely important. We report an investigator-sponsored study of nilotinib 300 mg twice daily in 130 chronic myeloid leukemia patients in early chronic phase. A deep molecular response was achieved in 46% (MR4.0) and 17% (MR4.5) of patients at 2 years; 58% of the enrolled patients achieved a MR4.0 at least once, with a sustained MR4.0 in 52% of them. With a median observation of 29 months (range 24–37 months), 77% of patients were still on treatment with nilotinib. The reasons for permanent discontinuation were: 3% progression, 5% failure or suboptimal response, 8% adverse events, 1% treatment-free remission, and 5% other reasons. Thirteen thrombotic arterial events were reported in 12 patients. A prospective evaluation of metabolic effects showed an increase of fasting glucose without significant variations of glycated hemoglobin, an increase of total cholesterol (both low density lipoprotein and high density lipoprotein fractions) and a decrease of triglycerides. This study confirms a high and rapid efficacy of nilotinib 300 mg twice daily and provides detailed information on the type and incidence of non-hematologic and metabolic adverse events (clinicaltrials.gov identifier: 01535391).

Pegfilgrastim has equivalent efficacy to daily G-CSF in enhancing neutrophil recovery after chemotherapy, but conclusive data concerning its use for peripheral blood stem cell (PBSC) mobilization are lacking. From 2003 to 2008 we used high-dose chemotherapy in 64 lymphoma patients. At mobilization chemotherapy (ESHAP) the first 26 patients used unconjugated G-CSF, while the remaining 38 patients received Pegfilgrastim. At the time of harvest 25 patients collected stem cells after the use of G-CSF and 36 in the Peg group. No statistical by significant differences were observed in median peripheral CD34+ cells mobilized (77 μL versus 71 μL) and in collected PBSC (12.3 × 10(6)/kg versus 9.4 × 10(6)/kg p = 0.76). In the PEG group all patients collected the target PBSC with a single apheresis with a greater proportion of "optimal" mobilizers (83% versus 64%; p = 0.05). In conclusion a single dose of Pegfilgrastim could be a valid alternative to unconjugated G-CSF to mobilize PBSC in lymphoma patients.

In 2004, we reported the short-term results of a multicentric, phase 2 study of imatinib 400 mg daily and pegylated interferon-alpha in the treatment of 76 early chronic phase Philadelphia-positive chronic myeloid leukemia patients. In this report, we update the results with an observation time of five years. After two years of treatment, all but 10 patients (13%) had discontinued pegylated interferon-alpha. The complete cytogenetic response rate at five years was 87%, and 94% of complete cytogenetic responders maintained the complete cytogenetic response after five years. All but one complete cytogenetic response also achieved a major molecular response. These data confirm the excellent response to imatinib front-line and the stability of the complete cytogenetic response. Any possible additional benefit of the combination with interferon-alpha remains uncertain, due to low patient compliance.

Background. The salivary gland is one of the most common sites involved by nongastric, extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT). A large series of patients with long-term follow-up has not been documented. This multicenter, international study sought to characterize the clinical characteristics, treatment, and natural history of salivary gland MALT lymphoma. Methods. Patients with biopsy-confirmed salivary gland MALT lymphoma were identified from multiple international sites. Risk factors, treatment, and long-term outcomes were evaluated. Results. A total of 247 patients were evaluated; 76% presented with limited-stage disease. There was a history of autoimmune disorder in 41%, with Sjögren disease being the most common (83%). Fifty-seven percent of patients were initially treated with local therapy with surgery, radiation, or both; 37 of patients were treated with systemic therapy initially, with 47% of those receiving rituximab; and 6% of patients were observed. The median overall survival (OS) was 18.3 years. The median progression-free survival (PFS) following primary therapy was 9.3 years. There was no difference in the outcomes between patients receiving local or systemic therapy in first-line management. On multivariate analysis, age Conclusion. Salivary gland MALT lymphoma has an excellent prognosis regardless of initial treatment, and patients with Sjögren disease have improved survival. Risks for long-term complications must be weighed when determining initial therapy. Implications for Practice: Patients with salivary gland extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) have an excellent prognosis, particularly those with associated Sjögren's disease. A wide range of available therapies may provide similar durable rates of disease control and survival. Therefore, an important goal in selection of therapy should be to minimize morbidity from treatment. When determining initial therapy for these patients, practitioners should consider the potential side effects and long-term toxicities of treatment.

Myelofibrosis (MF) is a clonal disorder of the hemopoietic stem/progenitor cells (HSPCs). Together with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of MF pathogenesis. It is argued that the up-regulated production of pro-inflammatory cytokines selects for the malignant clone. However, the key players linking inflammation and cancer in MF are still poorly known. Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived HSPCs. Plasma TIMP-1, TNF-α and IL-1β were measured by ELISA assay. Circulating CD34+ cells from MF patients (JAK2V617F (10 cases) and CALR (10 cases) mutations) or cord blood (CB; 8 samples) were functionally characterized after incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), cell cycle and clonogenic capacity. Migration was assessed towards a C-X-C motif chemokine 12 (CXCL12) gradient in the presence or absence of the pro-inflammatory factors. MF-derived CD34+ cells were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence of the pro-inflammatory factors and then evaluated for survival. Regardless mutation status, the plasma levels of IL-1β, TNF-α and TIMP-1 are increased in MF patients and the presence of pro-inflammatory cytokines confers a survival advantage of MF-derived CD34+/CD34+CD38- cells. In addition, MF-derived CD34+ cells promoted cell-cycle progression to the S-phase. Whereas in the JAK2V617F mutated group, the addition of IL-1β or TNF-α ± TIMP-1 impaired the erythroid clonogenic output of the CALR mutated patients. Of note, IL-1β + TNF-α ± TIMP-1 highly promoted the in vitro migration of MF-derived CD34+ cells in the presence of CXCL12. Intriguingly, MF-derived CD34+ cells revealed increased clonogenic ability after migration toward CXCL12 and IL-1β + TNF-α ± TIMP-1, suggesting a pivotal role of inflammation in the selection of the malignant clone. Finally, co-cultures of normal MSCs with MF-derived CD34+ cells sustained the survival effects of IL-1β+TNF-α±TIMP-1. Overall, our data indicate that the behavior of the MF-derived HSPCs can be influenced by regulatory signals provided by the microenvironment through the cooperation between various pro-inflammatory factors. The interplay of these pro-inflammatory factors promotes and selects the circulating MF-derived HSPCs with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach. Citation Format: Daria Sollazzo, Dorian Forte, Nicola Polverelli, Marco Romano, Margherita Perricone, Sofia Fatica, Francesco Lapi, Emanuela Ottaviani, Martinelli Giovanni, Nicola Vianelli, Michele Cavo, Maria Pantaleo, Francesca Palandri, Lucia Catani. Crucial factors of the inflammatory microenvironment (IL-1β/TNF-α/TIMP-1) promote the selection of highly malignant hemopoietic clone of myelofibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4077.

Additional chromosomal abnormalities (ACAs) in Philadelphia-positive cells have been reported in ∼ 5% of patients with newly diagnosed chronic myeloid leukemia (CML) in chronic phase (CP). Few studies addressing the prognostic significance of baseline ACAs in patients treated with imatinib have been published previously. The European LeukemiaNet recommendations suggest that the presence of ACAs at diagnosis is a “warning” for patients in early CP, but there is not much information about their outcome after therapy with tyrosine kinase inhibitors. To investigate the role of ACAs in early CP CML patients treated with imatinib mesylate, we performed an analysis in a large series of 559 patients enrolled in 3 prospective trials of the Gruppo Italiano Malattie Ematologiche dell'Adulto Working Party on CML: 378 patients were evaluable and ACAs occurred in 21 patients (5.6%). The overall cytogenetic and molecular response rates were significantly lower and the time to response was significantly longer in patients with ACAs. The long-term outcome of patients with ACAs was inferior, but the differences were not significant. The prognostic significance of each specific cytogenetic abnormality was not assessable. Therefore, we confirm that ACAs constitute an adverse prognostic factor in CML patients treated with imatinib as frontline therapy. This study was registered with clinicaltrials.gov as NCT00514488 and NCT00510926.

