bluuebunny/crossref_metadata_2025 huggingface dataset:
All IPI Factors Are Equal, but LDH Is More Equal Than Others.
Authors: van Imhoff Gustaaf W. , Graveland W. , Van der Holt B. , Van Glabbeke M. , Verdonck L. F. , Sonneveld P. , Kluin-Nelemans J. C.
Published: October 2019

The International Prognostic Risk Index (IPI) offers the most important prognostic information in patients with aggressive non-Hodgkin Lymphoma (NHL). Risk classification is based on presence or absence of age >60 yr, stage III or IV, more than one extranodal site, performance score 2–4, or elevated serum LDH above the upper limit of normal (ULN). Because each factor confers a more or less equal independent risk for treatment outcome, they are summed to generate a final IPI risk score (Shipp model). Dichotomization of the continuous variable age is practical, as treatment for patients >60 yr often differs from younger ones. However, as LDH also is a continuous variable we wondered if risk based on actual LDH, especially in patients with highly elevated levels, would have additional prognostic impact. IPI risk factors including actual serum LDH at diagnosis were retrieved from 1286 patients (28% >60 yr) with advanced aggressive NHL and treated with curative intent in 6 clinical trials conducted by HOVON (5 trials) and EORTC (1 trial). All LDH ULNs from the participating centers were verified. LDH levels were divided by the ULN of each center to generate normalized ratios. Six % of patients had LDH >5 times ULN, 8% 3–5 times, 11% 2–3 times, 34% 1–2 times, 20% 0.75–1 times and 20% had levels below 0.75 of ULN. In a multivariate Cox regression model similar independent hazard ratios (HR) ranging from 1.6 to 2.6 were found for the individual dichotomized risk factors according to the Shipp model except for the number of extranodal sites which turned out to be non-significant. This factor was with a HR of 1.4 also the least predictive factor in the Shipp model. In contrast to the dichotomized LDH variable (normal versus elevated), risk for inferior outcome increased linearly with actual LDH levels. Five year OS estimates were 71% for patients with LDH <0.75 x ULN; 53% for 0.75–1; 49% for 1–2; 37% for 2–3; 31% for 3–5; and 25% for LDH >5 x ULN. The HR for these groups were respectively 0.54, 1, 1.45, 2.46, 2.54 and 5.15. This analysis using actual LDH values gave a better discrimination as compared to the HR of 2.6 (95% CI 2.1–3.1) for the dichotomized LDH (i.e. normal versus elevated) in multivariate analysis. Interestingly, the 235 patients with very low LDH (<0.75 x ULN) had much better outcomes with a HR of 0.54 as compared with patients with an LDH-ratio between 0.75 and 1. In conclusion, highly elevated LDH levels in aggressive NHL confer a worse prognosis and suggest the application of a modified IPI risk index adding extra risk points for patients with highly elevated LDH levels.
Analysis of EBV-Specific T Cell Responses in Transplant Recipients with PTLD.
Authors: Sebelin Kathrin , Meier Antje , Papp-Vary Matthias , Oertel Stefan , Pezzutto Antonio , Doerken Bernd , Riess Hanno , Subklewe Marion
Published: October 2019

EBV causes a chronic infection in >95 % of the population and despite its strong growth transforming capacity the majority of EBV infected individuals remain asymptomatic. In contrary, in immunosuppressed patients (pts) the risk of EBV reactivation and development of posttransplant lymphoproliferative disease (PTLD) is high. This is assumed to be due to a defective T cell response. Here we analyzed the EBV-specific CD8 and CD4 T cell response to different EBV latent and lytic antigens in pts with newly diagnosed PTLD. A prospective study of 10 pts after solid organ transplantation at time of diagnosis of PTLD was performed. EBV-specific CD8 T cells were examined by flow cytometric analysis using HLA-A2, HLA-B7 and HLA-B8 restricted tetramers incorporating BMLF1 (lytic), EBNA3 and LMP2 (both latent)-derived peptides. Staining was done in conjunction with mAbs against CD8 and CCR7. The ability of CD8 T cells to produce IFN-γ in response to the same EBV-derived peptides was measured by cytokine secretion assay. In healthy, EBV+ donors, we previously have found a consistent CD4 T cell response to the latent EBV antigen EBNA1. Therefore, EBNA1-specific CD4 T cell responses were monitored for IFN-g / IL-4 secretion after protein stimulation. T cell analysis was combined with EBV-DNA quantiation by real time PCR. We found EBV-specific CD8 T cell responses at low frequency in most pts with PTLD (8/10). Half of the pts showed low frequency EBNA1 specific CD4+ T cell responses. All pts with an EBNA1 specific CD4 T cell response showed an EBV-specific CD8 T cell response. In 2/10 pts we found no EBV-specific CD4 and CD8 T cell responses and both pts died under initial therapy. EBV-viral load was found to inversely correlate to absolute CD4 T cell counts. In comparison to healthy normal donors, no significant differences in EBV-specific T cell response could be observed. However, pts EBV-specific T cells were decreased in comparison to pts with high EBV viral load after TX and no PTLD as well as in comparison to pts with infectious mononucleosis. These results indicate that impairment of EBV-specific T cells is not due to clonal depletion, but rather seems to be due to impaired functional activation and expansion. We therefore conclude that pts with PTLD have an inadequatly low EBV-specific T cell responses which correlates to a low absolute CD4 T cell count. We propose a combined immunomonitoring of EBV viral load, absolute CD4 T cell count and EBV-specific T cell enumeration in pts at risk for development of PTLD. Further studies are needed to evaluate the role of EBV-specific T cell monitoring in immunosuppressed pts for prediction of PTLD and the potential usefulness of T cell monitoring as a prognostic marker in PTLD.
Interluekin-10 (IL-10) Promoter Gene Polymorphism (−819*C) Associated with Poor Clinical Outcome in DLBCL Patients Treated with R (Rituximab)-CHOP Regimen as a First Line.
Authors: Kim Yeo-Kyeoung , Kim Hee-Nam , Lee Je-Jung , Yang Deok-Hwan , Lee Il-Kwon , Park Kyeoung-Soo , Kim Hyeoung-Joon
Published: October 2019

