Pietro Leoni

Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia

BACKGROUND Allogeneic or autologous bone marrow transplantation and intensive consolidation chemotherapy are used to treat acute myelogenous leukemia in a first complete remission. METHODS After induction treatment with daunorubicin and cytarabine, patients who had a complete remission received a first course of intensive consolidation chemotherapy, combining intermediate-dose cytarabine and amsacrine. Patients with an HLA-identical sibling were assigned to undergo allogeneic bone marrow transplantation; the others were randomly assigned to undergo autologous bone marrow transplantation (with unpurged bone marrow) or a second course of intensive chemotherapy, combining high-dose cytarabine and daunorubicin. Comparisons were made on the basis of the intention to treat. RESULTS A total of 623 patients had a complete remission; 168 were assigned to undergo allogeneic bone marrow transplantation, and 254 were randomly assigned to one of the other two groups. Of these patients, 343 completed the treatment assignment: 144 in the allogeneic-transplantation group, 95 in the autologous-transplantation group, and 104 in the intensive-chemotherapy group. The relapse rate was highest in the intensive-chemotherapy group and lowest in the allogeneic-transplantation group, whereas the mortality rate was highest after allogeneic transplantation and lowest after intensive chemotherapy. The projected rate of disease-free survival at four years was 55 percent for allogeneic transplantation, 48 percent for autologous transplantation, and 30 percent for intensive chemotherapy. However, the overall survival after complete remission was similar in the three groups, since more patients who relapsed after a second course of intensive chemotherapy had a response to subsequent autologous bone marrow transplantation. Other differences were also observed, especially with regard to hematopoietic recovery (it occurred later after autologous transplantation) and the duration of hospitalization (it was longer with bone marrow transplantation). CONCLUSIONS Autologous as well as allogeneic bone marrow transplantation results in better disease-free survival than intensive consolidation chemotherapy with high-dose cytarabine and daunorubicin. Transplantation soon after a relapse or during a second complete remission might also be appropriate.

Itraconazole Oral Solution as Prophylaxis for Fungal Infections in Neutropenic Patients with Hematologic Malignancies: A Randomized, Placebo-Controlled, Double-Blind, Multicenter Trial

To evaluate the efficacy and safety of itraconazole oral solution for preventing fungal infections, a randomized, placebo-controlled, double-blind, multicenter trial was conducted: 405 neutropenic patients with hematologic malignancies were randomly assigned to receive either itraconazole, 2.5 mg/kg every 12 hours (201 patients), or placebo (204 patients). Proven and suspected deep fungal infection occurred in 24% of itraconazole recipients and in 33% of placebo recipients, a difference of 9 percentage points (95% confidence interval [CI], 0.6% to 22.5%; P = .035). Fungemia due to Candida species was documented in 0.5% of itraconazole recipients and in 4% of placebo recipients, a difference of 3.5 percentage points (95% CI, 0.5% to 6%; P = .01). Deaths due to candidemia occurred in none of the itraconazole recipients compared with 4 placebo recipients, a difference of 2 percentage points (95% CI, 0.05% to 4%; P = .06). Aspergillus infection was documented in four itraconazole recipients (one death) and one placebo recipient (one death). Side effects causing drug interruption occurred in 18% of itraconazole recipients and 13% of placebo recipients. Itraconazole oral solution was well-tolerated and effectively prevented proven and suspected deep fungal infection as well as systemic infection and death due to Candida species.

Dasatinib as first-line treatment for adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia

