bluuebunny/crossref_metadata_2025 huggingface dataset:
Non Invasive Screening for Fetal RHD-Genotype in All D-Negative Women Is Reliable and Cost-Effective.
Authors: Van der Schoot C. Ellen , Soussan Aicha Ait , Bonsel Gouke J. , de Haas Masja
Published: April 2020

About 40% of D-negative pregnant women receive antenatal anti-D despite a D-negative fetus. We have developed an automated assay for fetal genotyping using cell-free fetal DNA from maternal plasma to restrict antenatal prophylaxis to women carrying a D-positive fetus. This will save anti-D of which is worldwide shortage, and will prevent the unnecessary administration of a blood product. 1 ml of plasma is automatically presented (Tecan) to a DNA isolation-robot (Roche). The DNA-eluate is tested (after automatically pipetting) in triplicate in a real-time RHD exon7-PCR (ApplBios).

Results

Plasma from 2415 D-negative pregnant women, whose blood was sent in for 28–30th week antibody screening, has been tested. In 35 cases (1.44%) the women (10 weak D and 25 normal D-positive) were typed as D-positive by repeated serological tests, and excluded from the study.

1465 of the plasma’s (61.59%) were typed RhD positive and 915 RhD negative. Because the RHD exon7-PCR will give positive results in D-negative women carrying D-negative variant RHD genes such as RHDΨ, we determined the incidence of these genes in our studygroup. In all cases (n=39) with Ct values below 33.2 DNA was isolated from maternal leukocytes. Of the 19 cases with extremely low Ct values (<32), 6 women carried an RHDΨ gene, 5 an RHD variant type VI, 3 weak D (type 1, 11 and 17) and 1 RHDdel. The 24 other women did not carry RHD genes.

To compare the plasma PCR results with cord blood serology, all women and obstetric caregivers were sent questionnaires on cord blood serology. 55% responded (n=1257). For cases with discrepant results(n=21) or incompleted questionnaires(n=31) the laboratory which performed the cord blood typing was contacted. Finally, concordant results were obtained in 1249 of the cases. In 3 cases the genotype suggests D-positivity while serology is D-negative. In 5 cases no RHD-sequences were detected in plasma but cord blood was typed D-positive. For the discrepant cases it cannot be concluded yet, which assay gave the correct phenotype.

Conclusion

This is the first large scale automated study demonstrating the feasibility of screening D-negative women to restrict antenatal anti-D to women carrying D-positive fetuses. For the recognition of women at risk for immunization, the plasma-PCR test seems to be at least as reliable as the serological test. Furthermore, postnatal prophylaxis could have been given directly after delivery in all women with a positive PCR-result, saving cord blood serology tests and possibly increasing the effectiveness of postnatal prophylaxis.

An economic evaluation demonstrated that this screening policy is cost effective in the Netherlands.
Health Care-Associated Infection (HAI) Rates Are More Pertinent Quality Indicators Than Nosocomial Infection (NI) Ones for Hematology Programs.
Authors: Chehata Sami , Grira Chiraz , Legrand Patrick , Pautas Cecile , Maury Sebastien , Kuentz Mathieu , Carlet Jean , Cordonnier Catherine
Published: April 2020

In hematology patients, HAI are a complex mixture of infections due to therapy, especially to transplant procedures, conditioning regimen, immunosuppressive drugs, and invasive procedures. These HAI may occur within the hospital and be related to the hospital environment (i.e., NI), or not. These different kinds of infections require very different preventive measures. The objective of our study was to assess the incidence, overlap, and causes of HAI and NI in our hematology ward over a 6 month period.

Methods and Definitions: Infections were considered to be HAI, irrespectively of their site of occurrence (hospital or not) when they were directly related to the therapeutic procedures and subsequent immune deficiency. This included: febrile neutropenia (FN) subsequent to chemotherapy, catheter-related infections, herpes virus reactivations in patients previously sero+ to the virus, primary CMV infection in CMV− patients transplanted with a CMV + donor, toxoplasmosis reactivation, invasive fungal infections, and any infection occurring within 6 months of transplant, or later in case of immunosuppressive treatment after this date. Infections were considered to be NI when they occurred ≥ 48 h after hospitalization without any symptom of infection at hospitalization. Aspergillosis was considered to be NI when there was a delay of 7 days between hospitalization and first symptoms.We retrospectively assessed 223 consecutive infectious episodes occurring between 7–12/2000 in 137 patients, including 59 stem cell transplant recipients (allogeneic: 23; autologous: 36).

Results : 204/223 infectious episodes (91,4%) were HAI while 94/223 (42%) were NI. 7/223 (3,6%) were causes of death. Among the 42 bacterial documented infections, 20 (47.6%) were due to potentially hospital-acquired strains: 16/23 (69.5%) of HAI-NIs, and 4/19 (21%) of the non NI-HAIs.

Conclusion: In a preventive infection program, the assessment of NI clearly underestimates half of the HAI in hematology patients. Although hospital environment control is of paramount importance in transplant programs, we suggest that HAI should be a better quality indicator for hematology programs than NI.
Quality of Life of Patients with Acute Myeloid Leukemia and Myelodysplatic Syndrome.
Authors: Kuehnbach Robert , Hiddemann Wolfgang
Published: April 2020

Background: Over the last decades, outcome from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) has improved due to better treatment. However, little is still known about Quality of Life (QoL) of patients treated.

Methods: QoL was evaluated in patients who were randomized between April 2003 and September 2004 for treatment for AML and MDS in the German AMLCG99 trial at the university hospital of Munich-Grosshadern, Germany. QoL was evaluated using the EORTC QLQ-C30 questionnaire. During inhouse and out-patient treatment, QoL was evaluated at eight consecutive time points. Besides leukemia-specific data, duration of inhouse treatment, duration of leukocytopenia and maximal c-reactive protein (CRP max), hemoglobine levels were evaluated. QoL results were compared to already published data from normal German population and patients with small cell lung cancer.

