Rosella Matera

High risk of early resistant relapse for leukaemic patients with presence of multidrug resistance associated P-glycoprotein positive cells in complete remission

Summary. The immunocytochemical detection of multidrugresistance (MDR) associated P‐glycoprotein (P‐170) was longitudinally performed on bone marrow smears from 32 responsive patients with acute leukaemia in the different phases of the disease (at diagnosis, in complete remission, at relapse) by means of APAAP technique and monoclonal antibody C219.

Clinical results of recombinant erythropoietin in transfusion-dependent patients with refractory multiple myeloma: role of cytokines and monitoring of erythropoiesis

Abstract:  Recombinant erythropoietin (r‐EPO) was administered to 37 patients with advanced, transfusion‐dependent and chemo‐resistant multiple myeloma (MM), at the fixed dose of 10,000/U s.c, 3 times a week, for 2 months. Thirteen patients (35.1%) achieved a significant response in terms of complete abolition of red cell transfusions. Factors significantly predictive of response were: a) inappropriate production of endogenous EPO, as expressed by a reduced observed/predicted ratio; b) presence of a consistant number of circulating erythroid precursors BFU–E; c) low serum levels of tumor necrosis factor (TNF) and interleukin‐1 (IL‐1), cytokines with inhibitory activity on erythropoiesis; d) a single line of previously received chemotherapy. Renal failure, bone marrow plasma cell infiltration, serum levels of IL‐6 and other main clinical and laboratory parameters did not affect significantly the response to r‐EPO. High fluorescence reticulocytes (HFR) and soluble transferrin receptor (sTfR) values were useful to detect an early stimulation of erythropoiesis in responders, while a high percentage of circulating hypochromic erythrocytes (HE), as assessed by an automated counter, identified those patients developing functional iron deficiency during r‐EPO treatment. We conclude that about one‐third of severely anemic patients with advanced MM, unresponsive to chemotherapy, may benefit by r‐EPO therapy. The clinical management of these patients can be accomplished using non‐invasive parameters, such as sTfR, HFR and HE.

Predictive parameters for mobilized peripheral blood CD34+ progenitor cell collection in patients with hematological malignancies

In order to investigate what is the best single parameter to predict the leukapheretic yield of circulating CD34+ progenitor cells, we retrospectively analyzed data from 68 patients with hematological malignancies who underwent mobilizing therapy. Three main parameters were monitored: total white blood cell (WBC), CD34+ cells, and monocyte counts in peripheral blood (PB) at the same day and at the preceding day of the apheretic procedure. Linear regression analysis revealed a strong correlation between CD34+ cell value in PB just before harvest and the number of CD34+ cells collected (P < 0.0001), but not at the preceding day. Monocyte PB concentration and absolute WBC count did not correlate with CD34+ cells harvested, at the preceding day of leukapheresis as well as at the same day of the procedure. The number of CD34+ cells in mobilized PB at the same day of harvest evidenced a very good capacity of predicting the value of harvested CD34+ cell number after collection, while WBC and monocyte count displayed quite a wide dispersion of results. In particular, an amount greater than 50/μL of circulating CD34+ cells ensured the best collections. Finally, CD34+ and CFU‐GM content evaluated for each apheresis showed a strong reciprocal correlation (r 0.78; P < 0.0001). We conclude that the absolute number of CD34+ cells at the day of leukapheresis is the only parameter for identifying the exact timing for apheresis and predicting the amount of peripheral blood progenitor cells (PBPCs) that will be collected. In this setting, WBC and monocyte counts, at the day of collection or at the preceding day, are not useful tools. Am. J. Hematol. 58:255–262, 1998.

Minimally differentiated acute myeloid leukemia (AML M0): clinico-biological findings of 29 cases.

Twenty-nine cases of minimally differentiated acute myeloid leukemia or AML MO identified among 441 AML diagnosed in the last 12 years are reported. In all cases, flow cytometric analysis using a large panel of monoclonal antibodies and cytogenetic and molecular studies (IgH, TcRβ, BCR/ABL, AML1/ETO and CBFB-MYH11 rearrangements) were performed. Of the 29 patients, 27 were treated with intensive chemotherapy based on GIMEMA protocols. We noted a greater incidence of older (over 60 years) and male patients (52% and 65%, respectively). CD33, CD13, CD7 and TdT were expressed in 79.3%, 82.7%, 58.6% and 42.8% of cases, respectively. Antigenic MPO was present in 17 of 22 cases (77.3%). Most cases expressed CD34 (93.1%), HLA-DR (93.1%), CD117 (80%) and CD45RA (87%). CD45RO and CD90 were consistently negative. In all cases, we observed an up-expression of bcl-2 and a down-expression of CD95 with an inverse trend between the two markers (r -5253; p 0.03). Karyotypic abnormalities were demonstrated in 53.6% of cases. Of these, 6 involved chromosomes 5, 7 and 8, t(9;22), confirmed by the BCR/ABL transcript, was detected in one case. Rearrangements of the TcRβ and IgH chains were observed in 3 and 2 cases, respectively. No AML1/ETO and CBFB-MYH 11 transcripts were found. Twelve out of 27 patients (44%) achieved a complete remission (CR) (in 2 cases after rescue therapy). Seven early (range 1–9 months) and one late (32 months) relapses were observed. Five patients are alive, but only the 4 who underwent bone marrow transplantation are in persistent first CR. In conclusion, AML MO is a subtype of AML antigenically well detectable, endowed with many adverse parameters (older age, TdT and CD34 expression, resistance to apoptosis, unfavorable cytogenetic abnormalities) and poor prognosis. A very aggressive consolidation treatment can be useful to improve the outcome.