IL-10 is an anti-inflammatory cytokine inhibiting Th1 functions, which may facilitate development of NHL by supporting tumor escape from the immune response. Among the various IL-10 promotor genes, the IL-10-1082G, −819C and −592C alleles were reported to be associated with high serum IL-10 level and there were a few reports about their roles in treatment outcome of DLBCL patients. In the present study, we studied the frequency of these 3 alleles and their influence to the clinical outcome in DLBCL patients treated with standardized regimen; 8 cycles of R (375 mg/m2 on day 1 of each cycle)-CHOP every 3 weeks or 4 cycles of R-CHOP with involved field radiotherapy. 60 patients with newly diagnosed DLBCL were included and all of them represented CD20 positivity. At diagnosis, median age was 60 (19~95) and 30 (57.7%), 13 (25.0), 7 (13.5%) and 2 (3.8%) patients belonged to the low, low-intermediate, high-intermediate and high risk group according to IPI reports, retrospectively. Of total 60 patients, 9 (15%), 23 (38.3%) and 27 (45%) were found to carry IL-10-1082G, −819C and −592C alleles, retrospectively. As compared with -1082AA genotype, -1082G allele (GA/GG) failed to show better outcome (eg. higher CR or longer progression-free survival, PFS) in interim results after 4 cycles of treatment. On the other hands, after completion of 4 R-CHOP, patients with −819C allele (TC/CC) showed significantly lower response rate (CR or PR) than in patients carrying -819TT (82.6% vs. 100%, p<0.05). Furthermore, the progression-free survival (PFS) was significantly shorter in patients carrying −819C allele than TT genotype (18.4 ms. vs. 26.9 ms. p<0.05). Patients with −592C allele (AC/CC) also showed lower interim response rate than with AA genotype (85.2% vs. 100%, p<0.05). PFS also decreased in −592C allele compared with AA genotype (18.8 ms. vs. 26.8 ms., p<0.05). In multivariate analysis including IPI, stage, IL-10-1082G, −819C and −592C allele frequencies, −819C allele remained as an independent prognostic factor predicting shorter PFS (RR=4.3, p<0.05). Additionally, in a haplotype analysis, there was a trend of longer PFS in patients carrying ATA/ATA than in other haplotypes but there was no statistical significance (p=0.08). As is well known, after introduction of rituximab in the treatment regimen, patients with DLBCL have been shown better treatment outcome than before. However, it is still a matter of problem to find and to manage the patients with refractory or progressive disease despite of these reinforced regimens. This study suggests that the detection of IL-10 gene promoter polymorphism such as −819C allele helps us to predict the prognosis and to establish a stratified treatment plan in DLBCL patients.
Assessment of Favorable (F) Versus Unfavorable (U) Early Stage Hodgkin’s Disease (HD); the Stanford V + Radiotherapy (RT) Experience.
Authors: Advani Ranjana , Maeda Lauren , Hoppe Richard T. , Breslin Sheila , Rosenberg Saul A. , Baer David , Mason Joseph , Horning Sandra J.
Published: October 2019

Early stage favorable HD is defined at Stanford and most US centers as stage I/II A and a mediastinal mass ratio (MMR) < 1/3 of the intrathoracic diameter. Within these parameters, we treated 101 patients (pts) with 8 weeks of Stanford V chemotherapy followed by 30 Gy (n = 84) or 20 Gy (n = 17) RT to involved sites. Seven pts had sites >10 cm (2 mediastinal and 5 nodal). At a median followup (f/u) of 5.5 years (y) the freedom from progression (FFP) was 96% with an overall survival (OS) of 98%. Stage I/II patients with MMR >1/3 of the intrathoracic diameter (n = 54) were treated with 12 weeks of Stanford V chemotherapy and 36 Gy RT to sites ≥5 cm. In this large MMR group the FFP and OS were 92% at a median f/u of 8 y. Other groups (EORTC, GHSG, NCI-C and GELA) have stratified early stage patients as favorable (F) and unfavorable (U) based on several risk factors. In addition to MMR and symptoms, these systems include age, # of nodal sites, sedimentation rate (ESR), extranodal lesions and in the GELA, elements of the international prognostic score for advanced HD. Phase III clinical trials have employed different therapies for F and U early stage pt based on these factors. We therefore retrospectively applied these factors to our “favorable” Stanford V pt to determine if they were prognostic as shown.

Group (% U) % FFP % OS F U p value F U p value EORTC (33) 98 86 0.02 100 92 0.03 GHSG (64) 100 91 0.08 100 96 0.27 NCI-C (60) 100 91 0.06 100 96 0.2 GELA (61) 97 92 0.4 100 96 0.3

ifferences in FFP and OS were significant using EORTC criteria. Notably, the majority of our pt were U (including all from Kaiser) according to GHSG criteria. Among 5 relapsed pt, 4 had ≥ 3 nodal sites and 3 had ESR ≥50. All relapses were in the RT field, one combined with distant disease. Secondary therapy was successful in 4 pts although one transplant-related death occurred. In conclusion, stage I-IIA pts with MMR <1/3 treated with Stanford V + 20–30 Gy had excellent overall outcome but application of stratification factors used by other groups, particularly the EORTC, defined a less favorable subset. As HD therapy moves to further reductions in chemotherapy and RT or chemotherapy alone for early stages, these risk factors take on added importance in the interpretation of current trials results and design of future studies.
Phosphatidylserine (PS) Binding Sites in Kringle Modules Regulate the Domain Organization and Conformation of Bovine Prothrombin.
Authors: Sen Shalmali , Banerjee Mou , Koppaka Vishwanath , Zhou Chaoming , Lentz Barry R.
Published: October 2019

