IRE1a-Induced FilaminA Phosphorylation Enhances Migration of Mesenchymal Stem Cells Derived from Multiple Myeloma Patients.

Multiple myeloma (MM) is an aggressive malignancy that shapes, during its progression, a pro-tumor microenvironment characterized by altered protein secretion and the gene expression of mesenchymal stem cells (MSCs). In turn, MSCs from MM patients can exert an high pro-tumor activity and play a strong immunosuppressive role. Here, we show, for the first time, greater cell mobility paralleled by the activation of FilaminA (FLNA) in MM-derived MSCs, when compared to healthy donor (HD)-derived MSCs. Moreover, we suggest the possible involvement of the IRE1a-FLNA axis in the control of the MSC migration process. In this way, IRE1a can be considered as a good target candidate for MM therapy, considering its pro-survival, pro-osteoclast and chemoresistance role in the MM microenvironment. Our results suggest that IRE1a downregulation could also interfere with the response of MSCs to MM stimuli, possibly preventing cell-cell adhesion-mediated drug resistance. In addition, further investigations harnessing IRE1a-FLNA interaction could improve the homing efficiency of MSC as cell product for advanced therapy applications.

Romidepsin-CHOEP followed by high-dose chemotherapy and stem-cell transplantation in untreated Peripheral T-Cell Lymphoma: results of the PTCL13 phase Ib/II study.

The standard treatment for young patients with untreated PTCLs is based on anthracycline containing-regimens followed by high-dose-chemotherapy and stem-cell-transplantation (HDT + SCT), but only 40% of them can be cured. Romidepsin, a histone-deacetylase inhibitor, showed promising activity in relapsed PTCLs; in first line, Romidepsin was added with CHOP. We designed a study combining romidepsin and CHOEP as induction before HDT + auto-SCT in untreated PTCLs (PTCL-NOS, AITL/THF, ALK-ALCL), aged 18-65 years. A phase Ib/II trial was conducted to define the maximum tolerated dose (MTD) of Ro-CHOEP, and to assess efficacy and safety of 6 Ro-CHOEP as induction before HDT. The study hypothesis was to achieve a 18-month PFS of 70%. Twenty-one patients were enrolled into phase Ib; 7 dose-limiting toxicities were observed, that led to define the MTD at 14 mg/ms. Eighty-six patients were included in the phase II. At a median follow-up of 28 months, the 18-month PFS was 46.2% (95%CI:35.0-56.7), and the 18-month overall survival was 73.1% (95%CI:61.6-81.7). The overall response after induction was 71%, with 62% CRs. No unexpected toxicities were reported. The primary endpoint was not met; therefore, the enrollment was stopped at a planned interim analysis. The addition of romidepsin to CHOEP did not improve the PFS of untreated PTCL patients.

Efficacy and toxicity of Decitabine in patients with acute myeloid leukemia (AML): A multicenter real-world experience.

BACKGROUND: The hypomethylating agent Decitabine (DAC) is a valuable treatment option in acute myeloid leukemia (AML), particularly in elderly patients (pts) not suitable for intensive chemotherapy (CHT). However, limited data are available about efficacy and safety of DAC in clinical practice. PATIENTS AND METHODS: We retrospectively reviewed data of 104 AML pts treated with DAC in eight Italian Hematological Centers from 2015 to 2017. The objective of this study was to evaluate the efficacy and safety of DAC in older AML pts outside of clinical trial. Seventy-five (75%) pts received DAC as first line treatment (Cohort 1) and 29 pts as salvage therapy (Cohort 2). All pts received a DAC schedule of 20 mg/sqm IV for 5-days, every 28 days. The median age was 72.5 years (74 in cohort 1 and 66 in cohort 2) and 16% of pts had an ECOG performance status >2 at the start of DAC treatment (with non-significant difference in the two cohorts). The cumulative illness rating scale (CIRS) was > 6 in 27% of pts. Forty-five pts (43%) had secondary AML. Bone marrow blast count was > 30% in 64% of patients (67/104). In the relapsed cohort 17/29 (59%) patients were treated with DAC after conventional CHT, 5/29 (17%) after allo-SCT and 7/29 (24%) after azacitidine therapy. RESULTS: A total of 469 DAC cycles were given to the 104 pts with a median of 3 cycles (range 1-21) and 45/104 (43%) pts received > 4 cycles. The Overall Response Rate (ORR = Complete Remission-CR plus Partial Remission-PR) was 33%, significantly higher in Cohort 1 (42%) compared to Cohort 2 (14%) (p = 0.009). The median duration of response was 6 months (range 1-20). In Cohort 1 the best response (CR or PR) was obtained between 3th and 6th cycle. In multivariate Cox regression analysis, achievement of CR or PR (HR = 0.78; p = 0.0004), CIRS < 6 (HR = 0.9; p = 0.04) and complex karyotype (HR = 0.8; p = 0.03) were significant predictors of better overall survival (OS). Median OS from the start of DAC therapy was 11 months for the whole population with a significant OS advantage in Cohort 1 (median OS 12.7 mths vs 6.3 mths; p = 0.003); median OS was significantly longer in responders compared to non-responders (22.6 mths vs 5.7 mths; p < 0.0001). At the last follow-up, 56 patients (54%) are still alive and 48 (46%) are dead (71% due to disease progression). The most common toxicities were myelosuppression and documented infectious complications that occurred mainly during the first 4 cycles. CONCLUSION: These data confirm the efficacy (ORR 33%) and the acceptable safety profile of DAC in the real life management of AML in elderly pts unsuitable for intensive CHT, with a significant better performance in first line therapy (ORR 42%, median OS 12.7 mths). The efficacy of DAC, both in first line and as salvage therapy, may probably be improved with combined treatment strategies and/or with different DAC schedules that could increase its anti-leukemic effect.

