Massimiliano Bonifacio

Bone mineral density, bone turnover markers and fractures in patients with indolent systemic mastocytosis

OBJECTIVE We systematically assessed bone mineral density (BMD), bone turnover markers (BTM), and fractures in a large cohort of patients with Indolent Systemic Mastocytosis (ISM). METHODS Eighty-two patients (mean age 48 years, 37 women) with ISM were studied. BMD was measured by dual X-ray absorptiometry at the lumbar spine and proximal hip. The serum markers of bone turnover included bone-specific alkaline phosphatase, C-telopeptides of type I collagen, and serum osteocalcin. Previous clinical fractures were registered and spine X-ray was obtained from all patients. RESULTS Three women were excluded for concomitant diseases associated to osteoporosis. Osteoporosis according with the WHO classification (T-score<-2.5) was found in 16 patients (20.0%) (7 females and 9 men). Mastocytosis-related low BMD (Z-score at either the spine or the hip<-2) was found in 3 women (9%) and 13 men (28%). The BMD was generally lower at the spine than at the hip. No significant correlation was observed between serum tryptase levels and T or Z-score BMD. One or more moderate or severe vertebral fractures were found in 17 patients (12 men); in 11 of them Z-score values were>-2 or not valuable at the spine. No significant difference was found in the prevalence of mastocytosis-related low BMD and/or vertebral fractures between patients with or without skin involvement. Two patients had radiographic and densitometric osteosclerosis-like characteristics. In osteoporotic patients higher, normal or lower serum BTM were found, without correlations with serum tryptase levels, while in patients with osteosclerosis both BTM and serum tryptase values were particularly increased. CONCLUSIONS Vertebral osteoporosis and fractures are frequent in patients with ISM. Spine X-ray and densitometric examination are warranted in all patients, also without skin involvement and particularly in males; Z-score other than T-score BMD must be evaluated. Patients with idiopathic osteoporosis should be evaluated for mast cell disease. Both high than low BTM can be observed in patients with osteoporosis while osteosclerosis is characterized by high bone turnover and serum tryptase levels.

Macrophages may promote cancer growth via a GM-CSF/HB-EGF paracrine loop that is enhanced by CXCL12

BackgroundIncreased numbers of tumour-associated macrophages correlate with shortened survival in some cancers. The molecular bases of this correlation are not thoroughly understood. Events triggered by CXCL12 may play a part, as CXCL12 drives the migration of both CXCR4-positive cancer cells and macrophages and may promote a molecular crosstalk between them.ResultsSamples of HER1-positive colon cancer metastases in liver, a tissue with high expression of CXCL12, were analysed by immunohistochemistry. In all of the patient biopsies, CD68-positive tumour-associated macrophages presented a mixed CXCL10 (M1)/CD163 (M2) pattern, expressed CXCR4, GM-CSF and HB-EGF, and some stained positive for CXCL12. Cancer cells stained positive for CXCR4, CXCL12, HER1, HER4 and GM-CSF. Regulatory interactions among these proteins were validated via experiments in vitro involving crosstalk between human mononuclear phagocytes and the cell lines DLD-1 (human colon adenocarcinoma) and HeLa (human cervical carcinoma), which express the above-mentioned ligand/receptor repertoire. CXCL12 induced mononuclear phagocytes to release HB-EGF, which activated HER1 and triggered anti-apoptotic and proliferative signals in cancer cells. The cancer cells then proliferated and released GM-CSF, which in turn activated mononuclear phagocytes and induced them to release more HB-EGF. Blockade of GM-CSF with neutralising antibodies or siRNA suppressed this loop.ConclusionsCXCL12-driven stimulation of cancer cells and macrophages may elicit and reinforce a GM-CSF/HB-EGF paracrine loop, whereby macrophages contribute to cancer survival and expansion. The involvement of mixed M1/M2 GM-CSF-stimulated macrophages in a tumour-promoting loop may challenge the paradigm of tumour-favouring macrophages as polarized M2 mononuclear phagocytes.

