Mauricio Ocqueteau

Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia.

PURPOSE The identification of immunophenotypic aberrancies through multiparametric flow cytometry makes the differentiation between normal and leukemic cells relatively simple and quick, and is therefore an attractive method for the investigation of minimal residual disease (MRD). In this report, we have analyzed the impact on relapse and relapse-free survival (RFS) of detecting immunophenotypical aberrant cells in acute lymphoblastic leukemia (ALL) patients in cytomorphologic complete remission (CR). MATERIALS AND METHODS Two hundred eleven bone marrow (BM) samples from 53 consecutive ALL (37 precursor B-ALL and 16 T-ALL) patients were analyzed. The only selection criteria were to have at least one aberrant immunophenotypic feature at diagosis and to have achieved cytomorphologic CR after induction therapy. For MRD detection, all follow-up samples were analyzed with triple labelings using a two-step acquisition procedure, in which 106 cells were screened for the possible persistence of residual leukemic cells with the same phenotypic aberrancy as that identified diagnosis. RESULTS Patients who displayed a gradual increase in MRD levels showed a higher relapse rate (90% v22%; P < .00001) and shorter median RFS (12 months v not reached; P < .0001) than those with stable or decreasing MRD levels. This adverse prognostic influence also was observed when children and adults, as well as B-ALL and T-ALL patients, were analyzed separately. An MRD level > or = or greater than 10(-3) discriminated two risk groups of ALL patients with significantly different relapse rates and RFS at all treatment phases (end of induction, consolidation, maintenance, and out of treatment). CONCLUSION Multiparametric flow cytometry of MRD in ALL patients is a valuable tool for relapse prediction and for the identification of a cohort of patients with very poor prognosis.

Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state.

The risk for thrombosis is significantly increased in systemic lupus erythematosus (SLE), affecting both venous and arterial vessels. Activated platelets are known to participate in thrombus formation and growth. A general feature of activated cells is the shedding of microparticles (MP) which support coagulation by exposure of negatively charged phospholipids and possibly tissue factor (TF). In this work we characterized circulating MP in patients with SLE and their relationship with a procoagulant state. Thirty patients with SLE (aged 15-72 years, mean age 38 years) and 20 healthy controls (aged 22-54 years, mean age 34 years) were studied; patients fulfilled 4 revised criteria for SLE. The number and cellular source of circulating MP were determined by flow cytometry using double labeling with specific monoclonal antibodies and annexin V. Thrombin generation was measured as the endogenous thrombin potential (ETP) without the addition of exogenous phospholipids and TF; under these conditions the generation of thrombin depended directly on the number of MP present in plasma. Thrombin anti-thrombin (TAT) and plasmin-antiplasmin (PAP) complexes were measured by ELISA. Compared to the controls, circulating MP were significantly elevated in the patient group (1218 +/- 136 vs 653 +/- 74 x 10(3)/ml plasma, p: 0.0007). In both groups, most of these MP were of platelet origin (927 +/- 131 vs 517 +/- 72 x 10(3)/ml plasma, p:0.009 ). ETP was higher among patients as compared to the controls (804 +/- 64 vs 631 +/- 37 nM thrombin, p: 0.025). Plasma levels ofTAT in patients and controls were 3.4 +/- 0.8 and 1.4 +/- 0.5 microg/L, respectively (p:0.04), and of PAP complexes were 62.5 +/- 14 and 24.05 +/- 2.5 microg/ml, respectively (p: 0.014). The number of platelet-derived MP correlated significantly with thrombin generation (r: 0.42; p: 0.038) and TAT levels (r: 0.40; p: 0.035). We did not find an association of circulating MP with disease activity nor with the presence of antiphospholipid antibodies. The increased number of circulating platelet-derived microparticles and their association with high ETP and activation of the coagulation system suggest that these microparticles play an important role in the pathogenesis of the prothrombotic state in SLE patients.