Results: In total, 57 patients were treated, of which 76,3% had a de-novo AML, 20,3% a secondary AML and two patients an MDS. Median event-free survival was 15,4 months.

QoL data from 49 patients, who filled in a total number of 238 QLQ-C30 questionnaires, was available for analysis. Ten patients received allogeneic stem cell or bone marrow transplantation (BMT) instead of cyclic maintainance therapy.

Scores for global health status were low in the beginning, but improved significantly during treatment, finally reaching levels, that were not significantly different from normal population. No differences were seen for patients who subsequently went off protocol due to relapse or death, for karyotype or initial lactate dehydrogenases (LDH). There were neither any differences in between age groups, induction therapy groups, for patients receiving g-csf priming and those who did not or in between older patients receiving a 2nd induction chemotherapy and those who did not. Global health status did not correlate with duation of inhouse treatment, with duration of Leukocytopenia or CRP max.

Physical functioning and fatigue mainly accounted for global health status and were correlating with other functional and symptome scores. However, hemoglobin levels did not show high correlation with physical functioning or fatigue. Social functioning was significantly better for older patients (60 years and older) at the end of treatment than it was for younger ones. BMT was worsening physical and social functioning at the end of inpatient treatment, however, differences disappeared during further evaluation of QoL.

Conclusion: Rather because of than despite treatment, patient’s QoL was improving during time, partially even reaching levels of normal population.
Cardiac Toxicities after Treatment of Primary Hodgkin’s Lymphoma (HL). Analysis of the LENT-SOMA Questionnaire Registered in the Database of the German Hodgkin Study Group (GHSG).
Authors: Behringer Karolin , Elena Gilman , Anesa Lihovac , Lucia Nogova , Heinrich Seegenschmiedt M. , Volker Diehl , Andreas Engert
Published: April 2020

Introduction:

As a consequence of the impressive long-term remission rates in Hodgkin’s Lymphoma (HL), the reduction of treatment-related complications is becoming increasingly important. The most important treatment-related toxicities are secondary malignancies, infertility, pulmonary, thyroid and cardiac dysfunction. Here, we report results of the prospectively planned LENT-SOMA questionnaire to determine cardiac toxicities in patients successfully treated for HL.

Patients and methods:

Between 1998 and 2002, the GHSG conducted the fourth generation of clinical trials for the treatment of early, intermediate and advanced stage HL (HD 10–12) involving a total of approximately 4000 evaluable patients. The LENT-SOMA questionnaire was sent out to the participating centers 2, 5 and 10 years after randomisation of participating patients to evaluate cardiac, pulmonary, and gonadal dysfunction after HL treatment. To focus on cardiac toxicities, questions referred to cardiac risk factors, subjective and objective cardiac dysfunction, cardiac intervention and therapy.

Results:

Overall, approximately 1.900 (50%) LENT-SOMA questionnaires after 2 years and 10% LENT-SOMA questionnaires after 5 years were identified. Among all documented toxicities, oedema were most frequently reported, followed by arrythmia, cardiac failure, myocardial ischaemia, cardiomegaly, and pericardial dysfunction. The frequency of cardiac dysfunction will be evaluated according to treatment modality (chemotherapy, chemo- and radiotherapy) and it will be analysed with respect to age, gender, and cardiac risk factors. Furthermore, the amount of patients suffering from more cardiac toxicities and the probability of recovery, once a cardiac dysfunction has been detected, will be presented.

Conclusion:

Cardiac toxicities after HL treatment include oedema, cardiac failure, myocardial ischaemia, cardiomegaly, arrythmia, and pericardial dysfunction. The first analysis of the LENT-SOMA questionnaire focussing on cardiac dysfunction will be presented.
Euthanasia: History, Definitions and Clinical Guidelines from Classical Jewish Sources.
Authors: Gesundheit Benjamin , Jotkowitz Alan , Or Reuven
Published: April 2020

Background : End-of-life care is a central ethical problem for clinicians. The definition of euthanasia is complex and difficult causing confusion for its practical application. Historically, the term was abused leading to medical atrocities. We trust that during medical training and in continuous medical education programs ethical topics relevant for clinical practice should be addressed systematically in their cultural context. The awareness for these crucial topics will improve clinical performance.

Objective : To define philosophical concepts, to present historical events, to discuss classical attitudes in modern bioethics and to analyze classical Jewish sources which are helpful to elaborate practical guidelines for clinicians in euthanasia and end-of-life care. Reflection on these concepts by discussing classical Jewish sources might help clinicians in their judgment and process of decision-making.

Methods : A historical overview with philosophical definitions of the concepts of active versus passive euthanasia, physician assisted suicide etc are given. Sources from the classical Jewish literature are presented and analyzed also according to later traditional interpretation. Their relevance and application in modern clinical medicine is elaborated.

Results : Philosophically, the distinction between active versus passive euthanasia is crucial. Jewish sources certainly support the obligation to ease the ongoing process of dying; there is no duty to prolong the agony of the actively dying patient, which is actually prohibited. On the other hand, no practical involvement is allowed to actively induce death of the terminal patient.