Variant Philadelphia (Ph) chromosome translocations have been reported in 5%-10% of patients with newly diagnosed chronic myeloid leukemia (CML). Variant translocations may involve one or more chromosomes in addition to 9 and 22, and can be generated by 2 different mechanisms, 1-step and 2-step rearrangements, as revealed by fluorescence in situ hybridization. The prognostic significance of the occurrence of variant translocations has been discussed in previous studies. The European LeukemiaNet recommendations do not provide a “warning” for patients with variant translocations, but there is limited information about their outcome after therapy with tyrosine kinase inhibitors. To identify the role of variant translocations in early chronic phase (CP) CML patients treated with imatinib mesylate, we performed an analysis in a large series of 559 patients enrolled in 3 prospective imatinib trials of the Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA) Working Party on CML. Variant translocations occurred in 30 patients (5%). Our data show that the presence of variant translocations has no impact on the cytogenetic and molecular response or on outcome, regardless of the involvement of different mechanisms, the number of involved chromosomes, or the presence of deletions. Therefore, we suggest that patients with variant translocations do not constitute a “warning” category in the imatinib era. This study is registered at www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Purpose Deletions of the derivative chromosome 9 [der(9)] have been associated with a poor prognosis in chronic myeloid leukemia (CML) across different treatment modalities. In the imatinib era, the prognostic impact of der(9) deletions has been evaluated mainly in patients with late chronic-phase (CP) CML, giving partially conflicting results. Few data are available in the early CP setting. For this reason, in 2006, the European LeukemiaNet recommendations still considered der(9) deletions as a candidate adverse prognostic factor and required a careful monitoring of the patient. Patients and Methods To investigate the prognostic value of der(9) deletions in early CP CML, we performed an analysis of three prospective imatinib trials of the Italian Group for Hematological Malignancies of the Adult (GIMEMA) CML Working Party. Results A fluorescent in situ hybridization (FISH) analysis of bone marrow cells was performed at diagnosis; der(9) deletions were detected in 60 (12%) of 521 evaluable patients. At 60 months, the cumulative incidence of complete cytogenetic response and major molecular response—and the probability of event-free survival, failure-free survival, progression-free survival, and overall survival—in patients with and without deletions were not statistically different. Conclusion Our data strongly support the notion that, when investigated by FISH, der(9) deletions are not a poor prognostic factor in patients with early CP CML treated with imatinib.

Contains fulltext : 88340.pdf (Publisher’s version ) (Closed access) The stem cell source for autologous transplantation has shifted from bone marrow to peripheral blood (PB). We previously showed that relapse incidence in patients with acute myelocytic leukemia autografted in first remission (CR1) was greater with PB than bone marrow, and a poorer outcome was associated with a shorter CR1 to PB transplantation interval ( 7.16 x 10(6)/kg) as the cutoff point. By multivariate analysis, relapse was more probable in patients who received the highest dose (hazard ratio = 1.48; 95% confidence interval, 1.12-1.95; P = .005), and leukemia-free survival was worse (hazard ratio = 0.72; 95% confidence interval, 0.55-0.93; P = .01). In conclusion, in patients autografted in first remission, relapse was higher and leukemia-free survival lower for those who received the highest CD34(+) PB doses.

Imatinib mesylate is considered standard of care for first-line treatment of chronic phase chronic myeloid leukemia (CML-CP). In the phase III, randomized, open-label International Randomized Study of Interferon vs STI571 (IRIS) trial, previously untreated CML-CP patients were randomized to imatinib (n=553) or interferon-alpha (IFN) plus cytarabine (n=553). This 6-year update focuses on patients randomized to receive imatinib as first-line therapy for newly diagnosed CML-CP. During the sixth year of study treatment, there were no reports of disease progression to accelerated phase (AP) or blast crisis (BC). The toxicity profile was unchanged. The cumulative best complete cytogenetic response (CCyR) rate was 82%; 63% of all patients randomized to receive imatinib and still on study treatment showed CCyR at last assessment. The estimated event-free survival at 6 years was 83%, and the estimated rate of freedom from progression to AP and BC was 93%. The estimated overall survival was 88% -- or 95% when only CML-related deaths were considered. This 6-year update of IRIS underscores the efficacy and safety of imatinib as first-line therapy for patients with CML.

Imatinib mesylate has become the treatment of choice for chronic myeloid leukemia (CML): the standard dose for chronic- phase (CP) CML is 400 mg daily. Response rates are different according to Sokal score, being significantly lower in intermediate and high Sokal risk patients. Phase 1 and 2 trials have shown a dose-response effect and high-dose imatinib trials in early CP CML showed better results compared with standard dose. Our study is the first prospective trial planned to evaluate the efficacy and tolerability of high-dose imatinib in previously untreated intermediate Sokal risk CML patients. Seventy-eight patients were treated with 400 mg imatinib twice daily: complete cytogenetic response (CCgR) rates at 12 and 24 months were 88% and 91%; moreover, at 12 and 24 months 56% and 73% of CCgR patients achieved a major molecular response. The incidence of adverse events was slightly higher than reported by the most important standard-dose trials. With a median follow-up of 24 months, 3 patients progressed to advanced phase. In intermediate Sokal risk newly diagnosed CML patients, high-dose imatinib induced rapid and high response rates, apparently faster than those documented in the International Randomized Study of IFN and Imatinib for the same risk category. These clinical trials are registered at www.clinicaltrials.gov as no. NCT00510926.

This phase 3, multicenter, open-label study evaluated the efficacy and safety of tipifarnib compared with best supportive care (BSC), including hydroxyurea, as first-line therapy in elderly patients (≥70 years) with newly diagnosed, de novo, or secondary acute myeloid leukemia. A total of 457 patients were enrolled with 24% 80 years of age or older. Tipifarnib 600 mg orally twice a day was administered for the first 21 consecutive days, in 28-day cycles. The primary endpoint was overall survival. The median survival was 107 days for the tipifarnib arm and 109 days for the BSC arm. The hazard ratio (tipifarnib vs BSC) for overall survival was 1.02 (P value by stratified log-rank test, .843). The complete response rate for tipifarnib in this study (8%) was lower than that observed previously, but with a similar median duration of 8 months. The most frequent grade 3 or 4 adverse events were cytopenias in both arms, slightly more infections (39% vs 33%), and febrile neutropenia (16% vs 10%) seen in the tipifarnib arm. The results of this randomized study showed that tipifarnib treatment did not result in an increased survival compared with BSC, including hydroxyurea. This trial was registered at www.clinicaltrials.gov as #NCT00093990.

Nilotinib has a higher binding affinity and selectivity for BCR-ABL with respect to imatinib and is an effective treatment of chronic myeloid leukemia (CML) after imatinib failure. In a phase 2 study, 73 early chronic-phase, untreated, Ph+ CML patients, received nilotinib at a dose of 400 mg twice daily. The primary endpoint was the complete cytogenetic response (CCgR) rate at 1 year. With a median follow-up of 15 months, the CCgR rate at 1 year was 96%, and the major molecular response rate 85%. Responses were rapid, with 78% CCgR and 52% major molecular response at 3 months. During the first year, the treatment was interrupted at least once in 38 patients (52%). The mean daily dose ranged between 600 and 800 mg in 74% of patients, 400 and 599 mg in 18% of patients, and was less than 400 mg in 8% of patients. Dose interruptions were mainly due to nonhematologic and biochemical side effects. Myelosuppression was irrelevant. One patient progressed to blastic crisis after 6 months; one went off-treatment for lipase increase grade 4 (no pancreatitis). Nilotinib is safe and very active in early chronic-phase CML. These data support a role for nilotinib for the frontline treatment of CML. This study was registered at ClinicalTrials.gov as NCT00481052.

BACKGROUND: Imatinib mesylate is the first line treatment for chronic myeloid leukemia. The advent of imatinib increased survival significantly in patients in an advanced phase of the disease. However, few long-term data on the outcome of these patients based on large, prospective and controlled trials are available. DESIGN AND METHODS: A phase 2 multicenter trial of the use of imatinib 600 mg/daily in patients with accelerated phase chronic myeloid leukemia was sponsored and promoted by the Italian Cooperative Study Group on Chronic Myeloid Leukemia in 2001. RESULTS: One hundred and eleven patients were enrolled; the median follow-up of the 41 living patients is 82 months (range, 73-87). One hundred and seven patients (96%) returned to chronic phase and 79 patients (71%) achieved a complete hematologic response. Cumulative best rates of major cytogenetic response and complete cytogenetic response were 30% and 21%, respectively. All responses were maintained for a minimum of 4 weeks. At last follow-up, four patients were alive in complete remission after allogeneic transplant, 16 patients (14%) had switched to a second generation tyrosine kinase inhibitor and 21 patients (19%) were alive on imatinib therapy. No late toxicities were observed. Progression-free survival and event-free survival rates were 36.5% and 15%, respectively, at 7 years. The median survival time was 37 months, and was significantly associated with the achievement of a complete hematologic response or a complete cytogenetic response. CONCLUSIONS: Imatinib may induce durable responses, associated with prolonged survival, in patients with accelerated phase chronic myeloid leukemia.

Purpose Imatinib mesylate (IM) has rapidly become the front-line treatment of Philadelphia-positive (Ph-pos) chronic myeloid leukemia, but the number of patients who were treated and are being treated with IM second-line is still substantial. Patients and Methods We have monitored and analyzed the cytogenetic and molecular response to IM 400 mg/d in a cohort of 277 late chronic phase (LCP) patients who were resistant or intolerant to interferon-α and were observed for 48 to 79 months (median, 72 months). Results One hundred fifty-three patients (55%) achieved a complete cytogenetic response (CCgR). Seventy-seven percent of them were still in CCgR after 5 years. The rate of response loss did not increase over time. The 6-year progression-free survival and overall survival of these 153 complete cytogenetic responders were 90% and 91%, respectively. Molecular response was less than major in 21%, major in 78%, and complete in one patient only. Conclusion These data confirm that, in LCP the CCgR rate to IM is 50% to 60%, and show that CCgR is stable and is associated with a prolonged survival, even if leukemia continues to be molecularly detectable.

