BM Samples Research Articles

Early Evaluation of Bone Marrow Minimal Residual Disease by Flowcytometry on Day 15 Is Feasible on a Multicenter Basis and Bears Strong Prognostic Value in Childhood Acute Lymphoblastic Leukemia. The AIEOP-BFM Experience.

Early measurement of blast clearance is a relevant prognostic indicator in childhood acute lymphoblastic leukemia (ALL). To this purpose we measured, by four-colour flowcytometry (FC), the percentage of blast cells in bone marrow samples from Italian patients enrolled in the multicentre AIEOP-BFM ALL 2000 trial. Samples were collected on day 15 (after 14 days of steroids, and one dose of IT-MTX, vincristine, daunorubicine, asparaginase) and shipped overnight to the reference laboratory. The data were compared to PCR-MRD performed, by study design, on day +33 and +78 BM samples. We report the results of patients enrolled between December 2000 and October 2004. The 561 patients studied were not different from the remaining ones (with no available material) including their cumulative incidence of relapse (SE): 17.3% (1.9) vs. 18.1% (1.5) in 850 patients not studied. According to the results of FC-MRD, 5 groups were defined: negative (blast count <0.01%, n=143), <0.1% (n=94), <1% (n=149), 1–10% (n=119), >10% (n=56). Their cumulative 5-year risk of relapse was: 4.1% (1.9), 9.3% (4.0), 14.3% (3.2), 26.5% (5.5), 53.7% (7.4), respectively. By PCR-MRD, the same patients were stratified as follows: 177 were standard risk and had 5-year risk of relapse of 4.1% (1.7), 233 at intermediate risk had a relapse risk of 24.2% (3.4), 37 at high risk had a relapse risk of 58.1% (9); the remaining 124 patients (21.6%) were not stratified by PCR-MRD due to lack of 2 sensitive (≥10−4) markers. Of 177 patients classified as standard risk by PCR (double negative), 110 fell within the 2 subgroups with lower FC-MRD (<0.1%), 46 had <1%, 19 had <10%, only 2 >10% of blasts. Of the 233 patients stratified as PCR-MRD intermediate risk (d78 <10−3), FC-MRD related groups had the following probabilities of EFS: 93.5% (3.6; n=47), 83.3%(8.0; n=30), 80.5%(5.1; n=70), 66.5%(10.8; n=57), 39.2%(11.8; n=29). We conclude that very early measurement of FC-MRD on day 15 bone marrow is feasible in our multicentre cooperative setting. On the basis of our data we suggest the following risk groups: standard, when <0.1% blasts on day 15 BM; intermediate for 0.1 to <10%; high, for >10% blasts. These groups had a risk of relapse of 6.2% (1.9), 19.5% (3), and 53.7% (7.4), respectively. Since it is fast, reproducible, relatively cheap and applicable to virtually all patients, our group decided to apply it prospectively on all ALL patients to integrate PCR-based stratification. Our findings showed that:i.early (d15) MRD detection by FCM identifies different patients than PCR on d33 and d78;ii.FCM may be very useful to identify earlier the highly sensitive ALL with low relapse risk (even though long-term follow-up is still missing), whereas later timepoints may be accessible for PCR and the identification of HR patients.

Successful Preemptive Treatment of Molecular Relapse after Autologous Stem Cell Transplantation in Mantle Cell Lymphoma

Autologous stem cell transplantation (ASCT) leads to induction of molecular remission in mantle cell lymphoma (MCL). A large proportion of the patients, however, relapse after ASCT. Increasing levels of minimal residual disease (MRD) in consecutive BM samples after ASCT have been observed prior to relapse by us and others, whereas patients in continuous clinical remission have remaining low levels of MRD. In the present 2nd Nordic MCL Phase 2 trial we aimed to direct preemptive treatment to patients in clinical remission but with increasing levels of MRD at high risk of relapse after ASCT. We used a combined standard nested and quantitative real-time PCR analysis to estimate MRD levels (Andersen et al, 2002, Exp. Hematol). According to the protocol consecutive BM and PB samples were procured and shipped for central PCR analysis every 3–4 months post-transplant. Preemptive therapy consisted of four weekly doses 375 mg/m2 of Rituximab. Of the 161 MCL cases included in the trial 81 cases underwent ASCT and had PCR markers available. CR-rate after ASCT was 92%. In total 852 post-transplant BM/PB samples were monitored for MRD. 47 of 81 (58%) cases remained standard nested PCR negative after ASCT for a median follow-up time of 2.7 years (range: 0.14–5.7 years). In 4 (8%) of these a clinical relapse was observed without any PCR detectable MRD present in BM or PB after ASCT, including at time of clinical relapse. In 34 (42%) of 81 cases standard nested PCR was positive at least once after ASCT. The majority of the PCR positive cases (26/34 cases, 76%) converted from standard nested PCR negative to positive during post-transplant follow-up, thus, these cases relapsed molecularly. 8 (24%) of 34 cases remained standard nested PCR positive after ASCT. In these, a rising level of MRD was detected by real-time PCR analysis in 4 cases. The remaining 4 cases either had stable low or declining levels of MRD. Of the 30 cases which relapsed molecularly 8 cases simultaneously underwent a clinical relapse leaving no therapeutical window for preemptive treatment. One case refused preemptive treatment. All molecular relapse occurred within 3 years after ASCT, except in 1 case. In total 21 cases have received preemptive treatment. 19/21 (90%) cases became standard nested PCR negative (18 cases) or reduced to low MRD level (1 case). 2/21 cases remained PCR positive and relapsed after 3 and 6 months, respectively. 16/21 cases remain in clinical CR for a median follow-up time of 1.4 years after preemptive treatment (range: 0.25 to 3.8 years) and 5/21 cases have relapsed. Of the latter cases the 3 of the 5 became PCR negative for 6–9 months before relapse. Of note, two cases have received preemptive treatment twice after a second molecular relapse after which they again became PCR negative. Preemptive treatment has not been reported in lymphoma before. Our results in MCL suggest that the large number of cases who remain in molecular remission after intensified ASCT may be followed by MRD monitoring and treated at molecular relapse instead of receiving maintenance therapy. However, 4 of these cases relapsed. Here, more than PCR methods are needed for early stage disease detection. Our results indicate that preemptive treatment using Rituximab can successfully reinduce molecular remission and prolong time to relapse. Finally, more patients may have PCR markers available by applying frozen diagnostic lymph node material.