Rituximab plus bendamustine as front-line treatment in frail elderly (>70 years) patients with diffuse large B-cell non-Hodgkin lymphoma: a phase II multicenter study of the Fondazione Italiana Linfomi.

We conducted a phase II study to assess activity and safety profile of bendamustine and rituximab in elderly patients with untreated diffuse large B-cell lymphoma (DLBCL) who were prospectively defined as frail using a simplified version of the Comprehensive Geriatric Assessment (CGA). Patients had to be over 70 years of age, with histologically confirmed DLBCL. Frail patients were those younger than 80 years with a frail profile at CGA or older than 80 years with an unfit profile. Treatment consisted of 4-6 courses of bendamustine [90 mg/m2 days (d)1-2] and rituximab (375 mg/m2 d1) administered every 28 days. Other main study end points were complete remission rate and the rate of extra-hematologic adverse events. Forty-nine patients were enrolled of whom 45 were confirmed eligible. Overall, 24 patients achieved a complete remission (53%; 95%CI: 38-68%) and the overall response rate was 62% (95%CI: 47-76%). The most frequent grade 3-4 adverse event was neutropenia (37.8%). Grade 3-4 extra-hematologic adverse events were observed in 7 patients (15.6%; 95%CI: 6.5-29.5%); the most frequent was grade 3 infection in 2 patients. With a median follow up of 33 months (range 1-52), the median progression-free survival was ten months (95%CI: 7-25). The study shows promising activity and manageable toxicity profile of BR combination as first-line therapy for patients with DLBCL who are prospectively defined as frail according to a simplified CGA, as adopted in this trial (clinicaltrials.gov identifier: 01990144).

Management of elderly patients with diffuse large B-cell lymphomas.

Life expectancy has impressively increased over the past century and the US population over 65 years is rapidly growing, especially those over 80 years. In fact, persons older than 80 years have increased by more than 250% between the years 1960 and 2000, and it is expected that the population aged >75 years will triple by the year 2030. With the increase of the geriatric population, there is a need for the development and validation of treatment strategies for NHL for these patients. Therapy in elderly patients needs special attention because older patients usually suffered of several co-morbidities and their management represents a challenge for physicians. In fact, older patients treated for lymphoma may not tolerate the high-dose therapies used in younger patients and have increased risk of therapy-related toxicity as a result of age-related physiological changes and frequent co-morbidities. The widespread use of a comprehensive geriatric assessment tool might overcome the difficulty to run prospective clinical trial in elderly patients with lymphoma.

Cytological and histological detection of amyloid deposits in bone marrow of patients affected by multiple myeloma.

We recently published a study aiming to verify the frequency of amyloid deposits in the bone marrow of patients with multiple myeloma (MM) who did not present any signs or symptoms of systemic amyloidosis, applying the Congo red technique on bone marrow smears obtained by aspiration from the posterior iliac spine. The results suggested that nearly 40% of patients affected by MM may have amyloid deposits in their bone marrow. Subsequently, this finding has not been confirmed by another study performed with histological specimens of bone marrow in a similar clinical setting. To explain this discrepancy, we performed a comparative study on the bone marrows of 36 patients affected by MM, evaluated by both cytological and histological techniques. The results of this study confirm the high frequency of amyloid deposits in the bone marrow of patients affected by MM when the analysis is made on cytological smears, and indicate that the presence of amyloid on marrow smears is confirmed by core biopsies simultaneously performed in only 25% of cases. Should further studies confirm our findings, cytological assessment could be considered a sensitive technique to detect bone marrow amyloid deposits.