To assess the effect of age on response and compliance to treatment in patients with chronic myeloid leukemia (CML) we performed a sub-analysis within a phase II trial of the GIMEMA CML Working Party (CML/002/STI571). Since the WHO cut-off age to define an older patient is 65 years, among the 284 patients considered, we identified 226 (80%) younger patients (below 65 years) and 58 (20%) older patients (above 65 years) before starting imatinib. Response rates (hematologic and cytogenetic) were lower in the older age group but the probabilities of progression-free survival and overall survival (median observation time 3 years) were the same. Moreover, among complete cytogenetic responders, no differences were found in the level of molecular response between the two age groups. As might be expected, older patients experienced more adverse events, both hematologic and non-hematologic: this worsened compliance did not, however, prevent a long-term outcome similar to that of younger patients.

Purpose: ABL kinase domain mutations have been implicated in the resistance to the BCR-ABL inhibitor imatinib mesylate of Philadelphia-positive (Ph+) leukemia patients. Experimental Design: Using denaturing high-performance liquid chromatography and sequencing, we screened for ABL kinase domain mutations in 370 Ph+ patients with evidence of hematologic or cytogenetic resistance to imatinib. Results: Mutations were found in 127 of 297 (43%) evaluable patients. Mutations were found in 27% of chronic-phase patients (14% treated with imatinib frontline; 31% treated with imatinib post-IFN failure), 52% of accelerated-phase patients, 75% of myeloid blast crisis patients, and 83% of lymphoid blast crisis/Ph+ acute lymphoblastic leukemia (ALL) patients. Mutations were associated in 30% of patients with primary resistance (44% hematologic and 28% cytogenetic) and in 57% of patients with acquired resistance (23% patients who lost cytogenetic response; 55% patients who lost hematologic response; and 87% patients who progressed to accelerated phase/blast crisis). P-loop and T315I mutations were particularly frequent in advanced-phase chronic myeloid leukemia and Ph+ ALL patients, and often accompanied progression from chronic phase to accelerated phase/blast crisis. Conclusions: We conclude that (a) amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) account for 85% of all resistance-associated mutations; (b) the search for mutations is important both in case of imatinib failure and in case of loss of response at the hematologic or cytogenetic level; (c) advanced-phase chronic myeloid leukemia and Ph+ ALL patients have a higher likelihood of developing imatinib-resistant mutations; and (d) the presence of either P-loop or T315I mutations in imatinib-treated patients should warn the clinician to reconsider the therapeutic strategy.

Purpose Point mutations within the ABL kinase domain of the BCR-ABL gene have been associated with clinical resistance to imatinib mesylate in chronic myeloid leukemia (CML) patients. To shed further light on the frequency, distribution, and prognostic significance of ABL mutations, we retrospectively analyzed a homogeneous cohort of late chronic phase CML patients who showed primary cytogenetic resistance to imatinib. Patients and Methods Using denaturing high-performance liquid chromatography (D-HPLC) and sequencing, we screened for ABL mutations in a total of 178 bone marrow and/or peripheral blood samples from 40 late chronic phase CML patients homogeneously treated with imatinib 400 mg/d, who did not reach a major cytogenetic response at 12 months. Results Mutations were found in 19 of 40 patients (48%). Mutations were already detectable by D-HPLC at a median of 3 months from the onset of therapy. The presence of a missense mutation was significantly associated with a greater likelihood of subsequent progression to accelerated phase/blast crisis (P = .0002) and shorter survival (P = .001). Patients carrying mutations falling within the P-loop seemed to have a particularly poor outcome in terms of time to progression (P = .032) and survival (P = .045). Conclusion Our results show that, irrespective of the hematologic response, monitoring for emerging mutations in the first months of therapy may play a role in detecting patients with worse prognosis, for whom a revision of the therapeutic strategy should be considered.

Since interferon-α and imatinib (IM; STI571, Glivec, Gleevec) are effective for the treatment of chronic myeloid leukemia (CML), and their mechanisms of action are different, we designed an exploratory study investigating the effects of a standard IM dose (400 mg/d) and a variable pegylated interferon-α (PegIFN) dose (50 μg/wk, 100 μg/wk, and 150 μg/wk). The criteria for dose adjustment were designed so as to ensure the delivery of the IM dose and to protect life quality. There were 76 patients with previously untreated Philadelphia (Ph)–positive CML enrolled in the study. There were 3 patients who discontinued IM and 45 patients who discontinued PegIFN. The severity of adverse events increased with increasing PegIFN dose. The IM dose could be administered to the patients who were assigned to receive 50 μg/wk or 100 μg/wk PegIFN but not to those who were assigned to receive 150 μg/wk. The median administered dose of PegIFN ranged between 32 μg/wk and 36 μg/wk. The cytogenetic response was 70% complete (Ph-neg 100%) and 83% major (Ph-neg > 65%). The BCR/ABL transcript was reduced by at least 3 logs in 68% of complete cytogenetic responders. These data of toxicity, compliance, and efficacy may assist in the design and preparation of prospective studies.

No abstract available

Abstract 4704 Background and aim of the study. Emerging trends emphasize the importance of both innate and adaptive immune system in the response against infections, in the pathogenesis of autoimmune diseases and graft-versus-host disease (GVHD). In the cross-talk between innate and adaptive immune system, pattern recognition receptors such as Toll-like receptors (TLRs) play a key role. TLRs recognize common protein, carbohydrate or DNA/RNA pattern motifs leading to signaling for cytokine production and T cell and dendritic cell maturation. These receptors may act as tuner of inflammatory and immunologic reactions. Very little is known about the expression and the function of TLRs in vivo in patients who underwent to allogeneic stem cell transplantation (SCT). The aim of this study was to evaluate the expression and the function of TLRs on lymphocytes and monocytes in relation to infection (CMV and HHV-6, especially) and the onset of GVHD. Patients and methods. The expression of TLRs on T cells and monocytes was analyzed by flow cytometry at day +30, +90, +180 after SCT and at the onset of GVHD. The expression of receptors for lipid-based pathogen-associated molecular patterns (PAMPs: TLR 1,2,4 and 6 surface receptors), receptors for nucleic acid based PAMPs (TLR 3,7,8 and 9 located in cytoplasmic compartments), TLR5 and, TLR10 (surface receptors) was evaluated as mean fluorescence intensity (MFI). Ex vivo induction of cytokines (TNFalpha, MCP1, IFNgamma, IL-10) by TLR ligands was analyzed in the cell supernatant by ELISA. Since the beginning of the study, we have analyzed data of 12 healthy donors and 14 patients. Median age was 46 years (range, 25–64) and 7 patients were male. Results. Acute GVHD developed in 7 patients. Patients without acute GVHD after SCT and healthy donors showed different MFI of TLR3 on T cells (5,8±1,4 vs 4,2±1,05 p=0,02), of TLR4 on monocytes (26,1±1,01 vs 15,8±4,9 p=0,004), and of TLR6 on T-lymphocytes (7,3±3,2 vs 4,6±1,1 p=0,02) and monocytes (27±12,1 vs 14,9±4,6 p=0,01). TLR3 expression was significantly decreased on T-lymphocytes and monocytes in patients with acute GVHD in comparison to those without GVHD (4,06±0,8 vs 5,8±1,4 p=0,02; 9,3±7,2 vs 38,02±30 p=0,04). The levels of TLR5 on T cells and monocytes were higher in patients with acute GVHD compared to healthy donors (8,4±2,1 vs 6,4±1,6 p=0,04; 54,2±20,2 vs 33,2±16,5 p=0,04). An increased induction of IFNgamma upon TLR1 ligand activation was observed in patients without GVHD in comparison to healthy donors and patients with GVHD (p=0,04). TLR6 ligand induced significantly the production of IFN gamma in patients with GVHD in comparison to controls and the other patients (p=0,03). Patients without GVHD showed a trend toward a decreased induction of MCP1 upon TLR4 ligand activation (p=0,07). The rate of infections (especially CMV reactivation), clinical and transplant characteristic were not significantly different between patients with and without GVHD. Conclusion In our study, a different expression profile of TLRs was found in healthy donors, in patients after SCT without acute GVHD and in those with GVHD. These results suggest that the innate immune response via TLRs activation could be involved in the development of GVHD. The assessment of a larger number of patients would be useful to understand the complex interplay between pathogens, self or non-self DNA and RNA and the immune system after SCT. Disclosures: Off Label Use: In Italy the use of azacitidine for Low-risk Myelodysplastic patients is off label. The use of azacitidine in our study is part of a Phase II clinical trial.

