bluuebunny/crossref_metadata_2025 huggingface dataset:
Prognostic Significance of Immunohistochemical Markers in Classical Hodgkin Lymphoma Response to Treatment A Study of 59 Cases.
Authors: Canioni Danielle , Deau Benedicte , Taupin Pierre , Bosq Jacques , Ribrag Vincent , Delarue Richard , Vasiliu Viorel , Brousse Nicole , Hermine Olivier
Published: April 2020

Classical Hodgkin lymphoma (cHL) belongs to the most curable lymphomas in adults. Some cHL however, are primary refractory to usual treatments including anthracyclins regimen. Currently, only clinical factors are considered as relevant for prognosis. In a previous study of a small cohort of patients, we showed that some immunohistochemical markers could help for predicting the treatment response of cHL. In this study, we extended the markers and increased the number of included patients. We performed a retrospective study on pre-treatment biopsy specimen of 59 patients, 18 with primary refractory cHL and 41 responders to chemotherapy and free of disease for at least 3 years. Most refractory cHL had a nodular sclerosis (NS) histological type, except one which was a mixed cellularity type. Thirty six responders had a NS type, 3 patients had a mixed cellularity type and the 2 others an interfollicular cHL. The semi-quantitative immunohistochemical study used CD20, CD3, CD30, bcl2, p53, Ki67, TiA1 and c-kit antibodies. The results were statistically evaluated using a Fisher ’s exact test or a Wilcoxon sum rank test depending on the variable studied. CD30 and Ki67 stained strongly Hodgkin (Hg) and Reed-Sternberg (RS) cells regarless the response status. In contrast, these cells expressed significantly less frequently CD20 in refractory cHL than in responders (p= 0.032) and were never stained with CD3. P53 and bcl2 had a significantly higher expression on Hg or RS cells in refractory cHL (median = 63% & 51%) compared to responders (median = 40% & 12%) (p=0.004 & p=0.015 respectively). The cytotoxic marker TiA1 stained significant higher number of small lymphocytes in refractory cHL (median=42.5 per high power field (hpf)) compared to responders (median= 21 per hpf) (p= 0.0006). C-kit antibody was negative in Hg or RS cells but stained significant more mastocytes in refractory cHL (median=9 per hpf) comparing to responders (median=3.8 per hpf) (p= 0.001). These results indicate that immunohistochemical markers are useful in cHL and should be used in association with clinical parameters for predict the cHL treatment response. The prognostic significance of CD20 expression in cHL is controversial but in this study seems predictive of a better treatment response and is merely a marker of different gene expression program that may be associated with a more favorable outcome. A high bcl2 and p53 expression in refractory cHL supports the notion that an intact apoptosis cascade is essential for cell killing effect of chemotherapy. The increasing of TiA1 and c-kit positive cells raises the importance of the environmental non-neoplastic cells in cHL and suggests that targeted therapy against mast cells could improve prognosis of refractory cHL.
Cloning of B Cell Lymphoma-Associated Antigens Using Modified Phage Displayed Expression cDNA Library and Immune Patient Sera.
Authors: Cha Soung-chul , Biragyn Arya , Ruffini Pier Adelchi , Qin Hong , Neelau Sattva S. , Kwak Larry W.
Published: April 2020

Active immunization of follicular lymphoma patients with idiotypic vaccines elicits antigen-specific antibody responses, T-cell responses, and antitumor effects (Bendandi, M. et al., Nat Med5, 1171). We hypothesized that these vaccinated patients could generate tumor specific immune responses, not only against idiotype, but also against other tumor associated antigens (TAA) by a mechanism of epitope spreading. We further hypothesize that these antigens may represent tumor rejection targets for immunotherapy. To identify potential antigens, a phage surface expressed cDNA library derived from primary tumor cells was screened with sera from idiotype-vaccinated patients. Consistent with our hypothesis, we identified two immunogenic peptides (FL-aa-7 and 18), unrelated to idiotype, which were recognized by postvaccine sera but not by prevaccine or normal human sera. These peptide sequences derived from the 5′-untranslated regions of the human GTPase, IMAP family member 7 gene (FL-aa-7) and an alternative reading frame of U1-snRNP 70 (FL-aa-18), respectively, suggesting that epitope spreading had occurred. To further determine the specificity of these patients’ post vaccine sera, the corresponding FL-aa-7 and FL-aa-18 peptides were synthesized and tested for serum binding. As expected, post-vaccine but not pre-vaccine or normal, specifically bound the FL-aa-7 and FL-aa-18 peptides, respectively. Furthermore, both peptides specifically inhibited the binding of post-vaccine sera to the respective FL-aa-7 and FL-aa-18 phage clones in a dose-dependent manner. These data suggest that Id vaccinated patients generated specific antibodies to novel peptides FL-aa-7 and FL-aa-18, unrelated to Id. Overall, these data indicate that Id-vaccinated B cell lymphoma patients mounted antibody responses specifically for FL-aa-7-1 and FL-aa-18 peptides, which may represent a potential immunotherapeutic tumor antigen. The clinical and biological relevance of the response is to be elucidated.
A Distinct Entity of Ocular Adnexal MALT Lymphoma with Trisomy 18.
Authors: Tanimoto Kazuki , Sekiguchi Naohiro , Yokota Yukiko , Kaneko Akihiro , Suzuki Shigenobu , Maruyama Dai , Kim Song-Wong , Watanabe Takashi , Maeshima Akiko , Matsuno Yoshihiro , Tobinai Kensei , Kobayashi Yukio
Published: April 2020

Background: It remains unknown whether primary ocular adnexal MALT lymphoma is a homogenous disease, because there have been few reports on cytogenetic or molecular analysis of this disease.