Phosphatidylserine (PS) specifically regulates prothrombin activation during blood coagulation by binding to specific on factor Xa (Koppaka et al. Biochemistry, 7483, 1996) and its cofactor, factor Va (Zhai et al. Biochemistry, 5675, 2002). Binding to PS-containing membranes also alters prothrombin conformation (Lentz et al., Biochemistry, 5460, 1994; Chen et al. ibid. 4701, 1997). We ask here whether specific PS binding sites on prothrombin also control these structural changes, and, if so, where these sites are located. Four methods (Trp fluorescence, circular dichroism [CD], differential scanning calorimetry [DSC], and quasi-elastic light scattering [QELS]) were used to define the structural consequences of soluble 1,2-dicaproyl-sn-glycero-3-phospho-L-serine; (C6PS) binding both to whole prothrombin and to its proteolytically generated fragments. Intrinsic fluorescence titrations suggested the existence of two linked C6PS binding sites in fragment 1.2 (F1.2) and prethrombin 1, both of which contain prothrombin’s kringle pair. The existence of two sites was supported by direct equilibrium binding measurements with F1.2. CD measurements at increasing C6PS concentrations with both F1.2 and the kringle pair (F1.2 minus the γ-carboxy-glutamic acid [GLA] domain) were consistent with formation of a structure akin to an anti-parallel β sheet. Thermal denaturation profiles of F1.2 suggested calcium-independent, C6PS-induced domain reorganization within this fragment. Denaturation profiles and Trp fluorescence of the N-terminal, membrane binding domain (F1) and fragment 2 (F2) did not reveal any C6PS-induced changes. F2 consists mainly of the second cys-rich kringle module. The hydrodynamic radius of prothrombin was also found to decrease substantially (from 3.3 nm to 2.6 nm) in the presence of saturating (1mM) C6PS. Other lipids (phosphatidylglycerol and phosphatidyl-D-serine) did bind to prothrombin but did not produce comparable structural changes. The results show that C6PS 1) binds specifically to linked, calcium-independent sites within prothrombin’s two kringle domains and 2) induces thereby a conformational reorganization in the whole molecule. Supported by USPHS grant HL072827 to BRL.
Inhibition of Thrombin Cleavage of Factor VIII by Fibrinogen γ’ Chain Carboxyl Terminus.
Authors: Farrell David H. , Lovely Rehana S. , Boshkov Lynn K.
Published: October 2019

Fibrinogen can contain two types of γ chains, γA and γ’, that have identical sequences for the first 407 amino acids, but differ at their carboxyl termini. The γA chain contains four amino acids from γA 408–411, whereas the γ’ chain contains a different, highly anionic sequence of twenty amino acids from γ’ 408–427. This small change results in profound differences in the fibrinogen isoforms containing these distinct chains. The γ’ chain contains high affinity binding sites for zymogen factor XIII and for thrombin, and fibrinogen containing γ’ chains forms fibrin clots that are resistant to fibrinolysis. We have shown previously that the fibrinogen γ’ chain carboxyl terminus binds to thrombin exosite II, an anion-binding exosite known for its heparin binding activity. Recent reports show that the presence of the γ’ chain in fibrinogen reduces prothrombin activation in whole plasma assays. This inhibition may correspond to an activity originally described by Seegers in 1945 as “antithrombin I”, the adsorption of thrombin to fibrin. We have therefore investigated the enzymatic effects of γ’ peptide binding to thrombin in order to determine the role of the γ’ chain in the inhibition of prothrombin activation. In whole plasma, the γ’410–427 peptide prolonged the activated partial thromboplastin time (aPTT) in a dose-dependent manner, causing a delay in clotting from 29 seconds to 47 seconds at a concentration of 500 μM. A scrambled control peptide at the same concentration had little effect, increasing the clotting time to only 31.4 seconds. A series of γ’ deletion peptides were tested for their ability to prolong the aPTT. The inhibitory effect of the peptides corresponded in rank order to their thrombin binding affinity. In contrast to the aPTT, the prothrombin time increased only slightly from 11.7 seconds to 12.8 seconds (INR from 1.04 to 1.14) with the γ’410–427 peptide. This suggested that the γ’ peptide exerted its inhibitory effect on (a) component(s) of the intrinsic pathway, rather than the extrinsic pathway. We therefore investigated the effect of the γ’ peptide on thrombin activation of factor VIII, a thrombin substrate that, unlike factor V, is unique to the intrinsic pathway. Using purified factor VIII and thrombin in vitro, we showed that the γ’ peptide inhibited thrombin cleavage of factor VIII. However, this inhibitory effect was not due to direct inhibition of the thrombin active site, since the free γ’ peptide had little effect on thrombin cleavage of a small peptidyl substrate, tosyl-glycyl-prolyl-arginine-4-nitranilide acetate. These results suggest that factor VIII interactions with thrombin exosite II are essential for efficient cleavage of factor VIII, and support recent findings by others that interactions between thrombin exosite II and the A2 domain of factor VIII facilitate thrombin-catalyzed cleavage. These findings may explain, at least in part, the inhibitory effect of the γ’ chain on prothrombin activation in plasma.
The CXCR4 Antagonist AMD3100 Mobilizes a More Primitive Subset of CD34+ Cells Than G-CSF.
Authors: Seeger Timon , Veldwijk Marlon , Bridger Gary , Calandra Gary , Goldschmidt Hartmut , Ho Anthony , Fruehauf Stefan
Published: October 2019