Abstract 3805 Background: Mastocytosis is a rare disease characterized by an abnormal proliferation and accumulation of mast cells (MC) in several organs and tissues such as skin, bone marrow, liver, gastrointestinal tract and lymphnodes. The reported prevalence of mastocytosis (cutaneous or systemic) is lower than 1/50,000. Mastocytosis encompasses a wide range of clinical entities, extremely heterogeneous for symptoms, clinical course and prognosis. The heterogeneity and the complexity of its clinical signs lead to the definition of mastocytosis as a multidisciplinary pathology, involving different specialists such as hematologists, internists, dermatologists, immunologists and pediatricians. Mastocytosis is a MC clonal disease associated to a somatic mutation (D816V) of the proto-oncogene c-kit (KIT), which codifies for the stem cell factor (SCF) receptor. SFC is the main factor stimulating the proliferation, chemotaxis and activation of human mast cells. Different KIT mutations have been found in 15% of patients. Clinical signs and symptoms of mastocytosis mainly depend on the liberation of chemical mediators produced by the mast cells, on the tissue infiltration of the mast cells and on other associated hematological diseases. Aim of the Registry: promoting studies on mastocytosis in Italy aimed at investigating the epidemiology of the disease, its prognostic factors and health technology assessment (HTA) aspects associated to the management of a rare “orphan” disease. Methods: The Italian Mastocytosis Registry was constituted in 2009, with the aim of promoting communication between specialists and collecting data about patients diagnosed with mastocytosis at a national level. Anagraphical, anamnestic, clinical, biological, treatment and follow-up data of patients with mastocytosis are being routinely collected in 15 Italian centers after written informed consent. An on-line database (www.registroitalianomastocitosi.it) has been set up for this purpose. The collected data will allow specialists to: Results: At present, data on 175 patients have been collected. Seventy-nine (45%) have been diagnosed with systemic mastocytosis and 40 (50%) of them progressed from a cutaneous disease. Ninety-four (54%) are females; 81 (46%) are males. Among 49 patients for whom data on familiarity were available, 12 (24%) reported familiar cases of autoimmune diseases (n=3), allergies (n=5) or interestingly mastocytosis (n=4). Sixty-one (35%) patients reported allergies. Of 121 reported lines of therapy, 37 (31%) were described as not specified anti chemical mediators, 51 (42%) anti-H1, 21 (17%) anti-H2, 30 (25%) corticosteroids, 22 (18%) phototherapy, 7 (6%) alpha-interpheron, 8 (7%) chemotherapy and 13 (11%) tyrosine-kinase inhibitors (total exceeds 100% because multiple choice is allowed). As to the histological findings, 82 (47%) patients have data on bone marrow biopsy: 48 (59%) had a positive finding, with a median mast cells infiltrate of 30% (range 3–90%). Among 43 patients tested for tryptase serum level, 41 (95%) had levels above normal values (12.5 ng/ml). Conclusions: This is the first spontaneous observational study on mastocytosis in Italy. The on-line database is a useful tool for data collection at a national level. The Registry is an opportunity to carry out epidemiological studies aimed at estimating occurrence and geographical distribution of the disease. It will also allow specialists to investigate possible prognostic factors and provide a starting point for the research into ad hoc therapies and HTA studies. It will hopefully provide a link with other international registries to improve understanding of this disease. Last but not least, the Italian Registry may support a National Government policy to provide assistance by the Public Health System to patients with mastocytosis. Disclosures: No relevant conflicts of interest to declare.

Tyrosine kinase inhibitors have improved survival for patients with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL). However, prognosis for old or unfit patients remains poor. In the INCB84344-201 (formerly GIMEMA LAL 1811) prospective, multicenter, phase 2 trial, we tested the efficacy and safety of ponatinib plus prednisone in newly diagnosed patients with Ph+ ALL ≥60 years, or unfit for intensive chemotherapy and stem cell transplantation. Forty-four patients received oral ponatinib 45 mg/d for 48 weeks (core phase), with prednisone tapered to 60 mg/m2/d from days-14-29. Prophylactic intrathecal chemotherapy was administered monthly. Median age was 66.5 years (range, 26-85). The primary endpoint (complete hematologic response [CHR] at 24 weeks) was reached in 38/44 patients (86.4%); complete molecular response (CMR) in 18/44 patients (40.9%) at 24 weeks. 61.4% of patients completed the core phase. As of 24 April 2020, median event-free survival was 14.31 months (95% CI 9.30-22.31). Median overall survival and duration of CHR were not reached; median duration of CMR was 11.6 months. Most common treatment-emergent adverse events (TEAEs) were rash (36.4%), asthenia (22.7%), alanine transaminase increase (15.9%), erythema (15.9%), and γ-glutamyltransferase increase (15.9%). Cardiac and vascular TEAEs occurred in 29.5% (grade ≥3, 18.2%) and 27.3% (grade ≥3, 15.9%), respectively. Dose reductions, interruptions, and discontinuations due to TEAEs occurred in 43.2%, 43.2%, and 27.3% of patients, respectively; 5 patients had fatal TEAEs. Ponatinib and prednisone showed efficacy in unfit patients with Ph+ ALL; however, a lower ponatinib dose may be more appropriate in this population. This trial was registered at www.clinicaltrials.gov as #NCT01641107.

The 2017 version of the European LeukemiaNet (ELN) recommendations, by integrating cytogenetics and mutational status of specific genes, divides patients with acute myeloid leukemia into 3 prognostically distinct risk categories: favorable (ELN2017-FR), intermediate (ELN2017-IR), and adverse (ELN2017-AR). We performed a post hoc analysis of the GIMEMA (Gruppo Italiano Malattie EMatologiche dell’Adulto) AML1310 trial to investigate the applicability of the ELN2017 risk stratification to our study population. In this trial, after induction and consolidation, patients in complete remission were to receive an autologous stem cell transplant (auto-SCT) if categorized as favorable risk or an allogeneic stem cell transplant (allo-SCT) if adverse risk. Intermediate-risk patients were to receive auto-SCT or allo-SCT based on the postconsolidation levels of measurable residual disease as measured by using flow cytometry. Risk categorization was originally conducted according to the 2009 National Comprehensive Cancer Network recommendations. Among 500 patients, 445 (89%) were reclassified according to the ELN2017 criteria: ELN2017-FR, 186 (41.8%) of 455; ELN2017-IR, 179 (40.2%) of 445; and ELN2017-AR, 80 (18%) of 455. In 55 patients (11%), ELN2017 was not applicable. Two-year overall survival (OS) was 68.8%, 51.3%, 45.8%, and 42.8% for the ELN2017-FR, ELN2017-IR, ELN2017-not classifiable, and ELN2017-AR groups, respectively (P < .001). When comparing the 2 different transplant strategies in each ELN2017 risk category, a significant benefit of auto-SCT over allo-SCT was observed among ELN2017-FR patients (2-year OS of 83.3% vs 66.7%; P = .0421). The 2 transplant procedures performed almost equally in the ELN2017-IR group (2-year OS of 73.9% vs 70.8%; P = .5552). This post hoc analysis of the GIMEMA AML1310 trial confirms that the ELN2017 classification is able to accurately discriminate patients with different outcomes and who may benefit from different transplant strategies. This trial was registered as EudraCT number 2010-023809-36 and at www.clinicaltrials.gov as #NCT01452646.

Thrombosis and neoplasms are strictly linked, and the diagnosis of a malignancy is a relevant risk factor for venous thromboembolism (VTE). In particular, between gammopathies, the VTE risk is known to be increased in both monoclonal gammopathy of uncertain significance and in multiple myeloma, with a 3- and 9-fold increase respectively, when compared to the general population. The risk appears to be further increased in patients treated with immunomodulating drugs, such as thalidomide, especially when in combination with dexamethasone or conventional cytotoxic chemotherapies, and lenalidomide. In 2008 the International Myeloma Working Group put out thrombosis prophylaxis recommendations for myeloma patients treated with IMiDs. Current recommendations for thromboprophylaxis suggest the use of low-dose acetylsalicylic acid in patients with low risk for thrombosis and therapeutic dose anticoagulation with LMWH or warfarin for high-risk patients. However, these recommendations have been frequently not followed in the clinical practice, due to various reasons that involve the patients' will, the level of evidence of the recommendations and some selection biases in the studies that were taken as basis for writing down the indications. The new direct oral anticoagulants have been preliminarily evaluated for the prophylaxis of thrombotic events in IMiDs-treated myelomas, being promising, even if more expensive. Currently, the most reliable tool for a correct thrombotic risk stratification appears to be the complete clinical and anamnestic evaluation of the myeloma patients added to a strong physician awareness of the evidences that the literature contains until now.