Patients and Methods: In 34 cases of ocular adnexal MALT lymphoma, we performed interphase fluorescence in situ hybridization (FISH) analysis to detect IgH/MALT1 and API2/MALT1 fusion genes. Aneuploidy of chromosome 3, 7, 12 and 18 was also identified using corresponding centromere probes. We defined trisomy when three centromeres were recognized in FISH analysis. Histopathologic features were reviewed and categorized according to the degree of plasma cell differentiation, monocytoid B cell feature, nodularity, abundance of reactive germinal centers, multikaryocytic cells, contaminated T cells, Duthcer bodies, and lymphoepithelial lesions (LELs). Correlations among FISH analysis, histopathologic features, and clinical data were analyzed.

Results: FISH analysis showed 1 (3%) IgH/MALT1 fusion, 21 (62%) trisomy 3, 16 (47%) trisomy 18, and 3(9%) trisomy 7 cases. No cases showed API2/MALT1 fusion or trisomy 12. Existence of these cytogenetic change did not influence the degree of morphological features significantly, except for trisomy 18; Cases with trisomy 18 had significantly more abundant LEL cells, monocytoid B cells and residual reactive germinal centers, but less nodules, contaminated plasma cells and large cells. Cases with trisomy 18 showed distinct features of female dominance, younger age, and included more cases originated from conjunctiva. In total, five (15%) of 34 patients relapsed between 21 and 103 months, and all of them were found to have trisomy 18.

Conclusions: Aneuploidy is found in a ceratin subset of ocular adnexal MALT lymphoma by FISH analysis. Cases with trisomy18 may make a distinct clinicopathlogic entity.
Reactive Oxygen Species (ROS) Are Induced by vFLIP and Regulate NF-kB Activation in KSHV-Infected Primary Effusion Lymphoma Cells.
Authors: Bubman Darya , Csernus Balazs , Guasparri Ilaria , Cesarman Ethel
Published: April 2020

Reactive Oxygen Species (ROS) act as second messengers in various cellular signaling cascades. While high levels of ROS induce oxidative stress response and cause cell death, lower levels of ROS in cancer cells can actually promote neoplastic growth and survival. ROS can activate the NF-kB pathway either through activation of the canonical IKK complex or through tyrosine phosphorylation of IkB alpha in a cell-specific manner. Recently it was shown that Epstein-Barr Virus (EBV) increased the amount of ROS in Burkitt’s lymphoma cells through the actions of its EBER mRNA and viral proteins EBNA2 and LMP1. Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human gamma-herpesvirus that is the etiologic agent of Kaposi’s sarcoma, multicentric Castleman’s disease (MCD) and primary effusion lymphoma (PEL). We investigated whether KSHV also increases ROS levels in PEL cells. We demonstrate that PEL cell lines have higher levels of intracellular ROS than uninfected cells, and highly activated MAPK, a downstream target of ROS. Since KSHV viral FLICE-inhibitory protein (vFLIP) activates NF-kB in PELs, we examined whether ROS is involved in this signaling pathway. We show that vFLIP potently induced ROS after treatment with doxycycline in vFLIP-inducible Namalwa cells. Inhibition of ROS by an antioxidant PDTC led to a reduction in NF-kB activity in PEL cells. These data suggest that reactive oxygen is a possible signaling mechanism downstream of vFLIP, which mediates NF-kB activation and PEL survival. Furthermore, antioxidants may be useful as pharmacological agents in the treatment of PEL.
IgM-Expressing Waldenstrom’s Macroglobulinemia Tumor Cells Reveal a Potential for Isotype Switch.
Authors: Sahota Surinder S. , Babbage Gavin , Townsend Mark , Mockridge Ian C. , Zojer Niklas , Garand Richard , Shaughnessy John , Stevenson Freda K.
Published: April 2020

In Waldenstrom’s macroglobulinemia (WM), which locates primarily in the bone marrow (BM), VH gene analysis had previously suggested origins from a post-follicular B-cell arresting prior to isotype switch. Using more sensitive assays, facilitated by amplified cDNA from BM cells, nested PCR unexpectedly revealed tumor-derived isotype-switch transcripts in 7/7 cases. In 5/7 cases, both Cγ and Cα variant transcripts were identified, and Cγ or Cα only in 2/7. Detection of activation induced cytidine deaminase (AID) and germline and circle transcripts confirmed switching activity. Selected gene expression profiles established the memory B-cell marker CD27 as highly expressed in all cases. These findings were evaluated further in additional WM cases where availability of tumor material allowed detailed analysis. In 2/2 cases, phenotype suggested a variable CD27 expression within the tumor clone. In these, tumor IgM transcripts were readily detected in both the CD19+CD27+ and CD19+CD27− fractions, and in 1 of the 2 cases, post-switched tumor-derived Cα transcripts were also identified in each fraction. In this WM case, the frequency of tumor-derived transcripts was then assessed at the single cell level. Switch transcripts were identified in 3/96 cells with no co-expression of the IgM isotype. Similarly, AID transcripts were observed in some cells, not always correlating with switch events or with ongoing somatic mutation, which was apparent in VDJ-Cμ sequences. These findings reveal a dynamic intratumoral diversification, with AID activated and ongoing mutational and switch activity occurring post-transformation in a proportion of the tumor clone. Heterogeneity in CD27 expression is also evident within tumor cells, revealing phenotypic change. Interestingly, these data indicate that although WM tumor cells have arrested at the IgM stage and do not express isotype switched Ig, they retain the capacity to initiate events critical for isotype switch.
The Unfolded Protein Response Is a Determinant of Disease Actvity in Waldenstrom’s Macroglobulinemia (WM).
Authors: Leleu Xavier , Xu Lian , Santos Daniel D. , Ho Allen W. , Hunter Zachary R. , O’Connor Kelly , Manning Robert J. , Tournilhac Olivier , Chemaly Mariana , Branagan Andrew R. , Patterson Christopher J. , Anderson Kenneth C. , Treon Steven
Published: April 2020

