Baseline [18F]FDG PET features are associated with survival and toxicity in patients treated with CAR T cells for large B cell lymphoma.

PURPOSE: Chimeric antigen receptor (CAR) T cells have established themselves as an effective treatment for refractory or relapsed large B cell lymphoma (LBCL). Recently, the sDmax, which corresponds to the distance separating the two farthest lesions standardized by the patient's body surface area, has appeared as a prognostic factor in LBCL. This study aimed to identify [18F]FDG-PET biomarkers associated with prognosis and predictive of adverse events in patients treated with CAR T cells. METHODS: Patients were retrospectively included from two different university hospitals. They were being treated with CAR T cells for LBCL and underwent [18F]FDG-PET just before CAR T cell infusion. Lesions were segmented semi-automatically with a threshold of 41% of the maximal uptake. In addition to clinico-biological features, sDmax, total metabolic tumor volume (TMTV), SUVmax, and uptake intensity of healthy lymphoid organs and liver were collected. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. The occurrence of adverse events, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), was reported. RESULTS: Fifty-six patients were included. The median follow-up was 9.7 months. Multivariate analysis showed that TMTV (cut-off of 36 mL) was an independent prognostic factor for PFS (p < 0.001) and that sDmax (cut-off of 0.15 m-1) was an independent prognostic factor for OS (p = 0.008). Concerning the occurrence of adverse events, a C-reactive protein level > 35 mg/L (p = 0.006) and a liver SUVmean > 2.5 (p = 0.027) before CAR T cells were associated with grade 2 to 4 CRS and a spleen SUVmean > 1.9 with grade 2 to 4 ICANS. CONCLUSION: TMTV and sDmax had independent prognostic values, respectively, on PFS and OS. Regarding adverse events, the mean liver and spleen uptakes were associated with the occurrence of grade 2 to 4 CRS and ICANS, respectively. Integrating these biomarkers into the clinical workflow could be useful for early adaptation of patients management.

Molecular and clinical diversity in primary central nervous system lymphoma.

BACKGROUND: Primary central nervous system lymphoma (PCNSL) is a rare and distinct entity within diffuse large B-cell lymphoma presenting with variable response rates probably to underlying molecular heterogeneity. PATIENTS AND METHODS: To identify and characterize PCNSL heterogeneity and facilitate clinical translation, we carried out a comprehensive multi-omic analysis [whole-exome sequencing, RNA sequencing (RNA-seq), methylation sequencing, and clinical features] in a discovery cohort of 147 fresh-frozen (FF) immunocompetent PCNSLs and a validation cohort of formalin-fixed, paraffin-embedded (FFPE) 93 PCNSLs with RNA-seq and clinico-radiological data. RESULTS: Consensus clustering of multi-omic data uncovered concordant classification of four robust, non-overlapping, prognostically significant clusters (CS). The CS1 and CS2 groups presented an immune-cold hypermethylated profile but a distinct clinical behavior. The 'immune-hot' CS4 group, enriched with mutations increasing the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and nuclear factor-κB activity, had the most favorable clinical outcome, while the heterogeneous-immune CS3 group had the worse prognosis probably due to its association with meningeal infiltration and enriched HIST1H1E mutations. CS1 was characterized by high Polycomb repressive complex 2 activity and CDKN2A/B loss leading to higher proliferation activity. Integrated analysis on proposed targets suggests potential use of immune checkpoint inhibitors/JAK1 inhibitors for CS4, cyclin D-Cdk4,6 plus phosphoinositide 3-kinase (PI3K) inhibitors for CS1, lenalidomide/demethylating drugs for CS2, and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) inhibitors for CS3. We developed an algorithm to identify the PCNSL subtypes using RNA-seq data from either FFPE or FF tissue. CONCLUSIONS: The integration of genome-wide data from multi-omic data revealed four molecular patterns in PCNSL with a distinctive prognostic impact that provides a basis for future clinical stratification and subtype-based targeted interventions.

Systemic relapses of primary CNS lymphomas (PCNSL): a LOC network study.