Human Bone Marrow as a Source of Multifunctional CMV-Specific CD4+ T Cells for Adoptive Cell Therapy.

Introduction Adoptive cell transfer of ex vivo primed and expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising approach to treat both infectious and malignant diseases in humans. First clinical studies have shown that transfer of Cytomegalovirus (CMV)-specific CD8+ T cells is safe and effective in reconstitution of cellular immunity against CMV disease. Efficacy of adoptive T cell therapy is limited by the numbers of CTLs in vitro and the survival and function after infusion. CD4+ T cells may enhance activity via direct or indirect effector functions (Matloubian 1994 [1]). In this study we have analysed whether bone marrow is superior to peripheral blood for expansion of CMV-specific T cells. Experimental design Paired peripheral blood and bone marrow samples were obtained from patients who underwent total hip arthroplasty. By using two different protocols T cells were expanded in the presence of IL-2 and IL-7 either from bulk culture with exposure of two different peptide pools (IE1 and pp65) or after selection via IFN-γ secretion by stimulation with pp65. CMV specific immune responses were assessed by using multiparameter flow cytometry staining cells for CD3, CD4, CD8, CCR7 and CD45RA and for the cytokines IFN-γ IL-2 and TNF at day 0 and after 10 days of in vitro expansion.Results Similar frequencies of cytokine-producing pp65– and IE1-specific CD4+ and CD8+ T cells were found in unmanipulated paired PB and BM samples. Expansion of CMV-specific T cells from BM resulted in significantly higher frequencies of specific CD4+ T cells than from PB, whereas no difference in frequencies of CMV-specific CD8+ T cells was observed. Interestingly, significantly higher frequencies of BM pp65 and IE1-specific CD4+ T cells were multifunctional, characterized by producing simultaneously IFN-γ, TNF and IL-2 (IE1: BM mean 0.44% ± 0.16; PB mean 0.09% ± 0.05, p=0.031; pp65: BM mean 3.87% ± 2.46; PB mean 1.24% ± 0.90, p=0.031). Expansion of multi-functional CD4+ T cells from BM was observed with both the bulk and selection assay protocol. Both PB and BM CMV-specific CD4+ and CD8+ T cell lines had a predominant CD45RA-CCR7- effector memory phenotype.Conclusions This study implicates the use of human bone marrow as a source for expansion of multifunctional CMV-specific CD4+ T cells. Recent studies in HIV and Leishmania support the crucial role of multifunctional T cells in disease control (Darrah 2007 [2]).

Detection and Treatment of Molecular Relapse in Acute Myeloid Leukemia with RUNX1, CBFB or MLL Gene Translocations. Frequent Quantitative Monitoring of Molecular Markers in Different Compartments and Correlation with WT1 Gene Expression.

Background. It seems that the population of leukemia stem cells (LSCs) have fundamental importance in the origin and maintenance of the acute myeloid leukaemia (AML). Eradication of LSCs is a new goal of AML therapy. We hypothesized, that by monitoring of minimal residual disease (MRD) and its dynamics in different compartments (peripheral blood, PB; bone marrow, BM; sorted CD34+ BM cells; and CD34- BM cells) it would be possible to find some patterns reliably predicting clinical relapse. Aims. To find which compartment is the best for MRD monitoring and whether it would be possible to treat the disease in the phase of molecular relapse in order to prevent the hematological relapse.Methods. MRD monitoring, in average once or twice per month, was performed in all phases of treatment, and was done even more frequently in the cases of unstable MRD. RQ PCR for fusion transcripts (CBFB/MYH11, RUNX1(AML1)/ETO or fusion transcripts of MLL gene) and WT1 gene was used. Molecular relapse was defined as reappearance of the fusion transcript detection or its 10-fold increase, repeatedly detected. Some patients with already known MRD dynamics and high probability of imminent hematological relapse were treated at the time of molecular relapse. Results. In 67 AML patients and 6 healthy volunteers, 2352 BM or PB samples were examined, including 265 samples from CD34+ BM cells. Follow up was 31–287 weeks (median: 113 w). The correlation between the fusion transcripts levels in BM and PB was excellent (r=0,9676). The correlation between WT1 PB and BM levels was far less satisfactory. Since the WT1values did not mostly reach zero values even if the level of fusion transcript was 0, we wanted to find some “normal” value for WT1. Using the ROC curves, however, we were not able to find any WT1 level being a confidential marker of molecular remission in either compartment (PB, BM, CD34− or CD34+). In relapsed patients, the time from molecular to haematological relapse was 8 – 79 days (median: 25 d). In the cases of subsequent development of haematological relapses, the levels of fusion transcript in CD34+ BM cells were one order of magnitude higher than in the BM or PB, even in the case of CD34- blasts. Nine patients were treated for 17 molecular relapses with following results: chemotherapy, CR=2, PR=3, NR=1; gemtuzumab ozogamicin, CR=3, PR=1, NR=3; IL-2±DLI, CR=3, NR=1 (PR was defined as a decrease in fusion transcript level at least 10-fold, CR as a decrease to 0). Patients with CD33- blasts at diagnosis did not respond to gentuzumab ozogamicin. Non-responsiveness to one treatment option did not mean non-responsiveness to another treatment.Conclusion: Frequent quantitative monitoring (especially in CD34+ BM cells) of fusion transcripts (in contrast to WT1) is useful for reliable prediction of haematological relapse in AML patients. PB seems to be sufficient for frequent outpatient MRD monitoring. Efficient targeting of LSCs will be essential for AML cure, however, the best method is currently not known. Some now available procedures are sometimes surprisingly successful.

Kinetics of Molecular Response to Imatinib in Chronic Myeloid Leukemia Patients May Predict Persistent Polymerase Chain Reaction Negativity after Imatinib Discontinuation.