The unfolded protein response (UPR) maintains the quality of newly synthesized secretory and transmembrane proteins such as immunoglobulins in the endoplasmic reticulum (ER) of eukaryotic cells. The most proximal sensors of the UPR are the ATF6, IRE1a-Xbp1, and PERK proteins. Bip, an ER stress-induced pro-survival molecular chaperone, controls the trafficking of Atf6 and is considered as an indicator of the onset of the UPR. EDEM, downstream of Xbp1 splicing, regulates the extraction of misfolded proteins into the cytosol for proteasome-mediated destruction. Ultimately, the initially cytoprotective UPR triggers an apoptotic cascade if ER stress is not corrected. We studied the UPR stress system in patients with Waldenstrom’s Macroglobulinemia (WM), a B-cell disorder characterized by excess secretion of IgM. As part of these efforts, we examined the UPR genes Bip-Atf6, Ire1a-Xbp1, Perk, as well as genes downstream of the UPR pathway including Xbp1 spliced-Edem, eIF2a and Gadd34 using semi-quantitative PCR analysis of CD19+ selected bone marrow lymphoplasmacytic cells (LPC) from 46 patients with the consensus panel diagnosis of WM. We found that UPR gene expression was highly heterogeneous with 16 (35%) patients expressing all genes, whilst 11 patients (24%) expressed only Xbp1 and Edem. Among patients expressing all UPR genes, median serum IgM (p=0.025) and B2M (p=0.05) levels, as well as bone marrow involvement (p=0.06) were higher, with levels of Bip showing greatest correlation to serum IgM and B2M levels, and BM involvement (p=0.01). These results confirm that UPR gene expression is related to disease activity in WM, and suggest a particular role for Bip as a prognostic factor in this disease.
Mast Cells in the Diffuse Large B-Cell Lymphoma; Their Role for Fibrosis.
Authors: Fukushima Hiroko , Ohsawa Masahiko , Ikura Yoshihiro , Inoue Takeshi , Naruko Takahiko , Shirai Nobuyuki , Sugama Yoshimi , Suekane Takehisa , Kitabayashi Chizuko , Muraguchi Takashi , Hino Masayuki , Ueda Makiko
Published: April 2020

Mast cells (MCs) play an important role in tissue remodeling in inflammation. According to content of tryptase and chymase, MC is divided into two phenotypes; MCTC phenotype contains tryptase and chymase, while MCT phenotype contains only tryptase. Recent studies have revealed that interleukin-4 (IL-4) promotes the maturation of MCs and the development of MCTC phenotype from MCT phenotype. Although MCs infiltration into diffuse large B-cell lymphoma (DLBCL) has been reported, MC phenotypes infiltrating into lymphomas remain unknown. We examined MC phenotype infiltrating into lymph nodes in 50 DLBCL and 20 reactive lymph nodes, and evaluated their association with fibrosis. We also evaluated IL-4-expressing cells in DLBCL. Reviewing hematoxylin-eosin stain and Azan-Mallory stain, 50 cases with DLBCL were divided into 3 groups; no fibrosis type in 32, reticular type in 8, showing fine reticular fibrosis in focal areas, and bundle type in 10, showing thick collagenous bundle through the parenchyma. Using these formalin-fixed and paraffin-embedded specimens, immunohistochemistry for tryptase and chymase was performed. Furthermore, immunodouble staining methods were performed to detect the MC phenotypes and the cells that express IL-4 in DLBCL, using frozen sections. Tryptase-positive, and chymase-positive MCs were found in all groups of DLBCL and density of tryptase-positive MCs were higher than that of chymase-positive MCs. Densities of trytase-positive, and chymase-positive MCs in fibrotic areas were significantly higher than those in the areas without fibrosis (reticular type; 12.681 ± 11.307 / mm2 vs. 2.082 ± 2.260 / mm2, p = 0.018, bundle type; 22.407 ± 11.989 / mm2 vs. 1.319 ± 1.937 /mm2, p = 0.005, in tryptase-positive MCs, reticular type; 4.758 ± 5.468 / mm2 vs. 0.440 ± 1.164 / mm2, p = 0.028, bundle type; 4.114 ± 5.216 / mm2 vs. 0.220 ± 0.374 / mm2, p = 0.008; in chymase-positive MCs). Numbers of tryptase-positive MCs in reactive lymph nodes were not different from those in no fibrosis type of DLBCL. There were few chymase-positive MCs in reactive lymph nodes. Immunodouble staining revealed that MCs in DLBCL comprised MCT and MCTC phenotypes. Chymase-positive MCs and T-lymphocytes expressed IL-4. These findings suggest that T-lymphcytes play a role in the development of MCTC phenotype from MCT phenotype in DLBCL and that tryptase-positive MCs and chymase-positive MCs are associated with the development of fibrosis in DLBCL.
Is There an Association between Ocular Adnexal Lymphoma and Chlamydia psittaci Infection? The University of Rochester Experience.
Authors: Rothberg Paul G. , Vargas Roberto L. , Fallone Enzo , Andersen Arthur A. , Felgar Raymond E.
Published: April 2020