Clinical relevance of immunocytochemical detection of multidrug-resistance-associated P-glycoprotein in hematologic malignancies.

P-glycoprotein (P-170) is the phenotypic marker of tumoral cells that show the phenomenon of multidrug resistance (MDR). Using an immunocytochemical approach, we employed the monoclonal antibody C219 (which recognizes an epitope of such a glycoprotein) to evaluate in cytologic samples the expression of P-170 on neoplastic cells from 52 patients affected by different hematologic malignancies and its eventual correlation to clinical outcome. Longitudinal studies were also performed in 14 patients. Results obtained demonstrated that a) the so-called « MDR phenotype » may be heterogeneously represented (from « 1 to 100% of positive cells) in hemopoietic tumors at diagnosis (without exposure to pharmacologic agents), as well as during the course of the disease, although a more substantial presence of P-170 occurred in treated patients. There was no correlation between neoplastic kinetic activity (such as expression of Ki 67 recognized nuclear proliferation-associated antigen) and P-170-positive cells. b) Percentage of positive cells as well as intensity of staining seemed to be important in determining MDR; in general, there was a strong correlation between expression of P-170 in more than 20% of neoplastic cells and a lack of response to chemotherapy. However, some false-positive and false-negative cases were observed. c) The detection of scattered P-170-positive cells may predict a pharmacologic selection of intrinsic or mutant-resistant clones.

Prevention of high-dose melphalan-induced mucositis by cryotherapy.

Oral mucositis is a frequent and significant sideeffect of both chemotherapy and radiation treatment. It is an especially severe problem in the setting of patients undergoing high-dose chemotherapy (HDC) followed by peripheral blood progenitor cell transplantation. In fact, in the presence of neutropenia, severe mucositis predisposes patients to infections. Rapaport et al. [1] reported that a higher peak of oral mucositis and the duration of parenteral nutrition support are correlated with the occurrence of bacteremias and transplant-related mortality; a relationship between oral mucositis and hepatic veno-occlusive disease has been reported by Wingard et al. [2]. Furthermore, the management of infectious complications and mucositis-related pain result in increased hospitalization and increased cost [3]. There are many grading scales for diagnosing oral mucositis and assessing its severity. The most commonly used is that of the World Health Organization, which is similar to that of the National Cancer Institute and the Eastern Cooperative Oncology Group [4 – 6]. Recently, a new scoring system, the Oral Mucosal Assessment Score, was developed and validated in a large study [3]. Considering that once mucositis has occurred the main treatment consists of measures to palliate symptoms, the need for prevention or early intervention is obvious. Recommendations that can be made include oral decontamination, oral cryotherapy, the administration of amifostine and agents directed to the epithelial mucosa, such as mitogens and epithelial growth factors. Oral cryotherapy, through vasoconstriction, can decrease the mucosal damage induced by agents with short half-lives, such as 5-fluorouracil (5-FU) or melphalan (L-PAM) [7]. Here we report our experience regarding the prevention of high-dose L-PAM-induced mucositis by cryotherapy. From December 2001 22 patients (5 with multiple myeloma, 9 with non-Hodgkin’s lymphoma, 7 with breast cancer, 1 with ovarian cancer) underwent HDC including L-PAM; 5 patients received L-PAM (140 – 160 mg/m) alone and 17 patients a L-PAM including regimen. All these patients were asked to suck on ice chips for 30 min, starting 5 min prior to the L-PAM infusion. These patients also received anti-mycotic and antibiotic prophylaxis with fluconazole 200 mg/ day and ciprofloxacin 500 mg 6 2/day, respectively. We observed oral mucositis (NCI-CTC) G0 in 2/22 patients (9%), G1 in 12/22 patients (55%), G2 in 3/22 patients (13%) and G3 in 5/22 patients (23%). In our experience, the incidence of severe mucositis was significantly lower with respect to data reported in the literature. Therefore, we suggest that prophylaxis with cryotherapy should be considered in patients receiving HDC including L-PAM.