Achieving a molecular response after imatinib treatment is considered today the gold standard on Ph+ CML therapy. However, only a restrict group of patients is able to achieve a complete molecular response (CMR). Based on a mathematic model, it has been suggested that imatinb reduces the rate at which differentiated cells are produced from leukaemic stem cells, with no significant effect in reduction of Ph+ stem cells (Michor F et al, Nature 2005). So, cure of the disease is still considered to be restricted to allogeneic bone marrow transplant. Clinical data obtained in patients who stop treatment can be of great value in evaluating the degree of suppression of the neoplastic clone. We report 6 cases of patients with CML in complete cytogenetic remission (CCyR) who interrupted imatinib treatment. Stop was due in 5 cases to some degree of toxicity whereas one male patient decided to stop for personal reasons. All patients were in chronic phase and previously achieved a CCyR within 12 months after imatinib start; major molecular remission (MMR) was observed in 5 cases, and 4 of them also showed a CMR in at least one occasion. After discontinuation, three patients had a molecular or cytogenetic relapse in a period of 3 to 6 months. In these cases, CMR was never reached, or in one case it was delayed in time, being observed after 3 years of treatment, and not confirmed in all subsequent samples. All patients responded again to imatinib treatment. Three other patients did not relapse and are PCR-negative with a follow-up of 18, 22 and 39 months respectively. One of them received imatinib for cytogenetic relapse after IFN + ARA-C, whereas two patients were treated with Peg-IFN and imatinib front-line; Peg-IFN was stopped for toxicity in both cases. At imatinib stop, they all were in sustained complete molecular remission, as defined by undetectable BCR-ABL transcript at RT- and RQ-PCR both on BM and PB samples, in multiple serial samples. In these three patients, CCyR was reached whithin 3 months of imatinib therapy and CMR was achieved after 1 year, 6 months, and 9 months respectively, and never lost. The median duration of treatment before discontinuation was 45,5 months, while the median time of PCR negativity on imatinib was 39 months. In most reported cases, imatinib discontinuation results in relapse after few months (Cortes J et al, Blood 2004; Michor F et al, Nature 2005; Mauro MJ et al, Leuk Res 2004). However, both time of treatment and duration of molecular response were relatively short in these reports. Recently, 12 patients who stopped treatment when in CMR for more than two years have been published (Rousselot P et al, Blood 2007): 6 of them relapsed early whereas 6 remain in CMR after a median follow-up of 18 months. We describe 3 cases who are persistently PCR-negative after imatinib discontinuation, with a median follow-up of 26 months; all of them were characterized by a rapid clearance of leukemic cells after start of imatinib, which allowed to obtain a stable CMR within the first 12 months of treatment. Less than 5% of early CP patients on imatinib achieve CMR in the first year (Brandford S et al, Blood 2006). These data suggest that kinetics of molecular response achievement may predict prolonged response after imatinib discontinuation, possibly reflecting a decline in leukemic stem cell compartement.

TH17 Pathway and Associated Pro-Inflammatory Cytokines Promote Immune Dysfunction in Myeloma.

Multiple myeloma (MM) is associated with significant immune dysfunction. Although various mechanisms mediating immune dysregulation in MM have been studied, its molecular and cellular basis is ill defined. IL-6, TGF-β and IL-1β have been implicated in this process, but their mechanism of effects on immune function have not been studied in MM. Together, IL-6 and TGF-β enhance the generation of TH17 cells, important in the development of immunity and auto-immunity. Additionally, TH17 cells are differentiated by number of inflammatory cytokines including, IL-21, IL-22, IL-23, and IL-27. Therefore, we evaluated the immune dysfunction and the role of TH17 cells and associated pro-inflammatory cytokines in myeloma. We have previously characterized that the production of TH1 mediated cytokines including IFN-γ following anti-CD3-mediated activation is significantly lower in myeloma PBMC compared to normal PBMC. We hypothesize that this may be regulated via skewing the immune system towards TH17 pathway. We observed that TH17 cells, measured by intra-cellular flow cytometry, are significantly increased in number in myeloma (16.9%) and MGUS (6.2%) compared to normal (3.3%). Furthermore, we analysed supporting pro-inflammatory cytokine network for the generation of TH17 cells in myeloma, which may be responsible for the observed TH17 skewing of T cell subsets. Sera from MGUS (n=12) and myeloma (n=17) patients were evaluated for the presence of these pro-inflammatory cytokines compared with normal sera (n=6) using ELISA. We observed significant increase in serum IL-21, IL-22 and IL-23 in MGUS (373 pg/ml, 14 pg/ml and 147 pg/ml respectively; p<0.05) and myeloma (296 pg/ml, 12 pg/ml and 215 pg/ml respectively; p<0.05) compared with normal (63 pg/ml, 1.5 pg/ml and 39 pg/ml respectively). In addition, we also observed that the myeloma PBMC stimulated in the presence of IL-6 and TGF-β, both of the cytokines present at a high level in myeloma, induced significant IL-23 production compared with normal. Importantly, IL-23 levels were 10 fold higher in myeloma BM samples compared with matching blood samples. These results indicate that the cytokines from myeloma BM microenvironment may be responsible for the observed T cell subset abnormality by favouring TH17 cells via IL-23/IL-21 production. These cytokines thus may be targets to modulate immune responses in myeloma to enhance immune function and devise effective vaccination strategies in the future.