Conclusion : The historical overview and the review of the literature support the need to define concepts of euthanasia for clinicians dealing with end-of-life issues, since this term caused historically and philosophically major confusion. Therefore, ethical issues should be included in formal training for physicians and other health care providers, in order to improve the clinical approach for these challenging aspects of clinical medicine. We believe, that classical Jewish sources might contribute to elaborate clinical definitions and to provide meaningful approaches for practical guidelines for clinicians.
Identification of Prognostic Factors in Patients with Early Stage Unfavorable Hodgkin’s Lymphoma: An Individual Patient Data Meta-Analysis.
Authors: Bohlius Julia , Haverkamp Heinz , Diehl Volker , Eghbali Houchingue , Franklin Jeremy , Pfistner Beate , Raemaekers John , Engert Andreas , Henry-Amar Michel
Published: April 2020

Background: In patients with early stage unfavorable Hodgkin’s lymphoma (HL) event-free-survival (EFS) is low compared to patients with early favorable or advanced stage disease. An international collaborative study was initiated to identify prognostic factors that could help to define among early stage unfavorable patients those who might benefit from more intensive treatment.

Methods: Medline and Cochrane Library were systematically searched for randomized controlled trials in stage I/II HL. Trials should concern patients with one or more risk factors (age, sex, stage, B-symptoms, bulky disease, number of areas involved, laboratory parameters) receiving 4–6 cycles of ABVD or similar chemotherapy and radiotherapy. Only trials with ≥ 100 patients per study arm were considered. Individual patient data were collected.

Results: Six studies were identified: GHSG: HD5, HD8, HD11; EORTC: H6U, H7U, H8U. Data from 4,235 patients enrolled between 08/1982 and 01/2003 were available for analysis. The proportion of missing data was very low for demographic and clinical characteristics (< 1%); it was acceptable (1.5–9%) for laboratory parameters with the exception of albumin (41% missing values, not recorded in 3 trials). Patient characteristics are listed below. The median follow up was 58.4 months (95% CI 56–61). At the time of analysis 612 patients had experienced an event (disease progression, relapse or death) leading to a 5-year EFS rate of 85%. Using an univariate Cox-regression stratified by study and treatment arm several parameters showed significant influence on EFS.

Conclusion: The data set available is sufficiently powered to identify relevant prognostic factors. A multivariate analysis using updated data will be presented.

Baseline parameter of inlcuded studies Age (median years) Male sex Sage (I/II) B symptoms Bulky disease Hb (median g/dL) All patients, n=4,325 32 2,039 (48.1%) I: 546 (12.9%), II: 3,688 (87.1%) 1,490 (35.2%) 1,936 (45.7%) 12.9 Missing data 1 (< 1%) 0 1 (< 1%) 35 (< 1%) 0 82 (1.9%)
The Mechanism of Rh-Immune Globulin Prophylaxis Involves Significant Increases in the Immunosuppressive Cytokines TGFβ and PGE2.
Authors: Branch Donald R. , Lund Nicole , Shabani Fariba , Denomme Gregory A.
Published: April 2020

For more than 40 years, hemolytic disease of the newborn (HDN) caused by anti-Rh(D) antibody produced by a pregnant mother to her Rh(D)+ baby has been prevented by Rh(D)-immune globulin prophylaxis. Surprisingly, although a number of theories have been proposed, the mechanism of action of this immunosuppressive treatment remains completely unknown (Kumpel BM, Immunol Lett.2002;82:67–73). One potential mechanism of action is the production of one or several strongly immunosuppressive cytokines capable of blocking a primary immune response. Thus, to begin to unravel the possible mechanism(s) of the immunosuppression induced by administration of Rh(D)-immune globulin, we have examined production of a number of immunomodulatory cytokines following administration of Rh(D)-immune globulin. After informed consent, we measured levels of 17 different cytokines pre- and 48 hours post-administration of 300 μg of anti-Rh(D)(WinRhoSDF) in 10 healthy women candidates for Rh(D)-immune globulin prophylaxis. We used platelet-poor plasma from citrated whole blood and quantitative ELISA having the most sensitive detection limits. We observed no striking changes in levels of the cytokines IL-1 sRII, IL-12 p40, IL-16, or MCP-1. Levels of IL-4, IL-5, IL-10, IL-13, IL-17, MIP-1α, GM-CSF, TNFβ, and IFNγ remained below detection levels both pre- and post-testing. However, IL-1ra levels showed a slight to moderate decrease in 7 of 10 women after Rh(D)-immune globulin administration. In contrast, levels of TGFβ-1 increased, >1.3-fold in 7 of 10 women and >2-fold in 4 of 10 women. In one instance, the increase in TGFβ-1 (42, 855 pg/mL) was >5-fold and this woman also had a significant increase in TGFβ-2 (352 pg/mL). In addition to TGFβ, 4 of 10 women had increases of >1.5-fold in PGE2. Statistical analyses using ANOVA, paired Student’s t-test, and Wilcoxon signed-rank, for the combined results of all 10 women receiving Rh(D)-immune globulin indicated that only TGFβ and PGE2 were significantly elevated post administration of Rh(D)-immune globulin. These results indicate that Rh(D)-immune prophylaxis can induce high levels of the strongly immunosuppressive cytokines TGFβ and PGE2. Both of these molecules have been shown to induce profound immunosuppression including prevention of primary immune responses (Ludviksson BR, et al., Eur J Immunol.2000;30:2101–2111; Zhou H, Lamont SJ, Int J Fertil.1986;31:305–310). Our findings represent the first clear evidence for a mechanism of action of Rh(D)-immune prophylaxis.
Incidence of Aplastic Anemia and Agranulocytosis in Brazil and Argentina - The LATIN Study.
Authors: Hamerschlak Nelson , Maluf Eliane , Pasquini Ricardo , Moreira Frederico R. , Cavalcante Alexandre B. , Okano Iria R. , Falcao Roberto P. , Pita Marimilia T. , Looggetto Sandra R. , Rosenfeld Luiz G. , Irene Lorand-Metze G. , Jose Eluf Neto
Published: April 2020

CONTEXT: Aplastic anemia and agranulocytosis are rare but life-threatening disorders, often caused by drugs and other environmental exposures. Reported incidences of these diseases seem to vary between different geographic regions, and few data on the incidence are available for Latin American countries.