Immunophenotypic Profile of AC133-Positive Cells in Bone Marrow, Mobilized Peripheral Blood and Umbilical Cord Blood

AC133 is a molecule whose expression in the human hematopoietic system is restricted to a subset of CD34+ progenitor/stem cells with long-term repopulating ability. The antigenic features of these cells, like CD34+ cells, are described heterogeneous. The immunophenotypic profile of AC133+ cells, detected by means of dual-color flow cytometry, in bone marrow (BM), cytokine-mobilized peripheral blood (PB) and umbilical cord blood (UCB) was evaluated. The highest percentage of AC133+ cells was detected in mobilized PB despite not significantly different from that found in BM, but both are higher than that found in UCB. In addition, the highest percentage of CD34, HLA-DR and CD33 co-expressing AC133+ cells was observed in mobilized PB. Furthermore, UCB was found to be enriched in CD7+ and CD19+ cells and BM was found to be enriched in AC133+ cells co-expressing CDw90 and CD71. Our data confirm the immunophenotypic heterogeneity of cells expressing AC133 antigen, a promising new stem cell marker to be increasingly used as additional target for alternative identification and separation of early hematopoietic cells.

Late and Long-lasting Response in an Adult Chronic Idiopathic Thrombocytopenic Purpura after Extended Course of Rituximab

Stasi et al. recently reported on response to rituximab treatment in adults with chronic idiopathic thrombocytopenic purpura (ITP) [1,2]. Using the classical schedule of rituximab administration (375 mg/sqm four time weekly), authors found that about half of patients experienced a long-lasting complete or partial response. In addition, they described two main pattern of response to rituximab. The first one with a rise of the platelet count occurring early, usually after the first antibody administration. On the other hand, some patients had a late response with platelet count increasing between 6 and 10 weeks after starting therapy. In agreement with this latter observation, we hereby report on a patient with chronic ITP who achieved a late but sustained and long-lasting platelet count recovery after a six course of weekly administration of standard dose of rituximab. In November 2001, a 50-year-old woman was first referred to Surgical Department of our Institution because of a right breast lesion evidenced by a mammography. In her clinical story, there was a diagnosis of chronic ITP in 1983. She was initially treated with prednisone with a transient complete response. In 1984, the patient underwent to splenectomy which induced a partial and short response. Further cyclic re-treatment with oral prednisone gave transient platelet count rise. At admission in our Hospital physical examination was unremarkable except for right breast nodule, serum biochemistry tests were within the normal ranges while peripheral blood cell count shown thrombocytopenia (31,000/ml). To perform surgical ablation of breast nodule, treatment with intravenous Immunoglobulins (400 mg/kg daily for five consecutively days) was started with a platelet rise to 66,000/ml. Nodulectomy was safe and histologic examination of nodule gave the diagnosis of fibroadenoma. In December 2001, patients was admitted to Hematology/Oncology Department for persistent thrombocytopenia (14,000/ml) without bleeding syndrome. Platelet-associated IgG were positive and bone marrow biopsy was consistent with ITP. Thus the patient was started on rituximab (Mabthera, Roche) at the standard dose of 375 mg/m. No side-effects occurred during the course of therapy and the follow-up period. The patient’s course is shown in Fig. 1. As shown, a dramatic increase of platelet count was observed after the first administration of rituximab, followed by reduction to pre-treatment value. Despite a slight but continue rise in platelet count, no partial or complete response were seen until an unexpected and fast complete response starting from tenth week was recorded. At the last follow-up (August 2002) there was evidence of persistent B lymphocytopenia with slightly reduced serum IgA and IgM levels. Eight months following cessation of rituximab therapy she continues in remission with a complete response. Autoreactive lymphocytes producing autoantibodies give the rationale for the use of rituximab, a monoclonal antibody against CD20 antigen, in ITP a disease for which is not well established the first choice therapy [3].

Role of parenteral nutrition in cancer patients undergoing high-dose chemotherapy followed by autologous peripheral blood progenitor cell transplantation.

High-dose chemotherapy followed by autologous bone marrow or peripheral blood progenitor cell transplantation represents a recognized option in the treatment of solid tumors and hematologic diseases. Patients receiving high-dose chemotherapy are traditionally supported with parenteral nutrition with the aim to prevent malnutrition secondary to gastrointestinal toxicity and metabolic alterations induced by the conditioning regimens. Nevertheless, well-defined guidelines for its use in this clinical setting are lacking and there are several areas of controversy.

Usefulness of RA and RO isoforms of common leukocyte antigen (CD45) for early distinction between normal and abnormal promyelocytes.

Normal promyelocytes express CD45RO, while acute promyelocytic leukemia (APL) blasts express CD45RA, both isoforms of common leukocyte antigen with mutually exclusive expression. Here we report a patient with accumulation of promyelocytes in the bone marrow and the diagnostic strategy is described along with the ability to quickly discriminate between normal and abnormal cells using the flow cytometric detection of expression of RA/RO isoforms of CD45.