Dasatinib is a potent BCR-ABL inhibitor effective in chronic myeloid leukemia and Ph(+) acute lymphoblastic leukemia (ALL) resistant/intolerant to imatinib. In the GIMEMA LAL1205 protocol, patients with newly diagnosed Ph(+) ALL older than 18 years (with no upper age limit) received dasatinib induction therapy for 84 days combined with steroids for the first 32 days and intrathecal chemotherapy. Postremission therapy was free. Fifty-three patients were evaluable (median age, 53.6 years). All patients achieved a complete hematologic remission (CHR), 49 (92.5%) at day 22. At this time point, 10 patients achieved a BCR-ABL reduction to < 10(-3). At 20 months, the overall survival was 69.2% and disease-free survival was 51.1%. A significant difference in DFS was observed between patients who showed at day 22 a decrease in BCR-ABL levels to < 10(-3) compared with patients who never reached these levels during induction. In multivariate analysis, BCR-ABL levels of < 10(-3) at day 85 correlated with disease-free survival. No deaths or relapses occurred during induction. Twenty-three patients relapsed after completing induction. A T315I mutation was detected in 12 of 17 relapsed cases. Treatment was well tolerated; only 4 patients discontinued therapy during the last phase of the induction when already in CHR. In adult Ph(+) ALL, induction treatment with dasatinib plus steroids is associated with a CHR in virtually all patients, irrespective of age, good compliance, no deaths, and a very rapid debulking of the neoplastic clone.

Imatinib for refractory chronic graft-versus-host disease with fibrotic features

We previously reported that patients with fibrotic, chronic graft-versus-host disease (cGVHD) have antibodies activating the platelet-derived growth factor receptor pathway. Because this pathway can be inhibited by imatinib, we performed a pilot study including 19 patients with refractory cGVHD, given imatinib at a starting dose of 100 mg per day. All patients had active cGVHD with measurable involvement of skin or other districts and had previously failed at least 2 treatment lines. Patient median age was 29 years (range, 10-62 years), and median duration of cGvHD was 37 months (range, 4-107 months). The organs involved were skin (n = 17), lung (n = 11), and bowel (n = 5); 15 patients had sicca syndrome. Imatinib-related, grade 3 to 4 toxicity included fluid retention, infections, and anemia. Imatinib was discontinued in 8 patients: in 3 because of toxicity and in 5 because of lack of response (n = 3) or relapse of malignancy (n = 2). Overall response rate at 6 months was 79%, with 7 complete remissions (CRs) and 8 partial remissions (PRs). With a median follow-up of 17 months, 16 patients are alive, 14 still in CR or PR. The 18-month probability of overall survival is 84%. This study suggests that imatinib is a promising treatment for patients with refractory fibrotic cGVHD.

Health-related quality of life in chronic myeloid leukemia patients receiving long-term therapy with imatinib compared with the general population

The main objective of this study was to investigate whether patients with chronic myeloid leukemia (CML) in treatment with long-term therapy imatinib have a different health-related quality-of-life (HRQOL) profile compared with the general population. In total, 448 CML patients were enrolled, and the SF-36 Health Survey was used to compare generic HRQOL profiles. Symptoms were also assessed. HRQOL comparisons were adjusted for key possible confounders. The median age of patients was 57 years and the median time of imatinib treatment was 5 years (range 3-9 years). The largest HRQOL differences were found in younger patients. In particular, patients aged between 18 and 39 years had marked impairments in role limitations because of physical and emotional problems, respectively: -22.6 (P < .001), -22.3 (P < .001). Patients with CML age 60 or older had a HRQOL profile very similar to that reported by the general population. Women had a worse profile than men when each were compared with their peers in the general population. Fatigue was the most frequently reported symptom. The HRQOL of CML patients is comparable with that of population norms in many areas, however, younger and female patients seem to report the major limitations.

Human Dedifferentiated Adipocytes Show Similar Properties to Bone Marrow‐Derived Mesenchymal Stem Cells

