Clonal hemopoiesis and risk of thrombosis in young female patients with essential thrombocythemia.

OBJECTIVE: Several studies demonstrated a high prevalence of nonrandom X-chromosome inactivation pattern (X-CIP) in essential thrombocythemia (ET). This study explored the incidence of clonal hemopoiesis in myeloid precursors and endogenous erythroid colonies (EECs) in ET patients and its correlation with thrombotic manifestations. MATERIALS AND METHODS: Clonal analysis of hemopoiesis using X-CIP was performed in 40 female patients with ET. Median age was 40.5 years (range 20-64), and median platelet count at testing time was 700 x 10(9)/L (range 220-1300 x 10(9)/L). Patients older than 65 years were excluded to reduce age-related skewing. Clonality was assessed on neutrophils, platelets, EECs, and bone marrow CD34(+) cells. RESULTS: Eight (20%) of 40 patients developed thrombosis mainly at diagnosis. Clonal hemopoiesis was found in 17 (42.5%) patients, 15 (37.5%) had polyclonal hemopoiesis, and 8 (20%) were considered uninterpretable due to constitutive skewing. Clonality was confirmed on purified CD34(+) subpopulations from bone marrow, documenting that clonality does not appear lineage-restricted. There were no statistical differences in age at diagnosis, median platelet count at testing time, and length of follow-up. Thrombotic episodes were significantly more frequent in the monoclonal group (p = 0.04, Fisher exact test). CONCLUSIONS: Young female patients with ET exhibiting a clonal pattern of hemopoiesis by X-CIP analysis are at higher risk for thrombosis. X-CIP analysis may contribute to defining the individual risk leading to appropriate treatment. X-CIP will allow a correct diagnosis in patients with latent myeloproliferative disorders and thrombosis in unusual sites. Clonal hemopoiesis is easily recognized by X-CIP, but its applicability is limited to the female sex and is hampered by the presence of age-related or constitutive skewing.

Molecular and clinical follow-up after stem cell transplantation for multiple myeloma.

Molecular follow-up has been carried out using immunoglobulin heavy-chain (IgH) gene finger-printing, a polymerase chain reaction (PCR)-based technique with a sensitivity of 0.1-0.01% (10(-3)-10(-4)), in 22 patients affected by multiple myeloma and submitted to stem cell transplantation (SCT). Twelve patients were submitted to either single or double autologous unselected peripheral blood progenitor cell transplantation, eight patients were submitted to autologous CD34+ immunoselected transplantation and two patients were submitted to allogeneic bone marrow (one patient) or peripheral blood CD34+ stem cell (one patient) transplantation. At diagnosis, all patients showed clonal CDIII rearrangement. The molecular analysis performed on leukapheresis products and CD34+ purified fractions proved to be contaminated by myeloma cells. During follow-up after autografting, all but one patient retained clonal rearrangement despite clinical complete remission (CR) in ten of them. These ten patients either relapsed (Rel) or showed progressive disease (PD) after transplantation; four of them died. Only one patient did not retain clonal rearrangement after autologous transplantation; she is currently alive in CR after a follow-up of 100 months. One patient submitted to allogeneic transplantation is currently alive with no evidence of the disease, but still retains clonal rearrangement after a follow-up of 47 months. Another patient died 4 months after transplantation after succumbing to fatal pneumonia showing myeloma progression.

MiCMA: an alternative treatment for refractory or recurrent Hodgkin's disease.

BACKGROUND: We determined the response rate to MiCMA (mitoxantrone, carboplatinum, methylprednisolone and aracytin) in a group of 29 patients with Hodgkin's disease (HD) and poor prognostic factors either resistant to first line or relapsing after conventional chemotherapy and subsequently evaluated the role of autologous stem-cell transplantation (ASCT) in these patients after MiCMA. PATIENTS AND METHODS: The treatment was intended as a brief tumor debulking program before ASCT. Twenty-nine patients with primary refractory HD or relapsed HD were submitted to two courses of MiCMA (mitoxantrone 10 mg/m2 day 1; carboplatinum 100 mg/m2 days 1-4; aracytin 2 g/m2 day 5; methylprednisolone 500 mg/m2 days 1-5) and subsequently evaluated for response. Those with responding or stable disease, received one or two other courses of MiCMA followed by ASCT. RESULTS: There were 10 complete responses (34% CR), 15 partial responses (52% PR) and 4 treatment failures with disease progression (14% PD). In total there were 25 evaluable responses out of 29 patients (86% CR + PR). Myelosuppression was the main toxicity of this treatment. At this time 20 patients (69%) are alive with a median follow-up of 26.5 months (7-100), 13 patients in CR (45%), 8 patients died, 7 of them from disease progression and one due to multi-organ failure, one patient is lost to follow-up. All but one of the patients who achieved CR after MiCMA are alive. Only the number of extranodal sites was found to predict a poor response to MiCMA. CONCLUSIONS: A short pre-transplantation treatment with MiCMA is an effective tumor debulking approach in patients with refractory or relapsed HD.