Primary central nervous system lymphomas (PCNSLs) classically remain confined within the CNS throughout their evolution for unknown reasons. Our objective was to analyse the rare extracerebral relapses of PCNSL in a nationwide population-based study. We retrospectively selected PCNSL patients who experienced extracerebral relapse during their follow-up from the French LOC database. Of the 1968 PCNSL included in the database from 2011, 30 (1.5%, median age 71 years, median KPS 70) presented an extracerebral relapse, either pure (n = 20) or mixed (both extracerebral and in the CNS) (n = 10), with a histological confirmation in 20 cases. The median delay between initial diagnosis and systemic relapse was 15.5 months [2-121 months]. We found visceral (n = 23, 77%), including testis in 5 (28%) men and breast in 3 (27%) women, lymph node (n = 12, 40%), and peripheral nervous system (PNS) (n = 7, 23%) involvement. Twenty-seven patients were treated with chemotherapy, either with only systemic targets (n = 7) or mixed systemic and CNS targets (n = 20), 4 were consolidated by HCT-ASCT. After systemic relapse, the median progression-free survival and overall survival (OS) were 7 and 12 months, respectively. KPS > 70 and pure systemic relapses were significantly associated with higher OS. Extracerebral PCNSL relapses are rare, mainly extranodal, and frequently involve the testis, breast, and PNS. The prognosis was worse in mixed relapses. Early relapses raise the question of misdiagnosed occult extracerebral lymphoma at diagnostic workup that should systematically include a PET-CT. Paired tumour analysis at diagnosis/relapse would provide a better understanding of the underlying molecular mechanisms.

[Cytopenia under topical imiquimod in two patients treated with oral hydroxyurea]. / Cytopénies sous imiquimod topique chez deux patients traités par hydroxyurée.

BACKGROUND: Aldara® is a topical immunomodulatory treatment. The risks of systemic passage are minimal. There have been rare reports of systemic adverse effects. PATIENTS AND METHODS: Case 1. Five sachets weekly of imiquimod were prescribed for Bowen's disease on the forearm in a patient known to have essential thrombocytosis under Hydrea®. His CBC was normal (6000 leukocytes/mm3, 2200 PMN/mm, 230,000 platelets/mm3). Imiquimod was given in 15 sachets weekly. Fifteen day later, the patient presented bicytopenia (3000 leukocytes/mm3, 1400 PMN/mm3, 119,000 platelets/mm3). Hydroxyurea and imiquimod were suspended until normalization of CBC. Hydroxyurea was resumed without recurrence of the bicytopenia. The patient's history included an identical episode following application of imiquimod. Case 2. Five sachets weekly of imiquimod were prescribed for actinic keratosis on the scalp in a patient known to have primary polycythemia under hydroxyurea. Her CBC was normal except for anemia (Hb 11.5g/L, 160,000 platelets/mm3, 1100 lymphocytes/mm3). Imiquimod was given in 12 sachets weekly. Ten days later, anemia increased (Hb 10g/dL) with lymphopenia (800/mm3) and thrombocytopenia (115,000/mm3). Suspension of imiquimod resulted in normalization of the previous CBC values. DISCUSSION: . The literature review identified reports of dose-dependent lymphopenia under oral imiquimod but not under Aldara®. The National Pharmacovigilance Database listed 10 cases of hematological disorders most likely caused by Aldara®. Hydroxyurea may induce cytopenia, and while it was not considered the sole causative agent in this case, it is likely to have had a triggering role in these patients with blood dyscrasias. Our findings show that misuse of imiquimod carries a potential risk of hematologic abnormality in patients receiving concomitant hydroxyurea, a commonly combined drug.

Alterations in bone mineral density and bone turnover markers in newly diagnosed adults with lymphoma receiving chemotherapy: a 1-year prospective pilot study.