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. Here, we studied the gene expression and functional properties of mature adipocytes isolated from human omental and subcutaneous fat tissues. We also focused on dedifferentiated adipocytes in culture and their morphologies and functional changes with respect to mature adipocytes, stromal‐vascular fraction (SVF)‐derived mesenchymal stem cells (MSCs) and bone marrow (BM)‐derived MSCs. Isolated mature adipocytes expressed stem cell and reprogramming genes. They replicated in culture after assuming a fibroblast‐like shape and expanded similarly to SVF‐ and BM‐derived MSCs. During the dedifferentiation process, mature adipocytes lost their lineage gene expression profile, assumed the typical mesenchymal morphology and immunophenotype, expressed stem cell genes and differentiated into multilineage cells. Moreover, during the dedifferentiation process, we showed changes in the epigenetic status of mature adipocytes, which led dedifferentiated adipocytes to display a similar DNA methylation condition to BM‐derived MSCs. Like SVF‐ and BM‐derived MSCs, dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in coculture while mature adipocytes stimulated their growth. Furthermore, dedifferentiated adipocytes maintained the survival and complete differentiation characteristic of hematopoietic stem cells. This is the first study that in addition to characterizing isolated and dedifferentiated adipocytes also reports on the immunoregulatory and hematopoietic supporting functions of these cells. This structural and functional characterization might have clinical applications of both mature and dedifferentiated adipocytes in such fields, as regenerative medicine. STEM CELLS 2012;30:965–974

Targeting Mutant BRAF in Relapsed or Refractory Hairy-Cell Leukemia

BACKGROUND BRAF V600E is the genetic lesion underlying hairy-cell leukemia. We assessed the safety and activity of the oral BRAF inhibitor vemurafenib in patients with hairy-cell leukemia that had relapsed after treatment with a purine analogue or who had disease that was refractory to purine analogues. METHODS We conducted two phase 2, single-group, multicenter studies of vemurafenib (at a dose of 960 mg twice daily)--one in Italy and one in the United States. The therapy was administered for a median of 16 weeks in the Italian study and 18 weeks in the U.S. study. Primary end points were the complete response rate (in the Italian trial) and the overall response rate (in the U.S. trial). Enrollment was completed (28 patients) in the Italian trial in April 2013 and is still open (26 of 36 planned patients) in the U.S. trial. RESULTS The overall response rates were 96% (25 of 26 patients who could be evaluated) after a median of 8 weeks in the Italian study and 100% (24 of 24) after a median of 12 weeks in the U.S. study. The rates of complete response were 35% (9 of 26 patients) and 42% (10 of 24) in the two trials, respectively. In the Italian trial, after a median follow-up of 23 months, the median relapse-free survival was 19 months among patients with a complete response and 6 months among those with a partial response; the median treatment-free survival was 25 months and 18 months, respectively. In the U.S. trial, at 1 year, the progression-free survival rate was 73% and the overall survival rate was 91%. Drug-related adverse events were usually of grade 1 or 2, and the events most frequently leading to dose reductions were rash and arthralgia or arthritis. Secondary cutaneous tumors (treated with simple excision) developed in 7 of 50 patients. The frequent persistence of phosphorylated ERK-positive leukemic cells in bone marrow at the end of treatment suggests bypass reactivation of MEK and ERK as a resistance mechanism. CONCLUSIONS A short oral course of vemurafenib was highly effective in patients with relapsed or refractory hairy-cell leukemia. (Funded by the Associazione Italiana per la Ricerca sul Cancro and others; EudraCT number, 2011-005487-13; ClinicalTrials.gov number NCT01711632.).

Characterization and expansion of mesenchymal progenitor cells from first-trimester chorionic villi of human placenta

BACKGROUND Mesenchymal stromal cells (MSC) have been identified in a variety of fetal and adult tissues, including bone marrow (BM), fetal blood and liver. We report on the isolation, expansion and differentiation in vitro of MSC-like cells from chorionic villi (CV). METHODS We evaluated 10 samples of CV collected at the first trimester (gestational age 11-13 weeks). We only used cells taken from back-up culture after a successful karyotype analysis. CV cells were characterized by morphologic, immunophenotypic and molecular analysis. The differentiation ability of mesenchymal and neural lineages was detected using specific culture conditions. Cell expansion was assessed after plating cells at different densities in different media, supplemented with animal and human serum. RESULTS CV cells showed a homogeneous population of spindle-shaped cells after the first passage. Cells expressed CD90, CD105, CD73, CD44, CD29 and CD13 but not CD45, CD14, CD34 and CD117. They expressed Oct-4, Rex-1, GATA-4 and nestin, which characterize the undifferentiated stem cell state. They differentiated into osteocytes, adipocytes, chondrocytes and neuronal cells. Cell expansion was greater than that of adult BM-derived MSC, 9 logs with fetal bovine serum and 6 logs with human serum. Despite their high proliferative capacity, we did not observe any karyotypic abnormalities after culture. DISCUSSION Our study shows that CV cells have better potential for expansion than adult stem cells. They can proliferate in a medium with human allogeneic serum and can differentiate into mesenchymal and neural lineages. CV cells may be an excellent cell source for therapeutic applications.

