bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Factors Influencing Skin Toxicity after Autologous Hematopoeitic Stem Cell Transplant.
Authors: Mahindra Anuj , Bolwell Brian , Sobecks Ronald , Rybicki Lisa , Brown Stacey , Pohlman Brad , Kalaycio Matt , Dean Robert , Andresen Steve , Sweetenham John , Copelan Edward
Published: October 2019

Skin toxicity is a known but understudied complication of autologous stem cell transplantation (ASCT). Its assessment is complicated by the development of graft-vs-host disease following allogeneic transplantation. We chose therefore to study skin toxicity following autologous transplantation. We retrospectively reviewed the records of 392 patients undergoing ASCT to analyze risk factors for skin toxicity, its association with mucositis and its effect on survival. Skin toxicity was assessed three times a week during the transplant admission and was classified as none, mild, moderate, severe or life threatening. The most severe skin toxicity is used in this analysis. Mucositis was assessed using the modified oral mucositis assessment scale (OMAS). Median patient age was 53 years; 63% of patients had non-Hodgkin s lymphoma (NHL), 17.6% multiple myeloma, 13.5% Hodgkin disease (HD) and 5.9% acute leukemia. Peripheral blood progenitor cells were mobilized with G-CSF alone (36%) or the combination of etoposide plus G-CSF (64%). The preparative regimen was busulfan (Bu)/cyclophosphamide (Cy) /etoposide in 82% and Bu/Cy alone in 18%. Two hundred and sixty patients (67%) developed skin toxicity of which 143 patients (36.5%) had mild, 105 (26.8%) had moderate and 16 (3.9%) had severe skin toxicity. Factors associated with the development of any skin toxicity in univariable analysis were male gender (p=0.028), diagnosis of NHL (p<0.001) or HD (p=0.001), higher number of prior chemotherapy regimens (p<0.001), mobilization regimen containing etoposide (p< 0.001) and ASCT preparative regimen containing etoposide (p<0.001). Moderate skin toxicity correlated with the above as well. Only the inclusion of etoposide in the mobilization regimen was associated with an increased risk of severe skin toxicity (p=0.035). Etoposide in the preparative regimen remained the most significant risk factor for the development of skin toxicity in multivariable analysis. Higher OMAS score was associated with increased severity of skin toxicity (p<0.001) as shown in the boxplot below:

Patients with severe skin toxicity appeared to have worse overall survival (p=0.054) and relapse-free survival (p=0.08), but these findings did not reach statistical significance.

Conclusion: A substantial proportion of patients develop skin toxicity following autotransplantation. The inclusion of etoposide in the preparative regimen is associated with a significant increased frequency in multivariable analysis. The severity of skin toxicity correlates closely with the severity of mucositis.

Figure Figure
Integrin αvβ3 Is Involved in the Stabilization of Ultra Long von Willebrand Factor Strings under Fluid Shear Stress on Human Endothelial Cells.
Authors: Huang Jing , Heuser John E. , McEver Rodger P. , Sadler J. Evan
Published: October 2019

Von Willebrand factor (VWF) multimers attached to endothelial cells can provide a platform for thrombosis, especially when accompanied by ADAMTS13 deficiency. We characterized the structural features of ultralarge VWF (ULVWF) molecules acutely secreted from Weibel-Palade bodies and identified a receptor responsible for their binding to the surface of cultured human umbilical vein endothelial cells (HUVECs). Using fluorescence microscopy on live cells, VWF multimers formed extended strings within minutes after stimulation. String formation did not require exogenous platelets and occurred over a range of shear stress from 2.5 dyn/cm2 to 40 dyn/cm2. A subset of ULVWF strings spontaneously bound formalin-fixed platelets via platelet GPIb. Quick-freeze, deep-etch electron microscopy showed that ULVWF strings often merged to form bundles and networks. Each string was tethered to the endothelial membrane by a limited number of anchorage sites, many of them located on small membrane projections, suggesting a specific mode of interaction. Several independent approaches implicated integrin αvβ3 in anchoring ULVWF strings to the HUVEC surface. Either “RGDS” peptide or function blocking antibody (LM609) to integrin αvβ3 specifically and dose dependently inhibited ULVWF string formation 62 ± 0.7% and 53 ± 4%, respectively. Furthermore, integrin αv was seen decorating the extending ULVWF strings using a non-functional blocking antibody (LM142) in live-cell immunofluorescence. In addition, a lentiviral vector encoding shRNA against integrin αv resulted in approximately 70% reduction in cell surface αv expression by FACS analysis with antibody LM609. HUVEC infected with this lentivirus were significantly impaired in their ability to form ULVWF strings compared to cells infected with a control virus. In multiple experiments, shRNA knockdown of αv expression reduced ULVWF strings 75.3 ± 4.4% at 2.5dyn/cm2 and 81.6 ± 2% at 7.5dyn/cm2. These results indicated that ULVWF strings bind tightly to endothelial cells via very few anchorage sites and are relatively resistant to fluid shear stress. Although integrin αvβ3 does not appear to be required to stabilize ULVWF strings on mouse endothelium, these data suggest that integrin αvβ3 may participate in the stabilization of ULVWF strings on human endothelial cell surfaces.
Reversible Self-Assembly of Weibel-Palade Body-Like Tubules from Recombinant N-Terminal Domains of von Willebrand Factor.
Authors: Huang Ren-Huai , Wang Ying , Roth Robyn , Yu Xiong , Purvis Angie R. , Heuser John E. , Egelman Edward H. , Sadler J. Evan
Published: October 2019