The CXCR4-antagonist AMD3100 mobilizes peripheral blood progenitor cells (PBPC) in 8–10 hours while a standard mobilization regimen with granulocyte colony-stimulating factor (G-CSF) takes 4 days before significant numbers of PBPC can be detected. Recently it was shown that AMD3100-mobilized PBPC have a higher reconstitution potential in the NOD/SCID mouse model than G-CSF mobilized PBPC. Clinically they also mediate rapid and sustained engraftment after transplantation. Prospective comparative studies are yet lacking. It can be expected that the functional differences are also reflected on the phenotypic level. We used 6-color and 3-color flow cytometry to study the co-expression of primitive and lineage-associated markers on mobilized PBPC of patients with multiple myeloma and non-Hodgkin’s lymphoma (n=5). Mobilization treatment consisted of 5 days G-CSF (10 μg/kg, s.c. AM) and a single dose of AMD3100 (240 μg/kg, s.c.) in the evening of day 4. CD34+ cells of day 4, before AMD3100-dosing (G-CSF group) and on day 5, 10-hours after AMD3100 (AMD3100+G-CSF group) were compared. Primitive CD34+/CD38- cells which solely mediate hematopoietic reconstitution in the NOD/SCID mouse model were increased after addition of AMD3100 in all patients by a median of 2,58 fold (range 1,19–6,66) while the myeloid lineage committed CD34+/CD117+ and the actively proliferating CD34+/CD71+ cell subsets were significantly downregulated (p<0,05 each). The myeloid CD34+/CD33+ cell population showed a similar trend (median 0,60-fold change, range 0,24–1,08). Adhesion molecules were significantly regulated (CD49b) or showed such a trend (CD49d). Quantification of aldehyde dehydrogenase (AlDH) positive cells in A+G vs. G-CSF-mobilized samples did not show significant differences between these groups. We conclude that AMD3100 mobilizes a more primitive subset of CD34+ progenitor cells than G-CSF alone. Whether this translates into lower CD34+ threshold values for transplantation to achieve a similarly rapid and sustained engraftment after transplantation or into a shorter reconstitution period if similar amounts of CD34+ cells are used as after standard G-CSF-mobilization needs further study.

CD34 coexpression analysis: AMD3100+G-CSF mobilized CD34+ cells/G-CSF mobilized CD34+ cells (fold-change) CD34+/ median min max P value CD38− 2.58 1.19 2.09 0.073 HLA-DR− 1.14 0.14 2.09 0.568 CD90+ 0.42 0.05 26.47 0.417 CD7+ 1.17 0.58 2.9 0.433 CD10+ 0.87 0.11 6.66 0.513 CD19+ 1.11 0.72 12.69 0.368 CD33+ 0.6 0.24 1.08 0.075 CD45RA+ 5.84 1.54 7.46 0.155 CD71+ 0.51 0.43 0.64 0.002 CD117+ 0.36 0.02 1.11 0.046 CD18+ 1.55 0.56 4.09 0.347 CD49b+ 6.91 2.74 11.75 0.08 CD49d+ 0.63 0.25 0.89 0.061 CD58+ 5.82 2.83 21.88 0.261 CD62L 2.9 1.43 4.36 0.419 CD184+ 0.99 0.25 1.79 0.955
Immunodeficient Mice Are Poor Mobilizers -Novel Evidence That Demonstrates a Pivotal Role of Complement in Triggering Mobilization of HSPC.
Authors: Reca Ryan , Wysoczynski Marcin , Hansen Richard , Kucia Magda , Janowska-Wieczorek Anna , Ratajczak Janina , Ratajczak Mariusz Z.
Published: October 2019

Complement (C) is activated by immunoglobulin-dependent classical and immunoglobulin-independent alternative pathways, and we recently reported that the activation of C enhances the responsiveness of hematopoietic stem/progenitor cells (HSPC) to an SDF-1 gradient cascade by releasing C3a and desArgC3a cleavage fragments (Blood2003;101:3784; Blood2004; 103: 2071). We also found that bone marrow (BM) concentration of C3a and desArgC3a increases during mobilization with G-CSF or cyclophosphamide (CY). To explain this phenomenon we envisioned that these mobilizing agents expose a neo-antigen in BM tissue by turning BM into a highly proteolytic microenvironment. As a consequence of this, the newly exposed neo-epitope becomes bound by natural IgM antibodies leading to the activation of the classical C cascade. In our studies to elucidate the role of C activation in triggering the mobilization of HSPC, we found that C is effectively activated in the BM of G-CSF- or CY- mobilized wild-type (wt) but not IgM-deficient (RAG2null) mice. More importantly we found that several immunodeficient murine strains (RAG2null, SCID and Xid) displayed severely reduced G-CSF-induced mobilization of HSPC which we believe is a result of lack of B lymphocytes and complement-activating immunoglobulins. Supporting this, we found T cell depletion in wt mice did not affect mobilization. Moreover, G-CSF-induced mobilization in RAG2null, SCID and Xid animals was restored after infusion of murine inmmunoglobulins. Furthermore, since mobilization by zymosan or sulfated glycans activates C via the alternative immunoglobulin-independent pathway, we focused on the role of C activation during zymosan-induced mobilization. As expected, zymosan-induced immunoglobulin-independent C activation and mobilization of HSPC were unaffected in immunodeficient (RAG2null, SCID and Xid) mice which have a normal serum level of C3. However, these processes were severely reduced in C3-deficient animals. Thus our data strongly support the notion that C and innate immunity is an important trigger of mobilization of HSPC. While G-CSF and cyclophosphamide activate C by a classical IgM-dependent pathway, zymosan and sulfated polyglycans activate it by employing an alternative pathway. In conclusion, mobilization of HSPC could be envisioned as part of a more global immune response that is triggered/mediated by C3 activation/cleavage.
Mobilization, Harvesting and Selection of Peripheral Blood Stem Cells in Patients with Autoimmune Diseases Undergoing Non-Myeloablative Autologous Hematopoietic Stem Cell Transplantation.
Authors: Statkute Laisvyde , Verda Larissa , Oyama Yu , Villa Marcelo , Shook Thomas , Clifton Ronnie , Krosnjar Nela , Quigley Kathleen , Yaung Kimberly , Weppner Catherine , Burt Richard K.
Published: October 2019