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor cure rate for relapsed/resistant patients. Due to the lack of T-cell restricted targetable antigens, effective immune-therapeutics are not presently available and the treatment of chemo-refractory T-ALL is still an unmet clinical need. To develop novel immune-therapy for T-ALL, we generated an afucosylated monoclonal antibody (mAb) (ahuUMG1) and two different bispecific T-cell engagers (BTCEs) against UMG1, a unique CD43-epitope highly and selectively expressed by T-ALL cells from pediatric and adult patients.MethodsUMG1 expression was assessed by immunohistochemistry (IHC) on a wide panel of normal tissue microarrays (TMAs), and by flow cytometry on healthy peripheral blood/bone marrow-derived cells, on 10 different T-ALL cell lines, and on 110 T-ALL primary patient-derived cells. CD43-UMG1 binding site was defined through a peptide microarray scanning. ahuUMG1 was generated by Genetic Glyco-Engineering technology from a novel humanized mAb directed against UMG1 (huUMG1). BTCEs were generated as IgG1-(scFv)2 constructs with bivalent (2+2) or monovalent (2+1) CD3ε arms. Antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and redirected T-cell cytotoxicity assays were analysed by flow cytometry. In vivo antitumor activity of ahUMG1 and UMG1-BTCEs was investigated in NSG mice against subcutaneous and orthotopic xenografts of human T-ALL.ResultsAmong 110 T-ALL patient-derived samples, 53 (48.1%) stained positive (24% of TI/TII, 82% of TIII and 42.8% of TIV). Importantly, no expression of UMG1-epitope was found in normal tissues/cells, excluding cortical thymocytes and a minority (2 log lower as compared with ahuUMG1, with significant mice survival advantage in different T-ALL models in vivo.ConclusionAltogether our findings, including the safe UMG1-epitope expression profile, provide a framework for the clinical development of these innovative immune-therapeutics for this still orphan disease.

Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.

Simple Summary Gemtuzumab Ozogamicin (GO) is a drug approved for the treatment of acute myeloid leukemia (AML). It targets leukemic cells that express the CD33 molecule on their surface and brings the toxic agent calicheamicin inside the cell to kill it. Several studies have shown that AML patients can benefit of the addition of GO to chemotherapy during induction regimens, pre- and post-transplantation. Moreover, some disease features have been addressed or are under investigation for their capacity to predict response to GO, with the future aim of selecting AML patients that can mostly benefit of GO treatment. Abstract Acute myeloid leukemia (AML) is a complex hematological malignancy characterized by genetic and clinical heterogeneity and high mortality. Despite the recent introduction of novel pharmaceutical agents in hemato-oncology, few advancements have been made in AML for decades. In the last years, the therapeutic options have rapidly changed, with the approval of innovative compounds that provide new opportunities, together with new challenges for clinicians: among them, on 1 September, 2017 the Food and Drug Administration granted approval for Gemtuzumab Ozogamicin (GO) in combination with daunorubicin and cytarabine for the treatment of adult patients affected by newly diagnosed CD33+ AML. Benefits of GO-based regimens were also reported in the pre- and post-transplantation settings. Moreover, several biomarkers of GO response have been suggested, including expression of CD33 and multidrug resistance genes, cytogenetic and molecular profiles, minimal residual disease and stemness signatures. Among them, elevated CD33 expression on blast cells and non-adverse cytogenetic or molecular risk represent largely validated predictors of good response.

Recent comparisons between plant and animal viruses reveal many common principles that underlie how all viruses express their genetic material, amplify their genomes, and link virion assembly with replication. Cauliflower mosaic virus (CaMV) is not infectious for human beings. Here, we show that CaMV transactivator/viroplasmin protein (TAV) shares sequence similarity with and behaves like the human ribonuclease H1 (RNase H1) in reducing DNA/RNA hybrids detected with S9.6 antibody in HEK293T cells. We showed that TAV is clearly expressed in the cytosol and in the nuclei of transiently transfected human cells, similar to its distribution in plants. TAV also showed remarkable cytotoxic effects in U251 human glioma cells in vitro. These characteristics pave the way for future analysis on the use of the plant virus protein TAV, as an alternative to human RNAse H1 during gene therapy in human cells.

No abstract available

Circulating tumor cells (CTCs) are a rare population of cells representing a key player in the metastatic cascade. They are recognized as a validated tool for the identification of patients with a higher risk of relapse, including those diagnosed with breast cancer (BC). However, CTCs are characterized by high levels of heterogeneity that also involve copy number alterations (CNAs), structural variations associated with gene dosage changes. In this study, single CTCs were isolated from the peripheral blood of 11 early-stage BC patients at different time points. A label-free enrichment of CTCs was performed using OncoQuick, and single CTCs were isolated using DEPArray. Libraries were prepared from single CTCs and DNA extracted from matched tumor tissues for a whole-genome low-coverage next-generation sequencing (NGS) analysis using the Ion Torrent S5 System. The analysis of the CNA burden highlighted that CTCs had different degrees of aberration based on the time point and subtype. CTCs were found even six months after surgery and shared CNAs with matched tumor tissue. Tumor-associated CNAs that were recurrent in CTCs were patient-specific, and some alterations involved regions associated with BC and survival (i.e., gains at 1q21-23 and 5p15.33). The enrichment analysis emphasized the involvement of aberrations of terms, associated in particular with interferon (IFN) signaling. Collectively, our findings reveal that these aberrations may contribute to understanding the molecular mechanisms involving CTC-related processes and their survival ability in occult niches, supporting the goal of exploiting their application in patients&rsquo, surveillance and follow-up.

In chronic myeloid leukemia (CML) patients, tyrosine kinase inhibitors (TKIs) may select for drug-resistant BCR-ABL1 kinase domain (KD) mutants. Although Sanger sequencing (SS) is considered the gold standard for BCR-ABL1 KD mutation screening, next-generation sequencing (NGS) has recently been assessed in retrospective studies. We conducted a prospective, multicenter study (NEXT-in-CML) to assess the frequency and clinical relevance of low-level mutations and the feasibility, cost, and turnaround times of NGS-based BCR-ABL1 mutation screening in a routine setting. A series of 236 consecutive CML patients with failure (n = 124) or warning (n = 112) response to TKI therapy were analyzed in parallel by SS and NGS in 1 of 4 reference laboratories. Fifty-one patients (22 failure, 29 warning) who were negative for mutations by SS had low-level mutations detectable by NGS. Moreover, 29 (27 failure, 2 warning) of 60 patients who were positive for mutations by SS showed additional low-level mutations. Thus, mutations undetectable by SS were identified in 80 out of 236 patients (34%), of whom 42 (18% of the total) had low-level mutations somehow relevant for clinical decision making. Prospective monitoring of mutation kinetics demonstrated that TKI-resistant low-level mutations are invariably selected if the patients are not switched to another TKI or if they are switched to a inappropriate TKI or TKI dose. The NEXT-in-CML study provides for the first time robust demonstration of the clinical relevance of low-level mutations, supporting the incorporation of NGS-based BCR-ABL1 KD mutation screening results in the clinical decision algorithms.

Bone marrow aspiration and biopsy (BMAB) is a painful procedure, and the routinely used local infiltration anesthesia (LIA) with lidocaine is unable to provide pain relief during the most uncomfortable phases. The primary endpoint of the present randomized, patient-blinded trial was to evaluate the efficacy of an opioid and benzodiazepine combination plus LIA (sedoanalgesia) in patients undergoing BMAB for hematological malignancies. The secondary endpoint was the safety of the procedure in an outpatient setting. Ancillary assessments were anticipatory anxiety related to pain recall in the event of re-biopsy, and adequacy of bone tissue harvested. Patients were randomly assigned to one of 2 arms to receive either sedoanalgesic placebo plus LIA (standard group) or oral fentanyl citrate 200 &mu, g plus oral midazolam 5 mg plus LIA (combo group) during BMAB. Pre-procedural anxiety and procedural pain were assessed according to the Numerical Rating Scale (NRS: 0&ndash, 10), dividing the time of the procedure into five intervals (T0, T1, T2a, T2b and T3) and evaluating the degree of discomfort at each time (T) in both groups. One hundred and sixteen patients were eligible for the study. At T2b (time of biopsy) and T3 (time after biopsy), a significantly lower perception of pain was registered in the combo group. Moreover, there were no significant sedoanalgesia-related side-effects. Finally, histological specimens were higher in quality in the combo group. Sedoanalgesia was highly effective in reducing pain during biopsy, diminished anticipatory anxiety in patients undergoing re-biopsy and led to fewer non-diagnostic specimens being harvested.