Clonal mast cell disorders in patients with severe Hymenoptera venom allergy and normal serum tryptase levels

BACKGROUND Systemic mastocytosis is a clonal mast cell (MC) disease that can lead to potentially fatal anaphylactic reactions caused by excessive MC mediator release. The prevalence of mastocytosis in patients with Hymenoptera venom allergy is high, and thus the disease should be suspected in patients with severe reactions caused by Hymenoptera stings and increased serum basal tryptase (SBT) levels. OBJECTIVE We sought to evaluate the presence of clonal MC disorders in patients seen at our mastocytosis center with Hymenoptera sting-induced anaphylaxis, documented hypotension, absence of urticaria pigmentosa, and normal SBT levels. METHODS Twenty-two patients with Hymenoptera sting-induced anaphylaxis, without skin lesions, and with tryptase levels of less than 11.4 ng/mL underwent bone marrow evaluation. Bone mineral density was assessed in those patients with ascertained mastocytosis. RESULTS In 16 of 22 patients, a diagnosis of indolent mastocytosis could be established, and 1 patient had a monoclonal MC activation syndrome. Patients with mastocytosis had higher SBT levels (P = .03) but only rarely had angioedema/urticaria associated with hypotension (P = .004). CONCLUSIONS The absence of urticaria or angioedema in severe reactions to Hymenoptera stings with hypotension might represent the most relevant factor in identifying patients with mastocytosis, regardless of their serum tryptase levels.

Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells

BackgroundWe previously demonstrated that the plant-derived agent α-bisabolol enters cells via lipid rafts, binds to the pro-apoptotic Bcl-2 family protein BID, and may induce apoptosis. Here we studied the activity of α-bisabolol in acute leukemia cells.MethodsWe tested ex vivo blasts from 42 acute leukemias (14 Philadelphia-negative and 14 Philadelphia-positive B acute lymphoid leukemias, Ph-/Ph+B-ALL; 14 acute myeloid leukemias, AML) for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34+, CD33+my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours.ResultsA clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 μM α-bisabolol IC50) included mainly Ph-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 μM IC50). Ph+B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent via a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential.ConclusionOur study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells.

Isolated bone marrow mastocytosis: an underestimated subvariant of indolent systemic mastocytosis

Systemic mastocytosis (SM) is a heterogeneous disorder characterized by the proliferation and accumulation of atypical mast cells (MC) in tissues, principally in the bone marrow (BM) and skin. The diagnosis of systemic mastocytosis requires the presence of multifocal dense mast cell infiltrates in

TP53 mutations are frequent in adult acute lymphoblastic leukemia cases negative for recurrent fusion genes and correlate with poor response to induction therapy

Acute lymphoblastic leukemia (ALL) is a disease of either B-cell (80–85%) or T-cell (20–25%) derivation. Several molecular aberrations (i.e. BCR-ABL1, MLL/AFF1, SIL/TAL1 and E2A/PBX1 ) confer an overall poor outcome.[1][1],[2][2] However, a proportion of patients do not carry known genetic

DNA methylation-profiling identifies two splenic marginal zone lymphoma subgroups with different clinical and genetic features

Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

Intensive short-term chemotherapy regimen induces high remission rate (over 90%) and event-free survival both in children and adult patients with advanced sporadic Burkitt lymphoma/leukemia.

The optimal treatment of advanced sporadic Burkitt lymphoma in adults is still a matter of debate. The salutary results of pediatric therapies did open the road for improving the adult outcome. Between May 1988 and March 2009, 71 consecutive patients—46 adults, 25 children—affected by Burkitt lymphoma/leukemia were treated with the same intensive pediatric protocol alternating vincristine, adriamycine and fractionated ciclophosphamide (phase A) with high dose methotrexate and high dose cytarabine (phase B) in four Italian institutions. Eighty‐nine per cent of patients were in Stage III–IV or had L3 leukemia. Complete remissions were 67/71 (94.4%), 24/25 (96%) in children, and 43/46 (93.5%) in adults. Toxic deaths were 3/71 (4.2%), all in adults. There were nine relapses (one in children, eight in adults), all but one observed early. After a median observation of 94 months (range 23–275), the Event–Free Survival rate is 92% in children and 71.7% in adults (P = 0.067). The 23 more recent adults received also rituximab, without differences in outcome as compared to patients who did not. Our experience confirms that such an intensive pediatric‐derived chemotherapy is feasible and improves the long‐term outcome of adults with advanced Burkitt lymphoma. Am. J. Hematol., 2012.