High‐sensitive immunophenotyping and DNA ploidy studies for the investigation of minimal residual disease in multiple myeloma

Sensitive techniques for monitoring minimal residual disease (MRD) in multiple myeloma (MM) are needed to evaluate the effectiveness of new intensive treatment strategies. The aim of the present study was to explore the applicability and sensitivity of flow cytometry immunophenotyping and DNA ploidy studies for the investigation of residual myelomatous plasma cells (PC) in MM patients. Bone marrow (BM) samples from 61 untreated MM patients were immunophenotypically analysed with a panel of 21 monoclonal antibodies, using a high‐sensitive method based on a two‐step acquisition procedure through a SSC/CD38+++‐CD138+‘live‐gate’. Overall, in 87% of MM cases, PC displayed an aberrant phenotype at diagnosis. The most important aberrant criteria were: antigen over‐expression of CD56 (62%), CD28 (16%) and CD33 (6%) and asynchronous expression of CD117 (28%), sIg (21%) and CD20 (10%). DNA aneuploidy was found in 62% of cases. The simultaneous use of these two techniques allowed the detection of aberrant/aneuploid PC in 95% of the cases. Based on dilutional experiments, the detection limit of both techniques ranged from 10−4 to 10−5. In 29 stem cells harvests and 19 BM samples obtained 3 months after autologous transplantation, we have investigated the presence of residual myelomatous PC; they were detected in 44% of the stem cell collections and in 61% of the BM samples obtained after transplant. The percentage of pathological PC did not significantly change during the days of harvest. In summary, the present study shows that the combined use of immunophenotyping and DNA ploidy studies is a suitable approach for MRD investigation in MM patients based on their applicability (95% of cases) and sensitivity (up to 10−5).

Expression of the CD117 antigen (C‐Kit) on normal and myelomatous plasma cells

Summary. The surface expression of CD117 on plasma cells (PCs) from normal individuals and patients with multiple myeloma (MM) has been analysed using triple‐stained cells for flow cytometry. In addition, the clinical significance of CD117 expression in MM patients and its possible value for the evaluation of minimal residual disease was explored. A total of 11 healthy volunteers and 56 untreated MM patients were included in the study. The expression of CD117 was analysed by flow cytometry, using simultaneous staining with the MAbs BB4, CD117 and CD38. Cell acquisition was performed in two consecutive steps using a live gate drawn on SSC/CD38+++ cells and a total of 300000 events were acquired. For data analysis, the Paint‐a‐Gate Plus software (Becton Dickinson) was used. PCs were identified according to their strong reactivity for CD38 and their positivity for BB4, as well as by their light scatter distribution. Dilution experiments of CD117+ myelomatous PCs with normal bone marrow (BM) cells were performed in order to assess the sensitivity level of the technique for detection of CD117+ residual PCs. None of the PCs from normal BM samples showed reactivity for the CD117 antigen. In contrast, CD117 antigen was present in 18/56 MM patients (32%), the proportion of positive cells in these cases being as high as 92·1 ± 9%. Therefore, within PC lineage the c‐Kit antigen would be restricted to the myelomatous population and thus could be considered as a “tumour‐associated marker” for monitoring minimal residual disease in about one third of MM patients. Dilution experiments indicate that the detection limit with this marker would be 10−4 (one myelomatous PC/104 normal BM cells). Upon comparing the clinical and haematological disease characteristics of CD117‐positive and CD117‐negative cases, no significant differences were found.

Bone marrow stromal cells modulate mouse ent1 activity and protect leukemia cells from cytarabine induced apoptosis

Background Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. Methods Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of 3H-adenosine. Results Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. Conclusion The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML.