OBJECTIVE: To determine the incidence of agranulocytosis and aplastic anemia in Brazil and Argentina.

TYPE OF STUDY: Incidence study.

SETTING: Seven centers in Brazil and one in Argentina took part in the study.

METHODS: Each center conducted active search for new cases in a defined region by means of regular contacts with all hematologists, main clinical laboratories and clinicians in hospitals of the region.

RESULTS: 191 patients with aplastic anemia and 63 with agranulocytosis were identified from 2002 to 2005. The incidence of agranulocytosis was estimated to be 0.6 cases per million people per year. The incidence of aplastic anemia was 1.5 cases per million people per year.

DISCUSSION: Aplastic anemia and agranulocytosis are rare diseases in Brazil and Argentina. The overall incidence of agranulocytosis was lower than generally reported for other countries, while the incidence of aplastic anemia was similar to previous studies. However, there was considerable incidence variability among the regions involved in the study, more markedly for agranulocytosis.

Incidence of Agranulocytosis and Aplastic Anemia/region Region Agranulocyyosis Aplastic Anemia a Rate calculated as population-weighted average of the region specific rates Parana 0.6 1.5 Ribeirao Preto 0.8 0.8 Uberaba e Juiz de Fora 0.7 1.4 Recife 0.3 2.0 Manaus 0.3 1.5 Goiania 0.1 1.6 Buenos Ayres 2.5 1.5 Total a 0.6 1.5
The Use of a Bone Marrow Transplant Program Public Website for Patient Education and Reporting of Outcomes: Increased Use Resulting in Improved Patients’ Trust and Assurance.
Authors: Malcolm Elissa F. , Patchett Linda J. , Fitzmaurice Thomas F. , Hill John M. , Meehan Kenneth R.
Published: April 2020

To promote patient education, the Dartmouth Bone Marrow Transplant (BMT) Program created a website focusing on Quality Reports, highlighting outcomes and patient satisfaction data. In an attempt to address areas most important to potential patients, the development of the website involved Transplant physicians and nurses, outside reviewers and recently transplanted patients. Based on these assessments and interviews, the BMT website was designed to provide various outcome data, including disease-specific overall- and disease-free survival, length of stay, and incidence of nausea. The website became public in January 2004. The use of the public website was evaluated 18 months after initiation. There was a 300% increase in external user visits to the Dartmouth BMT Website (comparing 3 month averages). Eighty percent of the information accessed was in the topic of “Safe and Effective Care”, which addresses patient-specific data, including survival statistics, length of stay, or toxicities during the transplant. The remaining 20% of interest was satisfaction measures addressing patients’ view of their overall care, opinions addressing nursing care or physician interaction. Of the top ten measures that were evaluated by users, the first 9 were disease-specific survival statistics and the remaining was “Average Length of Hospital Stay”. Individual interviews with patients (n=16) indicate that the information provided on the web site increased the trust of the institution. In addition, patients felt re-assured concerning their selection of our institution. In conclusion, the initiation of the Dartmouth BMT Quality Reports Website (http://www.dhmc.org/QualityReports) that focuses on outcome data resulted in a marked increased use of the Dartmouth BMT Website within 18 months. Patients were most interested in disease-specific survival statistics, followed by transplant patients’ opinions of their care. Patients report that the “transparency” of outcome results and the patient education provided by the website improve their trust of our BMT program/hospital and foster assurance of our institution’s reputation.
Prognostic Factors in Patients with Fanconi Anemia (FA) after Alternate Donor (AD) Hematopoietic Cell Transplantation (HCT).
Authors: Alsweedan Suleimman A. , Wagner John E. , Tan Ye , DeFor Todd E. , MacMillan Margaret L.
Published: April 2020

The treatment of choice for the hematological complications in FA patients is a histocompatible sibling donor HCT. However, as FA is inherited disorder, most patients do not have an HLA-matched non-affected sibling donor and therefore require AD (HLA-mismatched sibling or unrelated) HCT with inherent increased morbidity and mortality. To identify prognostic factors for survival after AD HCT, we evaluated outcomes in 83 FA patients transplanted at the University of Minnesota from 1990 to 2004. Median age at HCT was 11.8 years (range, 0.8–48.5). 82% received bone marrow (BM) grafts and 18% received unrelated cord blood (UCB). In univariate analysis, factors associated with significantly higher 3-year survival included: age [42% (<18 years, n=67) vs 16% (>18 years, n=16) p=0.01]; recipient CMV serostatus [45% (negative, n=55) vs 19% (positive) p=0.01]; blood product transfusion 82% [(none, n=11) vs 28%(any, n= 67) p= 0.03], prior infection (gram negative bacteria or fungal) [46% (absent, n=62) vs 8%(present, n=19) p= 0.01], and use of fludarabine (FLU) in preparative regimen [45% (present, n=54) vs 24% (absent, n=29) p=0.03]. In multivariate analysis, factors associated with improved survival were young age, negative recipient CMV serostatus, no prior infections and no severe acute GVHD (Table 1). If age <18 yrs, CMV negativity, and absence of prior infections defines ‘standard risk’, 3 year survival is 68% (95% CI, 50–85%) compared to 20% (95% CI, 7–32%) for those with ‘high risk’ disease (p =0.01). Disease status at HCT, androgen use, number of malformations, gender, donor source and HLA disparity did not have a demonstrable effect on survival.