Cancer metastasis is the leading cause of morbidity and cancer-related death. Bone is one of the most favored sites of metastatic spread, particularly from breast cancer. However, our understanding on how the host tissue can impact tumor growth is weak. This is hampered by the lack of a reliable in vitro model ables to replicate the complex microenvironment. Indeed, bone microenvironment provides physical support to cells but it can also affect cell behaviour and phenotype. We developed a hybrid 3D collagen-based scaffold functionalized with hydroxyapatite material (HA) in order to mimic natural bone features. Scaffolds were synthesized in our laboratory through collagen solution mixed and cross-linked with hydroxyapatite. Then, we cultured MDA-MB-231, a triple negative breast cancer cell line and MCF-7, an ER+ breast cancer cell line in collagen or hybrid scaffold, in order to evaluate how extracellular matrix affects cell behavior. Cell growth was evaluated by MTT cell-proliferation assay in a time course analysis. Moreover, we evaluated cell morphology and cell disposition within the scaffold by confocal microscopy. Finally, gene profiling was evaluated by qRT-PCR. MCF-7 cells acquire an organized and linear structure, whereas MDA-MB-231 an epithelial-like morphology, when cultured in hybrid scaffold. MDA-MB-231 cells proliferate faster than MCF-7 in both models. However, this is not affected by hydroxyapatite since the proliferation rate is similar when cells are cultured in collagen or hybrid scaffold. MDA-MB-231 grown in hybrid scaffold show a significant increase in RANKL/OPG ratio, pathway strictly involved in bone homeostasis. Moreover, they show a significant increased expression in JAG1, MMP2 and SNAIL1. For MCF-7, we observe a decrease in CDH1/VIM expression ratio and a significant decrease in OPG, osteoclastogenesis inhibitory factor. Overall, these results suggest a shift induced by hydroxyapatite scaffold towards an osteolytic phenotype. Whereas collagen-scaffold could better simulate in vivo primary tumor (Liverani C. et al, Sci Rep 9, 12263, 2019), we highlight that hybrid hydroxyapatite/collagen scaffold is more suitable to study breast cancer cells behaviour in bone metastasis. Then, we will implement it with a direct co-culture of osteoclast and osteoblast cells, in order to better investigate the contribution of the host microenvironment in bone metastasis. This model could provide a reliable 3D in vitro model for the study of bone metastasis mechanisms and for drug screening assays. Citation Format: Chiara Spadazzi, Laura Mercatali, Alessandro De Vita, Chiara Liverani, Claudia Cocchi, Giacomo Miserocchi, Alberto Bongiovanni, Federica Recine, Sebastiano Calpona, Nada Riva, Giandomenico Di Menna, Lorena Gurrieri, Valentina Fausti, Silvia Angela De Bonis, Giovanni Martinelli, Nicola Baldini, Toni Ibrahim. Development of a biomimetic 3D scaffold to study breast cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 333.

Cytarabine, the 4-amino-1-(β-D-arabinofuranosyl)-2(1H)-pyrimidinone, (ARA-C) is an antimetabolite cytidine analogue used worldwide as key drug in the management of leukaemia. As specified in the manufacturers' instructions, once the components-sterile water and cytarabine powder-are unpackaged and mixed, the solution begins to degrade after 6 hours at room temperature and 12 hours at 4°C. To evaluate how to avoid wasting the drug in short-term, low-dose treatment regimens, the reconstituted samples, stored at 25°C and 4°C, were analyzed every day of the test week by reversed-phase HPLC and high-field NMR spectroscopy. All the samples remained unchanged for the entire week, which corresponds to the time required to administer the entire commercial drug package during low-dose therapeutic regimens. The drug solution was stored in a glass container at 4°C in an ordinary freezer and drawn with sterile plastic syringes; during this period, no bacterial or fungal contamination was observed. Our findings show that an cytarabine solution prepared and stored in the original vials retains its efficacy and safety and can, therefore, be divided into small doses to be administered over more days, thus avoiding unnecessary expensive and harmful waste of the drug preparation. Moreover, patients who require daily administration of the drug could undergo the infusion at home without need to go to hospital. The stability of the aliquots would help decrease hospitalization costs.

The use of hydroxychloroquine (HCL) has been very common in countries with a rapid spread of coronavirus disease 2019 (COVID-19), although controversial and the subject of heated scientific discussions with implications for the whole society. This article is protected by copyright. All rights reserved.

Background Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. Methods The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. Results We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. Conclusions Our data suggest that WEE1 plays a role in ALL blast’s survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL. Electronic supplementary material The online version of this article (10.1186/s13045-018-0641-1) contains supplementary material, which is available to authorized users.

Deregulated activity of BCR-ABL1, a nonreceptor tyrosine kinase encoded by the fusion gene resulting from the t(9;22)(q34;q11) chromosomal translocation, is thought to be the driver event responsible for initiation and maintenance of chronic myeloid leukemia (CML). BCR-ABL1 was one of the first tyrosine kinases to be implicated in a human malignancy and the first to be successfully targeted. Imatinib mesylate, the first tyrosine kinase inhibitor (TKI) to be approved for therapeutic use, was hailed as a magic bullet against cancer and remains one of the safest and most effective anticancer agents ever developed. Second- and third-generation TKIs were later introduced to prevent or counteract the problem of drug resistance, that may arise in a small proportion of patients. They are more potent molecules, but have been associated to more serious side effects and complications. Patients achieving stable optimal responses to TKI therapy are predicted to have the same life expectancy of the general population. However, TKIs do not ‘cure’ CML. Only a small proportion of cases may attempt therapy discontinuation without experiencing subsequent relapse. The great majority of patients will have to assume TKIs indefinitely – which raises serious pharmacoeconomic concerns and is now shifting the focus from efficacy to compliance and quality of life issues. Here we retrace the steps that have led from the biological acquisitions regarding BCR-ABL1 structure and function to the development of inhibitory strategies and we discuss drug resistance mechanism and how they can be addressed. Electronic supplementary material The online version of this article (10.1186/s12943-018-0780-6) contains supplementary material, which is available to authorized users.

Summary Background Internal tandem duplication mutations in FLT3 are common in acute myeloid leukaemia and are associated with rapid relapse and short overall survival. The clinical benefit of FLT3 inhibitors in patients with acute myeloid leukaemia has been limited by rapid generation of resistance mutations, particularly in codon Asp835 (D835). We aimed to assess the highly selective oral FLT3 inhibitor gilteritinib in patients with relapsed or refractory acute myeloid leukaemia. Methods In this phase 1–2 trial, we enrolled patients aged 18 years or older with acute myeloid leukaemia who either were refractory to induction therapy or had relapsed after achieving remission with previous treatment. Patients were enrolled into one of seven dose-escalation or dose-expansion cohorts assigned to receive once-daily doses of oral gilteritinib (20 mg, 40 mg, 80 mg, 120 mg, 200 mg, 300 mg, or 450 mg). Cohort expansion was based on safety and tolerability, FLT3 inhibition in correlative assays, and antileukaemic activity. Although the presence of an FLT3 mutation was not an inclusion criterion, we required ten or more patients with locally confirmed FLT3 mutations ( FLT3 mut+ ) to be enrolled in expansion cohorts at each dose level. On the basis of emerging findings, we further expanded the 120 mg and 200 mg dose cohorts to include FLT3 mut+ patients only. The primary endpoints were the safety, tolerability, and pharmacokinetics of gilteritinib. Safety and tolerability were assessed in the safety analysis set (all patients who received at least one dose of gilteritinib). Responses were assessed in the full analysis set (all patients who received at least one dose of study drug and who had at least one datapoint post-treatment). Pharmacokinetics were assessed in a subset of the safety analysis set for which sufficient data for concentrations of gilteritinib in plasma were available to enable derivation of one or more pharmacokinetic variables. This study is registered with ClinicalTrials.gov, number NCT02014558, and is ongoing. Findings Between Oct 15, 2013, and Aug 27, 2015, 252 adults with relapsed or refractory acute myeloid leukaemia received oral gilteritinib once daily in one of seven dose-escalation (n=23) or dose-expansion (n=229) cohorts. Gilteritinib was well tolerated; the maximum tolerated dose was established as 300 mg/day when two of three patients enrolled in the 450 mg dose-escalation cohort had two dose-limiting toxicities (grade 3 diarrhoea and grade 3 elevated aspartate aminotransferase). The most common grade 3–4 adverse events irrespective of relation to treatment were febrile neutropenia (97 [39%] of 252), anaemia (61 [24%]), thrombocytopenia (33 [13%]), sepsis (28 [11%]), and pneumonia (27 [11%]). Commonly reported treatment-related adverse events were diarrhoea (41 [16%] of 252]), fatigue (37 [15%]), elevated aspartate aminotransferase (33 [13%]), and elevated alanine aminotransferase (24 [10%]). Serious adverse events occurring in 5% or more of patients were febrile neutropenia (78 [31%] of 252; five related to treatment), progressive disease (43 [17%]), sepsis (36 [14%]; two related to treatment), pneumonia (27 [11%]), acute renal failure (25 [10%]; five related to treatment), pyrexia (21 [8%]; three related to treatment), bacteraemia (14 [6%]; one related to treatment), and respiratory failure (14 [6%]). 95 people died in the safety analysis set, of which seven deaths were judged possibly or probably related to treatment (pulmonary embolism [200 mg/day], respiratory failure [120 mg/day], haemoptysis [80 mg/day], intracranial haemorrhage [20 mg/day], ventricular fibrillation [120 mg/day], septic shock [80 mg/day], and neutropenia [120 mg/day]). An exposure-related increase in inhibition of FLT3 phosphorylation was noted with increasing concentrations in plasma of gilteritinib. In-vivo inhibition of FLT3 phosphorylation occurred at all dose levels. At least 90% of FLT3 phosphorylation inhibition was seen by day 8 in most patients receiving a daily dose of 80 mg or higher. 100 (40%) of 249 patients in the full analysis set achieved a response, with 19 (8%) achieving complete remission, ten (4%) complete remission with incomplete platelet recovery, 46 (18%) complete remission with incomplete haematological recovery, and 25 (10%) partial remission. Interpretation Gilteritinib had a favourable safety profile and showed consistent FLT3 inhibition in patients with relapsed or refractory acute myeloid leukaemia. These findings confirm that FLT3 is a high-value target for treatment of relapsed or refractory acute myeloid leukaemia; based on activity data, gilteritinib at 120 mg/day is being tested in phase 3 trials. Funding Astellas Pharma, National Cancer Institute (Leukemia Specialized Program of Research Excellence grant), Associazione Italiana Ricerca sul Cancro.