BACKGROUND: Bone mineral density (BMD) loss is poorly defined in lymphoma patients. The aim of this study was to measure the extent of BMD loss in newly diagnosed lymphoma patients receiving chemotherapy. PATIENTS AND METHODS: This was a prospective, single-center study conducted in patients aged≥18 years with previously confirmed lymphoma treated by chemotherapy. Patients with low baseline BMD defined as Z/T-score less than or equal to -2.5 and/or history of osteoporotic fractures were excluded. BMD was measured at baseline before initiating chemotherapy and 1 year later. Predictive factors of BMD loss were investigated. RESULTS: Forty-one lymphoma patients (31 males and 10 females) receiving chemotherapy were enrolled. The median age at diagnosis was 59 (range: 19-86) years. Histological subtypes were predominantly diffuse large B-cell lymphoma (58%), mostly stage III-IV (54%). All patients received chemotherapy and 22% of patients received second-line treatment due to relapse or progressive disease. Thirty-two patients were evaluable at 1 year. The mean BMD changes were: -2.7%±3.9% for lumbar spine (P<0.001), -2.2%±7.6% for femoral neck (P<0.01) and -2.6%±4.5% for total hip (P<0.0001). In multivariate analysis, predictive factors of BMD loss at baseline were (i) at lumbar spine: female gender (P=0.01), higher lactate dehydrogenase level (P=0.04) and lower creatinine clearance (P=0.01); (ii) at total hip: lower albumin (P=0.01), higher corrected serum calcium (P<0.01), lower alkaline phosphatase (AP) (P<0.01) and autologous stem cell transplant (P=0.03); and (iii) at femoral neck: higher corrected serum calcium (P=0.02) and lower bone AP (P=0.01). CONCLUSION: Adult patients with known lymphoma receiving chemotherapy experienced significant BMD loss at 1 year.

Effect of single-unit transfusion in patients treated for haematological disease including acute leukemia: A multicenter randomized controlled clinical trial.

BACKGROUND: Retrospective studies in hematological unit have suggested that single red blood cell (1-RBC) unit transfusion policy may reduce the number of RBC used without negative clinical impact. METHOD: Acute leukemia patients requiring intensive chemotherapy or patients receiving autologous or allogeneic transplantation were randomly assigned to receive either single RBC (1-RBC arm) or double RBC (2-RBC arm) per transfusion with a hemoglobin trigger of 8 g/dL. The primary composite endpoint was the percentage of patients experiencing serious complications, such as a non-hematological adverse event grade ≥ 3 or intensive care admission or death. FINDINGS: A total of 981 and 592 RBC transfusions were required in the 1-RBC arm (n = 125) and the 2-RBC arm (n = 120), respectively. The mean pre-transfusion hemoglobin levels were 7.49 ± 0.83 g/dL in the 1-RBC arm and 7.46 ± 0.67 g/dL in the 2-RBC arm (p = 0.275). The predefined non-inferiority criteria was achieved with 28/125 patients reaching the primary endpoint in the 1-RBC arm (22.4 %) and 28/120 patients in the 2-RBC arm (23.3 %) (Risk difference 0.009; 95 %, Confidence interval [-0.0791 to 0.0978], p = 0.021). The median (IQR) of RBC units transfused per patient was 7 (4-12) in the 1-RBC arm and 8 (4-12) in 2-RBC arm. Hemoglobin levels at discharge were also comparable in both arms. INTERPRETATION: The results of this trial indicate that a single RBC transfusion policy is not inferior to a double RBC transfusion policy for patients receiving a bone marrow transplant or intensive chemotherapy in a hematological intensive care unit. However, the single RBC transfusion policy did not reduce the number of RBC units transfused per stay. FUNDING: This trial was funded by a grant from the French Ministry of Health.

Is Hematopoietic Clonality of Indetermined Potential a Risk Factor for Pulmonary Embolism?