Admission of Hematopoietic Cell Transplantation Patients to the Intensive Care Unit at the Pontificia Universidad Católica de Chile Hospital

Patients undergoing hematopoietic cell transplantation (HCT) can have complications that require management in the intensive care unit (ICU). We conducted a retrospective study of patients undergoing HCT between 2007 and 2011 with admission to the ICU. We analyzed 97 patients, with an average age of 37 (range, 15 to 68). The main indications for HCT were hematologic malignancies (84%, n = 82). Ninety percent (n = 87) received myeloablative conditioning. Thirty-one percent were admitted (autologous transplant recipients 15%, allogeneic transplant recipients 34%, and umbilical cord blood [UCB] transplant recipients 48%) with an average length of stay of 19 days (range, 1 to 73 days). The average time between transplantation and transfer was 15 days. The main causes of admission were acute respiratory failure (63%) and septic shock (20%). ICU mortality was 20% for autologous transplantations and 64% for allogeneic transplantations (adult donor and UCB combined). On average, patients died 108 days after the transplantation (range, 4 to 320 days). One-year overall survival, comparing patients entering the ICU with those never admitted, was 16% versus 82% (P < .0001) for allogeneic transplantations (adult donor and UCB combined) and 80% versus 89% (P = not significant) for autologous transplantations. Acute graft-versus-host disease was significantly associated with death in ICU after UCB HCT. ICU support is satisfactory in about one half of patients admitted, characterized by a short and medium term prognosis not as unfavorable as has been previously reported.

Outcomes in relapsed Hodgkin's lymphoma treated with autologous and allogeneic hematopoietic cell transplantation at the Pontificia Universidad Católica de Chile

Introduction Hodgkins lymphoma is a highly curable disease. Autologous and reduced intensity allogeneic hematopoietic cell transplantations are alternatives to treat relapsed patients. Here, we report on the results of one service using these procedures. Methods All patients who underwent transplantations in our institution between 1996 and 2014 were retrospectively studied and demographics, toxicities and survival rate were analyzed. Results This study evaluated 24 autologous and five reduced intensity allogeneic transplantations: the median ages of the patients were 29 and 32 years, respectively. At the time of autologous transplantation, ten patients were in complete remission, nine had chemosensitive disease but were not in complete remission, three had refractory disease and the status of two is unknown. In the allogeneic group, two were in complete remission and three had chemosensitive disease. The 5-year overall survival after autologous transplantation was 42% (66% patients were in complete remission, 37% had chemosensitive disease with incomplete remission and 0% had refractory disease) and 1-year overall survival after allogeneic transplantation was 80%. Transplant-related mortality was 0% in patients conditioned with the ifosfamide/carboplatin/etoposide (ICE), carmustine/etoposide/cyclophosphamide (BEC) and carmustine/etoposide/cytarabine/melphalan (BEAM) regimens, 37% in patients conditioned with busulfan-based regimens and 20% in allogeneic transplantations. Conclusions Hematopoietic cell transplantation for relapsed Hodgkins lymphoma is a potentially curative procedure especially in patients in complete remission at the time of autologous transplantations, and possibly after allogeneic transplantations. Further studies are necessary to clarify the role of allogeneic transplantations in the treatment of relapsed Hodgkins lymphoma.

Resistance of leukemia cells to cytarabine chemotherapy is mediated by bone marrow stroma, involves cell-surface equilibrative nucleoside transporter-1 removal and correlates with patient outcome

The interaction between acute myeloid leukemia cells (AML) with the bone marrow stroma cells (BMSCs) determines a protective environment that favors tumor development and resistance to conventional chemotherapy. We showed that BMSCs secrete soluble factors that protect AML cells from Ara-C induced cytotoxicity. This leukemia chemoresistance is associated with a decrease in the equilibrative nucleoside transporter (ENT1) activity by inducing removal of ENT1 from the cell surface. Reduction of cell proliferation was also observed with activation of AKT and mTOR-dependent cell survival pathways, which may also contribute to the tumor chemoprotection. Analysis of primary BMSC cultures has demonstrated that AML patients with stroma capable to confer Ara-C resistance in vitro compared to AML patients without this stroma capacity were associated with a worse prognosis. The two year overall survival rate was 0% versus 80% respectively (p=0.0001). This is the first report of a chemoprotection mechanism based on the removal of a drug transporter from the cell surface and most importantly the first time that a stroma phenotype has correlated with prognostic outcome in cancer.