Background Aneuploidy occurs in more than 20% of acute myeloid leukemia (AML) cases and correlates with an adverse prognosis. Methods To understand the molecular bases of aneuploid acute myeloid leukemia (A‐AML), this study examined the genomic profile in 42 A‐AML cases and 35 euploid acute myeloid leukemia (E‐AML) cases. Results A‐AML was characterized by increased genomic complexity based on exonic variants (an average of 26 somatic mutations per sample vs 15 for E‐AML). The integration of exome, copy number, and gene expression data revealed alterations in genes involved in DNA repair (eg, SLX4IP, RINT1, HINT1, and ATR) and the cell cycle (eg, MCM2, MCM4, MCM5, MCM7, MCM8, MCM10, UBE2C, USP37, CK2, CK3, CK4, BUB1B, NUSAP1, and E2F) in A‐AML, which was associated with a 3‐gene signature defined by PLK1 and CDC20 upregulation and RAD50 downregulation and with structural or functional silencing of the p53 transcriptional program. Moreover, A‐AML was enriched for alterations in the protein ubiquitination and degradation pathway (eg, increased levels of UHRF1 and UBE2C and decreased UBA3 expression), response to reactive oxygen species, energy metabolism, and biosynthetic processes, which may help in facing the unbalanced protein load. E‐AML was associated with BCOR/BCORL1 mutations and HOX gene overexpression. Conclusions These findings indicate that aneuploidy‐related and leukemia‐specific alterations cooperate to tolerate an abnormal chromosome number in AML, and they point to the mitotic and protein degradation machineries as potential therapeutic targets., Aneuploid acute myeloid leukemia (A‐AML) is associated with genomic and transcriptional alterations in the cell cycle and protein degradation pathways. The upregulation of PLK1 and CDC20 and the downregulation of RAD50 and of a p53‐related signature are hallmarks of A‐AML.

Steroid nuclear receptors are known to be involved in the regulation of epithelial-mesenchymal transition process with important roles in invasion and metastasis initiation. Androgen receptor (AR) has been extensively studied, but its role in relation to breast cancer patient prognosis remains to be clarified. AR/ER ratio has been reported to be an unfavorable prognostic marker in early primary breast cancer, but its role in the patients with advanced disease has to be cleared. We retrospectively analyzed ER, PgR, and AR expression on a case series of 159 specimens of primary BC samples by using immunohistochemistry and 89 patients of these had luminal tumors for which AR and ER expression and survival data were available. For twenty-four patients both primary and metastatic tumors were available. A significantly shorter overall survival was observed in primary tumors with AR/PgR ratio ≥ 1.54 (HR = 2.27; 95% CI 1.30-3.97; p = 0.004). Similarly OS was significantly shorter when ER/PgR ratio ≥2 in primary tumors (HR = 1.89; 95% CI 1.10-3.24; p = 0.021). The analysis of the 24 patients who had biomarker determinations both in primary tumors and metastasis showed a better OS when AR/ER ratio in the metastasis was ≥ 0.90 (p = 0.022). Patients with a high AR/ER ratio in primary tumor that remained high in the metastasis had better prognosis in terms of OS (p = 0.011). Despite we suggested that the ratios AR/ER and AR/PgR could be used to identify patients with different prognosis, their real value needs to be better clarified in different BC settings through prospective studies.

Adults with relapsed/refractory acute lymphoblastic leukemia have an unfavourable prognosis, which is influenced by disease and patient characteristics. To further evaluate these characteristics, a retrospective analysis of 1,706 adult patients with Ph-negative relapsed/refractory B-precursor acute lymphoblastic leukemia diagnosed between 1990-2013 was conducted using data reflecting the standard of care from 11 study groups and large centers in Europe and the United States. Outcomes included complete remission, overall survival, and realization of stem cell transplantation after salvage treatment. The overall complete remission rate after first salvage was 40%, ranging from 35%-41% across disease status categories (primary refractory, relapsed with or without prior transplant), and was lower after second (21%) and third or greater (11%) salvage. The overall complete remission rate was higher for patients diagnosed from 2005 onward (45%, 95% CI: 39%-50%). One- and three-year survival rates after first, second, and third or greater salvage were 26% and 11%, 18% and 6%, and 15% and 4%, respectively, and rates were 2%-5% higher among patients diagnosed from 2005. Prognostic factors included younger age, longer duration of first remission, and lower white blood cell counts at primary diagnosis. This large dataset can provide detailed reference outcomes for patients with relapsed/refractory Ph-negative B-precursor acute lymphoblastic leukemia. clinicaltrials.gov identifier: 02003612.

The prognosis for adults with relapsed acute lymphoblastic leukemia is poor. We sought to determine whether inotuzumab ozogamicin, an anti-CD22 antibody conjugated to calicheamicin, results in better outcomes in patients with relapsed or refractory acute lymphoblastic leukemia than does standard therapy.In this phase 3 trial, we randomly assigned adults with relapsed or refractory acute lymphoblastic leukemia to receive either inotuzumab ozogamicin (inotuzumab ozogamicin group) or standard intensive chemotherapy (standard-therapy group). The primary end points were complete remission (including complete remission with incomplete hematologic recovery) and overall survival.Of the 326 patients who underwent randomization, the first 218 (109 in each group) were included in the primary intention-to-treat analysis of complete remission. The rate of complete remission was significantly higher in the inotuzumab ozogamicin group than in the standard-therapy group (80.7% [95% confidence interval {CI}, 72.1 to 87.7] vs. 29.4% [95% CI, 21.0 to 38.8], P0.001). Among the patients who had complete remission, a higher percentage in the inotuzumab ozogamicin group had results below the threshold for minimal residual disease (0.01% marrow blasts) (78.4% vs. 28.1%, P0.001); the duration of remission was longer in the inotuzumab ozogamicin group (median, 4.6 months [95% CI, 3.9 to 5.4] vs. 3.1 months [95% CI, 1.4 to 4.9]; hazard ratio, 0.55 [95% CI, 0.31 to 0.96]; P=0.03). In the survival analysis, which included all 326 patients, progression-free survival was significantly longer in the inotuzumab ozogamicin group (median, 5.0 months [95% CI, 3.7 to 5.6] vs. 1.8 months [95% CI, 1.5 to 2.2]; hazard ratio, 0.45 [97.5% CI, 0.34 to 0.61]; P0.001); the median overall survival was 7.7 months (95% CI, 6.0 to 9.2) versus 6.7 months (95% CI, 4.9 to 8.3), and the hazard ratio was 0.77 (97.5% CI, 0.58 to 1.03) (P=0.04). In the safety population, the most frequent grade 3 or higher nonhematologic adverse events with inotuzumab ozogamicin were liver-related. Veno-occlusive liver disease of any grade occurred in 15 patients (11%) who received inotuzumab ozogamicin and in 1 patient (1%) who received standard therapy.The rate of complete remission was higher with inotuzumab ozogamicin than with standard therapy, and a higher percentage of patients in the inotuzumab ozogamicin group had results below the threshold for minimal residual disease. Both progression-free and overall survival were longer with inotuzumab ozogamicin. Veno-occlusive liver disease was a major adverse event associated with inotuzumab ozogamicin. (Funded by Pfizer; INO-VATE ALL ClinicalTrials.gov number, NCT01564784.).

// Giorgia Giordani 1, 5, * , Marilena Barraco 1, 6, * , Angela Giangrande 2 , Giovanni Martinelli 3 , Viviana Guadagnuolo 3 , Giorgia Simonetti 3 , Giovanni Perini 1, 4 , Roberto Bernardoni 1, 4 1 Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy 2 Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP 67404 Illkirch, France 3 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Institute of Hematology "L. e A. Seragnoli", University of Bologna, Bologna, Italy 4 Health Sciences and Technology - Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Ozzano Emilia, Italy 5 Present address: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield, UK 6 Present address: Institute of Hematology, “L e A Seragnoli”, S. Orsola-Malpighi Hospital, Bologna, Italy * These authors contributed equally to this work Correspondence to: Roberto Bernardoni, email: roberto.bernardoni@unibo.it Giovanni Perini, email: giovanni.perini@unibo.it Keywords: PF-04449913, Smoothened inhibitor, leukemia, Drosophila, prohemocytes Received: December 09, 2015 Accepted: June 26, 2016 Published: July 28, 2016 ABSTRACT The efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells and the prevention of disease relapse through targeting pathways that stimulate generation and maintenance of these cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In Drosophila melanogaster the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. Using Drosophila as an in vivo model, we investigated the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. We conclude that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether our results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies.