Background Unprovoked pulmonary embolism (uPE) is a severe and frequent condition. Identification of new risk factors is mandatory to identify patients that would benefit from a long-term treatment. Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the acquisition of somatic mutations that drive clonal expansion in the absence of cytopenia. Its prevalence is estimated of 5% in the population above 65 years. Since inflammation and endothelial dysfunction may share a pathophysiological pathway(1), we hypothesized that CHIP, may be a risk factor for uPE. Methods We conducted a pilot retrospective observational study. Patients with iPE between 18 to 65 years old were included. PE was considered as unprovoked, when no transient nor persistant risk factor was present and when thrombophilia testing was negative. We excluded documented atherosclerosis, personal or familial history of VTE and presence of cytopenias. CHIP proportion in uPE patients were analyzed using next generation sequencing of the coding sequence of a custom panel composed by DNMT3A, ASXL1, SF3B1, TET2 and TP 53 . Results Upon 61 patients with uPE consecutively included, a total of 19 somatic mutations were found in 12 patients (20%) IC95% [10 - 20]. 15 mutations were found in DNMT3A gene, 3 in ASXL1 and one in TET2 . There was no diference in terms of age, PE location, DVT presence and risk stratification in CHIP carriers and non carriers. Conclusion We report for the first time, the presence of high rates of CHIP in patients presenting with uPE. Thus, CHIP may be a new risk factor for VTE. These results need to be confirmed in an ongoing prospective case-control study including more patients and using a more diverse gene panel to better determine CHIP incidence in uPE.

Tyrosine kinase inhibitors and solid tumours: case report and review of the literature.

Bcr-Abl tyrosine kinase inhibitors (TKIs) such as imatinib or dasatinib produce high cytogenetic response rates in patients with Philadelphia-positive chronic myeloid leukaemia (CML) with a good overall safety profile. Despite a complete molecular response, it is currently recommended to continue these targeted therapies to avoid relapse. The immediate and short-term TKI side effects are well known, but the long-term side effects have not yet been clearly identified. A preclinical study in rats treated with TKI showed a statistically significant increase in benign and malignant renal tumours. The authors report the case of a 61-year-old man with CML treated with imatinib with a good response, and they switched to dasatinib after grade 4 hepatic toxicity. He had received treatment with 400 mg of imatinib per day for 77 days, followed by dasatinib for 133 days. He developed a metastatic carcinoma of unknown origin during TKI therapy. Despite chemotherapy, the patient died 2 months after the diagnosis. Although several cases of solid tumours have been reported during TKI therapy, the link between cancer and TKIs is not yet clear. Imatinib has remarkably improved the prognosis of patients with CML. Monitoring of the long-term safety profile of TKIs is essential due to the prolonged survival of these patients.

[Bortezomib-induced eruption: Sweet syndrome? Two case reports]. / Toxidermie au bortézomib : syndrome de Sweet? Deux cas.

BACKGROUND: Bortezomib (Velcade) is a proteasome inhibitor used in the treatment of myeloma and other blood dyscrasias. We report the cases of two patients who developed a peculiar toxic rash suggestive of Sweet's syndrome while receiving bortezomib; one patient also presented giant mucous membrane ulcerations. PATIENTS AND METHODS: Case 1: bortezomib treatment was started in a 62-year-old man for mantle cell lymphoma. Ten days after the first treatment cycle, giant, painful oral ulcerations were noted but they resolved spontaneously. One week after the second cycle, further oral ulceration appeared, this time with a papulonodular skin rash. Histology showed neutrophilic dermal infiltrates in the skin with predominantly lymphocytic inflammation of the oral mucosa. Bortezomib was stopped and all lesions resolved with colchicine treatment. Case 2: a 46-year-old woman was receiving bortezomib treatment for plasma cell leukemia. A febrile skin rash appeared two days after the first treatment cycle but resolved spontaneously. After the first bortezomib injection during the next cycle, painful papules and nodules appeared on the trunk. The skin biopsy results were consistent with Sweet's syndrome. The lesions disappeared spontaneously. Dexamethasone was administered concomitantly with bortezomib in the ensuing cycles and there was no relapse of the skin lesions. DISCUSSION: Bortezomib-induced skin lesions are common and usually do not justify treatment withdrawal. Published observations of bortezomib-induced eruption occasionally show clinical and histological features of Sweet's syndrome, but there has been no mention of oral mucosal ulcerations. In our cases, these could be related to bortezomib-induced neutrophilic dermatosis.

Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture.

OBJECTIVE: The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations. METHODS: Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA). RESULTS: Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity. CONCLUSIONS: Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.

Phenotypic and functional characterization of bone marrow mesenchymal stem cells derived from patients with multiple myeloma.

Multiple myeloma (MM) is a B-cell neoplasia caused by the proliferation of clonal plasma cells, primarily in the bone marrow (BM). The role of the BM microenvironment in the pathogenesis of the disease has been demonstrated, especially for the survival and growth of the myeloma plasma cells. Functional characterization of the major component of the BM microenvironment, namely the recently characterized mesenchymal stem cells (MSCs), was never performed in MM. Based on a series of 61 consecutive patients, we evaluated the ability of MSCs derived from myeloma patients to differentiate into adipocytes and osteocytes, inhibit T-cell functions, and support normal hematopoiesis. MSCs phenotypic characterization and quantification of interleukin-6 (IL-6) secretion were also performed. As compared to normal MSCs, MSCs from MM patients exhibited normal phenotype, differentiation capacity and long-term hematopoietic support, but showed reduced efficiency to inhibit T-cell proliferation and produced abnormally high amounts of IL-6. Importantly, these characteristics were observed in the absence of any detectable tumor plasma cell. Chromosomal analysis revealed that MM patients MSCs were devoid of chromosomal clonal markers identified in plasma cells. MM MSCs present abnormal features that may participate in the pathogenesis of MM.

[Haematopoietic stem cell transplantation in the treatment of autoimmune diseases]. / Intensification thérapeutique dans les maladies auto-immunes.

PURPOSE: During the past ten years, more than 1000 patients suffering from severe autoimmune disease have received an autologous haematopoietic stem cell transplant. These new therapeutic have been used in systemic sclerosis (scleroderma), multiple sclerosis, rheumatoid arthritis, juvenile idiopathic arthritis and systemic lupus erythematosus. CURRENT KNOWLEDGE AND KEY POINTS: Autologous haematopoietic stem cell transplantation has become a curative option for condition with very poor prognosis as severe systemic sclerosis, lupus erythematosus or other systemic diseases. This review summarizes the current experience in the phase I and II clinical trials in Europe and North America. We describe the main results and the limits of stem cell transplantation in systemic diseases. FUTURE PROSPECTS AND PROJECTS: Autologous haematopoietic stem cell transplant in the treatment of autoimmune disease has evolved from a experimental concept to a clinically feasible and powerful therapy for selected patients with severe disease.

Recovery, viability and clinical toxicity of thawed and washed haematopoietic progenitor cells: analysis of 952 autologous peripheral blood stem cell transplantations.

Cryopreservation and thawing of haematopoietic stem cells are associated with cell loss and infusion-related toxicities. We analysed viability, total nucleated cell (TNC) and CD34+ cell recovery, and infusion-related toxicities of 952 thawed and washed products. Mean TNC and CD34+ viable cells recoveries were 55.9+/-18.6 and 98.0+/-36.5%, respectively. Mean cell viability was 68.25+/-18.9%. TNC recovery was correlated with viability but independent of the initial nucleated cell concentration. No difference in TNC recovery or viability was observed according to underlying diseases, except for myeloma, for which these variables were significantly lower (P<0.05). CD34+ cell recovery was not correlated with viability or CD34+ initial count and was similar for all diseases. Cryostorage duration was not associated with cell loss. Immediate adverse events occurred in 169 patients (19%) and were moderate (grade I or II) for the majority of patients. Clinical toxicity was associated with a higher infused cell number and the presence of clumps in infused bags. The washing procedure of cell products lead to a low rate of adverse events, but patients transplanted with high cell numbers or bags in which clumps were identified are predisposed to such complications.

High-dose thiotepa-based chemotherapy with autologous stem cell support in elderly patients with primary central nervous system lymphoma: a European retrospective study.