Low impact of cardiovascular adverse events on anagrelide treatment discontinuation in a cohort of 232 patients with essential thrombocythemia.

This retrospective study of the thrombocythemia Italian registry (RIT) documented that 71 (30.6%) out of 232 ET patients experienced 88 cardiovascular adverse events (CV-AEs) during anagrelide treatment (522 pt-y). The rate of CV-AEs was: 24.1% for palpitations, 4.3% for angina, 3.5% for arterial hypertension, 3.0% for congestive heart failure, 1.8% for arrhythmia, 0.9% for AMI, 0.4% for pericardial effusion. CV-AEs led to treatment discontinuation in nine (3.9%) patients, while in the remaining cases they were managed by pharmacological intervention and/or patient life style improvement. CV-AEs had no relationship with patient characteristics (including older age). A significant relationship was found only with a higher anagrelide induction dose. In the absence of any agreed protocol, a cardiovascular instrumental evaluation (CV-IE) was performed in 102 (44%) patients before commencement of anagrelide (with higher rate after the anagrelide/Xagrid EMA approval of 2004), and in 84 (36%) patients during treatment. Patients with and without CV-IEs, who resulted completely balanced for all their characteristics, did not significantly differ in the occurrence of CV-AEs. In conclusion, this study on ET patients treated with anagrelide shows that CV-AEs, equally distributed in younger and older subjects, were mostly mild and easily manageable, allowing safe treatment continuation in the majority of cases. Moreover, routinely performing a CV-IE did not appear to anticipate the occurrence of CV-AEs.

Outcome of 122 pregnancies in essential thrombocythemia patients: A report from the Italian registry.

Pregnancy is a high-risk event in women with essential thrombocythemia (ET). This observational study evaluated pregnancy outcome in ET patients focusing on the potential impact of aspirin (ASA) or interferon alpha (IFN) treatment during pregnancy. We retrospectively analyzed 122 pregnancies in 92 women consecutively observed in the last 10 years in 17 centers of the Italian thrombocythemia registry (RIT). The live birth rate was 75.4% (92/122 pregnancies). The risk of spontaneous abortion was 2.5-fold higher than in the control population (P < 0.01). ASA did not affect the live birth rate (71/93, 76.3% vs. 21/29, 72.4%, P = 0.67). However, IFN treatment during pregnancy was associated with a better outcome than was management without IFN (live births 19/20, 95% vs. 73/102, 71.6%, P = 0.025), and this finding was supported by multivariate analysis (OR: 0.10; 95% CI: 0.013-0.846, P = 0.034). The JAK2 V617F mutation was associated with a poorer outcome (fetal losses JAK2 V617F positive 9/25, 36% vs. wild type 2/24, 8.3%, P = 0.037), and this association was still significant after multivariate analysis (OR: 6.19; 95% CI: 1.17-32.61; P = 0.038). No outcome concordance between first and second pregnancies was found (P = 0.30). Maternal complications occurred in 8% of cases. In this retrospective study, in consecutively observed pregnant ET patients, IFN treatment was associated with a higher live birth rate, while ASA treatment was not. In addition, the JAK2 V617F mutation was confirmed to be an adverse prognostic factor.

Spectroscopical and mechanical characterization of normal and thalassemic red blood cells by Raman Tweezers.

In this work, the effects of thalassemia, a blood disease quite diffuse in the Mediterranean sea region, have been investigated at single cell level using a Raman Tweezers system. By resonant excitation of hemoglobin Raman bands, we have examined the oxygenation capability of beta-thalassemic erythrocytes. A reduction of this fundamental erythrocyte function has been found. The measurements have been performed on a significant number of red blood cells; the relative statistical analysis is presented. Moreover, the response to photo-induced oxidative stress of diseased cells with respect to the normal ones has been analyzed. Finally, the deformability of thalassemic erythrocytes has been quantified by measuring the membrane shear modulus by using a double-trap system: the measurements have revealed an increase in membrane rigidity of more than 40%, giving evidence that the genetic defect associated to thalassemia, which manly relies on hemoglobin structure, also strongly affects the erythrocyte mechanical properties. Our results demonstrate that the developed set-up may have potential for the monitoring of blood diseases and their response to drug therapies.

A methodology to study the deformability of red blood cells flowing in microcapillaries in vitro.