Hyperactivation of the Hedgehog (Hh) pathway, which controls refueling of multiple myeloma (MM) clones, might be critical to disease recurrence. Although several studies suggest the Hh pathway is activated in CD138- immature cells, differentiated CD138+ plasma cells might also be able to self-renew by producing themselves the Hh ligands. We studied the gene expression profiles of 126 newly diagnosed MM patients analyzed in both the CD138+ plasma cell fraction and CD138-CD19+ B-cell compartment. Results demonstrated that an Hh-gene signature was able to cluster patients in two subgroups characterized by the opposite Hh pathway expression in mature plasma cells and their precursors. Strikingly, patients characterized by Hh hyperactivation in plasma cells, but not in their B cells, displayed high genomic instability and an unfavorable outcome in terms of shorter progression-free survival (hazard ratio: 1.92; 95% confidence interval: 1.19-3.07) and overall survival (hazard ratio: 2.61; 95% confidence interval: 1.26-5.38). These results suggest that the mechanisms triggered by the Hh pathway ultimately led to identify a more indolent vs a more aggressive biological and clinical subtype of MM. Therefore, patient stratification according to their molecular background might help the fine-tuning of future clinical and therapeutic studies.

In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients who fail imatinib treatment, BCR-ABL1 mutation profiling by Sanger sequencing (SS) is recommended before changing therapy since detection of specific mutations influences second-generation tyrosine kinase inhibitor (2GTKI) choice. We aimed to assess i) in how many patients who relapse on second-line 2GTKI therapy next generation sequencing (NGS) may track resistant mutations back to the sample collected at the time of imatinib resistance, before 2GTKI start (switchover sample) and ii) whether low level mutations identified by NGS always undergo clonal expansion. To this purpose, we used NGS to retrospectively analyze 60 imatinib-resistant patients (CML, n = 45; Ph+ ALL, n = 15) who had failed second-line 2GTKI therapy and had acquired BCR-ABL1 mutations (Group 1) and 25 imatinib-resistant patients (CML, n = 21; Ph+ ALL, n = 4) who had responded to second-line 2GTKI therapy, for comparison (Group 2). NGS uncovered that in 26 (43%) patients in Group 1, the 2GTKI-resistant mutations that triggered relapse were already detectable at low levels in the switchover sample (median mutation burden, 5%; range 1.1%–18.4%). Importantly, none of the low level mutations detected by NGS in switchover samples failed to expand whenever the patient received the 2GTKI to whom they were insensitive. In contrast, no low level mutation that was resistant to the 2GTKI the patients subsequently received was detected in the switchover samples from Group 2. NGS at the time of imatinib failure reliably identifies clinically relevant mutations, thus enabling a more effective therapeutic tailoring.

// Carolina Terragna 1 , Daniel Remondini 2 , Marina Martello 1 , Elena Zamagni 1 , Lucia Pantani 1 , Francesca Patriarca 3 , Annalisa Pezzi 1 , Giuseppe Levi 2 , Massimo Offidani 4 , Ilaria Proserpio 5 , Giovanni De Sabbata 6 , Paola Tacchetti 1 , Clotilde Cangialosi 7 , Fabrizio Ciambelli 8 , Clara Virginia Vigano 9 , Flores Angela Dico 1 , Barbara Santacroce 1 , Enrica Borsi 1 , Annamaria Brioli 1 , Giulia Marzocchi 1 , Gastone Castellani 2 , Giovanni Martinelli 1 , Antonio Palumbo 10 , Michele Cavo 1 1 ”Seragnoli” Institute of Hematology, Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Bologna University School of Medicine, Bologna, Italy 2 Department of Physics and Astronomy (DIFA), Bologna University, Bologna, Italy 3 Clinica Ematologica, DISM, University of Udine, Udine, Italy 4 Clinica di Ematologia, A.O.U. Ospedali Riuniti di Ancona, Ancona, Italy 5 U.O Oncologia Medica, Ospedale di Circolo e Fondazione Macchi, Varese, Italy 6 Ematologia Clinica, A.O.U. Ospedali Riuniti, Trieste, Italy 7 Hematology Division UTMO, Azienda “Ospedali Riuniti Villa Sofia-Cervello” Presidio Ospedaliero V.Cervello, Palermo, Italy 8 S.C.Oncologia Medica, A.O. Sant’Antonio Abate, Gallarate, Varese, Italy 9 Unita Operativa di Ematologia, Istituti Ospitalieri di Cremona, Cremona, Italy 10 Myeloma Unit, Division of Hematology, University of Torino, A.O.U. Citta della Salute e della Scienza di Torino, Torino, Italy Correspondence to: Michele Cavo, e-mail: michele.cavo@unibo.it Keywords: multiple myeloma, gene expression profile, SNPs, VTD, complete response Received: August 06, 2015 Accepted: September 16, 2015 Published: November 09, 2015 ABSTRACT The prime focus of the current therapeutic strategy for Multiple Myeloma (MM) is to obtain an early and deep tumour burden reduction, up to the level of complete response (CR). To date, no description of the characteristics of the plasma cells (PC) prone to achieve CR has been reported. This study aimed at the molecular characterization of PC obtained at baseline from MM patients in CR after bortezomib-thalidomide-dexamethasone (VTD) first line therapy. One hundred and eighteen MM primary tumours obtained from homogeneously treated patients were profiled both for gene expression and for single nucleotide polymorphism genotype. Genomic results were used to obtain a predictor of sensitivity to VTD induction therapy, as well as to describe both the transcription and the genomic profile of PC derived from MM with subsequent optimal response to primary induction therapy. By analysing the gene profiles of CR patients, we identified a 5-gene signature predicting CR with an overall median accuracy of 75% (range: 72%–85%). In addition, we highlighted the differential expression of a series of genes, whose deregulation might explain patients’ sensitivity to VTD therapy. We also showed that a small copy number loss, covering 606Kb on chromosome 1p22.1 was the most significantly associated with CR patients.

Background In chronic lymphocytic leukemia (CLL) disease onset and progression are influenced by the behavior of specific CD4+ T cell subsets, such as T regulatory cells (Tregs). Here, we focused on the phenotypic and functional characterization of Tregs in CLL patients to improve our understanding of the putative mechanism by which these cells combine immunosuppressive and effector-like properties. Methods Peripheral blood mononuclear cells were isolated from newly diagnosed CLL patients (n = 25) and healthy volunteers (n = 25). The phenotypic and functional characterization of Tregs and their subsets was assessed by flow cytometry. In vitro analysis of TH1, TH2, TH17 and Tregs cytokines was evaluated by IFN-γ, IL-4, IL-17A and IL-10 secretion assays. The transcriptional profiling of 84 genes panel was evaluated by RT2 Profiler PCR Array. Statistical analysis was carried out using exact non parametric Mann–Whitney U test. Results In all CLL samples, we found a significant increase in the frequency of IL-10-secreting Tregs and Tregs subsets, a significant rise of TH2 IL-4+ and TH17 IL-17A+ cells, and a higher percentage of IFN-γ/IL-10 and IL-4/IL-10 double-releasing CD4+ T cells. In addition, we also observed the up-regulation of innate immunity genes and the down-regulation of adaptive immunity ones. Conclusions Our data show that Tregs switch towards an effector-like phenotype in CLL patients. This multifaceted behavior is accompanied by an altered cytokine profiling and transcriptional program of immune genes, leading to a dysfunction in immune response in the peripheral blood environment of CLL patients. Electronic supplementary material The online version of this article (10.1186/s12967-018-1545-0) contains supplementary material, which is available to authorized users.

open 17 no We thank Rita Bertoni and Antonella Orlando for the technical assistance. This work has been supported by Bologna AIL (Italian Association against Leukemia, Lymphoma and Myeloma), Roberto and Cornelia Pallotti Legacy for Cancer Research (University of Bologna) to M.B., and REUSE with Love ONLUS, Bologna, Italy. Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC > 0.5 × 109/L) and 70 ± 10% (platelet > 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ± 9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche. none none Bonifazi, Francesca; Dan, Elisa; Labopin, Myriam; Sessa, Mariarosaria; Guadagnuolo, Viviana; Ferioli, Martina; Rizzi, Simonetta; De Carolis, Sabrina; Sinigaglia, Barbara; Motta, Maria Rosa; Bontadini, Andrea; Giudice, Valeria; Martinelli, Giovanni; Arpinati, Mario; Cavo, Michele; Bonafé, Massimiliano; Storci, Gianluca Bonifazi, Francesca; Dan, Elisa; Labopin, Myriam; Sessa, Mariarosaria; Guadagnuolo, Viviana; Ferioli, Martina; Rizzi, Simonetta; De Carolis, Sabrina; Sinigaglia, Barbara; Motta, Maria Rosa; Bontadini, Andrea; Giudice, Valeria; Martinelli, Giovanni; Arpinati, Mario; Cavo, Michele; Bonafé, Massimiliano; Storci, Gianluca