Several subsets of extranodal marginal zone lymphomas of mucosa-associated lymphoid tissues (MALT lymphomas) have been associated with infectious organisms. This finding is of particular significance in the treatment of these conditions because it suggests that eliminating the source of the antigen may result in regression of the malignancy. For example, gastric MALT lymphoma is associated with Helicobacter pylori infection. Antibiotic therapy that eliminates the H. pylori infection frequently causes regression of the lymphoma. Ferreri et al (2004, J Natl Cancer Inst. 96:586) reported the presence of Chlamydia psittaci DNA in 80% of 40 ocular adnexal lymphomas. Consistent with the gastric MALT lymphoma data, a subset of these patients responded well to antibiotic treatment. We used a PCR based assay similar to that of Ferreri et al to analyze a set of ocular adnexal lymphomas and benign (non-neoplastic) lesions for evidence of C. psittaci DNA in patients from New York State. No evidence of C. psittaci DNA was seen in seven MALT-type ocular adnexal lymphomas, four non-MALT B-cell ocular adnexal lymphomas, one Langerhans histiocytosis, and five reactive lymphoproliferations. In addition, we found no evidence for C. psittaci in DNA samples from 21 monoclonal B-cell lymphomas from sites other than the ocular adnexa. We confirmed the negative results using an alternate PCR target in the 23S ribosomal RNA gene. In addition, we used appropriate controls to demonstrate that PCR inhibition and DNA template degradation did not affect our results. We conclude that C. psittaci infection did not play a role in the ocular adnexal lymphoproliferations in our survey. The difference in results with the previous report (Ferreri et al) suggests geographical differences in the incidence of C. psittaci infection, or other unknown disease cofactors.
Identification of Three Non-Overlapping Epitopes in the C2 Domain of Factor VIII.
Authors: Meeks Shannon , Healey John F. , Lollar John (Pete) S.
Published: April 2020

Factor VIII (fVIII) inhibitory antibodies (fVIII inhibitors) are a significant source of morbidity in patients with hemophilia A. Approximately 30% of patients with severe hemophilia A will develop inhibitors. Most inhibitors are directed against either the A2 or C2 domains of fVIII. The repertoire of antibodies to the C2 domain is functionally complex, including antibodies that inhibit fVIII binding to phospholipid and von Willebrand factor and display either type I or type II kinetics.

The goal of this study was to investigate the diversity of the immune response to the C2 domain of human fVIII in a murine hemophilia A model and to identify a set of non-overlapping epitopes recognized by these fVIII inhibitors. A panel of 55 murine anti-C2 antibodies was obtained, which included 53 antibodies from anti-fVIII hybridomas produced in our laboratory and previously described antibodies ESH-4 and ESH-8. Nine of the hybridomas were cloned by limiting dilution and the corresponding monoclonal IgG antibodies were purified. IgG from the remaining 44 hybridomas was isolated directly from the hybridoma supernatants and biotinylated. The 9 monoclonal antibodies along with ESH-4 and ESH-8 were used as primary antibodies in a competition ELISA using human fVIII as the antigen. The panel of 55 biotinylated antibodies was used as secondary antibodies. Antibody pairs were classified as having non-overlapping or overlapping epitopes based on whether the binding of the secondary antibody was present or absent, respectively.

A basis set of 3 antibodies, 1B5-1B, 3E6-1B, and (2)117-1B, was defined, which consisted of a set of non-overlapping antibodies that as a group competed for binding of human fVIII with all other antibodies. 1B5-1B and 3E6-1B exhibited type I kinetics with Bethesda titers of 950 and 30, respectively. The Bethesda titer of (2)117-1B could not be determined due to its type II behavior. ESH-4 and ESH-8 had the same epitope profiles as 3E6-1B and (2)117-1B, respectively. Of the 52 non-basis set antibodies, 85% overlapped with 1B5-1B, 29% with 3E6-1B, and 56% with (2)117-1B. The majority of the antibodies overlapped with more than one member of the basis set, leading to the identification of five classes of antibodies (see figure). Because of the large number of antibodies characterized, it is unlikely that the frequency of antibodies in any additional classes is high. The elucidation of the structural complexity of the anti-fVIII C2 repertoire should be useful in the characterization of the pathogenicity of C2 inhibitors.

Figure Figure
Prophylactic Therapy with Fibrogammin Is Associated with a Decreased Incidence of Bleeding Episodes: A Retrospective Case Control Study.
Authors: Lusher Jeanne M. , Pipe Steven W. , Alexander Sarah , Nugent Diane J.
Published: April 2020

In an ongoing Phase III study we are evaluating the prophylactic efficacy and long-term safety of Fibrogammin® P, a purified, pasteurized concentrate of Factor XIII, in patients (pts) with congenital Factor XIII deficiency (an extremely rare, hereditary, life-threatening bleeding disorder). The study is being conducted at 37 sites and has enrolled 61 pts; these 61 represent approximately 2/3 of US patients with FXIII deficiency. For this analysis, a retrospective chart review was undertaken to compare spontaneous bleed-event rates 24 months prior to (when possible) and after the initiation of therapy in a subset of 7 study subjects who did not receive any prophylactic therapy prior to entering the study. The table below presents data for each of these pts on dosing, bleeding episodes and treatment (Rx) during both observation periods (Obs Pd). An additional 4 pts had been treated prophylactically with regular infusions of FFP or cryoprecipitate and transitioned successfully to FXIII therapy; there were no seroconversions to HIV, Hepatitis B/C in any study pts.

Pre-Fibrogammin P Therapy On Fibrogammin P Therapy Pt # Age ♦ Obs Pd (mos) / # Bleeds; Bleed Type Rx Obs Pd mos; (# inf) / # bleeds Bleed Type Dose/Kg† ♦ Age at onset of Fibrogammin Prophylactic therapy; † Pts were dosed on a unit basis (1250 unit vials), at 5-wk intervals, variability in weight and vial size lead to a range of per Kg doses; ⋄ p = .01 vs pre-therapy 504 5 yr 24 / 2 Hematomas none 12 (12) / 0 27 2703 10 mo 9 / 2 Umbilical cord; extensive bruising FFP 25 (26) / 1 Head trauma and bruising 29.7 – 44.9 2704 20 mo 6 / 4 Forehead × 2; frenulum × 2; other FFP × 5 26 (26) / 2 Traumatic foot; traumatic head (extracranial) 18.3 – 43.1 2705 9 yr 24 / 3 Lip; ankle; forehead FFP 13 (12) / 0 none 19.9 – 29.7 3301 19 yr 24 / 5 Large hematomas none 9.5 (11) / 0 none 18.9 3302 17 yr 24 / 0 none none 9.5 (12) / 0 none 24.3 3303 14 yr 24 / 8 Intracranial; others × 7 FFP, Cryoppt 10 (11) / 0 none 15.6 Mean 2.5 bleeds/yr 0.2 bleeds/yr⋄