Infectious complications after autologous peripheral blood progenitor cell transplantation followed by G-CSF

Infectious complications after autologous peripheral blood progenitor cell transplantation (PBPCT) have been reported in a few studies including small patient numbers. The present study was performed to assess the incidence, types, outcome and factors affecting early and late infections in 150 patients aged 18 to 68 years (median 46.5) who underwent high-dose therapy, with G-CSF. Patients were kept in reverse isolation rooms and received antimicrobial chemoprophylaxis with oral quinolone and fluconazole. One hundred and fifteen patients (76.7%) developed fever (median 3 days, range 1–29); 20 patients (55.5%) had Gram-positive and 13 (36.2%) Gram-negative bacterial infections. There were no fungal infections or infection-related deaths. Mucositis grade II–IV (P = 0.0001; odds ratio 3.4) and >5 days on ANC<100/μl (P = 0.0001; odds ratio 2.3) correlated with development of infection. Only days with ANC <100/μl affected infection outcome (P = 0.0024) whereas the antibiotic regimen did not. After day +30 there were four cases of bacterial pneumonitis (2.7%), one case of fatal CMV pneumonia (0.8%) and 20 of localized VZV infection (13.3%). Reduction of neutropenia duration with PBPCT and G-CSF is not enough to prevent early infectious complications since only a few days of severe neutropenia and mucositis are related to development of early infections. However, no infection-related deaths were seen. Although Gram-positive organisms were the major cause of bacteremia, a glycopeptide in the empirical antibiotic regimen did not affect infection outcome. In PBPCT recipients, early and late opportunistic infections were notably absent, which was at variance with what was seen with bone marrow recipients. Efforts should be made to prevent mucositis and neutropenia and identify new strategies of antibacterial prophylaxis.

Human Mesenchymal Stem Cells from Chorionic Villi and Amniotic Fluid are not Susceptible to Transformation after Extensive in Vitro Expansion

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering. Increasing evidence suggests that MSCs isolated from fetal tissues are more plastic and grow faster than adult MSCs. In this study, we characterized human mesenchymal progenitor cells from chorionic villi (CV) and amniotic fluid (AF) isolated during the first and second trimesters, respectively, and compared them with adult bone marrow-derived MSCs (BM). We evaluated 10 CV, 10 AF, and 6 BM samples expanded until the MSCs reached senescence. We used discarded cells from prenatal analyses for all the experiments. To evaluate the replicative stability of these cells, we studied the telomerase activity, hTERT gene transcription, and telomere length in these cells. Spontaneous chromosomal alterations were excluded by cytogenetic analysis. We studied the expression of c-myc and p53, tumor-associated genes, at different passage in culture and the capacity of these cells to grow in an anchorage-independent manner by using soft agar assay. We isolated homogeneous populations of spindle-shaped CV, AF, and BM cells expressing mesenchymal immunophenotypic markers throughout the period of expansion. CV cells achieved 14 ± 0.9 logs of expansion in 118 days and AF cells achieved 21 ± 0.9 logs in 118 days, while BM cells achieved 11 × 0.4 logs in 84 days. Despite their high proliferation capacity, fetal MSCs showed no telomerase activity, no hTERT and c-myc transcriptions, and maintained long, stable telomeres. A constant expression level of p53 and a normal karyotype were preserved throughout long-term expansion, suggesting the safety of fetal MSCs. In conclusion, our results indicate that fetal MSCs could be an alternative, more accessible resource for cell therapy and regenerative medicine.