In this retrospective multicentre study, we investigated the outcomes of elderly primary central nervous system lymphoma (PCNSL) patients (⩾65 years) who underwent high-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) at 11 centres between 2003 and 2016. End points included remission, progression-free survival (PFS), overall survival (OS) and treatment-related mortality. We identified 52 patients (median age 68.5 years, median Karnofsky Performance Status before HDT-ASCT 80%) who all underwent thiotepa-based HDT-ASCT. Fifteen patients (28.8%) received HDT-ASCT as first-line treatment and 37 (71.2%) received it as second or subsequent line. Remission status before HDT-ASCT was: CR 34.6%, PR 51.9%, stable disease 3.8% and progressive disease 9.6%. Following completion of HDT-ASCT, 36 patients (69.2%) achieved CR (21.2% first-line setting and 48.1% second or subsequent line setting) and 9 (17.3%) PR (5.8% first-line setting and 11.5% second or subsequent line setting). With a median follow-up of 22 months after HDT-ASCT, median PFS and OS were reached after 51.1 and 122.3 months, respectively. The 2-year PFS and OS rates were 62.0% and 70.8%, respectively. We observed two HDT-ASCT-associated deaths (3.8%). In selected elderly PCNSL patients, HDT-ASCT, using thiotepa-based conditioning regimes, is feasible and effective. Further prospective and comparative studies are warranted to further evaluate the role of HDT-ASCT in elderly PCNSL patients.

Arsenic trioxide and melarsoprol induce apoptosis in plasma cell lines and in plasma cells from myeloma patients.

Recent data have renewed the interest for arsenic-containing compounds as anticancer agents. In particular, arsenic trioxide (As2O3) has been demonstrated to be an effective drug in the treatment of acute promyelocytic leukemia by inducing programmed cell death in leukemic cells both in vitro and in vivo. This prompted us to study the in vitro effects of As2O3 and of another arsenical derivative, the organic compound melarsoprol, on human myeloma cells and on the plasma cell differentiation of normal B cells. At pharmacological concentrations (10(-8) to 10(-6) mol/L), As2O3 and melarsoprol caused a dose- and time-dependent inhibition of survival and growth in myeloma cell lines that was, in some, similar to that of acute promyelocytic leukemia cells. Both arsenical compounds induced plasma cell apoptosis, as assessed by 4',6-diamidino-2-phenylindole staining, detection of phosphatidylserine at the cell surface using annexin V, and by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. As2O3 and melarsoprol also inhibited viability and growth and induced apoptosis in plasma-cell enriched preparations from the bone marrow or blood of myeloma patients. In nonseparated bone marrow samples, both arsenical compounds triggered death in myeloma cells while sparing most myeloid cells, as demonstrated by double staining with annexin V and CD38 or CD15 antibodies. In primary myeloma cells as in cell lines, interleukin 6 did not prevent arsenic-induced cell death or growth inhibition, and no synergistic effect was observed with IFN-alpha. In contrast to As2O3, melarsoprol only slightly reduced the plasma cell differentiation of normal B cells induced by pokeweed mitogen. Both pokeweed mitogen-induced normal plasma cells and malignant plasma cells showed a normal nuclear distribution of PML protein, which was disrupted by As2O3 but not by melarsoprol, suggesting that the two arsenical derivatives acted by different mechanisms. These results point to the use of arsenical derivatives as investigational drugs in the treatment of multiple myeloma.

Phenotypic and genotypic heterogeneity in large granular lymphocyte expansion.

The cellular heterogeneity of large granular lymphocyte expansions has been illustrated by the phenotypic and genotypic findings in five patients. In one patient whose circulating cells were CD2+, CD3-, CD5-, CD7+, CD8-, CD11+, Leu7+, CD16+, and displayed strong natural killer activity, no rearrangement of the T cell receptor beta-chain gene and T cell rearranging gene gamma was detected. The four other patients presented with neutropenia without overt lymphocytosis. In these patients the circulating lymphocytes expressed a predominant T cell phenotype CD2+, CD3+, CD5+, CD7+, CD8+, Leu7+. In three of them the presence of a T cell clone was demonstrated on the basis of a unique pattern of rearrangement of the T cell receptor beta-chain genes.