GATA1 Is Over-Expressed in Essential Thrombocytemia

GATA1, founding member of the GATA transcription factor family, is essential for cell maturation and differentiation within the erythroid and megakaryocyte (MK) lineages. Disruption of DNA- or protein-binding capacity of GATA1 causes severe hematopoietic dysfunction and plays a role in blood disorders such as thrombocytopenia, anemia or leukemia. GATA1 expression seems to be related to the MK commitment both in mice and in humans; indeed, similarly to the murine myeloid M1 cell line, in which the enforced expression of GATA1 induces the c-Mpl appearance and MK differentiation, transduction of human hematopoietic stem cells with a GATA1 highly expressing vector results in self-renewal block and in the exclusive generation of Meg-E lineages. More recently, a role for GATA1 also in myeloproliferative disorders (MPDs) was indicated by the “GATA1-low” mouse model which develop a disease closely resembling the human idiopathic myelofibrosis. Interestingly, patients affected by myelofibrosis was also shown to express decreased GATA1 levels by immunostaining of BM sections. In this study, we investigated by Real Time PCR the levels of GATA1 in a myeloproliferative disorders such as essential thrombocytemia (ET) tipically characterized by a neoplastic megakaryocitic proliferation. We have studied BM samples of 40 newly diagnosed patients (M:F ratio 1:1 - median age 53 years, range 18–84) affected by ET, as for the PVSG group criteria. These patients were selected from a cohort of 65 ET patients considering a similar erythroid/myeloid ratio at the FACS analysis to reduce a possible bias for the RT-PCR results due to the erythroid compartment interference. The median platelets count of the selected patients was 670,000/mL (range 493,000–1,400,000/mL), myelofibrotic index 0/1, and 18 out of 40 patients (45%) showed mild splenomegaly both at the physical examination and US scan (median spleen vol 550 ml - range 430–1400 mL). No chromosomal abnormalities were detected by cytogenetic analysis. JAK2 sequencing in 21/40 patients indicated that 9/21 patients (43%) were positive for the JAK2 V617F genomic mutation. At the end of observation time (median 18 mo.) no patients had evidence or signs of thrombotic or hemorrhagic complications. BM cells from six healthy donors were used as normal controls in the study. The relative GATA1 quantification was calculated in according to the DCt method with GAPDH as internal control. The results showed a significant increase of GATA1 expression in BM cells from ET patients (median DCt + 6,11 ; range −0,41/+18,11) compared to the controls (median DCt + 0,172 ; range −4,03/+1,7) (p < 0,003). Interestingly, the GATA1 overexpression is not a mere consequence of the proliferation and activation of MKs, indeed samples from three patients affected by idiopathic thrombocytopenic purpura, whose BM smears had the typical secondary megakaryocytic hyperplasia, showed GATA1 levels much lower than the ET patients (median DCt − 0,6 ; range − 3,21/−0,9). No significant difference in GATA1 level was found between patients harbouring a JAK mutation (median DCt +5,86 ; range 0,85/16,12) and those with wild type alleles (median DCt +4,75 ; range −0,41/10,21). In conclusion, our results suggest that GATA1 overexpression could be a trigger for MK neoplastic commitment and proliferation and, consequently, seems to have a central role for ET pathogenesis both in JAK2 mutated and in JAK2 WT patients.

Treatment with Sequential Valproic Acid (VPA) +/− Low Doses All Trans Retinoic Acid (LoATRA) and Low Doses Ara-C (LoDAC) for “De Novo” or Relapsed Acute Myeloid Leukaemia (AML).

Recent reports indicated the clinical feasibility and biological therapeutic potentiality of epigenetic treatments with VPA + ATRA or ATRA alone and suggested the capability of these agents in rendering neoplastic cells more sensitive to chemotherapy. Therefore, we designed treatments consisting of LoDAC preceded by the sequential administration of VPA+/LoATRA or LoATRA alone, according to the clinical condition of patients, to treat AML patients not eligible for intensive therapy. Aims of this study were to evaluate:i.the efficacy of these approaches in term of response rate and toxicity;ii.the biological changes occurring in the leukemic clone in relation to the clinical response, by a multiparametric in vivo translational experimental approach.From September 2006 to June 2007, 5 patients (pts) with de novo and 5 with relapsed AMLs were enrolled.The median age was 66 years (range: 46–81 yrs), the mean percentage of BM leukemic infiltration was 60% (range 30–81%);as cytogenetics 5 pts showed complex karyotype. Four patients (1= de novo; 3=relapsed) received an induction treatment with VPA+LoATRA+LoDAC (VPA):VPA at the initial dose of 10 mg/kg/die orally escalated, to reach the therapeutic VPA plasma levels (>50μg/ml),days 1–55, LoATRA 25 mg/m2/die orally, days 7–55 and LoDAC 40 mg/sc at day (d)10 and 45 for 7 d. In responders, therapy was repeated up to a maximum of 3 cycles with a 20 days rest period. Six patients (4 = de novo; 2 = relapsed), received LoATRA alone (days 1–55) because of coexisting co-morbidities , followed by LoDAC as described. Peripheral blood (Pb) and BM samples for biological studies were collected at d 0,7,14,21,35,55. Responses were evaluated after the first cycle of therapy according to the IWC criteria. A complete remission (CR) was observed in 4 cases (2 relapsed = VPA; 2 de novo= LoATRA), while 2 de novo pts (LoATRA) showed a major response (MR).In the 6 responders PB recovery (Hb>9g/dl; PMN>1000/m 3and PLTS>50.000/m3), was observed within a median time of 42 d (range: 35–50 d), while the median time of BM blast clearance was 35 d (range 25–50 d). Of the remaining 4 pts, 1 died during induction, at 45 d for cardiac failure, 3 maintained a stable disease and died 7, 5 and 3 months(mo) later. As to July 2007, 2 pts (Lo ATRA) persisted in CR at 2 and 4 mo, while 2 pts (VPA) relapsed at 3 and 4 mo from CR. Five pts are still alive at 4+, 5+, 5+, 5+, and 9+ mo.Biological assays showed that in mononuclear samples from the 4 CR patients, phenotypic changes including:i.the increased percentage of cells in S phase;ii.cytochemical changes (myelo-monocytic differentiation);iii.decreased expression of early myeloid/progenitor immunophenotypic markers (HLA-DR, CD34, CD117), which paralleled the increase of late myeloid differentiation markers (CD11b, CD15 or CD14), started to be measurable before LoDAC administration.In conclusion, the sequential therapy with VPA and/or LoATRA + LoDAC are well tolerated out-patient therapies that enabled a CR in a sizeable portion of our cases. In addition, these treatments induce AML blast phenotypical changes that may increase their sensitivity to LoDAC, whose molecular basis is currently under investigation.

Impact of Minimal Residual Disease (MRD) on Prognosis in Children with Acute Lymphoblastic Leukemia (ALL) According to WBC Count, Age and TEL/AML1 Status at Diagnosis. Results of the AIEOP-BFM ALL 2000 Study.