As shown in the table, these children had 2.5 bleeds per year in the period before Fibrogammin prophylaxis, and only 0.2 per year while on Fibrogammin. These results demonstrate a consistent and clinically significant reduction in spontaneous bleeding with prophylactic use of Fibrogammin® P in pts with symptomatic congenital Factor XIII deficiency.
Restoring Initial Thrombin Generation Does Not Normalize Cutaneous Wound Healing in Hemophilia B.
Authors: McDonald Anna G. , Hoffman Maureane , Hedner Ulla , Roberts Harold R. , Monroe Dougald M.
Published: April 2020

We recently described abnormal wound healing in a mouse model of hemophilia B (Hoffman et al, Blood 2006; DOI 10.1182/blood-2006-05-020495). Specifically: epithelial closure was delayed in hemophilia B mice; hemophilic animals, unlike wild type, developed subcutaneous hematomas; macrophage influx was delayed compared to wild-type mice; and, surprisingly, angiogenesis was enhanced in the hemophilia B mice. We hypothesized that restoring the initial hemostatic burst of thrombin generation following wounding by administration of a single dose of factor IX (FIX) replacement or factor VIIa (FVIIa) bypassing therapy would not only prevent bleeding, but correct the subsequent wound healing process. One dose of therapy was given thirty minutes prior to placement of a single three mm punch biopsy wound on the dorsal skin of each wild type, untreated hemophilia B, and treated hemophilia B mouse. The size of the wounds was measured daily until full epithelial closure. The time course of epithelial closure in treated hemophilia B was intermediate between wild type and untreated hemophilia B. FVIIa-treated hemophilia B began to heal earlier than FIX-treated. Skin from the wound site was collected at different days and examined histologically. Macrophage influx was earlier in treated hemophilia B mice compared to untreated hemophilia B, likely a due to the increased thrombin and fibrin acting as chemotactic agents. The macrophage influx in FVIIa-treated HB was significantly greater at certain time points than in FIX-treated mice, possibly reflecting some signaling effect of TF/FVIIa in addition to its effects on thrombin generation. With the earlier influx in macrophages, hemoglobin was degraded to storage iron at earlier time points. However, tissue iron continued to persist in treated hemophilia B mice similar to untreated hemophilia B, suggesting continued rebleeding. FIX treatment led to significantly more angiogenesis than FVIIa. The reasons for this difference remain to be determined. Untreated and some treated hemophilia B mice developed subcutaneous hematomas both before and after wound closure. The early hematomas are likely caused by a combination of the initial wounding trauma and vulnerability to bleeding related to the high level of vascularity within the granulation tissue. We propose the late hematomas are due to a cycle of bleeding, leading to more inflammation with production of more pro-angiogenic cytokines, leading to greater angiogenesis with its attendant risk of bleeding. In conclusion, restoring initial hemostatic thrombin generation did not normalize cutaneous wound healing in a hemophilia B mouse model. While the time frame of healing may not be the same in human and murine hemophiliacs, our findings suggest that this model could be helpful in rational determination of treatment schedules for replacement or bypassing therapy following injury or surgery.
Outcome Assessment in Hemophilia: A Pilot Study on Correlation of Different Instruments.
Authors: Fischer Kathelijn , Van Genderen Frank R. , De Kleijn Piet , Van den Berg Marijke
Published: April 2020

Hemophilia is a rare disease, requiring life-long treatment with expensive clotting factor concentrates. Outcome assessment in hemophilia is important to establish both results of treatment, and compare between strategies. Since the 1980s, hemophilia specific assessment tools were available only for radiological (Pettersson score) and clinical (orthopedic joint score) joint damage. Recently, the HAL (Hemophilia Activities List) was developed to assess functional limitations, and the orthopedic joint score was adapted into the HJHS (hemophilia joint health score) to assess more subtle joint damage.

To assess correlation of instruments for outcome assessment, a pilot study was conducted in 22 young adults with hemophilia (15 severe, 5 moderate, 2 mild). Joint damage (Pettersson score, HJHS), physical function (self-reported: HAL, performance-based: Figure 8 test; time taken to walk around two pilons at a distance of 5 meter at maximum speed in a pattern of figure 8), Health Related Quality of Life (SF36), and utility (EQ5D) were measured.

Median age was 17.8 years (range 14–30), 15 (68%) patients were treated with early prophylaxis, including all but one patient with severe hemophilia who had recently stopped taking prophylaxis. Overall outcome was good, with median HJHS score of 3.5 (range 0–30; max 140, optimum 0), median HAL score of 100 (range 62–100; max 100= optimum), and median figure 8 test 4.9 sec (range 4.2–7.4). Recent (< 2 years of testing) Pettersson scores were available in only 11 patients (9 with severe, 2 with moderate hemophilia); the median score was 3 (range 0–28, max 78, optimum=0), the median physical component score of the SF36 was 53 (range 24–62), median utility (EQ5D) was 1.0 (range 0.7–1.0, optimum=1.0).

Spearman’s correlations for EQ5D, and Pearson’s correlations for other instruments are shown in the table.