We have analyzed peripheral blood stem cell (PBSC) mobilization, harvesting and selection properties in 128 patients with severe autoimmune diseases undergoing non-myeloablative autologous hematopoietic stem cell transplantation (HSCT) (50 patients with systemic lupus erythematosus (SLE), 43 - with multiple sclerosis (MS), 15 - with Crohn’s disease (CD), 8 - with scleroderma (Scl), and 12 - with others). Female/male ratio and mean age (range) were 90/38 patients, and 34 (14 to 59) years old, respectively. Mobilization regimen included cyclophosphamide 2g/m2 and G-CSF 10 mcg/kg (except for SLE patients 5 mcg/kg). Forty one patients underwent stem cell collection using Baxter CS300, 78 patients - Spectra, and for 9 patients both apheresis machines were utilized. The mean number of aphereses was 1.8 (range 1–10). Patients with SLE required the largest number of apheresis sessions (mean 2.4), comparing to patients with CD (mean 1.9), Scl (mean 1.4), MS (mean 1.3). Five patients additionally required bone marrow harvest for collection of adequate numbers of stem cells. One patient failed to reach CD34+ cell number of 1.0x106/kg, therefore did not proceed to HSCT. The mean number of CD34+ cells in each apheresis unit was 6.07+−6.96x106/kg (the highest of 9.22+−8.52x106/kg in patients with MS, and the lowest of 3.93+−4.48x106/kg in patients with SLE). Ninety eight patients underwent stem cell selection with CEPRATE SC (N=18), Isolex 300iv1.12 (N=2) or Isolex 300iv2.5 (N=78) stem cell concentrator. The mean purity of selected products was 74.3% (the highest of 81.1% attained in patients with Scl); mean recovery of CD34+cells was 61.2%. T cell reduction by average of 3.7 logs was achieved. The mean number of infused CD34+ cells was 7.24+−5.5x106/kg. The highest mean number of CD34+ cells/kg were infused to patients with MS (9.04+−6.74x106/kg), the lowest - to patients with SLE (5.78+−4.13x106/kg). We found a moderate positive correlation between peripheral blood (PB) CD34+ cells/ul and PB WBC/ul (R=0.34, p<0.05), PB platelets/ul (R=0.51, p<0.05) and a strong positive correlation between PB CD34+ cells/ul and the number of CD34+ cells/kg/apheresis (R=0.67, p<0.05). A weak positive correlation was observed between the number of infused CD34+cells/kg and faster WBC engraftment (ANC>500) and platelet engraftment (platelet count>20K). There was no toxicity observed in our patient population during peri-mobilization period except for 1 patient with SLE who died of disseminated mucormycosis 1 week after stem cell collection. Mobilization and selection of PBSC are safe and efficient in patients with severe autoimmune diseases undergoing non-myeloablative autologous HSCT.
Quantification of Imatinib in Human Plasma by High Performance Liquid Chromatography-Tandem Mass Spectrometry: Applications to Therapeutic Drug Monitoring of Patients with Chronic Myelogenous Leukemia.
Authors: Mahon Francois-Xavier , Picard Stéphane , Titier Karine , Nicolini Franck , Ducint Dominique , Moore Nicolas , Marit Gerald , Berthaud Patrice , Molimard Mathieu
Published: October 2019

Imatinib (Gleevec® or Glivec®) is a competitive inhibitor of protein tyrosine kinase is currently used for the treatment of chronic myeloid leukaemia (CML), Philadelphia chromosome-positive acute lymphoblatique leukemia and for other malignant pathologies.

Although there is no clearly defined therapeutic window, the imatinib plasma quantification seems interesting to evaluate patient compliance to daily oral therapy, drug-drug interactions or pharmacokinetic/pharmacodynamic relationship. The aim of this study was to develop a simple and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the imatinib quantification, adapted to routine applications. After a liquid-liquid extraction, the imatinib and its deuterated internal standard were eluted on a XTerra® RP18 column with a gradient of acetonitrile-ammonium formiate buffer 4 mM pH 3.2. Imatinib was detected by electrospray ionisation mass spectrometry with multiple reaction monitoring mode. The calibration curves were linear over the range 10–5000 ng/ml. The limit of quantitation was set at 10 ng/ml. The bias was lower than 8 %. Intra-day and inter-day precisions were lower than 8 %. The extraction recovery was higher than 90 %.

The monitored population was defined as follows: patients treated for CML and receiving imatinib with one daily dose of 300, 400 or 600 mg. Blood samples were collected at steady state, 23 (±3) hours after drug administration, and were centrifuged at 4500 rpm for 5min. As shown on the figure 1, all patients treated with 600mg have a concentration more than 500 ng/ml which corresponds to the efficient concentration of imatinib according to the previously in vitro studies (i.e. the target plasma concentration required to induce the death of leukemic cells). Five patients - previously known as not having an efficient hematologic response or cytogenetic response showed an imatinib level lower than 500 ng/ml Two of these patients were not compliant and three patients were coadministered with enzymatic inducers: phenytoïn or phenobarbital or oxcarbazepine This method is simple, adapted to routine application and allows accurate therapeutic monitoring of imatinib. It can be used to evaluate patient adherence to daily oral therapy, drug-drug interactions or pharmacokinetic/pharmacodynamic relationship. In conclusion, regarding the definition imatinib resistance in CML, it seems necessary to take in account the value of the imatinib concentration. A study of correlation with the cytogenetic and molecular response is in progress.

Figure Figure
Use of Direct Sequence PCR for ABl Kinase Mutations in Patients with CML Blast Crisis, Treated Prior to the Availability of Imatinib Therapy.
Authors: Ghorashian Sara , Babb Anna , Khorasan Jamshid , Kaeda Jaspal , Marin David , Apperley Jane
Published: October 2019