Minimal residual disease (MRD), the most sensitive method to evaluate treatment response, has been adopted to stratify patients in study AIEOP-BFM ALL 2000. To assess whether PCR-MRD levels discriminate outcome in patients classified by WBC count, age at diagnosis, NCI criteria (Standard Risk, SR: WBC < 50,000/cmm and age 1–9 years; High Risk, HR: all others) and TEL/AML1 status. Between 07–2000 and 07–2006, 4,730 Ph-negative patients were enrolled in AIEOP-BFM ALL 2000 study. They were treated with BFM Induction (protocol IA) consolidation (protocol IB), extra-compartment/intensified consolidation (HD-MTX in non-HR patients, blocks in HR patients), reinduction therapy (one or more Protocols II or III), followed by maintainance. BM samples obtained at weeks 5 (Time Point 1, TP1) and 12 (TP2) of induction/consolidation therapy were used for PCR-based MRD analysis of patient specific gene targets. At least 2 sensitive markers (≥ 1 x 10−4) could be determined in 3,707 (78.4%) patients. SR was defined by MRD− at both TP1 and TP2; HR by MRD ≥1x10−3 at TP2; Intermediate Risk (IR): all others. Median follow-up was 3 years; 5-year percent EFS (SE) estimates are given.Patients at MRD-SR, IR or HR had, respectively, an EFS of 93.0 (1.0), 80.5 (1.5) and 43.4 (6.0) in patients with WBC <50,000/cmm vs 90.4 (2.6), 72.4 (3.0) and 47.0 (5.1) in patients with WBC ≥50,000/cmm. Patients at MRD SR, IR or HR had, respectively, EFS of 93.6 (1.0), 80.3 (1.5) and 44.1 (5.4) if aged 1–9 years vs 87.2 (3.4), 73.9 (3.0) and 49.3 (5.2) if aged ≥10 years. Patients at SR by NCI criteria [N= 2,355, EFS of 85.3 (1.0)] were stratified by PCR-MRD as SR (N=1046; 44.4%), IR (N=1198; 50.9%), or HR (N=111; 4.7%). EFS in these subgroups was 93.9 (1.0), 81.3 (1.6) and 43.9 (7.2), respectively (p<0.001). In patients at HR by NCI criteria [N=1,352, EFS of 75.6 (1.6)], 403 (29.8%), 774 (57.3%) and 175 (12.9%) respectively were at SR IR and HR by MRD. EFS was 89.4 (2.2) in MRD SR, 74.7 (2.3) in MRD IR and 47.9 (4.2) in MRD HR patients (p<0.001). Of 3,707 study patients, 3,410 were investigated for TEL/AML1 status: 771 (22.6%) were positive and 2,639 were negative. TEL/AML1+ patients were at SR (N=444; 57.6%) or IR (N=317; 41.1%) or HR (N=10; 1.3%) by PCR-MRD; EFS in this subgroup was 94.4% (1.5), 80% (3.7) and 60% (18.4), respectively (p<0.001). TEL/AML1− patients at SR (N=887; 33.6%) or IR (N=1497; 56.7%) or HR (N=255; 9.7%) had an EFS of 91.6% (1.3), 78.5% (1.4) and 45.7% (4.5), respectively (p<0.001). PCR-MRD in patients treated with BFM-oriented therapy overcomes the prognostic value of “historical” factors such as WBC count, age, NCI criteria or TEL/AML1 status, as it markedly discriminates prognosis within each subgroup defined by these variables. Study design for contemporary risk-directed therapy of childhood ALL should incorporate a technique for MRD determination.

Different Number of AML1/ETO Transcripts in Single Leukemic Cell, Estimated by Combined Measurement of MRD by FISH and Real-Time PCR, Is a Powerful Predictor of Relapse.

The t(8;21)(q22;q22) translocation is observed in about 40% of pediatric cases of AML M2. Although patients with t(8;21) AML have a relatively good prognosis, relapses still occur in about 30% cases. To predict relapse, several studies examining sequential quantitation of minimal residual disease (MRD) by real-time PCR after chemotherapy or BMT have been attempted. However, these studies have shown that only a very small number of AML1/ETO transcripts are detectable in most t(8;21) AML patients who maintain complete long-term remission (CR). A recent study clearly demonstrated the existence of nonmalignant stem cells with leukemia specific genes. In another study, the suppression of AML1/ETO by siRNA in vitro resulted in the increased expression of genes associated with myeloid differentiation as well as antiproliferative genes in both leukemic blasts and cell lines. This suggests that cells expressing lower level of AML1/ETO might be well-differentiated malignant cells with slower proliferation rates compared to those expressing higher levels of AML1/ETO. Taken together, we hypothesized that the translocated gene detected by PCR, even in long-term remission patients, might be derived from nonleukemic stem cells or well-differentiated leukemic cells. If we can estimate AML1/ETO transcripts in single cells possessing leukemic translocation genes in BM in CR, we can predict, with high reliability, whether early relapse would occur or not. To test this hypothesis, we simultaneously assessed MRD by FISH with locus-specific probes with higher sensitivity and by quantitative real-time PCR in BM samples obtained from seven pediatric patients with AML1/ETO during and after chemotherapy/BMT. We then evaluated the value which was calculated by dividing normalized AML1/ETO copies/μgRNA by the percentage of t(8;21)-positive cells by FISH (RQ-PCR/FISH), which ideally reflected actual number of AML1/ETO transcripts in single cell with leukemic translocation in BM cells. RQ-PCR/FISH of non-relapse patients ranged from 5.5 × 102 to 5.5 × 103 (mean 2.5 × 103 ) in BM in CR. In marked contrast, 8.0 × 103 to 1.8 × 105 (mean 5.9 × 104 ) was observed from the patients who finally relapsed. Indeed, we demonstrated that in some cases showing low value of RQ-PCR/FISH, the AML1/ETO was detected only in terminally differentiated basophils (CD34− CD203c+ ), not in stem cell fractions (CD34+ CD203c± ). In conclusion, we propose that RQ-PCR/FISH can be a powerful predictor of relapse, especially in patients with lasting MRD positive after chemotherapy/BMT.