Weibel-Palade bodies (WPBs) are elongated secretory granules of endothelial cells that are packed with tubules composed of von Willebrand factor (VWF), a multimeric protein required for hemostasis. Disruption of tubular packing prevents the orderly secretion of VWF multimers and blocks the subsequent binding of platelets. The cigar-like shape and tubular cross section of WPBs are conserved in all vertebrates, but little is known about how VWF specifies this packing arrangement. Starting from recombinant 82 kDa VWF propeptide (domains D1D2) and 114 kDa disulfide-bonded D’D3 dimer, we now have assembled tubules reversibly in vitro with the same dimensions as VWF tubules in WPBs. Assembly was induced at pH 6.2, reversed at pH 7.4, and required Ca2+. Recombinant D’D3 dimers did not self-associate at pH 7.4 or pH 6.2, with or without Ca2+. Without Ca2+, VWF propeptide did not bind to D’D3 dimers. At pH 7.4, with Ca2+, VWF propeptide formed noncovalent 160 kDa dimers and, when mixed with D’D3 dimers, assembled a 280 kDa complex of two propeptides and one D’D3 dimer as shown by gel filtration chromatography and multi-angle light scattering. Lowering the pH to 6.2 caused the formation of >3 MDa aggregates with the same stoichiometry, which dissociated upon adding EDTA or raising the pH to 7.4. Quick-freeze deep-etch EM showed that the large aggregates are hollow right-handed tubular helices. The iterative helical real space reconstruction method was used to make 3D reconstructions of the tubules at 22 Å resolution from negative stain EM images (Figure, left). Tubules consist of a right-handed helix with axial rise of 26.2 Å and twist of 85.6 degrees per subunit, or 4.2 subunits per 11 nm turn. The dimensions (outside diameter 25 nm, inside diameter 12 nm) are similar to those of tubules in WPBs in thin sections of endothelial cells by transmission EM (Figure, right and its insert). Each subunit contains one D’D3 dimer flanked by two D1D2 propeptides (Figure, center). Each D’D3 dimer makes a total of six contacts with D1D2 domains. Each D1D2 propeptide makes three contacts with D’D3 and just one end-to-end homotypic contact. The spatial arrangement of these building blocks and inter-domain contacts in tubules suggest a model by which decreasing pH along the secretory pathway coordinates the formation of intersubunit disulfide bonds with the tubular packaging of VWF multimers. Within the WPB, Ca2+-dependent and pH-dependent binding of D1D2 to D’D3 domains stabilizes the packing of VWF multimers into tubules, which behave as constrained springs. Upon secretion, the increased pH weakens these constraints and permits the helical tubules to unfurl into flowing blood without tangling.

Figure Figure
Trend in Prevalence of Human T-Lymphotropic Virus Type-1 (HTLV-1) Infection in Japanese Blood Donors, Nagasaki, 1999 to 2006.
Authors: Iwanaga Masako , Chiyoda Shin , Kusaba Eisuke , Kamihira Shimeru ,   (Intr. by Yasuaki Yamada )
Published: October 2019

To evaluate time-trend of HTLV-1 prevalence and the effect of preventative measure against the viral transmission are important in the virus endemic regions. In Nagasaki, Japan, an endemic area of HTLV-1, a routine serological virus screening for blood donors and a prefecture-wide intervention project (the ATL Prevention Program; APP) to prevent milk-borne transmission for the virus carrier mothers have been conducted since 1986 and 1987, respectively. However, the effects of both projects on the virus seroprevalence have not been well evaluated. In this study, we conducted trend analyses of age-specific, birth-year-specific, and period-specific seroprevalence of HTLV-1 for first-time blood donors who donated between January 1999 and December 2006. Among 55668 first-time donors (age at donation; 16–65 years, birth year; 1934–1990), 718 were test positive for HTLV-1, indicating that the overall seroprevalence was1.29% (95%CI, 1.20–1.39). Prevalence was significantly higher in women than men (1.53% vs. 1.13%; OR; 1.36, 95%CI; 1.17–1.57). Seroprevalence increased significantly with increasing age at donation from 0.70% at 16–25 years to 7.34% at over 56 years (Chi-square test, P < 0.0001). The annual prevalence was 1.32 in 1999, 1.31 in 2002, and 1.37 in 2006, indicating that there was no significant secular trend during 1999–2006 (P for trend=0.99). In analyses by age at donation, trends of HTLV-1 prevalence significantly declined among age over 56 years (P for trend=0.02) and age 16–25 years (P for trend=0.0007), whereas in birth-year-specific analyses, there was no apparent change of the prevalence over time, except in birth year 1981–90 group in which the prevalence declined from 1.22% in 1999 to 0.44% in 2006 (P for trend < 0.0001). In analyses for limited birth year from 1985 to 1990, the seroprevalence declined from 0.75% in birth year 1985–86 group, 0.31% in 1987–88 group, to zero% in 1989–90 group (P for trend =0.0002). HTLV-1 seroprevalence was significantly lower among donors born in 1987–90 (after APP) than 1985–86 (before APP). These results indicate that a birth-year-specific analysis for HTLV-1 prevalence may be appropriate to evaluate secular trend since the virus mostly transmit during infancy, and that a prefecture-wide intervention, the refraining from breast-feeding by the virus carrier mothers, contributes a declining HTLV-1 seroprevalence in our region.
Targeting 14-3-3 Sensitizes Native and Mutant BCR-ABL to Inhibition with U0126, Rapamycin and Bcl-2 Inhibitor GX15-070.
Authors: Dong Shaozhong , Kang Sumin , Lonial Sagar , Khoury Hanna Jean , Viallet Jean , Chen Jing
Published: October 2019