We identified all patients who developed blast crisis before the advent of imatinib therapy and in whom stored marrow samples were available from a database of CML patients in a major teaching hospital. mRNA was extracted and cDNA synthesized successfully in 20 of 22 cases. The cDNA was subjected to real time PCR for quantification of BCR-ABL transcripts. The ABL allele not involved in the t(9;22) translocation was excluded from mutational analysis by subjecting the cDNA to nested PCR. This was achieved by amplifying from exons 13 and 10 of the BCR and ABL moieties of the BCR-ABL fusion gene using primers B2A and JAMR, respectively. The resulting amplicon was then subjected to nested PCR using primers, NTPB+ and NTPE-, sited within exons 4 and 10 of the ABL gene. The nested PCR yielded an 863 bp fragment in length containing the entire BCR-ABL kinase domain. To test for successful amplification an aliquot of the PCR products were electrophoresed through 2.0% agarose gel. In all cases, a single amplicon was observed and the PCR reaction subjected to magnetic purification. The purified amplicon were then sequenced by Sanger’s dideoxy chain termination reaction using Big-Dye ABI 310 sequencer (Applied Biosystems, Foster City, USA). In each case, the sequence obtained was compared with the published ABL type 1a sequence, Genbank M14752, using Blast 2 software. No mutations were found in any patient. This suggests the absence of a mutated kinase in a proportion of greater than approximately 30% of the total BCR-ABL kinase, due to the limit of detection of the technique. Our results suggest that the predominant form of BCR-ABL in the setting of blast crisis (in the absence of imatinib) is wild type. This goes against theories that kinase mutations confer an intrinsic ‘gain of function’ or a proliferative advantage to a CML clone. Instead, it is likely that mutant forms of BCR-ABL which are relatively resistant to imatinib binding gain prominence through positive selection with the presence of imatinib within the cell milieu.
Enforced STAT3 and PU.1 Expression Activates Type I Interferon-Producing Cell and Dendritic Cell Differentiation Programs in Megakaryocyte/Erythrocyte Progenitors.
Authors: Onai Nobuyuki , Onai Aya , Tussiwand Roxane , Lanzavecchia Antonio , Manz Markus G
Published: October 2019

A standing question in early hematopoiesis is whether cytokine signaling is sufficient to induce cell fate decisions. Previously, we have reported that enforced expression of human Flt3 in Flt3 negative megakaryocyte/erythrocyte progenitors (MEPs) rescues their interferon producing cell (IPC) and dendritic cell (DC) developmental potential: Human Flt3-signaling in MEPs leads to up-regulation of IPC, DC, and myelomonocytic development affiliated genes such as, STAT3, PU.1, and G-CSFR/M-CSFR/GM-CSFR, and activates differentiation to these lineages. To test whether single Flt3 downstream activated genes would be sufficient to cause the same effects, we transduced murine STAT3 and PU.1 cDNA into MEPs using retrovirus expression vectors, respectively. Both STAT3- and PU.1-transduced MEPs were capable to differentiate into IPCs and DCs in SCF and TPO supplemented media, and, with even higher efficiency, in SCF, TPO, and Flt3L supplemented cultures. While human Flt3-transduced MEPs maintained megakaryocyte-erythrocyte developmental potential and gained IPC, DC, and myelomonocytic cell potential, STAT3- and PU.1-transduced MEPs lost both megakaryocyte and erythrocyte developmental options, indicating that strong signaling of these transcription factors fully converted MEPs to the IPC, DC, and myelomonocytic lineages. Consistently, GATA-1 expression was down-regulated, and C/EBPα mRNA was up-regulated in STAT3- and PU.1-transduced MEPs. Interestingly, STAT3- and PU.1 over-expression in MEPs led to up-regulation of Flt3 mRNA levels, suggesting a self-sustaining effect of Flt3 signaling-cascade induced Flt3 expression. Thus, enforced expression of STAT3 and PU.1 in MEPs reprogrammed them to differentiate into IPCs, DCs and myelomonocytic cell lineages and inhibit Meg/E-lineage potential. Based on these data, we propose a model where Flt3 positive progenitor cells that locate in Flt3L rich environments will be directed to develop into IPCs and DCs. This process might be enhanced by a self-sustaining mechanism where Flt3 downstream transcription factors as STAT3 and PU.1 in turn maintain Flt3 expression.
Dose-Escalated Donor Lymphocyte Infusions Post Allogeneic Transplantation in Non-Hodgkin’s Lymphoma.
Authors: Bloor Adrian J.C. , Thomson Kirsty , Cooper Nichola , Peggs Karl S. , Mackinnon Stephen
Published: October 2019

The role of donor lymphocytes infusions (DLI) in the treatment of non-Hodgkin’s lymphoma (NHL) following allogeneic haemopoietic stem cell transplant (HCST) has not been clearly characterised. We report the results of 51 infusions of dose-escalating donor lymphocytes to 24 patients following myeloablative (n=3) or reduced intensity (n=21) transplantation as treatment for NHL. The diagnoses were low grade NHL (n=19), transformed low grade NHL and high grade NHL (n=5). Twenty patients were transplanted from an HLA-matched sibling, and 4 from an unrelated donor. The indications for DLI were progressive or relapsed disease (n=17), with or without mixed chimersim, and persistent mixed chimersim alone (n=7). Eight of the patients treated for disease progression or relapse had both clinical and radiological evidence of disease, a further 8 cases had radiological evidence of disease alone (CT scan in 3; CT-PET scan in 6) and one case of progressive disease was diagnosed following a bone marrow biopsy. Histological confirmation of relapse or disease progression was confirmed in 8 cases. Eighteen patients received an initial dose of 1x106/kg CD3 positive cells at a median of 265 days post transplant (range 181–728) and 6 received an initial dose of 1x107/kg cells at a median of 402 days post transplant (range 130–2674). Escalating doses of cells were administered at 3-month intervals (3 x 106/kg, 1 x 107/kg, 3 x 107/kg, 1 x 108/kg) in the absence of development of graft-versus-host disease (GvHD), if mixed chimerism persisted or if there was no evidence of disease response. Four of the patients treated for disease progression also received antitumoural chemotherapy shortly before DLI. Of the 16 evaluable patients treated for disease, 12 (75%) showed a significant response to DLI (9 patients with low grade NHL and 3 with transformed low grade NHL). A complete remission (CR) was observed in 10 patients and partial remission in 2 patients. Three of the patients achieving a CR following DLI subsequently relapsed; the median duration of the ongoing complete remissions is 574 days (range 122–1479). One patient died of sepsis before the response to DLI could be assessed. Conversion from mixed to multilineage full donor chimersim occurred in 18 of 18 (78%) evaluable patients. The major toxicity resulting from the use of DLI was GvHD. Following DLI, acute GvHD (grade II–IV) occurred in 5 out of 20 evaluable patients (24%) and chronic extensive GvHD in eight patients (38%); 1 patient died during treatment for grade IV acute gut GvHD. The incidence of GvHD did not correlate with the dose of DLI infused and 5 patients had graft-versus-lymphoma responses without GvHD. These data support the existence of a clinically significant graft versus tumour effect in non-Hodgkin’s lymphoma and indicate that this is an effective treatment for progressive disease post allogeneic HSCT.
High Dose Busulfan and Fludarabine with Anti-Thymocyte Globulin (ATG) Followed by Unmodified Marrow Grafts from HLA-Matched Siblings for the Treatment of High Risk Hemoglobinopathies (HGBpathies).
Authors: Boulad Farid , Kernan Nancy A. , Small Trudy N. , Chewning Joseph , Prockop Susan , Scaradavou Andromachi , Keating Anna , O’Reilly Richard J.
Published: October 2019