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CMV‐specific central memory T cells reside in bone marrow

CMV-specific CD8(+) T cell responses in peripheral blood (PB) are characterized by a preponderance of effector and effector memory T cells. CMV-specific central memory T cells (T(CM)), which are considered crucial in maintaining long-term immunity, are rarely detectable in PB. In this study we have analyzed differentiation and function of CMV pp65-specific CD8(+) T cells in paired samples of human PB and BM using intracellular cytokine and tetramer staining. Overall frequencies of CMV pp65-specific T cells were similar in PB compared to BM; however, CMV-specific CD45RA(-)CCR7(+) T(CM) were almost exclusively detectable in BM, which was not related to a general accumulation of T(CM) in BM. In vitro, CMV-specific T cells could be more efficiently expanded from BM (median 128-fold, n=6) than from PB (median 72-fold, p=0.01). Taken together, these data show that the BM is a compartment harboring CMV-specific T(CM) and underline the concept of the BM as a secondary immune organ. CMV specific BM-derived T(CM) might be a valuable source for generating T cells for adoptive transfer.

Microstructure and hydrogen storage properties of the laser sintered [formula omitted] alloy

The Mg 2 Ni hydrogen storage samples were prepared by the laser sintering method. Before laser sintering, the Mg and Ni powders were pre-mixed by pestle milling (sample PM) or ball milling (sample BM). The microstructures and hydrogen storage properties of the laser sintered Mg 2 Ni samples were investigated. The sintered samples contained Mg 2 Ni and small amount of MgNi 2 , Mg, Ni and MgO. Compared with the sample PM, the amounts of Mg 2 Ni and MgNi 2 reduced and the amounts of Mg and Ni increased in the sample BM. The laser sintered Mg 2 Ni samples can be activated easily at 573 K. The maximum hydrogen storage capacities of the PM and BM samples were 3.23 and 3.44 wt%, respectively.

Genomic profile of chronic myelogenous leukemia: Imbalances associated with disease progression

The expression of the chimeric BCR/ABL1 fusion gene resulting from t(9;22)(q34;q11) in chronic myelogenous leukemia (CML) is necessary for malignant transformation, but not sufficient to maintain disease progression. The appearance of various chromosomal and molecular alterations in the accelerated and terminal phase of CML is well documented, but evidence for causal relationship is largely lacking. We carried out a genome wide screening at a resolution of 1 Mb of 54 samples at different stages of CML together with 12 CML cell lines and found that disease progression is accompanied by a spectrum of recurrent genome imbalances. Among the most frequent are losses at 1p36, 5q21, 9p21, and 9q34 and gains at 1q, 8q24, 9q34, 16p, and 22q11, all of which were located with higher precision within the genome than previously possible. These genome imbalances are unique to CML cases with clinically manifested or suspected accelerated/blast stage alike, but not seen in chronic phase samples. Previously unrecognized cryptic imbalances occurring within the Ph-chromosome were also detected, although further scrutiny is required to pin-point gene involvement and seek association with disease features. Importantly, some of these imbalances were seen in the CD34(+) cells but not in the whole BM samples of patients in accelerated phase. Taken together, these findings highlight the potential of screening CD34(+) cells for genome wide imbalances associated with disease progression. Finally, the numerous single copy number variations recorded, many unique to this cohort of patients, raise the possible association of genome polymorphism and CML.

Chromosome 13q deletion and IgH abnormalities may be both masked by near-tetraploidy in a high proportion of multiple myeloma patients: A combined morphology and I-FISH analysis

Ploidy status and chromosomal aberrations involving chromosome 13q and the immunoglobulin heavy chain locus (IgH) are important prognostic features in multiple myeloma (MM). However, conventional cytogenetic studies are often not reveling and determination of plasma cells (PC) ploidy status in MM is technically difficult. We have used a combined cell morphology and interphase FISH (I-FISH) analysis in 184 consecutive BM samples from 136 MM patients for the diagnosis of chromosome 13q deletion [del (13q)] and IgH abnormalities. We have found a high prevalence (37%) of near-tetraploid (NT) PC in the BM samples studied. NT status of PC was verified with DNA index (DI) measurements. del (13q) was found in 69% and a total absence of one IgH copy (loss of IgH) in 20% of NT samples. We have shown that the presence of del (13q) and loss of IgH can be masked in NT cases: in 12 NT samples originally identified as normal for del (13q) the abnormality was obscured in the majority of plasma cells due to the presence of NT. Similarly, loss of IgH was masked in four samples with a large population of NT cells. Moreover, in one case the appearance of a 100% tetraploidy during disease progression masked the presence of del (13q), originally present, and could therefore falsely appear as disappearance of this prognostic marker. In conclusion, we have shown that a combination of three abnormalities, i.e., del (13q), loss of IgH and NT, all of potential prognostic significance, can be overlooked unless NT is specifically searched for and ruled out. Therefore, we suggest that a search for NT should be added to the routine BM assessment in MM patients.

GMP-grade preparation of biomimetic scaffolds with osteo-differentiated autologous mesenchymal stromal cells for the treatment of alveolar bone resorption in periodontal disease

Periodontal disease is a degenerative illness that leads to resorption of the alveolar bone. Mesenchymal stromal cells (MSC) represent a novel tool for the production of biologic constructs for the treatment of degenerative bone diseases. The preparation of MSC differentiated into osteogenic lineage for clinical use requires the fulfillment of strict good manufacturing practice (GMP) procedures. MSC were isolated from BM samples and then cultured under GMP conditions. MSC were characterized phenotypically and for their differentiative potential. Cells were seeded onto collagen scaffolds (Gingistat) and induced to differentiate into osteogenic lineages using clinical grade drugs compared with standard osteogenic supplements. Alizarin Red S stain was used to test the deposition of the mineral matrix. Standard microbiologic analysis was performed to verify the product sterility. The resulting MSC were negative for CD33, CD34 and HLA-DR but showed high expression of CD90, CD105 and HLA-ABC (average expressions of 94.3%, 75.8% and 94.2%, respectively). Chondrogenic, osteogenic and adipogenic differentiation potential was demonstrated. The MSC retained their ability to differentiate into osteogenic lineage when seeded onto collagen scaffolds after exposure to a clinical grade medium. Cell numbers and cell viability were adequate for clinical use, and microbiologic assays demonstrated the absence of any contamination. In the specific context of a degenerative bone disease with limited involvement of skeletal tissue, the combined use of MSC, exposed to an osteogenic clinical grade medium, and biomimetic biodegradable scaffolds offers the possibility of producing adequate numbers of biologic tissue-engineered cell-based constructs for use in clinical trials.