Recurring chromosomal abnormalities in human leukemias result in expression of a wide spectrum of constitutively activated fusion tyrosine kinases, including BCR-ABL associated with t(9;22)(q34;q22) chronic myelogenous leukemia (CML). Small molecule tyrosine kinase inhibitors, such as imatinib, are effective therapies for BCR-ABL-mediated human leukemias. However, clinical drug resistance occurs, which warrants development of alternative and/or complementary therapeutic strategies to target critical downstream signaling molecules. We recently demonstrated that disrupting 14-3-3/ligand association by a peptide-based 14-3-3 competitive antagonist, R18 induces significant apoptosis, partially through reactivation of AKT-inhibited pro-apoptotic FOXO3a in FGFR1 fusion transformed hematopoietic cells [Dong et al, Blood, 2007, 110(1):360–9]. Here we report that targeting 14-3-3 by R18 effectively induced significant apoptosis in Ba/F3 and K562 cells expressing BCR-ABL, through liberation and reactivation of FOXO3a, but not by affecting 14-3-3/BCR-ABL association or BCR-ABL kinase activity. In addition, co-immunoprecipitation experiments revealed that R18 was not able to disrupt 14-3-3/BAD association, suggesting a model that targeting 14-3-3 by R18 induces apoptosis in BCR-ABL transformed cells, at least in part, through liberation and reactivation of FOXO3a phosphorylated and inhibited by activated AKT, but not BAD inhibited by ERK. Thus, we hypothesize that targeting 14-3-3 by R18 may potentiate the inhibition of BCR-ABL transformed cells induced by blocking the parallel MEK1/ERK pathway, pro-survival Bcl-2 proteins, or other signaling effectors downstream of AKT. Indeed, R18 sensitized BCR-ABL transformed cells to inhibition with MEK1 inhibitor U0126, Bcl-2 inhibitor GX15-070, or mTOR inhibitor rapamycin, and the combined treatment of R18 with these anti-cancer reagents synergistically induced apoptotic cell death. Moreover, treatment of these reagents potentiates R18-induced reactivation of pro-apoptotic FOXO3a with enhanced expression of downstream transcription targets including p27kip1 and Bim1, suggesting a potential molecular mechanism of the combined therapy. Furthermore, R18 induced apoptotic cell death in cells expressing diverse imatinib-resistant BCR-ABL mutants, including T315I. This inhibition was enhanced by treatment of R18 in combination with U0126 and rapamycin. Together, our findings suggest a novel therapeutic strategy that targeting 14-3-3 may potentiate the effects of conventional therapy for BCR-ABL associated hematopoietic malignancies, and overcome drug-resistance.
Array CGH Analysis at 60kb Resolution of CML Samples at Advanced Stage of Disease.
Authors: Grace Colin , Brazma Diana , Chanalaris Anastasios , Howard Julie , Virgili Anna , Valgañon Mikel , Cross Nick , Apperley Jane F. , Nacheva Elisabeth
Published: October 2019

In spite of the universal presence of the BCR/ABL1 fusion gene, chronic myelogenous leukemia (CML) shows remarkable clinical and genetic diversity. The consequences of der(9)t(9;22) chromosome deletions, associated with poor survival, as well as the mechanism behind their formation remain unclear, as does our understanding of the molecular events driving the disease evolution. The presence of these deletions fuelled the expectations that cryptic genome-wide aberrations may be accountable for the disease progression. Following a comprehensive BAC aCGH analysis of 48 CML samples (Brazma et al., Genes, Chromosomes & Cancer, 2007 in press) we report high-resolution oligo-nucleotide array study of a further 30 CML accelerated/ blast phase samples. We were unable to confirm the high frequency of particular single BAC imbalances (CNVs), reported both by ourselves and others, possibly due to the manufacturer’s array selection strategy. Never-the-less some of the CNVs and a wealth of new imbalances were obtained at 60kb resolution. It was possible to build a precise map of the amplicon affecting the sequences flanking the 3′ ABL1 breakpoint site, which include the LAMC3 and NUP214 genes. The presence of this amplicon was associated with therapy resistance. When assessed, at a resolution of 60 kb, the deletions of the regions flanking the ABL1/BCR breakpoint showed novel features:

the genome loss affects preferentially both flanking sites as seen in 5 of the 6 ‘deleted’ samples and the 120kb deletion identified is the smallest recorded so far.

Most of the major cytogenetic features of the samples were confirmed and a number of cryptic genome imbalances were detected, from 120kb to 10Mb in size, involving regions rich in genes, some already implicated in the pathogenesis of CML. Finally, recurrent micro aberrations of several adjacent oligo-nucleotides affecting non-coding sequences were detected in as many as 2/3 of the samples.
Survival Outcomes for Patients (Pts) with Chronic Myeloid Leukemia (CML) with Clonal Evolution (CE) Treated with 2nd Generation Tyrosine Kinase Inhibitors (TKI) after Imatinib Failure.
Authors: Verma Dushyant , Kantarjian Hagop , Estrov Zeev , Garcia-Manero Guillermo , Koller Charles , Borthakur Gautam , Cortes Jorge
Published: October 2019

Background: CE has been considered a criterion for accelerated phase (AP) CML, particularly when it appears during the course of therapy, when it is associated with a poor prognosis. CE may involve a variety of chromosomal abnormalities and may signal resistance to imatinib. The 2nd generation TKI (2nd TKI) dasatinib and nilotinib are effective in patients with AP after failure to imatinib, including those with CE. However, it is unclear whether different chromosomal abnormalities constituting CE may have the same outcome after therapy with 2nd TKI.

Methods: We analyzed the outcome after 2nd TKI therapy of 61 pts with CML with CE who had failed prior imatinib therapy.

Results: The median age was 55 years (range 23–76); the median follow-up after start of 2nd TKI was 18.9 months (mo) (range 5.3–39.3), and median CML duration 67.9 mo (0.4–206.6). Thirty-five pts had CE alone and 26 had CE with other AP features. At the time of this report 59 patients are evaluable for response: 30 treated with dasatinib and 29 with nilotinib. The accompanying table summarizes the findings.

Conclusion: CE constitutes a heterogeneous entity with variable outcome with 2nd TKI. Regardless of the percentage of metaphases with CE, those with trisomy 8 or with abnormalities in chromosome 17 may have the worse outcome. In all cases, the presence of other features of AP further worsens the outcome. The molecular events behind this worse outcome and potential therapeutic approaches directed at them need to be defined.