Blinatumomab for minimal residual disease in adults with B-precursor acute lymphoblastic leukemia

Approximately 30% to 50% of adults with acute lymphoblastic leukemia (ALL) in hematologic complete remission after multiagent therapy exhibit minimal residual disease (MRD) by reverse transcriptase-polymerase chain reaction or flow cytometry. MRD is the strongest predictor of relapse in ALL. In this open-label, single-arm study, adults with B-cell precursor ALL in hematologic complete remission with MRD (≥10-3) received blinatumomab 15 µg/m2 per day by continuous IV infusion for up to 4 cycles. Patients could undergo allogeneic hematopoietic stem-cell transplantation any time after cycle 1. The primary end point was complete MRD response status after 1 cycle of blinatumomab. One hundred sixteen patients received blinatumomab. Eighty-eight (78%) of 113 evaluable patients achieved a complete MRD response. In the subgroup of 110 patients with Ph-negative ALL in hematologic remission, the Kaplan-Meier estimate of relapse-free survival (RFS) at 18 months was 54%. Median overall survival (OS) was 36.5 months. In landmark analyses, complete MRD responders had longer RFS (23.6 vs 5.7 months; P = .002) and OS (38.9 vs 12.5 months; P = .002) compared with MRD nonresponders. Adverse events were consistent with previous studies of blinatumomab. Twelve (10%) and 3 patients (3%) had grade 3 or 4 neurologic events, respectively. Four patients (3%) had cytokine release syndrome grade 1, n = 2; grade 3, n = 2), all during cycle 1. After treatment with blinatumomab in a population of patients with MRD-positive B-cell precursor ALL, a majority achieved a complete MRD response, which was associated with significantly longer RFS and OS compared with MRD nonresponders. This study is registered at www.clinicaltrials.gov as #NCT01207388.

Somatic D816V KIT mutation in a case of adult-onset familial mastocytosis

Rank # Genes Highest OR (L95-U95) Smallest P-value # Genes Highest OR (L95-U95) Smallest P-value # Genes Highest OR (L95-U95) Smallest P-value 1 IRAK1 0.70 (0.55-0.90) 0.006 IRAK1 0.73 (0.54-0.99) 0.042 IRAK1 0.50 (0.28-0.86) 0.013 2 MKK3 1.28 (1.10-1.50) 0.002 IRAKM 0.53 (0.31-0.90) 0.019 SIGIRR 0.54 (0.35-0.85) 0.008 3 ERK2 1.22 (1.05-1.42) 0.010 RIP1 1.49 (1.04-2.12) 0.012 TRAF6 0.65 (0.46-0.90) 0.009 4 IKBKE 0.68 (0.51-0.91) 0.010 JNK2 0.78 (0.65-0.94) 0.010 MKK6 2.29 (1.23-4.25) 0.009 5 Mek1 1.33 (1.00-1.76) 0.025 MKK6 0.55 (0.31-0.95) 0.029 TAB2 0.63 (0.46-0.86) 0.004 6 TOLLIP 0.82 (0.70-0.96) 0.009 MKK3 1.27 (1.05-1.53) 0.014 IRAK4 0.42 (0.20-0.89) 0.024 7 JNK2 1.42 (1.07-1.87) 0.014 IL1RL1 0.79 (0.65-0.96) 0.015 MyD88 2.07 (1.10-3.90) 0.024 8 SIGIRR 0.77 (0.62-0.95) 0.014 IRF7 0.74 (0.56-0.98) 0.029 IKBKE 1.47 (1.08-1.98) 0.013 9 TAB2 0.82 (0.70-0.95) 0.009 NFKB1 0.61 (0.39-0.97) 0.034 RIP1 1.85 (1.05-3.25) 0.034 10 TAB1 0.83 (0.71-0.97) 0.022 TIRAP 1.65 (1.01-2.69) 0.045 TRAM1 1.75 (1.02-3.01) 0.043 11 AKT1 0.77 (0.61-0.98) 0.030 IRF3 0.76 (0.59-0.98) 0.031 P38 1.67 (1.03-2.72) 0.039 12 IRF3 0.79 (0.64-0.97) 0.026 Mek1 1.25 (1.02-1.53) 0.032 JNK1 1.60 (1.03-2.48) 0.038 13 IRAKM 1.24 (1.04-1.47) 0.015 AKT1 1.25 (1.01-1.55) 0.037 JNK2 1.51 (1.04-2.20) 0.030 14 RIP1 1.38 (1.03-1.84) 0.029 AP1 0.68 (0.46-1.00) 0.049 15 TAK1 1.30 (1.03-1.64) 0.026 16 TRAM1 1.37 (1.01-1.86) 0.045 17 NFKB1 0.68 (0.47-1.00) 0.048 18 IL1RL1 0.84 (0.72-0.99) 0.031 19 MKK4 1.25 (1.00-1.55) 0.047