Appearance of Bone Marrow Lymphocytosis as a Sign of Immunoactivation Is Associated with a Good Response to Imatinib Therapy in Patients with Chronic Myeloid Leukemia.

In addition to potent BCR-ABL -mediated anti-leukemia effects, growing evidence suggests that imatinib mesylate (IM) also acts as an immunomodulatory agent. IM modulates both innate and adaptive immunity with impact on dendritic, NK, and T cell activation. Current data implies an immunostimulatory effect of IM, which could be a factor in the long-term control of chronic myeloid leukemia (CML). Modest increase in BM lymphocyte count has often been observed during imatinib therapy. We speculated that this increase may be related to the immunoactivation induced by IM and studied the relation of lymphocyte counts in BM aspirates to therapeutic response to IM.A total of 147 BM aspirates were studied from 28 CML patients in chronic phase and on imatinib therapy (28 diagnostic/pretreatment and 119 follow-up samples taken generally 3 monthly). The samples were analyzed blinded to the imatinib response. The patient was classified as having BM lymphocytosis if the BM lymphocyte count was above normal (>25%) in more than one BM sample during IM therapy. Patients were treated according to clinical protocols and response to treatment was assessed according to recent guidelines (Baccarani et al, 2006). Lymphocyte immunophenotyping was done from BM and peripheral blood samples, when available.The median follow-up time for IM response was 18 months. During the IM treatment 45 of 119 samples (38%) had an increase in lymphocyte count in BM aspirates, whereas only 1 of 28 (4%) samples at diagnosis had a minor increase in lymphocyte count. The increase in lymphocyte count was related to the IM therapy response; patients with good response had more often lymphocytosis in BM aspirates.Response to IMBM lymphocytosisYesNoGood (n=13 patients)9 (69%)4 (31%)Suboptimal (n=7 patients)2 (29%)5 (71%)Failure (n=8 patients)1 (13%)7 (87%)p=0.036 (Fisher's Exact test)In the good response group most patients (7/9) had an increase in lymphocyte count already at 3 months after the start of therapy and the same phenomenon usually continued throughout the therapy. In the failure and suboptimal groups the BM lymphocyte increases occurred only in sporadic samples (median value for positive lymphocytosis samples per patient was 6%, 13%, and 80% in failure, suboptimal, and good therapy response groups, respectively).Peripheral blood leukopenia was more common in the failure group (69%) compared to good (14%) and suboptimal (26%) groups, but BM samples were not hypoplastic and the blood leukocyte/lymphocyte counts were not related to BM lymphocyte counts.Preliminary data on immunophenotyping indicated that the BM lymphocytes were a mixture of T- (normal CD4/CD8 ratio), NK-, and B-cells (T-cells predominating). B-cells were polyclonal and a minor percentage of B-cells were immature (CD10 positive). In conclusion, we found a clear association between an increased BM lymphocyte count and a good IM response. In patients responding to IM, increases in BM lymphocyte count could already be seen at 3 months. The observed lymphocyte proliferation in good-responding patients may reflect the concerted action of various types of lymphocytes mediating putative immunological anti-tumor effect of IM treatment.

Correlation between Flow Cytometry Immunophenotyping, Chimerism Quantification and IGH Gene Rearrangement Analysis in Disease Associated Leukocyte Lineages after Stem Cell Transplantation in an ALL Patient.

INTRODUCTION: Oligocloanality is a phenomenon shown in up to 40% of patients with childhood B-lineage acute lymphoblastic leukemia (ALL), and it might be associated with a poorer prognosis.OBJECTIVE: To present a case of oligoclonality based on the detection of IGH gene rearrangements in a patient with ALL and the correlation of the results with those of flow cytometry immunophenotyping and chimerism quantification.PATIENT AND METHODS: 26 years old female, pre-B ALL diagnosed in her childhood, in relapse after autologous and allogeneic HLA-identical unrelated stem cell transplantation (SCT). Immunophenotyping was done by multiparametric flow-cytometry, chimerism quantification was performed using fluorescent in situ hybridization (FISH) for the sex chromosomes, and IGH gene rearrangements were detected by nested-PCR revealed by capillary electrophoresis. Disease associated leukocyte lineages (CD19+, CD19+CD34+ and CD19+CD34−) were purified using immunomagnetic technology.RESULTUS: Flow-cytometry analysis in PB and BM samples at diagnosis showed expression of CD45 (weak), CD34, CD38, CD10, CD19, CD20, CD22, CD66, CD79acyt, TdT and aberrant expression of CD33, with a detectable clonal IGH rearrangement. At relapse, two distinct CD19+ cell populations were revealed from BM samples: one immature (CD34+, 15% of the total leukocytes) with similar phenotype at diagnosis, and another one mature (CD34−, 10% of the total leukocytes). FISH analysis showed mixed chimerism (MC)(31% recipient) in BM samples and complete chimerism (CC) in PB samples. Molecular analysis showed three different clones in BM and a polyclonal pattern in PB. In order to increase sensitivity, CD19+ cells were isolated from BM and PB. Cells selected from BM showed MC (98,5% recipient) and the three clonal IGH gene rearrangements while those from PB showed CC and a polyclonal pattern. Further studies exhibited persistence of the CD19+CD34+ population and significant reduction of the mature CD19+CD34− cells. A persistent three clone pattern was present in BM samples. In order to clarify the origin of the different IGH gene rearrangements, two consecutive immunomagnetic selections, one with CD19+ and other with CD34+ antibodies, were performed from BM samples to purify both CD19+CD34+ and CD19+CD34− populations. CD19+CD34+ cells showed all the three IGH clones and MC (88% recipient). Moreover, CD19+CD34− cells showed a monoclonal pattern with an unique peak, not detected in previous studies, and CC (Table 1).CONCLUSIONS: The clonal diversity of IGH gene rearrangements detected at relapse after SCT was generated from the original malignant CD34+ clone, possibly due to clonal evolution, and seems to be associated with the poor outcome of this case. After multiple therapy modalities, the CD19+CD34+ cells showed resistance to treatment. Surprisingly, the CD19+CD34− cells showed a new clonal rearrangement although its donor origin confirmed its reactive nature in the context of SCT. This observation has relevance in the further follow up and minimal residual disease detection in this case. [Display omitted]

Gene Expression Profiling of Paired Pre- and Post-Prednisolone (PRED) BM Samples from Childhood ALL Identifies Robust Signatures for PRED Response and Eventual Outcome.