Structural Functional Quality of Life HJHS Pettersson HAL Fig 8 SF 36 EQ5D HJHS 1 Pettersson 0.86 1 HAL −0.61 −0.26 1 Fig 8 0.51 0.51 −0.56 1 SF 36 −0.73 −0.41 0.81 −0.54 1 EQ5D −0.37 −0.04 0.76 −0.29 0.68 1

The HJHS scores showed good correlation with the Pettersson score and the physical component score of the SF36. Correlation with functional outcome assessment was only moderate, while the HAL showed a high correlation with both SF36 and EQ5D. In conclusion, the HJHS is a promising instrument for structural assessment, but functional assessment using the HAL showes higher correlation with Quality of Life and utility. For full assessment of results of treatment and to compare different treatment strategies, functional assessment, in addition to structural assessment, is warranted.
Assessment of Aneuploidy in Childhood Acute Lymphoblastic Leukaemia Using High Density Oligonucleotide Arrays.
Authors: Hall Andrew G. , Sulong Sarina , Harrison Christine , Minto Lynne , Bown Nick , Case Marian C. , Bailey Simon , Irving Julie
Published: April 2020

Chromosomal copy number (CN) is an important prognostic index in childhood acute lymphoblastic leukaemia (ALL). High hyperdiploidy (51–65 chromosomes) is associated with a good prognosis and near haploidy (23–29) a poor outcome. Conventional cytogenetics produces accurate estimates of CN in the majority of cases but sometimes cytogenetic fails or the morphology of chromosomes in ALL is too poor for accurate identification. We have used single nucleotide polymorphism arrays (SNPA) to determine CN in 86 childhood ALL patients and have compared the results with conventional cytogenetic analysis. Comparison of hybridisation intensity to a reference set of 110 normal individuals was used to derive CN estimates on 34 samples obtained during remission from ALL. Twelve were analysed using GeneChip® Human Mapping 10K Array Xba 131 and 22 using Xba 142 version 2.0 arrays (Affymetrix). Gene copy number estimates for individual SNPs were averaged across the autosomes and X chromosome. Results confirmed that SNPA gave accurate estimates of CN in samples of normal cells. Analysis by SNPA was successful in all 86 presentation leukaemic samples. The results of conventional cytogenetic analysis was available in 75, 11 having a failed cytogenetic result. Estimates of CN were identical in 34 and within 1 chromosome in a further 28. High hyperdiploidy was observed in 28 cases. In 23 of these the cytogenetic and SNPA results were concordant, while in the remaining five cases with a normal (n=2) or a failed (n=3) cytogenetic result, there was evidence from interphase FISH analysis of a hidden, non-dividing high hyperdiploid clone. One patient had a SNPA result indicative of near-haploidy (23–29 chromosomes) and another low hypodiploidy (33–39 chromosomes) with the SNPA confirming the appropriate chromosomal losses. There was marked discordance in one case of near tetraploidy (a total underestimate of 33 chromosomes) but this was not unexpected as the four copies of most chromosomes comprised no allelic imbalance. Analysis of concordance for individual chromosomes showed that, of the 1679 chromosomes compared, there was concordance of copy number in 1517 (90%). The lowest concordance was for the X chromosome (79%) and chromosome 21 (77%). This level of discordance, at least for chromosome 21, may be explained by multiple copies with no allelic imbalance. For all other chromosomes the concordance level was above 80%. We have shown that SNPA can reliably estimate CN in high hyperdiploidy and near haploid cases. This is of great potential value for genome-wide screening in cases in which cytogenetics is not available. It also provides validation of the applicability of the technique for the analysis of DNA from the majority of tumour types which do not yield metaphases.
Response to Anti CD20 Monoclonal Antibody Rituximab® and Epitope Mapping of Inhibitory Antibodies in Patients with Acquired Haemophilia.
Authors: Krause Manuela , Koenigs Christoph , Kessel Christoph , Scharrer Inge , Grossmann Ralf , Kreuz Wolfhard
Published: April 2020

Introduction: Acquired haemophilia (AcH) is associated with the development of polyclonal autoantibodies against FVIII, which affect directly the A2 or C2 domain of the FVIII molecule. Immunomodulatory therapy regimes to normalize FVIII levels and to eliminate the inhibitor are essential options in the treatment of patients (pts) with AcH. The aims of the present study were to investigate the response to Rituximab® and to localize the inhibitor epitopes on the FVIII domains.

Patients and Methods: In 5 pts with AcH (2 females,3 males; age: 64–81 yrs) the inhibitor titers ranged from 9 to 156 BU and the FVIII activities from <1 % to 6 %. Rituximab® was administered once weekly for 4 weeks (dosage: 375 mg/m2) in combination with a prednisone therapy (1–1.5 mg/kg).

Random peptide phage display libraries were used to identify peptide sequences specific for inhibitors. The short peptide sequences allow the mapping of conformational epitopes on the surface of the FVIII molecule.

Results: In all pts (median follow up of 24 months, range: 2–49 months) we documented complete remission (CR). The median time to achieve CR was 13 weeks (range: 2–32 weeks). The time to CR was much longer (23/32 weeks) in two pts, who showed high inhibitor titers (50/188 BU) compared to pts (2/4/5 weeks) with low inhibitor titers (9/22/29 BU). One pt with a high inhibitor titer (50 BU) relapsed 19 months after CR, an other pt died in septic shock 8 weeks after the initiation of therapy with Rituximab®. No other relevant side effects were observed. In the pt with a relapse we identified target epitopes in the A1 and A2 domain of FVIII, and in the other pts (n=3) the peptide sequences mapped to epitopes on the C2 domain of FVIII.