Table 1: Cox regression on survival Factors Relative Risk(95% CI) P-value Note:*-reference category Model(X2=33.5, p<0.01) Age at transplant(X10) 1.62 (1.15-2.28) <0.01 Recipient CMV Negative 1.0 * Positive 3.41(1.82-6.37) <0.01 Prior Infection No 1.0 * Yes 2.33 (1.24-4.36) <0.01 Grade 3-4 acute GvHD No 1.0 * Yes 5.04 (2.34-10.88) <0.01

These data suggest that patients with FA should receive AD HCT before the development of preventable high risk features, namely older age and serious infections. FLU based preparative regimens and measures to prevent acute GVHD should also be used to improve survival. Mismatched BM and UCB donor may provide greater donor options for timely transplants in FA patients.
Enzymatically-Active ADAMTS13 Variants Are Not Inhibited by Anti-ADAMTS13 Autoantibodies - A Potential Novel Therapeutic Strategy.
Authors: Zhou Wenhua , Dong Lingli , Motto David , Ginsburg David , Bouhassira Eric , Tsai Han-Mou
Published: April 2020

ADAMTS13, a reprolysin-type zinc metalloprotease consisting of a metalloprotease (MP), a disintegrin, a thrombospondin-1 (TSP), a cysteine-rich region (Cys), a spacer, seven additional TSP, and two CUB domains, is critical for preventing unwarranted VWF-platelet interaction in the circulation. Profound deficiency of ADAMTS13, due to genetic mutations of the ADAMTS13 gene or autoimmune inhibitors of the protease, causes microvascular thrombosis characteristic of TTP. Plasma exchange is a conventional but complex therapy of TTP that may be ineffective for patients with high inhibitor titers. In this study we produce recombinant ADAMTS13 variant proteins and analyze their binding with and suppression by TTP inhibitors. TTP IgG binds the ADAMTS13 variants ending downstream of the spacer domain but not those truncated upstream, consistent with recent reports that the spacer domain is essential for interaction between TTP IgG and ADAMTS13. Nevertheless, the spacer domain sequence alone may be insufficient since TTP IgG does not bind a fragment consisting solely of the spacer domain sequence but binds a fragment encompassing the sequence between the distal 2/3 of the MP and the spacer. ADAMTS13 ending at the TSP#1 or downstream are proteolytically active in cleaving either VWF multimers or a 73-amino acid fragment of the VWF A2 domain. Mixing studies show that each of ten TTP plasma samples tested suppresses the protease activity of ADAMTS13 ending downstream of the spacer domain, but not the activity of the variants truncated at the TSP#1 or Cys domain. The variant proteins truncated at TSP#1, when added to a series of plasma samples from a TTP patient with rising inhibitor titers, consistently yield the expected protease activity levels, even in those samples whose high inhibitor titers abolish the activity of the full-length ADAMTS13 at 15x of its concentration. These findings indiate that it is feasible to design ADAMTS13 variant proteins that are proteolytically active yet refractory to suppression by inhibitors of TTP. Such ADAMTS13 variants may be exploited to circumvent the inhibitors of TTP, potentially eliminating the need of performing plasma exchanges in the future.
The Fate of Human Langerhans Cells in Hematopoietic Stem Cell Transplantation.
Authors: Collin Matthew P. , Hart Derek N. , Jackson Graham H. , Cook Gordon , Cavet James , Mackinnon Stephen , Dickinson Anne M.
Published: April 2020

The fate of Langerhans cells (LC) and other antigen-presenting cells (APC) in haematopoietic stem cell transplantation (HSCT), their depletion by conditioning regimens, reconstitution and chimerism are important factors in understanding graft versus host disease and the outcome of transplantation. Hitherto untested predictions in humans state that depletion of recipient LC may prevent acute graft versus host disease (GVHD), the acquisition of donor chimerism in LC may drive the evolution of clinical GVHD from acute to chronic and the persistence of recipient LC may explain late acute GVHD phenomena and donor lymphocyte infusion toxicity.

Between April 2003 and June 2005 we obtained 184 skin biopsies from 76 patients at 4 UK centres. Using confocal microscopy of intact epidermal sheets to image up to 2000 LC at a time, we find that full intensity transplant (FIT) conditioning with TBI-based regimens or busulfan and cyclophosphamide depletes 55% of LC at day 0 (n 14; p 0.001). In contrast, fludarabine-based reduced intensity transplant (RIT) depletes only 9% of LC (n 23; p 0.061). A minimum LC density is reached at 14-21 days in both regimes with little difference at the nadir, suggesting that the effect on density at day 0 is primarily kinetic. Rapid recovery occurs by 40 days in the absence of acute cutaneous GVHD but is delayed beyond 100 days in the presence of GVHD (n 50; p 0.006)

We assessed LC chimerism in sex-mismatched transplants by allowing single cells to migrate from small epidermal sheets in vitro and applying a two-step Giemsa/FISH assay. At 40 days median LC donor chimerism is 97% after FIT and 36.5% after RIT (n 11; p 0.004). At 100 days median donor chimerism is 100% after FIT and 97.5% after RIT (n 28; p 0.133.). The majority of transplants - 16/28 (57%) - achieve 100% LC donor chimerism at day 100 and all are at least 90%. Attainment of full donor chimerism at 100 days shows only a trend with FIT (p 0.133), and with complete myeloid chimerism in the blood (p 0.080) but is strongly associated with prior acute cutaneous GVHD (p 0.002). At one year post transplant it is possible to detect rare recipient cells in a minority of transplants (12/1746 interphase nuclei - less than 1%), confirming that LC may regenerate in the skin under quiescent conditions.