The standard cytoreduction for bone marrow transplantation (BMT) of pts with HGBpathies has been the combination of Busulfan (BU) and Cyclophosphamide However, for pts with advanced disease, high rates of graft rejection and toxicity were reported. For such high risk pts, a BU and Fludarabine (FLU) cytoreductive combination was used at low dose in the context of non-myeloablative BMT and was associated with a poor outcome with high rates of graft rejection. In view of the low toxicity associated with the BU-FLU combination, we used both agents at higher, potentially myeloablative doses for the cytoreduction of six pts with high risk HGBpathies. Between 12/00 and 04/05, 4 pts with thalassemia (Thal) and 2 pts with sickle cell disease (SCD), including 2 males and 4 females aged 4.6–15.3 years were transplanted using this regimen. All 4 pts with Thal had advanced Lucarelli Class 2 disease, while the 2 pts with SCD had stroke, recurrent vaso-occlusive crises (VOC), sickle lung disease and alloimmunization (n=1) and recurrent VOC, acute chest syndrome and osteomyelitis (n=1). Cytoreduction included intravenous BU (0.8–1 mg/Kg/dose x 14), FLU (30 mg/m2/day x 5) and Rabbit ATG (2.5 mg/Kg x 2). GvHD prophylaxis consisted of Tacrolimus (n=3) or cyclosporine (n=3) and methotrexate (n=5) or Steroids (n=1). Pts received an unmodified BMT from their HLA-identical sibling with total nucleated cell doses of 0.7–5.7 x 108 cells/Kg. The regimen was well tolerated with minimal toxicity. With a median follow-up of 21.5 mo (range 3–55 mo), all 6 pts are disease- and transfusion- free. There was no graft rejection and no GvHD. Chimerism status for pts with Thal was 98–100% donor, 5 mo to 2 years post BMT, while for the 2 pts with SCD, it was mixed with 50 and 80% donor cells at 3 mo and 2 years post BMT. The pt with 50% donor cells received a low graft cell dose (0.7 x 108 nucleated cells/Kg). One additional pt with SCD and a history of 2 strokes and moya-moya disease received BU FLU + melphalan (70 mg/m2 x 2). This pt had minimal post BMT toxicity, and is engrafted with 100% donor cells at 1 year post BMT. In summary, the combination of high dose BU and FLU for pts with high risk HGBpathies was well tolerated and induced full engraftment for pts with Thal, but mixed chimerism for pts with SCD. Nevertheless, all pts so treated survive with normal hematologic function. The addition of melphalan to BU FLU was also well tolerated and could be beneficial in attaining complete myeloablation and full chimerism in pts with SCD.
ZAP-70 Status May Not Predict Outcome after Non-Myeloablative Allogeneic Transplantation (NMT) in Patients with Chronic Lymphocytic Leukemia(CLL) Who Failed Conventional Chemotherapy.
Authors: Khouri Issa F. , Saliba Rima M. , Keating Michael J. , Admirand Joan , O’Brien Susan , Champlin Richard E. , Buenos-Ramos Carlos
Published: October 2019

In aggressive disease, the CLL cells usually express the 70-kD zeta-associated protein (ZAP-70). We used immunohistochemical techniques and routinely fixed, paraffin-embedded tissue to survey ZAP-70 expression in patients (pts) with CLL who received a NMT, as previously described (Modem Pathol17:954, 2004). All cases were reviewed independently by two authors. Cytoplasmic staining of non-neoplastic, reactive T cells served as an internal control in each case. Neoplasms (Pax-5 +) demonstrating ZAP-70 cytoplasmic staining in greater than 20% of tumor cells were considered positive. Pts were eligible for NMT if they had failed fludarabine-based conventional therapy. The conditioning regimen prior to NMT consisted of fludarabine, cyclophosphamide and rituximab. Twenty nine pts were treated. The pts characteristics and outcome were as follows:

ZAP-70 Negative ZAP-70 Positive P value No. Pts 8 19 Age (range) 58 (45–69) yrs 53 (34–72) yrs 0.36 Time Dx to NMT 4.5 yrs 4.5 yrs 0.6 No. Prior Chemoregimens 3 (2–5) 3 (2–8) 0.7 No. Pts in Richter 1 6 0.3 Status at NMT CR/PR/NR 1/3/4 1/12/6 0.18 Donor: Matched Sibling 7 16 0.6 Unrelated 1 3 #Pts requiring DLI 3 9 0.4 Final Response CR/PR 6/1 15/0 0.6 NR/NE 1/0 3/1 Relapse Post CR 1 0 Follow-up Time 11 (9–47) mos 37 (13–75) mos Survival 1.5- year 83% 84% 0.7 4- year NA 64% Current PFS 56% (1.5 yr) 65% (4 yr) Causes of Death PD(1), cGVHD(1) PD(2), cGVHD(2), Infection(2) GVHD grade II–IV 37% 39%