Characteristics (n=59) CCyR n/no. evaluable(%) p EFS % (12mo) p OS % (12mo p CCyR: Complete Cytogenetic Response, EFS: Event Free Survival, OS: Overall Survival, Chr: Chromosome, Ph+: Philadelphia chromosome positive, n: number of patients % Cellls with CE <16 3/12(25) 62 77 16-35 4/10(40) 60 68 36-99 9/15(60) 73 80 100 7/22(32) 0.24 60 0.96 78 0.85 Other AP features No 18/34(53) 80 89 Yes 5/25(20) 0.02 40 <0.001 60 0.005 Double Ph+ No 9/28(32) 55 69 Yes 14/31(45) 0.42 71 0.93 84 0.72 Trisomy 8 No 21/49(43) 72 84 Yes 2/10(20) 0.29 20 <0.001 40 <0.001 Chr 17 Abnormalities No 19/45(42) 74 83 Yes 4/14(29) 0.53 29 0.003 56 0.02 Other Translocations No 20/42(48) 61 75 Yes 3/17(18) 0.04 69 0.71 82 0.81 Other abnormalities No 15/37(41) 62 81 Yes 8/22(36) 0.79 65 0.45 70 0.93
Relationship of Stem Cell Transplant and Risk of Atrial Fibrillation in Multiple Myeloma: A Case-Control Study.
Authors: Gertz Morie Abraham , Fatema Kaniz , Micallef Ivana N. , Eisinger Amy D. , Barnes Marion E. , Cha Stephen S. , Tsang Teresa S.
Published: October 2019

Background: Clinical observations within our tertiary care setting suggested that atrial arrhythmia is common following hematopoietic stem-cell transplant (SCT). Yet little is known with regard to the incidence of such arrhythmia and factors contributing to its development. We assessed the frequency of first atrial fibrillation/flutter (AF/flutter) development in multiple myeloma (MM) patients who underwent SCT at the Mayo Clinic over a 4 year period.

Method: Using a case-control design, we compared 395 consecutive patients who underwent SCT at the Mayo Clinic during the period 2002–2005 to control subjects who did not have SCT (2:1 matched by age, gender, and date of MM diagnosis) in terms of the baseline risk factors and the frequency of new AF/flutter after MM diagnosis. Patients who had a prior history or evidence of atrial arrhythmia were excluded from analyses. Factors predictive of atrial arrhythmia in the cases were also determined.

Results: First AF/Flutter developed in 38 of 395 cases (mean age 58 ± 9 years) and 47 of 790 controls subjects within 5 years of MM diagnosis (P=0.002). The 5-year Kaplan-Meier estimates of the risk of AF/flutter were 17% and 8% for cases and controls, respectively. Amongst the cases who developed new AF/flutter, the onset of the arrhythmia was within 21 days of SCT for the majority (94%). In multivariable analysis, body weight change of ≥3% in the first week post-transplant (HR 2.86; CI 1.13–7.24; P=0.026) and presence of diastolic dysfunction (HR 1.79; CI 0.99–3.23; p=0.054) at MM diagnosis were independent predictors of first AF/flutter in the cases, adjusting for age, sex, and well established clinical risk factors.

Conclusion: The risk for new AF/flutter was remarkably high in the early post- SCT period in MM patients. In addition to established risk factors for atrial arrhythmia, a weight change ≥3% during the first 7 days after SCT, and the presence of diastolic dysfunction at baseline, were independent novel risk factors for new AF/flutter. These findings warrant further investigation, as modification of fluid management and diastolic filling may potentially lower the risk of post-SCT atrial arrhythmia.
Efficacy of Azacytidine (5-AC) Given as Maintenance or Salvage Therapy for Patients (pts) with Acute Leukemia Post Allogeneic Stem Cell Transplantation (HSCT).
Authors: Jabbour Elias , Giralt Sergio , Kantarjian Hagop , Garcia-Manero Guillermo , Shpall Elizabeth , Kebriaei Patrow , de Padua Leandro , Champlin Richard , de Lima Marcos
Published: October 2019

Background. Relapse is the most frequent cause of treatment failure in pts with refractory acute leukemia undergoing HSCT. Modifications of the preparative regimen using multiple combinations of chemotherapy and physical agents have failed to reduce the risk of relapse without an increase in non relapse mortality. 5-AC is a DNA hypomethylating agent that may induce leukemic cell differentiation and increased immunogenicity, therefore potentially increasing the graft-versus-leukemia effect. 5-AC, at low doses, through its hypomethylation effect has shown significant activity in pts with myelodysplastic syndromes. Furthermore, low doses are likely to be better tolerated after ASCT. Patients 5-AC was given as salvage therapy for post HSCT relapse in 7 pts with acute myeloid leukemia (AML) and as maintenance therapy in 5 (4 AML, 1 acute lymphocytic leukemia) with high-risk disease. Median age was 52 years (range, 29–67 years). At the time of HSCT, 8 pts were refractory and 4 pts (AML) were in second (n=2) and third complete remission (CR) (n=2). Four pts received and failed previous HSCT following ablative regimen. Donors were HLA-compatible related (n=2), unrelated (n=7; cord blood, n=2), or syngeneic (n=1). Conditioning regimen was gemtuzumab ozogamicin 2 mg/m2, fludarabine and melphalan 140 mg/m2 in 3 pts, fludarabine and melphalan 140 mg/m2 in 6, cyclophosphamide, fludarabine and low dose TBI in 2, and busulfan and cyclophosphamide in 1.GVHD prophylaxis was tacrolimus and mini-methotrexate. ATG was administered to recipients of unrelated donor transplants.

Results. After HSCT all pts were in CR. 5-AC was administered at the time of relapse that occurred at a median of 11 months (range, 5–26), and at a median of 4 months (range, 1–12) post HSCT as a maintenance therapy. 5-AC was given at the dose of 16 mg/m2 daily × 5 repeated every 4 weeks (n=3) and 25 mg/m2 daily × 5 (n=9). A median of 7 cycles (range, 2–18 cycles) were delivered. Among 7 pts with relapsed disease [medullary relapse in 4 (median percentage of blasts, 39), extramedullary relapse in 3], 3 achieved a sustained CR for 7+ months, 2 had sustained partial remission for 3+ months, and 2 pts did not respond. All pts receiving 5 AC as maintenance therapy remain in CR for a median of 14+ months (4–28). There was no significant increase in the rates of GVHD. After a median follow-up of 14 months (range, 3–20 months) from the start of 5-AC therapy,11 out of 12 pts are alive, 9 in CR. The 12-month actuarial survival from HSCT is 91%. There were no extra-medullary toxicities and only grade I/II hematologic toxicities were noted.