Autologous graft-versus-host disease after CD34+-purified autologous peripheral blood progenitor cell transplantation.

Autologous graft-versus-host disease (GVHD) has been frequently reported after cyclosporine A (CsA) administration in the autologous setting. This complication is related to the disruption of self-tolerance mechanisms induced by CsA and may exert an antitumor effect. We report the spontaneous occurrence of autologous GVHD after CD34+-purified peripheral blood progenitor cell transplantation (PBPCT) in 5 out of 24 consecutive patients (20.8%). The syndrome was characterized by skin rash (5/5), pruritus (5/5), eosinophilia (5/5), and fever (2/5) occurring at a median of 37 days (range 22-60) after transplantation. Diagnosis was confirmed by skin biopsy in all patients. The syndrome was self-limiting, lasted a median of 25 days, and did not require treatment. The rate of autologous GVHD was high after CD34+-purified autologous PBPCT. In fact, no autologous GVHD was documented in an historical control of 100 consecutive patients submitted to unmanipulated PBPCT at the same institution. The manipulation of the graft by the purging procedure causes a profound T lymphocyte depletion, thus possibly perturbing the equilibrium between autoregulatory cells and autocytotoxic T cells. These observations add new interest to the antitumor efficacy of autologous GVHD and suggest new questions regarding the role of transplantation for autoimmune diseases.

Inappropriate antidiuretic hormone secretion after high-dose thiotepa.

High-dose thiotepa has been successfully included in a variety of conditioning regimens for stem cell transplantation in hematological and solid neoplasms. Toxicity of high-dose thiotepa mainly manifests as profound myeloablation and some degree of liver damage. We report a case of inappropriate secretion of antidiuretic hormone (SIADH) in a patient with primary CNS lymphoma who underwent therapy with high-dose thiotepa.

Optimal timing of G-CSF administration after CD34+ immunoselected peripheral blood progenitor cell transplantation.

G-CSF accelerates neutrophil recovery after autologous peripheral blood progenitor cell transplantation (aPBPCT), although the optimal timing for its administration is currently unknown. In order to establish the role and the optimal timing of administration of G-CSF after immunoselected CD34+ aPBPCT, we analyzed the data from 21 consecutive patients affected by haematological malignancies. Patients were randomized into three groups according to G-CSF administration after transplantation: day +1 (group B); day +7 (group C) or no G-CSF (group A). Serum G-CSF level was evaluated until engraftment. The CD34+ cell dose reinfused was similar (P = 0.48). G-CSF significantly reduced time to recovery of PMN >0.5 x 10(9)/l (11 vs 14 vs 20.5 days) (P= 0.00046); >1.0 x 10(9)/l (12 vs 15 vs 22) (P = 0.001). No difference was observed in the number of days with PMN <0.1 x 10(9)/l (5.5 vs 7 vs 8 days). Platelet count >50 x 10(9)/l and >100 x 10(9)/l, reticulocytes >1%, length of hospitalization, non-prophylactic antibiotic therapy, fever, incidence of sepsis and transfusion support did not differ. Early or delayed G-CSF after immunoselected CD34+ aPBPCT significantly accelerated PMN recovery but did not reduce the amount of supportive treatment or the duration of hospitalization. Delaying the initiation of G-CSF did not reduce the length of treatment (11.5 vs 12 days). Early or delayed G-CSF administration resulted in G-CSF peak serum levels 7 (early)-12 (delayed)-fold greater than an endogenous response to neutropenia.

Expression of p15INK4B in normal hematopoiesis.

The cyclin-dependent kinase inhibitor (CDKI) p15INK4B (p15) induces cell cycle arrest in G0/G1 phase. Several studies report deletion or transcriptional loss of the p15 gene in myeloid and lymphoid hematological malignancies, and a possible role as a tumor suppressor gene has been proposed for this CDKI. In this study we evaluated the expression of p15 by cytofluorometric, immunohistochemical, and reverse transcriptase-polymerase chain reaction (RT-PCR) methods in CD34+ progenitors (both during steady state and after chemotherapy and/or granulocyte-colony stimulating factor [G-CSF] administration) and in cells belonging to different hematopoietic differentiative lineages. We found that p15 is not expressed in normal G0/G1-arrested peripheral blood (PB)- or bone marrow (BM)-CD34+ cells. Moreover, p15 is expressed in G0/G1-blocked CD34+ cells mobilized by chemotherapy and G-CSF but not in CD34+ cells mobilized by G-CSF alone. To clarify the role of p15 in normal hematopoiesis, we used flow cytometry to investigate its expression in normal differentiating BM and PB cells. We found that p15 was expressed in cells belonging to the granulocyte-monocyte lineage and in B and T lymphocytes, whereas erythroid and megakaryocytic cells were p15 negative. These findings, which were confirmed both by immunohistochemical and RT-PCR analysis, definitely establish a linkage between p15 expression and granulocyte-monocyte differentiation.