Influence of CD34 cell selection on the incidence of mixed chimaerism and minimal residual disease after allogeneic unrelated donor transplantation.

Marrow transplantation from unrelated donors has been linked with an increased risk of graft-versus-host disease (GVHD). In an attempt to lower the risk of acute GVHD we used CD34 marrow cell selection for T cell depletion. Since T cell depletion has been linked to an increased risk of relapse and an increased risk of marrow failure, we used PCR amplification of minisatellite sequences to investigate donor cell engraftment and RT-PCR amplification of recurrent chromosomal translocations to investigate the residual disease post-transplant. Twenty-three patients who underwent BMT after positive selection of the CD34-positive cell population were studied. Results were then compared with those of 37 patients who underwent transplantation with unmanipulated marrow graft. Among the 23 patients who received CD34+ selected cell grafts, seven (30%) had evidence of full donor engraftment, 14 had evidence of residual recipient cells (61%), one had a non-take, and one autologous bone marrow recovery. Analysis of the chimaerism status post-transplant in 36 patients who received unmanipulated marrow grafts showed that 31 patients (86%) had evidence of full donor engraftment. The difference in the incidence of mixed chimaerism profile between patients who received unmanipulated marrow graft and those receiving CD34+ selected cell grafts was statistically significant (P< 0.01). Nine patients who received CD34+ selected cell grafts could be analysed for the presence of minimal residual disease post-transplant (one with t(9;22) acute lymphoblastic leukaemia and eight with CML). In the patient transplanted for a Ph-positive acute leukaemia, and in two out of the eight patients with CML, the search fora fusion transcript was consistently negative after transplantation. Among the six patients with evidence of residual disease, three patients also had a mixed chimaerism profile and were given donor lymphocyte infusions. Minimal residual disease study was performed post-transplant in 16 patients who received unmanipulated marrow grafts. In 10 of 14 patients with CML, and in two patients with acute leukaemia the search for a fusion transcript was consistently negative after transplantation. The difference in the incidence of minimal residual disease between patients who received an unmanipulated marrow graft and those receiving CD34+ selected cell grafts was not statistically significantly significant, but numbers of patients included in this analysis are still few. In conclusion, our study highlights the strong influence of graft manipulation on the incidence of mixed chimaerism after transplantation from an unrelated donor.

Influence of bone marrow graft lymphocyte subsets on outcome after HLA-identical sibling transplants.

OBJECTIVE: The aim of this study was to analyze bone marrow lymphocyte subsets and CD34 cell dose and their influence on the outcomes of bone marrow transplantation. MATERIALS AND METHODS: Forty-eight patients (median age 30 years, range 5-54) receiving HLA-identical sibling bone marrow transplantation for hematologic malignancies were analyzed. RESULTS: Median number (range) of nucleated cells and CD34+ cells infused were 2.4 (0.4-6.0) x 10(8)/kg and 3.5 (0.5-13.0) x 10(6)/kg, respectively. Probability of neutrophil recovery was 97%. In a multivariate analysis, time to neutrophil recovery was shortened when a higher number of CD3/CD8 cells was infused (> or =1.0 x 10(7)/kg) (hazard ratio [HR] = 2.13, p = 0.018); when the patient was female or had negative cytomegalovirus serology (HR = 2.03, p = 0.03; HR = 0.41, p = 0.009; respectively). The incidence of grade II to IV acute graft-vs-host disease (GVHD) was 47%. Infusion of >1 x 10(7) CD4 infused/kg increased the risk of acute GVHD (HR = 2.86, p = 0.03). Nineteen of 40 patients at risk experienced chronic GVHD, the risk of which was increased by diagnosis of chronic leukemia (p = 0.03), <2.0 x 10(8) nucleated cells infused/kg (p = 0.05), and a low number of all lymphocyte subsets, except CD19. Estimated 3-year survival rate was 54%. Risk of death was increased in patients receiving <3.5 x 10(6)CD34 infused/kg (HR = 0.37, p = 0.02). Only six patients relapsed. CONCLUSIONS: A high cell dose of CD3/CD8 is associated with faster neutrophil recovery, whereas a high cell dose of CD4+ cells increases the incidence of acute GVHD. A high number of nucleated cells and CD34+ cells infused was associated with decreased risk of chronic GVHD and improved survival, respectively.