In conclusion we find that: 1) recipient LC survive conditioning, especially after RIT, suggesting that targeted APC depletion might have potential as a novel means of preventing acute GVHD; 2) LC turnover is accelerated by full conditioning, complete myeloid chimerism and acute GVHD; 3) the majority of LC are donor-derived in all transplants at 100 days, consistent with the hypothesis that donor engraftment of APC drives the transition of acute to chronic GVHD. 4) there is little evidence using these conditioning regimens that acute GVHD or DLI-toxicity occurring after 100 days is related to the persistence of recipient APC.
CD4+ Effector Memory and Naive T Cells Mediate Graft-Versus-Leukemia Via Direct Recognition of MHCII on Leukemic Cells.
Authors: Zheng Hong , Matte Catherine C. , Venkatesan Srividhya , Anderson Britt E. , Shlomchik Mark J. , Shlomchik Warren D.
Published: April 2020

One of the major challenges in allogeneic stem cell transplantation (alloSCT) is to separate graft-versus-host-disease (GVHD) from graft-versus-leukemia (GVL). We and others have previously demonstrated, in both major histocompatibility complex (MHC)-compatible/multiple minor histocompatibility antigen-mismatched and MHC-mismatched murine models of alloSCT, that spontaneous effector memory (EM) CD4+T cells depleted of regulatory CD25+ cells (CD4+CD44+CD62L-CD25-) do not cause GVHD. We have also shown that these EM CD4+ T cells can mediate GVL against a model of murine chronic phase of CML (mCP-CML) induced via retroviral transduction of BM cells with the bcr-abl fusion cDNA without causing GVHD (Zheng, et al ASH meeting 2004). In the present study we analyzed the effector mechanisms of these EM CD4+ cells in the B6bm12 → B6 MHCII disparate bone marrow transplantation (BMT) model. First, we demonstrated that the GVL activity of both EM and naïve CD4+ T cells required cognate interactions with CML targets as GVL was ineffective against mCP-CML induced in bone marrow from B6.I-Ab−/− (MHCII−) mice. Recipients of MHCII− mCP-CML died from mCP-CML between day 15-20 post BMT, regardless of whether they received EM or naïve CD4+ cells or no T cells at all. In light of data in the same model that parenchymal MHCII expression is not required for GVHD (Teshima et al, 2002), these data demonstrate distinct mechanisms for the cytotoxicity by CD4+ cells in GVL and GVHD—direct in the former and indirect in the latter. To further investigate the specific mechanisms of T cell killing, we tested the effectiveness of EM CD4+ cells in eradicating mCP-CML induced in bone marrow cells from Fas−/− and TNFR1/R2−/− mice. Both EM and naïve CD4+ cells mediated GVL against these gene deficient leukemias that was similar to that against wild type mCP-CML. In summary, these results suggest that EM and naive CD4+ cells mediate GVL via direct cognate engagement with targets. Their killing, however, does not depend on either FasL or TNF-α which suggests a dominant role for perforin, TRAIL, or both. Interestingly, although the mechanisms of recognition and killing of mCP-CML by either naïve or EM CD4+ T cells are so far indistinguishable, whereas only the naïve cells cause GVHD. Whereas a number of investigators have been able to separate mechanisms of killing in GVHD vs. GVL, this is to our knowledge the first clear demonstration of a difference in the mechanism of recognition between GVHD and GVL.
Bone Marrow Derived, De Novo Generated CD4+ T Cells Are Previously Unsuspected Participants in CD8+ T Cell-Mediated Graft-Versus-Host Disease.
Authors: Zhang Yi , Hexner Elizabeth , Frank Dale , Gerard Joe , Kung Frank , Emerson Stephen G.
Published: April 2020

Although mature CD8+ T cells are known to be major effectors of acute GVHD, patients receiving T cell-depleted allografts remain at high risk for chronic GVHD. To what extent CD8+, CD4+ or both T cell subsets contribute to this chronic immunopathology is not known. We have recently demonstrated that alloreactive memory T cells develop in mice with acute GVHD and account for the persistence of host tissue injury (Journal of Immunology, 2005;174:3051). Based on these findings, we now ask whether de novo generated donor T cells from engrafted T-BM themselves contribute to persistent host tissue injury in GVHD. Confirming previous observations, we found that transplantation of lethally irradiated C57BL/6SJL (B6, CD45.1) mice with highly purified C3H.SW (CD45.2) CD4+ naïve T cells did not cause GVHD, but mice receiving highly purified CD8+ naïve T cells together with C3H.SW T-BM, suffered severe acute GVHD. Surprisingly, in these mice receiving only CD8+ T cells, a substantial number of donor CD4+ T cells as well as CD8+ T cells were detected in GVHD target tissues, indicating that these infiltrating CD4+ T cells had arisen de novo from the transplanted T-BM. Donor CD4+ T cells recovered from GVHD mice expressed surface markers of activated effector/effector memory T cells, including CD25, CD69, CXCR3, and CD44hiCD62Llo. In response to host DCs, purified GVHD CD4+ T cells proliferated and expanded 4-5X more, and produced 10X higher levels of IFN-γ than did CD4+ T cells derived from B6 mice receiving C3H.SW T-BM alone. Furthermore, adoptive transfer of these in vivo generated GVHD CD4+ T cells, without CD8+ T cells, into secondary irradiated B6 recipients induced clinical GVHD characterized by delayed onset, weight loss, diarrhea, and lymphopenia, but without cutaneous inflammation. Histologic examination demonstrated chronic inflammation in the liver and intestinal tract, including epithelial apoptosis. Thymic pathology was dramatic in secondary B6 recipients of GVHD CD4+ T cells, including thymic atrophy, loss of thymic cortex, and infiltration of large amount of tingible macrophages. Taken together, these results demonstrate that donor bone marrow derived, de novo generated CD4+ T cells also contribute to GVHD together with transferred mature CD8+ T cells. Moreover, they suggest that these CD4+ T cells, in concert with alloreactive memory CD8+ T cells that develop during the evolution of GVHD, cause the persistence of acute GVHD and its subsequent progression into chronic GVHD. Thus, donor BM-derived, de novo generated CD4+ T cells are the “Hidden Dragon” of CD8+ T cell-mediated GVHD. Understanding how these CD4+ T cells are generated and regulated will prove to be critical to the prevention and treatment of both acute and chronic GVHD.
G-CSF Potently Suppresses Osteoblast Activity in the Bone Marrow.
Authors: Christopher Matthew J. , Liu Fulu , Short Brenton , Simmons Paul J. , Winkler Ingrid , Levesque Jean-Pierre , Link Daniel C.
Published: April 2020