Conclusion: The therapy with Rituximab®shows high response rates and seems to be effective in the elimination of high and low inhibitors against FVIII in pts with AcH. Limited by the small number of pts, further studies are needed to confirm whether the different target epitopes found in patients with (A1/A2) or without a relapse (C2) might have a relevance in the specific immunomodulatory therapy.
Diagnostic Workup of Patients with Acquired Von Willebrand’s Syndrome.
Authors: Tiede Andreas , Priesack Jan , Bolte Oliver , Trummer Arne , Bohlmann Katrin , Helms Wiebke , Ganser Arnold , Eisert Roswith
Published: April 2020

Acquired von Willebrand’s syndrome (AVWS) is a rare but probably underdiagnosed hemorrhagic disorder often associated with hematological or cardiovalvular disorders. Diagnostic workup remains challenging, particularly in patients with normal or increased von Willebrand factor antigen (Ag) and ristocetin cofactor (RCo). Here, we present a retrospective single-center study of 35 patients diagnosed with AVWS based on (i) a history of recent onset of bleeding, (ii) a negative family history of von Willebrand’s disease, and (iii) abnormal plasma VWF multimers. AVWS was associated with monoclonal gammopathy (n=11), cardiovalvular disorders (n=16), or other conditions (n=8) including myeloproliferative and autoimmune disorders. The PFA-100® screening test was inconclusive due to anemia (hematocrit <30 %) or thrombocytopenia (<100/nl) in 10 patients (29 %); prolonged closure times were observed using collagen/epinephrine and collagen/adrenalin in 20 of 25 (80 %) and 18 of 25 (72 %) patients, respectively. Factor VIII:C was reduced below 50 IU/dl in 7 of 35 patients (20 %). VWF Ag and RCo were reduced below 50 IU/dl in 8 patients (23 %). VWF Ag and RCo were normal or increased in all patients with cardiovalvular disease and in four of eleven patients with gammopathy. Median VWF Ag was higher in cardiovalvular disease (median 202 IU/dl, range 90 to 608) compared to gammopathy (median 31 IU/dl, range 8 to 468, p<0.02 by Mann Whitney U test). Of 27 patients with normal or increased VWF Ag and RCo, 12 (44 %) had a reduced collagen binding activity (CBA) or CBA to Ag ratio <0.7; 10 (37 %) had a borderline CBA ratio between 0.7 and 0.8; 5 (19 %) had a normal CBA >0.8. A normal or increased VWF Ag together with a CBA ratio >0.7 was observed both in patients with cardiovalvular disease (n=9), gammopathy (n=2) and other disorders (n=4). However, in all patients the largest VWF multimers were decreased (n=14) or absent (n=21). In conclusion, no single test was sufficient to detect all cases of AVWS. The PFA-100® test is of limited use in this population because of its limitation in anemia or thrombocytopenia and because of its low sensitivity. A significant number of patients present with a normal or increased VWF Ag and RCo as well as a CBA ratio >0.7 emphasizing the importance of multimer analysis in all patients with suspected AVWS.
Platelet Aggregation Analysis of AMG 531 in a Randomized, Placebo-Controlled Study in Healthy Japanese Subjects.
Authors: Kumagai Yuji , Fujita Tomoe , Ozaki Machiko , Sahashi Kunihiko , Ohkura Masayuki , Ohtsu Tomoko , Arai Yoshihiro , Sonehara Yusuke , Nichol Janet
Published: April 2020

AMG 531 is a novel thrombopoiesis-stimulating peptibody currently being investigated for the treatment of chronic immune thrombocytopenic purpura. This study was a double-blind, phase 1 evaluation of safety (including a platelet aggregation analysis), pharmacodynamics, and pharmacokinetics of AMG 531 in healthy Japanese men. Thirty subjects were randomly assigned 4:1 (AMG 531:placebo) to each of 3 sequential dose cohorts (0.3, 1, and 2 μg/kg) of 10 subjects each. A single dose of AMG 531 or placebo was administered by subcutaneous injection, and subjects were evaluated for 6 weeks. Concentrations of 0.2 and 2 μg/mL collagen and 0.5 and 2 μmol/L adenosine phosphate were used as reagents in the platelet aggregation analysis conducted on day -1, day 2, day 15, and at the end of the study. No enhancement or reduction in platelet aggregation was detected in samples collected from subjects treated with AMG 531 compared with either pretreatment or placebo samples. Overall, adverse events were similar between the AMG 531 and placebo groups. Treatment-related adverse events (headache, “feeling hot,” migraine without aura, and/or malaise) were reported for 5 of 24 subjects treated with AMG 531. Four of 8 subjects who received the 1 μg/kg dose and 7 of 8 subjects who received the 2 μg/kg dose had platelet count increases ≥ 1.5-fold over baseline, and 3 of 8 subjects who received the 2 μg/kg dose had platelet count increases ≥ 2-fold over baseline, a pharmacodynamic effect similar to that seen in previous studies of non-Japanese subjects. Serum concentrations of AMG 531 were below the lower limit of quantification in all but 2 subjects who received the 2 μg/kg dose. In summary, platelets in subjects treated with AMG 531 functioned normally compared with pretreatment and placebo samples. AMG 531 was generally well tolerated and was effective at raising platelet counts after a single subcutaneous administration.
Deletion of Two Contiguous Genes, Platelet GPIbβ (Glycoprotein Ibβ) and Septin SEPT5, in a Boy with Bernard-Soulier Syndrome and Developmental Delay: A Possible New Contiguous Gene Syndrome.
Authors: Bartsch Ingrid , Hainmann Ina , Bläser Susanne , Pavlova Anna , Oldenburg Johannes , Busse Anja , Lanza Francois , Nurden Paquita , Superti-Furga Andrea , Zieger Barbara
Published: April 2020

Mutations or deletions in the genes for platelet GPIb/V/IX (consisting of GPIbα, GPIbβ, GPV, GPIX) cause the bleeding disorder Bernard-Soulier syndrome. Septins are GTP-binding proteins essential for active membrane movement such as vesicle trafficking. In non-dividing cells (i.e. platelets) septins are implicated in exocytosis. Platelets from a SEPT5 knockout mouse show altered serotonin secretion and platelet aggregation suggesting that SEPT5 is involved in secretion in platelets.