CD14+ CD16+ monocytes rather than CD14+ CD51/61+ monocytes are a potential cytological marker of circulating osteoclast precursors in multiple myeloma. A preliminary study.

INTRODUCTION: Osteolytic bone destruction is a major clinical problem in multiple myeloma patients. Osteoclasts can differentiate in vitro from bone marrow-resident monocyte progenitors, such as common monocyte progenitors, as well as circulating monocytes. Various types of monocytes, including osteoclast precursors, appear to circulate systemically. METHODS: We investigated the possibility of demonstrating, by in vitro differentiation and flow cytometry, a circulating osteoclast precursor population in multiple myeloma (MM) patients by studying the distribution of CD14(+/++) CD11b(+) CD51/61(+) and CD14(+/++) CD16(+/-) populations. RESULTS: Under short-term in vitro osteoclastic differentiation conditions, almost all CD14 monocytes acquired CD51/61 and CD16 expression. Flow cytometry studies failed to demonstrate a statistically significant increase in circulating CD14(+/++) CD11b(+) CD51/61(+) populations in 20 MM patients with osteolytic lesions. However, the minor circulating CD14(+/++) CD16(+) fraction was significantly increased in MM patients compared with healthy volunteers (109.3 ± 63.1/mm(3) vs. 65.3 ± 34.9/mm(3) ; P = 0.005), but with no correlation with markers of tumour burden. The CD14(+/++) CD16(+) to CD14(+/++) CD16(-) ratio was higher in MM patients. CONCLUSION: The circulating CD14(+/++) CD11b(+) CD51/61(+) fraction was not correlated with bone lesions in MM patients. However, CD14(+/++) CD16(+) monocytes may be a candidate marker. A larger study must be conducted to confirm these promising results for the diagnosis and follow-up of MM patients.

Efficient transduction of hemopoietic CD34+ progenitors of human origin using an original retroviral vector derived from Fr-MuLV-FB29: in vitro assessment.

A novel retroviral vector has been designed based on a Friend-murine leukemia virus (Fr-MuLV) FB29 strain. The latter has been selected according to characteristics of pathogenicity in mice where it induces a disease of the haemopoietic system affecting all lineages. Higher infectivity has also been demonstrated as compared to other strains. In accordance with these findings, the amphotropic producer clone used in this study carrying along the neomycine resistance gene (FOCH-Neo), harbors viral titers over 10(7) cfu/ml. To investigate the potential of genetically engineering hematopoietic precursors, CD34+ progenitors were selected from cord blood, bone marrow, and peripheral blood mobilized stem cells (patients + solid tumors) and transduced with FOCH-Neo. High transduction rates were achieved using virus supernatant and minimal doses of hematopoietic growth factors during pretransduction and transduction steps. A polymerase chain reaction (PCR) assay investigating the presence of both neomycin-encoding and viral vector sequences tested positive in 45-90% of granulocyte-macrophage colony-forming units (CFU-GM) generating cells (bone marrow and peripheral blood derived cells) following transduction. An average of 35% colonies showed resistance to G418. Such levels of transduction proved reproducible using only supernatants harboring over 10(7) cfu/ml. In those experiments where long-term in vitro cultures could be maintained over 5 weeks (all cord blood and 5 among 23 PBSC), efficient transduction of long-term culture initiating cell (LTC-IC) hematopoietic progenitors was demonstrated on the basis of both resistance to G418 and virus integration. In the latter case, the PCR assay tested positive in as much as 35-60% of late unselected CFU-colonies. This novel retroviral vector harbors interesting features toward genetic modification of hematopoietic progenitors.