Conclusion. Low dose 5-AC may be effective in the post HSCT setting. Further follow-up and larger number of patients will be necessary to confirm these observations.
Administration of Rituximab Is Feasible and Does Not Affect Engraftment of Allograft Recipients with Advanced CD20+ Malignancies.
Authors: Kharfan-Dabaja Mohamed A. , Tate Cheryl , Perkins Janelle , Fernandez Hugo F. , Field Teresa , Ayala Ernesto , Perez Lia , Raychaudhuri Jyotishankar , Alsina Melissa , Sullivan Dan , Brand Linda , Poling William , Anasetti Claudio
Published: October 2019

Rituximab is a chimeric murine/human IgG1 kappa anti-CD20 monoclonal antibody that has revolutionized treatment of patients with CD20+ malignancies because of its non-cross resistance with chemotherapy and favorable toxicity profile. There is limited data evaluating the feasibility of combining rituximab with conditioning chemotherapy (or chemo-radiotherapy) for patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) for advanced CD20+ expressing malignancies. Twelve patients (M= 7; F=5), with a median age of 54.5 (41–66) years, underwent allo-HCT for the following diagnoses (disease status at time of transplantation) [chronic lymphocytic leukemia (CLL)= 7 (CR2=2; PR2= 3; ≥PR3=2); mantle cell lymphoma (MCL)= 3 (CR1=1; ≥PR2= 2); follicular NHL=1 (CR3=1), diffuse large B-cell (DLBC)=1 (>PR3=1)]. Donors were as follows: matched-related donors (MRD) = 7; matched-unrelated donors (MUD) =3; mismatched unrelated donor (MMUD)= 2. Fludarabine-based conditioning regimens comprised fludarabine plus targeted doses of intravenous busulfan (FLU-BU=8), or total-body irradiation (FLU-TBI=3), or cyclophosphamide (FLU-CY=1). Anti-thymocyte globulin (ATG) was administered in two cases of MMUDs. GVHD prophylaxis consisted of tacrolimus plus mycophenolate mofetil= 8, or methotrexate = 4. Nine (75%) of 12 patients received rituximab 375 mg/m2 on day +1 (±3 days) and all (100%) patients received rituximab 375 mg/m2 on day +8 (± 3 days) without serious infusion reactions. All patients engrafted. Median time to neutrophil engraftment for the entire cohort was 15.5 (12–27) days. Median time to platelet engraftment in nine patients was 14.5 (10–17) days. Three patients never dropped their platelet counts below 20,000/uL. Median donor chimerism at day +90 (±10 days) for unsorted BM, CD3 and CD33 by PCR/STR method were 94% (70–100%), 86% (59–100%) and 100%, respectively. At a median follow up of 5 (0.6–30.3) months, the 100-day non-relapse mortality (NRM) in 11 evaluable patients was 10%. Acute GVHD, grades II, III, and IV, developed in 50%, 8.3% and 8.3%, respectively, at a median time of 27 (16–77) days. These findings demonstrate that administration of rituximab as part of conditioning chemotherapy is feasible and does not affect timely hematopoietic engraftment of allograft recipients with advanced CD20+ malignancies.
Graft-Versus-Leukemia Effect Is Equivalent in Recipients of Matched Sibling and Matched Unrelated Donor Conventional Hematopoietic Cell Transplant.
Authors: Arora Mukta , Pavletic Steven , Anasetti Claudio , Barrett John , Wang Tao , Antin Joseph H. , Di Bartolomeo Paolo , Bolwell Brian J. , Bredeson Christopher , Cairo Mitchell , Gale Robert P. , Giralt Sergio , Hahn Theresa , Hale Gregory , Halter Joerg , Jagasia Madan , Litzow Mark R. , Locatelli Franco , McCarthy Phillip L. , Mort Cowan , Petersdorf Effie W. , Russel James , Schiller Gary , Schouten Harry S. , Tallman Martin , Verdonck Leo F. , Wiley Joseph , Wingard John R. , Horowitz Mary M. , Ringden Olle
Published: October 2019

Anti-leukemia effects of allotransplants are largely mediated by immune mechanisms collectively termed graft-versus-leukemia (GvL). We postulated that allotransplants from HLA-matched unrelated donors (URD) have greater GvL activity than similar transplants from HLA-identical siblings because of greater genetic-disparity between donor and recipient. We tested this hypothesis in 4099 adult recipients of HLA-matched sibling (N= 3158) or 8/8 HLA-allele-matched URD (N= 941) transplants for acute leukemia (AML and ALL) and chronic myeloid leukemia (CML) receiving conventional transplants between 1995–2004 and reported to CIBMTR. Relapse, transplant-related mortality (TRM) and leukemia-free survival (LFS) were compared between the cohorts. Contrary to our hypothesis, relapse rates at 5 y were similar in patients receiving sibling and URD transplants. More TRM was seen in URD cohort in early AML [sibs 24% (20–27%) versus URD 40% (31–50%), p=0.001], advanced ALL or AML [sibs 31% (25–36%) versus URD 44% (35–53%), p= 0.01] and early CML [sibs 31% (28–33%) versus 38% (32–44%), p=0.02]. LFS was superior with sibling donors in early- and advanced- AML and CML. These data indicate comparable GvL-activity of HLA matched URD and HLA-identical sibling transplants in AML, ALL and CML receiving conventional transplants despite greater genetic disparity.