Differential sensitivity of leukemic and normal hematopoietic progenitors to the killing effect of hyperthermia and quercetin used in combination: role of heat-shock protein-70.

Autologous bone-marrow transplantation (ABMT) is widely used in the treatment of acute leukemias where a matched sibling donor is not available for allogeneic transplantation. However, a major problem in ABMT is relapse, and ex vivo purging may be very important in preventing it. We show here that quercetin enhances the growth-inhibitory effect of hyperthermia (HT) in AML (19 cases) and ALL (6 cases) leukemic blasts. Furthermore, the inhibitory effect of this combined treatment resulted in leukemic-cell apoptosis. On the contrary, normal hematopoietic progenitors were neither growth-inhibited nor induced to apoptosis by HT-plus-quercetin treatment. To explain this difference in sensitivity of leukemic and normal hematopoietic progenitors, we analyzed the effect of quercetin on heat-induced expression of heat-shock protein-70 (HSP-70), which has been shown to be important in regulating thermosensitivity. We found that quercetin inhibits heat-induced HSP-70 expression both at protein and at mRNA levels in AML and ALL blasts. In normal CD34+ progenitors, the combined treatment with HT and quercetin did not reduce HSP-70 expression and did not induce cell apoptosis. Considering the difference in heat sensitivity of normal CD34+ and leukemic progenitors in the presence of quercetin, the combined use of HT and quercetin could constitute a purging protocol for ABMT.

Comparative analysis of prognostic indicators of aspergillosis in haematological malignancies and HIV infection.

The objective of this study was to identify the prognostic factors influencing the outcome of aspergillosis in two models of immunodeficiency, namely haematological malignancies and HIV infection. The study is based on a 5 year prospective logistic regression analysis of risk factors, clinical features, radiological findings and therapy affecting the prognosis of aspergillosis in 43 patients, i.e. 27 haematological neoplastic patients (group A) and 16 HIV infected patients (group B). Univariate analysis indicated that neutropenia (P = 0.02), haemoptysis (P = 0.03) and concomitant AIDS (P = 0.02), negatively influenced the prognosis of aspergillosis. Comparing the two groups of patients, significant differences emerged in the prognostic indicators. In particular respiratory failure (P = 0.02) and radiological bilateral involvement of the lungs were associated with a poor prognosis in group A (P = 0.04) and low (2100/mm3) T CD4+ cell count in group B (P = 0.02). At variance, a better prognosis was documented in patients treated with sequential therapy (amphotericin B and itraconazole) only within the group of haematological patients (P = 0.003). On multivariate analysis sequential therapy (P = 0.01) and AIDS (P = 0.03) were independent prognostic indicators of aspergillosis. In conclusion, our prospective study indicates that aspergillosis, although an uncommon event in patients with HIV infection, has a more severe prognosis in comparison to haematological patients. Future prospective clinical trials are necessary to confirm the real importance of the sequential therapy, with amphotericin B and itraconazole, in patients with aspergillosis.

Localization of aspergillosis to the central nervous system among patients with acute leukemia: report of 14 cases. Gruppo Italiano Malattie Ematologiche dell'Adulto Infection Program.

We retrospectively studied a consecutive series of 100 patients with acute leukemia and aspergillosis to evaluate the clinical findings and risk factors for colonization of the central nervous system (CNS) by Aspergillus species. The diagnosis of CNS aspergillosis was made in 14 patients on the basis of the following criteria: neurological signs of CNS involvement (13 of 14 patients); cerebral CT scan findings (9 of 12); microbiological findings (6 of 12); and histological findings at autopsy (11 of 11). The majority of patients had severe neurological complications (i.e., hemiparesis or seizures), due mainly to brain abscesses or mycetomas. Autopsies were performed on 11 of 14 patients and provided evidence that CNS localization was secondary to invasive aspergillosis; in each case, the most likely primary focus of infection was the lung. Although all patients had received oral antimycotic prophylaxis and had received timely empirical antifungal treatment, they all died within a median time of 5 days from the onset of neurological symptoms. Analysis of the characteristics of patients with invasive aspergillosis did not reveal any difference between those with CNS localization and those without CNS localization.

Salvage chemotherapy with pentostatin in prolymphocytic leukemia.

We report 4 cases of prolymphocytic leukemia (PLL) resistant to conventional chemotherapy that were treated with pentostatin. We obtained 1 complete remission (CR) and 2 partial remissions (PR). Our data confirm the effectiveness of this drug in the treatment of prolymphocytic leukemia.