There is accumulating evidence that interaction of stromal cell derived factor-1 (SDF-1/CXCL12) with its cognate receptor, CXCR4, generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During G-CSF induced HPC mobilization, SDF-1 protein expression in the bone marrow decreases, thereby attenuating CXCR4 signaling. We recently reported that G-CSF treatment induced a decrease in bone marrow SDF-1 mRNA that closely mirrored the fall in SDF-1 protein, suggesting that G-CSF targets one or more SDF-1 producing cell population in the bone marrow. However, the identity of cell populations in the bone marrow that express SDF-1 is controversial. In the present study, we address this issue by sorting cells into mature hematopoietic, hematopoietic progenitor, endothelial, and osteoblast cell populations. Real time RT-PCR analyses showed that osteoblasts and to a lesser degree endothelial cells are the major sources of SDF-1 production in the bone marrow. Surprisingly, on a per cell basis, SDF-1 expression per osteoblast was only modestly (less than two-fold) reduced in mice treated with G-CSF. These data raised the possibility that, rather than affecting SDF-1 expression per osteoblast, G-CSF regulated the number of osteoblasts in the bone marrow. To explore this possibility, osteoblast number in the bone marrow was measured by histomorphometry. Indeed, after 5 days of G-CSF treatment, a significant reduction in the number of endosteal osteoblasts was observed [number of osteoblasts per mm bone perimeter ± SEM: 74.8 ± 13.5 (untreated) versus 33.3 ± 3.8 (G-CSF)]. Moreover, expression of osteocalcin (a specific marker of mature osteoblasts) in the bone marrow was sharply reduced during G-CSF treatment: a 47 ± 12 fold reduction in osteocalcin mRNA (relative to b-actin mRNA) was observed in the bone marrow of G-CSF-treated mice compared with untreated mice. Finally, calcein double-labeling experiments showed that the mineral apposition rate was significantly reduced in G-CSF-treated mice. However, RT-PCR analyses showed that the G-CSF receptor is not expressed on osteoblasts. Accordingly, G-CSF had no direct effect on osteoblast activity in vitro. Collectively, these data show that G-CSF potently suppresses osteoblast number/activity in the bone marrow through an indirect mechanism. Since osteoblasts are thought to play a key role in establishing and maintaining the stem cell niche in the bone marrow, these data raise the possibility that G-CSF, by regulating osteoblast function (including SDF-1 expression), may have profound effects on the stem cell niche that ultimately contribute to HPC mobilization.
Study of Lenalidomide Plus Dexamethasone Versus Dexamethasone Alone in Relapsed or Refractory Multiple Myeloma (MM): Results of a Phase 3 Study (MM-010).
Authors: Dimopoulos Meletios A. , Spencer Andrew , Attal Michael , Prince Miles , Harousseau Jean-Luc , Dmoszynska Anna , Yu Zhinuan , Olesnyckyj Marta , Zeldis Jerome , Knight Robert
Published: April 2020

High-dose dexamethasone (Dex) remains a standard therapy for relapsed or refractory MM. Lenalidomide is a novel, orally administered, immunomodulatory drug (IMiD) that has single-agent activity against MM and additive effects when combined with Dex. In this phase 3, multicenter, randomized, double-blind trial, 351 patients with relapsed or refractory MM were enrolled from clinical centers in Europe, Israel, and Australia. Patients were treated with Dex 40 mg daily on days 1–4, 9–12, and 17–20 every 28 days and were randomized to receive either lenalidomide 25 mg daily orally on days 1–21 every 28 days or placebo. Beginning at cycle 5, the dose intensity of Dex was reduced to 40 mg daily on days 1–4 only every 28 days. Patients resistant to Dex were excluded. Patients were stratified with respect to B2M (≤ 2.5 vs. > 2.5 mg/mL), prior stem cell transplant (none vs. ≥1), and number of prior regimens (1 vs > 1). A pre-planned interim analysis of time to progression (TTP; primary endpoint), response (Blade criteria), and safety data was performed by an independent Data Monitoring Committee (IDMC) when at least half the number of disease progressions required for full statistical power had occurred (50% information). The treatment arms were well balanced for prognostic features. With a study duration of 18 months, the median TTP for patients treated with the combination of lenalidomide plus Dex was 13.3 months compared to 5.1 months for patients treated with Dex and placebo (p < 0.000001). The overall response rate was greater in patients who received lenalidomide plus Dex than in patients who were given Dex alone (58% vs. 22%; p < 0.001). Grade 3 or 4 neutropenia adverse events were reported more frequently in patients given combination therapy than in patients treated Dex alone (16.5% vs. 1.2%), however grade 3 or 4 infections were reported with similar frequencies between treatment groups. Thromboembolic events occurred in 8.5% of patients in the lenalidomide/Dex group and in 4.5% of patients in the Dex alone group. Otherwise, the safety profile of lenalidomide/Dex was similar to that of Dex alone.