Early Disease (1stremission or 1st chronic phase) Outcome at 5 years Sibling donor URD P AML (N= 878) 760 118 Relapse 15 (13–18)% 22 (15–30)% 0.1 LFS 61 (57–65)% 38 (28–48)% <0.001 ALL (N= 333) 271 62 Relapse 23 (18–29)% 15 (7–25)% 0.13 LFS 46 (39–53)% 42 (29–55)% 0.6 CML (N=1443) 1152 291 Relapse 6 (5–8)% 5 (3–8)% 0.5 LFS 63 (60–66)% 57 (51–63)% 0.06 Intermediate Disease (2nd or subsequent remission or accelerated phase) AML (N= 268) 180 88 Relapse 22 (16–29)% 21 (13–30)% 0.8 LFS 48 (40–56)% 41 (30–52)% 0.3 ALL (N= 172) 111 61 Relapse 32 (24–42)% 36 (24–48)% 0.7 LFS 27 (18–36)% 24 (14–37)% 0.8 CML (N=263) 173 90 Relapse 22 (15–28)% 16 (9–24)% 0.5 LFS 36 (29–44)% 35 (26–46)% 0.9 Advanced Disease (Not in remission or blast phase) AML (N= 423) 294 129 Relapse 46 (40–52)% 43 (34–51)% 0.6 LFS 23 (18–29)% 13 (8–20)% 0.01 ALL (N= 152) 88 64 Relapse 52 (42–63)% 48 (35–60)% 0.6 LFS 10 (4–18)% 5 (1–12)% 0.2 CML (N=76) 49 27 Relapse 35 (23–49)% 39 (20–60)% 0.8 LFS 21 (11–35)% 0 <0.001
The Clinical Usefulness of Novel Prognostic Factors in CLL: A Study of 477 Patients with Low-Risk (Non-11q, Non-17p) FISH and Known IGVH Mutation Status.
Authors: Tam Constantine S. , Keating Michael J. , Tsimberidou Apostolia M. , O’Brien Susan , Tsimberidou Alessandra , Lerner Susan , Abruzzo Lynne , Schlette Ellen , Kantarjian Hagop , Wierda William
Published: October 2019

In order to develop integrated models utilizing commonly available prognostic factors, we studied the clinical signficance of IGVH mutation, CD38 and ZAP-70 in 477 CLL patients (pts) with low-risk (non-11q, non-17p) FISH findings. All pts were untreated at the time of FISH assessment, and were collected prospectively in the MD Anderson CLL database. Two hundred & fifteen pts (45%) had mono- (n=160) or bi-alleleic (n=55) deletion of 13q {DEL13Q}, 162 pts (34%) had a negative FISH panel {NEG}, and 100 pts (21%) had trisomy 12 as sole FISH abnormality (n=78) or in association with deletion 13q (n=22) {T12}. Compared to other FISH groups, DEL13Q pts had lower B2m (median 2.2 v 2.6mg/L, p=0.01) and were less likely to be IGVH unmutated (33% v 48%, p=0.001). In contrast, T12 pts were more likely to present with advanced stage disease (Rai≥2 36% v 23%, p=0.01), be CD38 positive (44% v 13%, p<0.001), and have karyotypic abnormalities (48% v 7%, p<0.001). One hundred and twenty-three pts had active disease requiring immediate therapy and 354 pts had stable disease, of whom 291 were evaluable for disease progression. At a median follow-up of 20 months, 73 pts had developed active disease with NCI-WG indication(s) for treatment. Actuarial 2 year time to treatment (TTT) was 26%, with no significant difference between 13q, NEG and T12 pts (p=0.27). TTT was associated with elevated B2m (≥1.5ULN), IGVH mutation status and ZAP-70 in DEL13Q and NEG pts, but not in T12 patients (Table). For DEL13Q/NEG pts, a simple model using IGVH mutation and B2m separated high risk pts (unmutated or high B2m, 2yr TTT 43%) from standard risk pts (mutated and low B2m, 2yr TTT 11%, p<0.0001). For T12 pts, a model based on CD38 positivity and karyotypic abnormalities separated high risk pts (2 factors, 2yr TTT 75%) from standard risk pts (0 or 1 factor, 2yr TTT 15%, p=0.008). These results show that the impact of prognostic factors on TTT is dependent on the underlying FISH karyotype, and underscores the need for future studies in CLL prognostic factors to take into account the complete risk profile of the pt.

NEGATIVE FISH DELETION 13Q TRISOMY 12 p-value hazard ratio p-value hazard ratio p-value hazard ratio IGVH Mutation <0.001 8.0 0.003 2.9 0.97 0.98 B2m ≥1.5ULN <0.001 4.5 0.07 2.2 0.54 0.68 CD38 Positivity 0.05 2.5 0.05 2.4 0.06 7.4 Abn Cytogenetics <0.001 11.0 0.27 2.2 0.09 2.8 ZAP-70 0.02 2.9 0.007 3.1 0.70 1.3

Figure Figure Figure Figure
ZAP70 Expression by Immunohistochemistry Correlates with IgVH Mutation Status in Chronic Lymphocytic Leukemia and Predicts Time to Progression, but Survival Is Decreased in p53-Positive CLL, Regardless of ZAP70 Expression.
Authors: Schlette Ellen J. , Tam Constantine , Admirand Joan , Wierda William , O’Brien Susan , Lerner Susan , Keating Michael J.
Published: October 2019

Background: IgVH mutation status (IgVH MS) in chronic lymphocytic leukemia (CLL) is accepted as a powerful prognostic indicator, however, this test is not widely available. Therefore, ZAP70 expression, first described as a surrogate for IgVH MS, but also described as an independent prognostic marker, is more commonly used. ZAP70 expression can by detected by flow cytometry or immunohistochemical staining (IHC) methods, but the optimal detection method is controversial. Though cytogenetic abnormalities of 17p in CLL have been reported as a poor prognostic indicator when compared to other abnormalities, the prognostic effect of p53 expression by CLL has not been well described, nor has it’s utility in combination with ZAP70 expression been reported.

Methods: The bone marrow specimens from 2 cohorts of CLL patients (pts), specifically 208 pts uniformly treated with FCR between 11/99–01/04 and 349 CLL pts entered in the Low Risk FISH (LRF) study (no 11q or 17p FISH abnormalities) between 1/04 - 05/07, were evaluated for ZAP70 expression by IHC. IgVH MS was available for 61/208 FCR pts and all LRF study pts. The FCR group was evaluable for complete response (CR) and CR duration. 215 LRF study pts were evaluable for time to first treatment and 114 pts were evaluable for response to either FCR-type therapy (74pts) or rituximab-based therapy (40pts). 193 BM specimens from the FCR cohort were also studied for p53 expression by IHC staining.