Early response to therapy is the most important prognostic factor for childhood ALL. CCG investigators have shown that Day-7 and Day-14 BM blast counts were prognostically important although there is great inter-observer variability. BFM group have shown that day 8 prednisolone (PRED) response is highly predictive of the treatment outcome. While gene expression profiling (GEP) of diagnostic marrow can discern a pattern of PRED sensitivity as determined by in vitro MTT assay, the accuracy was low at only 70%. We hypothesized that changes in global GEP after therapy have a higher likelihood to predict response as the signatures of sensitivity and resistance may be unmasked during the therapy. We prospectively studied the changes in GEP using Affymetrix HG-U133A or Plus 2 chips on paired BM samples before and after 7-day course of PRED and one dose IT MTX in 58 patients with newly diagnosed or relapsed ALL. Unsupervised hierarchical clustering revealed that pre- and post- PRED samples in the patients still tended to cluster together, indicating that expression profiles of molecular subgroups were still most important. To remove intrinsic influence of molecular subtypes and identify potential signatures independent of genetic abnormalities, we subtracted Day-0 GEP from its paired Day-8 profile and retained probe sets with significant changes (≥ 10-fold). To avoid the ambiguity of variation in BM blast counting at Day-8, we divided the samples into a stringently reproducible group where “Good” PRED response was defined as that Day-8 blast count in PBL < 109/L and BM lymphoblasts ≤ 30% (n=16). “Poor” response was when Day 8 PBL ≥ 109/L (n=11). This stringently reproducible group (n=27) formed the training group to help define a distinct signature while the rest (n=31 pairs) were used as a blinded test set. 54 and 19 discriminating genes were identified by 2 independent statistical methods respectively, and an integrated predictor model was constructed based on shortlisted entries. This model predicted the PRED response with 100% accuracy for the training set using the leave-one-out cross validation but was less accurate in predicting the BM blast count in blinded test set. But intriguingly, in the blinded test set, this model predicted correctly 19 out of 21 reliable “Good” PRED responses are in CCR (91%), while among 8 predicted as “Poor” responses, only 2 are in CCR (25%). This suggests that as gene expression profiling as early as day 8 of PRED could discern the beginning of leukaemia cell death even before morphological changes are discernable and is highly correlated to eventual outcome. In conclusion, we have shown that analyses on the relative changes of gene expression profile can identify real genetic signatures indicating the sensitivity to PRED administration which is highly correlated with outcome.

Real-Time Detection of PML-RARA and RARA-PML Fusion Transcripts in High Risk Acute Promyelocytic Leukemia (APL) Treated with Arsenic Trioxide (ATO): Implications for Realization of Pre-Emptive Therapy for Molecular Relapse.

A key challenge in the management of APL is to improve upon survival rates now achieved with ATRA and anthracycline-based chemotherapy. One approach involves minimal residual disease (MRD) monitoring to identify patients otherwise destined to relapse, whose outcome may be improved by pre-emptive therapy. There is also scope for improvement through reduction in induction deaths and treatment-related mortality, with recent studies suggesting that molecularly targeted therapy involving ATO can achieve long term remissions with limited toxicity. However, such strategies are critically dependent upon optimized schedules for MRD detection to rapidly identify those patients needing additional treatment. Yet, there are limited data concerning assay sensitivity, optimal sample type or kinetics of relapse, that are fundamental to delivery of MRD-directed therapy. We determined expression levels of PML-RARA and reciprocal RARA-PML transcripts in diagnostic BM samples from 102 patients using real-time quantitative PCR (RQ-PCR). Normalized PML-RARA transcript levels varied by 3-logs, equating with marked inter-patient variability in maximal achievable sensitivities for MRD detection, ranging between 1 in 102 and 1 in 105 (med. 1 in 103.8). RARA-PML fusion transcripts were expressed in 68/102 (67%). Parallel detection of both transcripts increased sensitivity for MRD detection in 35/102 (34%); yet, the detection limit was still <1 in 104 in 49% due to the low level of fusion transcript expression. Serial MRD monitoring was undertaken in patients (n=32) treated with ATO for relapsed APL in UK and Eire since Nov 02: 22 in hematologic relapse (HR) and 10 treated pre-emptively in 1st molecular relapse (MR). RQ-PCR assessment prior to MR/HR showed a median increase in PML-RARA transcripts of 0.8 log/month (range 0.2–1.9). Analysis of paired BM and blood (PB) samples revealed that molecular conversion in BM invariably preceded that in PB; including a patient testing negative in PB despite a significant level of disease in BM (6% by FISH). Parallel use of the RARA-PML assay in evaluable patients increased rates of MRD detection, in some instances providing earlier warning of impending relapse. ATO was given using the conventional schedule (0.15mg/kg/d - Regimen A) or a regimen involving loading followed by intermittent dosing (0.3mg/kg/d x 5d, then 0.25mg/kg 2x/wk - Regimen B). No difference in kinetics of molecular response was observed; however amongst patients treated in HR, hyperleucocytosis was less common with Regimen B (peak WBC 5 vs 126 x 109/l, p=0.007), indicating that this schedule may favor apoptosis over differentiation and merits evaluation in newly diagnosed APL. OS from time of relapse was 60% at 3yrs; of 10 patients treated pre-emptively, 7 are in 2nd molecular CR at median of 23mo (7–40mo), 2 died in relapse, treatment is on-going in 1. This study highlights sample type, assay sensitivity (which demonstrates inter-patient and -sample variability) and kinetics of relapse as critical factors that need to be taken into account for the successful delivery of MRD-directed therapy, which is becoming increasingly pertinent to the management of other subsets of acute leukemia.