Thymoma associated with hypogammaglobulinaemia and pure red cell aplasia

Thymomas are neoplasias that begin in the thymus and develop in the anterior mediastinum. They are commonly associated with a variety of systemic and autoimmune disorders, such as pure red cell aplasia, hypogammaglobulinaemia, pancytopaenia, collagen diseases, and, most commonly, myasthenia gravis. The presence of inter-current infections, especially diarrhoea and pneumonia, in the presence of lymphocyte B depletion and hypogammaglobulinaemia is known as Good’s syndrome and may affect up to 5% of patients with thymoma. While anaemia is present in 50%–86% of patients with Good’s syndrome, only 41.9% of cases present pure red cell aplasia. Concomitance of these two conditions has only been rarely studied. We report on the case of a 55-year-old man diagnosed with advanced thymoma, who, during the progression of his disease, developed signs and symptoms suggesting Good’s syndrome and pure red cell aplasia. We also performed a brief review of the literature concerning this association, its clinical characteristics, and treatment.

Detection of monoclonality in bone marrow plasma cells by flow cytometry: limitations for minimal residual disease detection

We would like to add to the recent correspondence regarding severe hypophosphataemiain autograft recipients reported by Raanani et al (1995). We too have noticed severe hypophosphataemia in two patients undergoing bone marrow transplantation (BMT). Case 1 was a 62-year-old male with acute myeloid leukaemia who underwent an autograft using cyclophosphamide/busulphan conditioning. He developed severe hypophosphataemia (plasma phosphate 0.29 mmol/l) within 1 week of his BMT. Case 2 was a 35-year-old male with a teratoma of the testis who underwent BMT with cyclophosphamide and busulphan conditioning. He also developed severe hypophosphataemia of 0.26 mmol/l within 1 week of his BMT. There are two major points that need to be emphasized in the context of severe hypophosphataemia and bone marrow transplantation. Firstly, hypophosphataemia can result in many clinical abnormalities (Betro & Pain, 1972). In many cases the effects are either the result of impaired cellular energy pathways that involve ATP or are due to reduced erythrocyte 2,3-DPG (Knochel, 1977). Rhabdomyolysis can occur, as may impaired skeletal muscle function, including weakness and myopathy. Cardiomyopathy is another possible complication. The nervous system can also be affected with seizures, perturbed mental state, and paraesthesia. Hypophosphataemia can lead to osteomalacia and, if prolonged, renal tubular impairment and acute tubular necrosis (Stoff, 1982). The haematological effects caused by severe hypophosphataemia include impaired leucocyte phagocytosis and chemotaxis. Haemolysis may occur, as can erythrocyte 2,3DPG depletion, resulting in a shift in the haemoglobin/oxygen dissociation curve to the left, i.e. haemoglobin has a greater affinity for oxygen (Knochel, 1977). This is of fundamental importance to a patient undergoing bone marrow transplantation, particularly as their haematological state is already compromised. Secondly, care is required when giving intravenous phosphate. This is usually not necessary unless the plasma phosphate concentration is <0.30 mmol/l or the patient is symptomatic. A Lancet editorial (1981) suggested that severe hypophosphataemia should be treated. The recommendation in severe hypophosphataemia was to use intravenous phosphate replacement by the Vannatta regime (Vannatta et al, 1981). This gives 9 mmol of monobasic potassium phosphate in half-normal saline by continuous intravenous infusion over 12 h. However, this should not be given to patients with hypercalcaemia, because of the risk of metastatic calcification, nor to patients with hyperkalaemia. Plasma phosphate, ionized calcium, magnesium and potassium should be closely monitored, and the infusion stopped once the plasma phosphate concentration exceeds 0.30 mmol/l (Coyle et al, 1992). Monitoring urine output is also important. Recently there has been continued debate about the treatment of severe hypophosphataemia,illustrated by a patient who had been given 50 mmol/l of intravenous phosphate as was earlier recommended in the British National Formulary in preference to the Vannatta regime (Young et al, 1993). This patient developed severe hyperphosphataemia with hypocalcaemia and needed haemodialysis. Clearly, attention should be paid to the safest phosphate replacement regimen in bone marrow transplant patients manifesting severe hypophosphataemia, so as to avoid possible iatrogenic complications.