Results: IgVH MS showed good concordance (80%) with ZAP70 IHC expression(table 1). The time to first therapy for the LRF cohort was 18% v 43% at 18 months for ZAP70+ versus ZAP70− CLL pts (p=0.0001). However ZAP70 IHC did not predict response in either the FCR cohort (n= 208) or the LRF cohort (n=114). In 193 FCR pts, a significant correlation between p53 expression and overall survival, regardless of ZAP70 expression, was seen (p<0.001) (Figure 1). ZAP70 IHC expression correlated with the time to progression for 165 FCR pts who had achieved complete response (p=0.0009)(Figure 2).

Conclusions: ZAP70 IHC expression shows good correlation with IgVH MS for the pooled cohorts. ZAP70 IHC expression has additional value in predicting time to first therapy and time to progression, but does not have an impact on CR rate. The decreased CR duration is due to the ZAP70 relationship with IgVH MS. When ZAP70 and p53 expression are combined, decreased survival is seen in pts with p53+ CLL, regardless of ZAP70 expression.

SH status in combined cohorts (N=410) versus ZAP70 expression by IHC Combined cohorts N=410 IHC ZAP− IHC ZAP+ Sensitivity 78%; Specificity 82% IgVH MS Mutated 178 39 Unmutated 42 151 Figure Figure Figure Figure
Transfection with mRNA for CD19 Specific Chimeric Antigen Receptor Restores Natural Killer Cell Mediated Killing of CLL Cells.
Authors: Boissel Laurent , Betancur Monica , Tuncer Hande , Weitzman James , Klingemann Hans
Published: October 2019

B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world and is characterized by monoclonal proliferation of functionally deficient lymphocytes, with relative decrease in T and Natural Killer (NK) cells. Available monoclonal antibody (mAb) therapies elicit a response in almost half of the patients, but durable remissions are rare. B-CLL cells are virtually impervious to T and NK cell-mediated cytotoxicity. However, a feasible approach to overcome this resistance is to redirect the specificity of T or NK cells by making them express chimeric antigen receptors (CAR). Such a method has been successfully tested in NK cells against a number of CD19-expressing cells; however the loading of cells with CD19-CAR relied on retrovirus-based transfection methods. We describe here a non-retroviral method for obtaining NK cells expressing high levels of CD19-CAR, based on electroporation of messenger RNA (mRNA). We used a CAR that is comprised of the single chain Fv portion of a mouse mAb against CD19 fused to the signaling intracellular domain of CD3ζ subunit. The corresponding cDNA was subcloned by PCR into a plasmid allowing T7-dependant mRNA synthesis. In vitro synthesis of the CD19-CAR mRNA was followed by a poly-adenylated tail elongation step in order to improve in vivo stability and translation of the messenger. Electroporation of NK-92 cells with this construct, using a Biorad GenePulser II apparatus, yields up to 78% expression efficiency and as high as 90% cell survival. Cytotoxicity of transfected NK-92 cells was tested against 3 target cell lines (NK sensitive, CD19 negative K562; CD19 expressing, NK resistant REH and SUPB15) as well as primary CLL cells from 6 untreated patients. A non-radioactive fluorochrome-based flow cytometric assay was used to determine NK-mediated cytotoxicity, at effector to target (E:T) ratios ranging from 1:1 to 10:1. Expression of the CD19-CAR on the surface of NK-92 cells do not change their cytolytic properties against K562: 70% ±5% killing compared to 74% ±2% killing for non-transfected NK-92, (E:T ratio of 10:1). Expression of CD19-CAR in NK-92 cells completely overcomes the resistance of REH and SUPB15 cell lines: 76% ±7% and 73% ±5% killing respectively, compared to 21% ±10% and 18% ±7% killing respectively with non-transfected NK-92 (E:T ratio of 10:1). Moreover, previously resistant primary CLL cells became highly sensitive to lysis by NK-92 expressing CD19-CAR: 46–82% killing, as opposed to 7–22% killing for non-transfected NK-92 (E:T ratio of 10:1). These results show that NK cells can be engineered using mRNA transfection by electroporation to target and effectively kill resistant primary CLL cells.
Pentostatin, Cyclophosphamide, Rituximab, and Mitoxantrone (PCRM): A New Highly Active Regimen for Patients with Chronic Lymphocytic Leukemia (CLL) Previously Treated with PCR or FCR.
Authors: Lamanna Nicole , Heaney Mark L. , Brentjens Renier J. , Jurcic Joseph G. , Weiss Mark A.
Published: October 2019

Combination therapy with purine analogs, alkylators, and/or monoclonal antibodies has markedly improved the quality of responses in patients with chronic lymphocytic leukemia (CLL). Most regimens have utilized fludarabine as the purine analog but the severe myelosuppression and immunosuppression of these combinations require careful attention to dosing and schedule to minimize these toxic complications. Of the purine analogs active in CLL, pentostatin is the least myelosuppressive. Previously, we reported that combination pentostatin, cyclophosphamide, and rituximab was very active and acceptably safe to administer to patients with CLL and in the salvage setting this regimen appeared to have less myelosuppression and less frequent infectious complications than comparable fludarabine-based combinations. The current study combines pentostatin 4mg/m2, cyclophosphamide 600mg/m2, rituximab 375mg/m2 (omitted from cycle 1) and mitoxantrone (dose escalated in a phase 1 portion starting at 6mg/m2, 8mg/m2, and 10mg/m2) all administered on day 1 of 28-day cycles for a total of 6 treatments. Supportive measures included prophylactic administration of pegfilgrastim, sulfamethoxazole/trimethoprim, acyclovir, and antiemetics. Renal function was closely monitored and all patients received at least 1.5 liters of intravenous hydration with the administration of chemotherapy. Twenty-one patients (median age 62, range 44–74) with CLL (17 patients) or other low grade B cell neoplasms (4 patients) have been enrolled. There were 16 men and 5 women. Of the CLL patients all had either high risk disease (71%) or “active” intermediate (29%) risk disease and their median pretreatment WBC count was 74,000/μl, HGB 9.9 g/dl, and PLT 144,000/μl. The median β-2 microglobulin was 3.3 mg/l. The median number of prior treatment regimens was two (range 1–6). Most of the CLL patients (65%) had previously been treated with chemoimmunotherapy utilizing PCR or FCR. Response data is currently available for 16/17 of the CLL patients. In this group there were 15 responses (94%), including 4 CRs (25%) and 11 PRs (69%). Prior therapy with PCR or FCR did not adversely affect the frequency of response with 91% of these patients responding (CR in 19% and PRs in 73%). These preliminary results indicate that PCRM therapy is very active and well tolerated even in patients who have previously received FCR or PCR.
Valproic Acid Induces Apoptosis in Chronic Lymphocytic Leukemia Cells by Targeting Several Pro- and Anti-Apoptotic Genes.
Authors: Stamatopoulos Basile , Meuleman Nathalie , Kentos Alain , Hermans Philippe , Martiat Philippe , Bron Dominique , Lagneaux Laurence
Published: October 2019