The deformability of red blood cells flowing in microvessels is essential to maintain optimal blood circulation and to allow gas transfer between blood and tissues. Here, we report on an experimental methodology to investigate the deformability of RBCs flowing in microcapillaries having diameter close to the average cell size. The microcapillaries are placed in a rectangular flow cell, where a suspension of RBCs, properly diluted in albumin-additioned ACD, is fed through a syringe under the action of a liquid head in the physiological range. Video microscopy images of the flowing RBCs are acquired at high magnification and later processed by an automated image analysis macro. It was found that RBCs from healthy donors exhibit the classical parachute shape observed in vivo. Furthermore, all the data of healthy RBC velocity vs liquid head are well represented by the same linear regression, independently on the donor. Preliminary results on beta-thalassemia RBCs are also presented and show, on the average, a reduced velocity compared to healthy samples.

Estimation of bulky lymph nodes by power Doppler ultrasound scanning in patients with Hodgkin's lymphoma: a prospective study.

The accuracy of standard methods in estimating bulky lesions requires validation. We used clinical/computed tomography (CT) evaluation and power Doppler ultrasound (US) to detect bulky disease in 137 consecutive Hodgkin's lymphoma patients, and analyzed the prognostic relevance of each method. Bulky disease was detected by clinical/CT evaluation in 47% of the patients and by power Doppler US in 20%. After treatment, at multivariate analysis power Doppler US-selected bulky disease was the parameter that best correlated with freedom from treatment failure (p<0.001). Power Doppler US, a readily available imaging technique, provides a better prognostic classification by detecting true bulky disease more accurately.

High number of circulating CD34+ cells in patients with myelophthisis.

Six patients with bone marrow micrometastases from solid cancers presented with increased numbers of circulating CD34+ cells; the CD34+ cell counts were very high in some cases. By contrast, no patient with metastatic cancer without bone marrow involvement showed raised numbers of circulating hemopoietic progenitors.

Interferon alfa treatment for pregnant women affected by essential thrombocythemia: case reports and a review.

OBJECTIVES: In the past essential thrombocythemia was considered a disease of the elderly. At present, the number of young people suffering from this disease is growing, with a slightly higher frequency in females. We investigated the effects of interferon alfa therapy in these patients. STUDY DESIGN: We describe 9 pregnancies in 4 women affected by essential thrombocythemia. RESULTS: Four pregnancies were carried out without interferon alfa therapy, and resulted in 2 intrauterine deaths, 1 spontaneous abortion, and 1 neonatal death. Interferon alfa was given during another 5 pregnancies; among them, 2 ended in preterm deliveries with normal infants, and 3 in full-term deliveries. The literature is reviewed. CONCLUSION: Our cases and published series suggest that fetal outcome is improved by therapy, and that interferon alfa may be the best therapeutic option.

Randomized comparison of power Doppler ultrasound-directed excisional biopsy with standard excisional biopsy for the characterization of lymphadenopathies in patients with suspected lymphoma.

PURPOSE: The sensitivity of lymph node excisional biopsy requires validation. Power Doppler ultrasound (US) helps predict the malignant status of lymphadenopathies. We used power Doppler US to select for biopsy the lymph node most suspected of malignancy. PATIENTS AND METHODS: One hundred fifty-two patients having lymphadenopathies with clinical suspicion of lymphoma were divided into two well-matched groups and randomly assigned to undergo either standard or power Doppler US-directed lymph node excisional biopsy. RESULTS: Histology showed a malignancy in 64% of patients in the standard group (lymphoma, 49 patients; carcinoma, two patients) and in 87% of patients in the US-assisted group (lymphoma, 62 patients; carcinoma, one patient). There were significantly fewer biopsy-related complications in the assisted group than in the standard group. During the follow-up of the patients with lymph nodes reported as being reactive, 14 of 29 patients in the standard group were rebiopsied and were found to have lymphoma (13 patients) or carcinoma at the subsequent lymph node histology, whereas none of the patients in the assisted group (nine patients) required a second biopsy. Thus, biopsy provided false-negative results for malignancy in 21% of patients affected by lymphoma in the standard group and never in the assisted group (P <.01). CONCLUSION: Power Doppler US is an accurate tool for screening lymphadenopathies to be removed by excisional biopsy in patients with suspected lymphoma.

Hepatitis B virus reactivation after fludarabine-based regimens for indolent non-Hodgkin's lymphomas: high prevalence of acquired viral genomic mutations.