Conclusion: Combination lenalidomide/Dex is a relatively well-tolerated and active oral regimen for patients with relapsed or refractory MM. The difference in TTP between the 2 arms surpassed the pre-specified O’Brien-Fleming boundary for superior efficacy (p < 0.0015) and the IDMC recommended the data be released to all study participants. Prophylactic antithrombotic therapy should be considered for patients undergoing treatment with lenalidomide/Dex. These data support further evaluation of this combination in previously untreated patients with MM.
Telomere-Based Pre-Clinical Therapy in a Murine Model of Non-Hodgkin’s Lymphoma of the Diffuse Large B Cell (DLCL)Type.
Authors: Longe Harold , Faller Douglas V. , Denis Gerald V.
Published: April 2020

The dual bromodomain Brd2 is closely related to the basal transcription factor TAFII250, which is essential for cyclin A transactivation and mammalian cell cycle progression. Constitutive expression of BRD2 (under Eμ control) in the lymphoid lineage of transgenic mice elevates basal transcription of cyclin A, destabilizes the cell cycle and leads to B cell leukemias and lymphomas that are monoclonal, morphologically uniform, transplantable and highly malignant. The surface immunophenotype of the lymphoma cells is: B220+, CD19+, sIgM+, CD5+, CD9+; B7-1 and B7-2 elevated, CD23low; CD11b-, Ly-6G- and CD49b-, supporting lymphoid-restricted lineage; CD117- (c-kit) and CD127- (interleukin-7 receptor α-chain), consistent with mature cells; CD25-, syndecan-1-, and CD69- and exhibit a high IgM/low IgD ratio, which taken together identify a B-1 cell type. Malignant, proliferating cells infiltrate lymph nodes, liver, lung and kidney, and at late stages, cause anemia and a fatal peripheral leukemia over a 14-day time course from tumor cell transplantation to death. Genome-wide transcriptional expression profiling of these lymphoma cells reveals a transcriptional fingerprint that is most similar to human diffuse large B cell lymphoma (DLCL) with an “activated B cell” signature, consistent with histopathology, and establishes a novel murine DLCL model. DLCL is the most common type of non-Hodgkin’s lymphoma (NHL) in humans; all lymphomas combined are the fifth most common type of cancer diagnosed and the sixth most common cause of death in the United States. We treated syngeneic transplanted mice with CHOP (i.e. cyclophosphamide 40 mg/kg i.v., doxorubicin 3.3 mg/kg i.v., vincristine 0.5 mg/kg i.v. and predisone 0.2 mg/kg p.o. every day for 14 days) and monitored individual lymphoma cells, tagged with human-CD4+, by flow cytometry of lymphoid and non-lymphoid organs. In a novel approach, we supplemented CHOP with DNA oligonucleotides that mimic the chromosomal telomere, which we call a “T-oligo.” Normal cells exposed to this drug in vitro undergo transient cell cycle arrest, but DLCL cells undergo p53-dependent apoptosis. The mechanism immediately suggests a novel method of chemotherapy for leukemia and lymphoma to supplement CHOP. In mice treated systemically with CHOP and T-oligo, we observed major reductions in the leukemic burden in peripheral blood, reduced lymphadenopathy, reduced leukemic infiltrates of non-lymphoid organs and splenomegaly in the combined (“CHOP+T”) regimen over CHOP alone. We also confirmed in normal B lymphocytes that T-oligo causes only cell cycle arrest, not apoptosis. Mice likewise showed low toxicity to T-oligo at the effective doses, opening the way to a more extensive pre-clinical trial of this novel approach to NHL therapy.
Insights into the Basis of Chromosomal Imbalances during the Clonal Evolution of Multiple Myeloma Using SNP Array Analysis.
Authors: Walker Brian A. , Leone Paola E. , Jenner Matthew , Gonzalez David , Ross Fiona M. , Li Cheng , Davies Faith E. , Morgan Gareth J.
Published: April 2020

On the basis of hyperdiploidy, often involving trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21, and chromosomal translocations multiple myeloma (MM) can be separated into two broad groups. Neither the mechanism leading to the development of these trisomies nor their effect on myeloma pathogenesis is fully understood. With the aim of understanding these two questions further we have analysed the zygosity status and chromosomal copy number of 39 samples from the MRC Myeloma IX trial (9 t(4;14), 15 t(11;14), 15 hyperdiploid) and two cell lines using the Affymetrix 50K SNP mapping arrays. Plasma cells were selected using CD138+ MACS beads to purity greater than 95%. There was a good correlation between the FISH based detection of copy number change and that defined using SNP mapping arrays indicating that the SNP array methodology was robust. In the 24 non-hyperdiploid cases there are examples of chromosomes that are diploid by FISH and by copy number analysis, using a WGSA programme, however in some of these chromosomes there is loss of heterozygosity (LOH) across the whole or part of the chromosome indicating uniparental disomy (UPD). In addition, we demonstrate where there are large regions of UPD on one chromosome there will often be regions of UPD on other chromosomes. In the hyperdiploidy cases we find that large regions of UPD are rare on the trisomic chromosomes, but small regions of LOH exist in all 15 hyperdiploid samples indicating uniparental trisomy which is in agreement to MM cell lines where uniparental trisomy can also be found. In four out of 15 hyperdiploid samples LOH is also seen on tetraploid chromosomes. UPD, therefore, represents an important new mechanism for gene inactivation in myeloma whereby tumour suppressor genes can be inactivated even in the presence of increased copy number. The mechanism underlying this gene activation relies on the loss of one functional allele leaving only a single allele which may be defective. Duplication of the defective allele gives a normal chromosomal number but an abnormal phenotype, which would not be detected by FISH or conventional CGH. Such regions of LOH may arise through gene conversion events and somatic recombination during mitosis. The order in which these events occur in hyperdiploid cells can help in the understanding of the clonal evolution of myeloma. We demonstrate that gene inactivation methods such as deletions or UPD occur prior to chromosome copy number gain as areas of LOH are present on diploid and trisomic chromosomes. In order to understand how such regions of LOH contribute to the pathogenesis of MM and to define critically deregulated genes within these regions, where mutation and methylation may be important, we are currently comparing the SNP mapping arrays with expression patterns using the Affymetrix U133 plus 2 expression microarrays.