These data suggest that NMT may overcome the negative prognostic impact of ZAP-positivity in CLL. Controlled trials are needed to confirm these results in a larger number of patients.
A Single Center Experience with Allogeneic Sibling Umbilical-Cord-Blood Transplantation in Children with Malignant and Non-Malignant Disease.
Authors: Song Yongping , Zhang Yanli , Fang Baijun , Wei Xudong , Lu Lulu , Zhu Xinhu , Wang Ping , Li Yufu , Gao Quanli , Wang Guiye , Qiu Lugui , Zhao Chunhua
Published: October 2019

To study the outcome of allogeneic sibling umbilical-cord-blood transplantation (CBT) in children in China, we studied the records of 38 recipients of cord blood from HLA-identical siblings from 1998 through 2004. The mean age at transplant was 10.3+/−5.5 years. Diseases of the patients were leukemia (28), neuroblastoma (3), severe aplastic anaemia (3) and inborn errors of metabolism (four). A volume of 76–208 ml of cord blood was collected from sibling infants soon after delivery. HLA antigens were identical in 32 and one to three antigens mismatched in six. The patients received busulphan and cyclophosphamide as conditioning, and antithymocyte globulin was given to prevent graft rejection. The median number of collected nucleated cells was 3.5 x 107/kg nucleated cells (range, 2.0 to 10.2). T-cell reconstitution was evaluated by using combined approaches of phenotyping analysis of lymphocytes and assessment of ex vivo thymic function by measuring T-cell receptor (TCR) rearrangement excision circles (TRECs). The overall survival is 89% at the median observation time of 72 months. The median time for neutrophil engraftment (absolute neutrophil count >500/mm3) was 16 days (range, 10 to 41days). The median time to become transfusion independent after CBT was 27 days for platelets (range, 15 to 54) and was 28 days for packed red blood cells (range, 19 to 128). Acute GVHD (aGVHD)was observed in 20/38 patients and involved only skin in 16 patients, skin and liver or gut in 2 patients and all 3 organs in another 2 patients. Seventeen of 20 patients had grade 1 to 2 aGVHD toxicity, whereas 3 patients experienced grade 3 to 4 aGVHD. Chronic GVHD (cGVHD) developed in 12 patients. Acute transplant related mortality was 5.5%. Cause of death was persisting non-engraftment till day +100 after transplant. Late mortality occurred in 2 patients: one cGVHD associated haemorrhage 22 months after CBT and one cGVHD associated septicaemia 4 years after CBT. Immunologic reconstitution demonstrated that CBT resulted in consistent and stable T-, B- and natural killer (NK) cell development. The kinetics of development was such that T-cell development occurred between 65 to 180 days. Initial T-cell engraftment consisted predominantly of CD45RO+, CD3+, and CD4+ T cells, and at 12 to 24 months changed to CD45RA+, CD3+, and CD4+ T cells, indicating de novo maturation of T cells. During the first 5 months after transplantation, TCR repertoires were highly abnormal and TREC values low. However, in a longer follow-up (one years or more after transplantation) TREC values and TCR diversity increased and became normal. This data confirms that CBT still should be the first treatment choice if an HLA-identical sibling is available.
Upfront Autologous Stem Cell Transplantation (SCT) Ameliorates the Prognostic Disadvantage of an Intermediate/High-Risk FLIPI Score in Patients with Advanced Follicular Lymphoma (FL): Evidence from Two Independent Data Sets.
Authors: Martin Kornacker , Suessmilch Simone , Gulla Hilde , Seyfarth Baerbel , Schoch Robert , Kneba Michael , Ho Anthony D. , Hensel Manfred , Schmitz Norbert , Dreger Peter
Published: October 2019

The Follicular Lymphoma International Prognostic Index (FLIPI) is a useful predictor for the prognosis of patients with FL. It relies on a score consisting of age, stage, hemoglobin, LDH, and number of nodal sites involved at diagnosis, which defines 3 risk groups with different 5-year survival (5-y OS): Low risk (0–1 factor; 5-y OS >90%); intermediate risk (2 factors; 5-y OS 78%); and high risk (>2 factors; 5-y OS 53%). We hypothesized that SCT in first remission might improve the course of patients with advanced FL and high/intermediate risk FLIPI, thereby abrogating the prognostic impact of the FLIPI.

Methods: Consecutive patients from two independent data sets (Kiel/Hamburg = training sample (T); Heidelberg = validation sample (V)) who received upfront SCT for FL between 1990 and 2002 and had sufficient baseline information available to assess the FLIPI were analyzed retrospectively. SCT eligibility criteria were similar for both series and comprised first remission of advanced FL, age <60, and performance status <2. Patients were treated with sequential therapy comprising 2–8 cycles of a CHOP-like regimen for remission induction, Dexa-BEAM (T) or HAM (V) for stem cell mobilization, and TBI/CY or BEAM for myeloablation prior to SCT.

Results: 49 (T) and 57 (V) patients were eligible. Time from diagnosis to transplant was 9 (5–21) and 14 (5–116) months, respectively. The FLIPI assigned 40 (82%) and 36 (63%) patients to the high/intermediate risk group. With a median follow-up from SCT of 64 (12–134) or 62 (1–132) months, respectively, outcome data were similar in both samples. 5-y OS from diagnosis was 94% (T) and 90% (V) for the high/intermediate risk group vs. 83% (T) and 87% (V) for the low-risk group (p=0.09 and p=0.4). 5-year progression-free survival (PFS) was 62% (T) and 77% (V) vs. 71% (T) and 77% (V) (p=0.35 and p=0.73). Combining all patients (n=106) allowed for examination of each prognostic group separately. Differences in 5-y OS from diagnosis (90% for high risk, 93% for intermediate risk and 85% for low risk) or in 5-y PFS (60% for high risk, 75% for intermediate risk and 78% for low risk) were not statistically significant.

Conclusions: Upfront SCT abrogates the prognostic impact of the FLIPI by improving the outcome of intermediate/high-risk patients but not of low-risk patients. Therefore the FLIPI might be a useful tool to select patients for prospective trials on SCT in FL who may particularly benefit from this procedure.