BACKGROUND AND OBJECTIVES: Chemotherapy can cause hepatitis flare-up through viral reactivation in patients who have had contact with hepatitis viruses. Few data are available on the genotype of the reactivated viruses. DESIGN AND METHODS: In 40 consecutive adult patients with indolent non-Hodgkin's lymphoma (NHL) receiving fludarabine-based front-line chemotherapy, we performed a prospective study on viral hepatitis reactivation and analyzed the genotype of the reactivated viruses. Before chemotherapy, 4 patients were healthy carriers of hepatitis B surface antigen (HBsAg), 2 had HB core antigen antibodies (anti-HBc), 6 anti-HBs and 6 anti-HCV; 22 were seronegative. RESULTS: Hepatitis flare-up occurred in the 4 HBsAg-positive patients and in 1 anti-HBc-positive patient at a median of 1 month (range 1-4) after chemotherapy, when the CD4/CD8 ratio was still inverted. HBV reactivation was documented in all 5 instances (HBV-DNA 2-8 x 10(6) copies/mL). Two of the 5 patients responded to lamivudine, whereas 1 died of acute liver failure and 2 had persistent severe hepatitis. HBV genome sequencing at hepatitis flare-up showed that deviation from the closest related published sequences was 1.0% and 1.1% in the 2 lamivudine-responsive patients, and 1.5%, 1.8% and 1.7% in the 3 lamivudine-resistant patients. The polymerase open reading frame (ORF) and the HBs ORF of lamivudine-resistant strains contained several novel amino acid substitutions. INTERPRETATION AND CONCLUSIONS: These results suggest that fludarabine treatment of HBV-infected patients is frequently associated with acute hepatitis due to viral reactivation, and that lamivudine may be less effective in this situation than in other settings of immunocompromised hosts because of the emergence of resistant mutant strains.

Intestinal toxicity during induction chemotherapy with cytarabine-based regimens in adult acute myeloid leukemia.

BACKGROUND: Cytotoxic regimens used in induction treatments for acute myeloid leukemia (AML) almost always include standard or high-dose cytarabine (Ara-C). During or soon after induction therapy, leukemic patients frequently develop gastroenteric complications, characterized by abdominal pain and diarrhea. The association of these symptoms with fever and melena is typical of necrotizing enterocolitis (NE), a life-threatening condition that can be documented by ultrasound abdominal scan. PATIENTS AND METHODS: We analyzed retrospectively the clinical course of 169 adult patients with AML treated by standard dose Ara-C-containing induction regimens, either by continuous venous infusion (group 1) or subcutaneous injection (group 2). Ultrasonography was employed as early diagnostic tool in a majority of patients with gastroenteric complications. Bowel wall thickening was accurately measured and used to confirm the diagnosis of necrotizing enterocolitis. RESULTS: In the first group of 115 patients (median age, 51 years), gastroenteric complications were observed in 55 patients (48%), and 10 patients (9%) received diagnosis of NE, which was fatal in four. Patients with NE had a median age older than that of patients without gastroenteric symptoms, and a more prolonged neutropenia. In the second group of 54 patients (median age, 60 years), gastroenteric events were observed in 14 patients (26%), and no case of NE was recorded. CONCLUSIONS: This retrospective analysis shows that NE is a serious complication occurring mainly in patients treated by Ara-C administered as continuous i.v. infusion.

Spleen enlargement following recombinant human granulocyte colony-stimulating factor administration for peripheral blood stem cell mobilization.

BACKGROUND AND OBJECTIVES: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used to mobilize peripheral blood stem cells (PBSC) for autologous or allogeneic transplants. Such treatment may cause spleen enlargement; exceptionally, spontaneous spleen rupture has been reported. We investigated changes in spleen size during stem cell mobilization. DESIGN AND METHODS: We evaluated spleen size, comparing palpation with ultrasound (US)-evaluated longitudinal diameter and volume, in 13 healthy donors and 22 patients with a hematological malignancy who were undergoing PBSC mobilization with rhG-CSF-including regimens. RESULTS: Intraobserver and interobserver variability of US-calculated spleen volume was very low; the correlation between the volume calculated by US and that measured by 3-dimensional computed tomography was excellent. During mobilization, spleen enlargement was detected by palpation in 17% of subjects, by US-measured longitudinal diameter in 60%, and by US-calculated volume in 91%. The median increase in spleen volume was 300 mL (range, 54-820; p<0.001) in healthy donors and 135 mL (range, 0-413; p=0.004) in the group of patients; the enlargement correlated with white blood cell count elevation (p=0.016) but not with circulating CD34+ cells. One month after the last administration of rhG-CSF, the median decrease was 160 mL (range, 35-800) in healthy donors and 58 mL (range, 0-310) in patients. INTERPRETATION AND CONCLUSIONS: When evaluated by sensitive methods, rhG-CSF caused spleen enlargement in almost all individuals treated. US-calculated volume proved to be an excellent method, much better than longitudinal diameter, for detecting non-palpable splenomegaly induced by rhG-CSF.