Histone deacetylase inhibitors have been shown to modulate the cell cycle, to induce apoptosis and to sensitize cancer cells to other chemotherapeutics. Among these inhibitors, valproic acid (VPA), an antiepileptic drug, is being discussed as a promising novel anti-cancer drug. Chronic Lymphocytic Leukemia (CLL) is a clinically heterogeneous disease remaining incurable despite introducing new promising treatments. The effects of VPA and its mechanism of action were evaluated on mononuclear cells isolated from 40 CLL patients. Exposure of CLL cells to increased doses of VPA (0.5–5mM) leads to a dose-dependent cytotoxicity and apoptosis in all CLL patients. VPA treatment induced apoptotic changes in CLL cells including phosphatidylserine externalization and DNA fragmentation. The mean apoptotic rates were similar between IGHV mutated and unmutated patients, the latter presenting a more aggressive clinical course. VPA induced apoptosis via the extrinsic pathway involving engagement of the caspase-8-dependent cascade. Interestingly, VPA increased the sensitivity of leukemic cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) even among resistant patients. Moreover, VPA at physiological concentration of 1mM can significantly increase the in vitro cytotoxic effects of fludarabine, bortezomib and the natural product honokiol allowing the reduction of effective concentration 50% (EC50). In order to understand the early mechanism of action of VPA, we investigated gene expression profiles of 14 CLL-patient samples (7 with a good prognosis and 7 with a bad prognosis regarding IGHV mutational status and Zap-70 expression) treated in vitro during 4 hours with a physiological dose (1mM) of VPA and compared with their untreated counterpart using Affymetrix technology. No difference in gene modulation was observed between poor and good prognosis patients after VPA treatment. Modulation of several pro- and anti-apoptotic mRNA expression was confirmed by a real-time reverse transcription-PCR. The molecular analysis of the apoptotic machinery involved in VPA response revealed the up-regulation of APAF1 (5.5 fold, P<0.0001), BNIP3 (2.2 fold, P=0.0006), PTEN (1.9 fold, P=0.0002), CASP6 (2.5 fold, P<0.0001) and the down-regulation of CFLAR/FLIP (2.0 fold, P<0.0001), BCL2 (1.6 fold, P=0.0222), AVEN (1.9 fold, P<0.0001), BIRC4/XIAP (1.7 fold, P<0.0001) and BIRC1/NAIP (1.6 fold, P=0.0007). In conclusion, VPA induced apoptosis of CLL cells at clinically relevant concentration by selective activation of the caspase-8 (extrinsic) pathway and by targeting several pro- and anti-apoptotic genes. Therefore, the combined application of VPA with other drugs might be considered as a potential strategy for CLL treatment.
Tissue Factor and Cancer Procoagulant as Predictors of Thrombosis or Progression in Cancer Patients.
Authors: Molnar Soledad , Guglielmone Hugo , Minoldo Salvador , Rizzi Maria L. , Lavarda Marcelo , Jarchum Gustavo
Published: October 2019

Background: Two procoagulant factors are associated with the tumor cell: tissue factor (TF) and cancer procoagulant (CP). The activation of the coagulation system can cause thrombosis and it has also been involved in the development of metastasis.

Aims: 1- to establish the relationship between the presence and the levels of both procoagulants and the development of thrombosis or disease progression. 2- to analyse if the treatment with chemotherapy increases the levels of TF or CP.

Methods: Patients with recent diagnosis of cancer were included in this prospective study. TF and CP determinations were done at diagnosis, at three months after chemotherapy, and at either event of progression or thrombosis. TF evaluation was done through ELISA test (normal value 159 pg/ml) and CP evaluation by a coagulometric method (Gordon et al, Thromb Res1989;56:431–40). In the last test negative values indicate high CP activity. Statistical analysis of the data was performed by two-tailed Fisher exact test and Mann-Withney/Wilcoxon test.

Results: 31 patients, 61% female sex, age median 54 years. Digestive cancer 49%, breast 32%, genitourinary 13%, lung 2%. Stages of the disease were: early (I and II) 48% and advanced (III and IV) 52%. Median follow-up was 11.6 months. High levels of TF and CP at diagnosis were seen in 48% and 84% respectively. No patients developed thrombosis. Nine patients had disease progression (29%) and 4 (13%) died. Eigthy eight percent patients of this last group had advanced stages of the disease. The type of tumor more frequently associated with the event was digestive cancer (45%) and genitourinary cancer (33%). TF and CP values were increased by 40% and 50% respectively during chemotherapy treatment. Level of CP at diagnosis predicted progression (-26 sec versus -8 sec in patients with and without progression p 0.006) and also mortality (-34 sec versus -10 sec p0.003). TF at diagnosis predicted neither progression nor mortality. All patients who had progressive disease showed increased levels of TF (388 pg/ml vs 217 pg/ml p0.002) and CP (-63 sec vs -23 sec p.006) with respect to the values at diagnosis.

Conclusions: Higher levels of CP at diagnosis are associated with a greater frequency of progression and mortality. No patients developed thrombosis. TF and CP levels were increased at the time of progression with respect to the values obtained at diagnosis. Chemotherapy increases the values of both procoagulants. A larger follow-up and a large number of patients could clarify the role of these procoagulants in the development of disease progression (metastasis).