We report on a 4 year old boy born to consanguineous parents who developed several life-threatening mucosal bleeding episodes (when he suffered from upper respiratory infections) and showed developmental delay. Giant platelets were present in the blood smear, and ristocetin-induced platelet aggregation was reduced indicating that the boy suffered from Bernard-Soulier syndrome. Immune transmission electron microscopy of the platelets showed reduced expression of GPIb. PCR- and Southern analysis demonstrated that exon 1 and partly exon 2 of GPIbβ was deleted on both alleles. Further experiments showed that the deletion extended 5′ from exon 1 of GPIbβ to include the whole gene coding for SEPT5 located 5′ of GPIbβ. After bone marrow transplantation from a HLA-identical unrelated donor the boy developed no further bleeding symptoms.

Conclusions: We identified a patient with Bernard-Soulier syndrome and mental retardation who had not only a homozygous deletion of exon 1 and partly exon 2 of GPIbβ but also a deletion of the whole adjacent SEPT5 gene, possibly indicating a novel contiguous gene syndrome. Whether absence of SEPT5 contributed to particularly severe bleeding symptoms and to developmental delay remains to be proven.
Anti-Apoptotic MCL-1 Plays a Critical Role in Maintenance of Normal and Malignant Human Hematopoietic Stem Cells through FLT3 Signaling.
Authors: Yoshimoto Goichi , Miyamoto Toshihiro , Iino Tadafumi , Kikushige Yoshikane , Niiro Hiroaki , Iwasaki Hiromi , Harada Mine , Akashi Koichi
Published: April 2020

Mcl-1 (myeloid cell leukaemia-1) is an anti-apoptotic member of the Bcl-2 family protein, originally identified through the analysis of differentiating myeloid cells. We have recently reported that disruption of murine MCL-1 in adult murine hematopoiesis resulted in a complete deficiency of hematopoietic cells in the bone marrow, and that MCL-1 expression was activated in response to activation of c-Kit tyrosine kinase receptor signaling (Science307;1101, 2005). Here we propose that human MCL-1 also plays a critical role in maintenance of normal and malignant human hematopoiesis. The analysis of FACS-sorted human HSCs and myeloid progenitors demonstrated that, as in case of murine hematopoiesis, HSCs expressed hMCL-1 at the highest level, and that its expression gradually downregulated in committed myeloid progenitors such as CMPs and GMPs. We also found that unlike murine long-term HSCs that express c-Kit but not FLT3 receptor tyrosine kinase, human long-term HSCs express both of these receptors. Ligation of FLT3 receptor in purified human HSCs immediately induced the expression of MCL-1, suggesting that FLT3 supports human HSC survival through stimulating MCL-1 expression. We then evaluated whether hMCL-1 is expressed in leukemic stem cells. Thirty patients with acute myeloid leukemia (AML) were enrolled in this study. Within the immature CD34+ blast population, the CD34+CD38− fraction expressed higher levels of MCL-1 than those in more differentiated CD34+CD38+ fraction. In most cases, CD34+CD38− AML cells expressed 2 to 10-fold higher levels of MCL-1 transcripts as compared to normal HSCs. Interestingly, the group of samples expressed highest levels of MCL-1 constituted mostly of FLT3/internal tandem duplications (ITDs) positive AML, suggesting that constitutive activation of FLT3 signaling by FLT3 mutations might induce overexpression of MCL-1. A high level expression of MCL-1 was also found in MOLM-13 and MV4-11, FLT3/ITD positive AML cell lines. Treatment of MOLM-13 and MV4-11 with the small molecule tyrosine kinase inhibitor PKC 412, resulted in induction of apoptosis, which was associated with decreased expression of MCL-1 transcripts and proteins. Based on these data, MCL-1 might play a critical role in maintenance of normal and malignant HSCs in human at least through FLT3 signaling. Our data also suggest that constitutive activation of FLT3 signaling by FLT3 mutation might contribute to leukemic transformation through enforcing survival of AML stem cells.
Haploinsufficiency of the Platelet P2Y12 Gene in a Family with Congenital Bleeding Diathesis.
Authors: Fontana Gessica , Cattaneo Marco , Ware Jerry
Published: April 2020

Two related patients (sisters) with a congenital bleeding diathesis associated with a severe defect of the platelet ADP receptor, P2Y12, have been described (Cattaneo et al, ATVB2000;20:883). Platelets from these patients are severely defective in ADP-dependent aggregation and in Gi-coupled ADP-mediated responses, but are normal in Gq-coupled ADP-mediated responses. Molecular analysis of the P2Y12 gene of each sister revealed an identical single base pair (bp) deletion (378delC) occurring just beyond the coding sequence for the third transmembrane domain in P2Y12. The mutation results in a frame shift (Thr126 frame shift X34) and premature truncation of the protein (Conley et al, Blood2001; 98:43b). In the present study, we focused on a 13-year old son (GL) of one of the affected sisters. GL has not suffered spontaneous bleedings; his bleeding time is moderately prolonged (13 min). His platelets display a phenotype that is compatible with partial defect of the platelet P2Y12 receptor: abnormal aggregation and ATP secretion induced by several agonists, moderate deficiency of platelet-binding sites for [33P]2MeS-ADP, and partial impairment of inhibition of adenylate cyclase by ADP. PCR analysis of GL’s P2Y12 gene revealed only normal DNA sequence. Therefore, we hypothesized that the abnormal platelet phenotype of GL is due to haploinsufficiency of his P2Y12 gene, and, accordingly, his mother and aunt suffer from P2Y12 deficiency owing to haploinsufficiency and the previously described single base mutation in their remaining P2Y12 allele. In order to test our hypothesis, we analyzed genomic DNA from the family by Southern blotting and real time quantitative PCR. The Southern blot results suggested that GL had one P2Y12 allele only, which he inherited from his father. Our P2Y12 gene quantification strategy was based on SYBR Green detection and normalization using two reference genes with a normal copy number, ZNF 80 (3q13.31) and GPR15 (3q12.1). P2Y12 gene copy numbers, calculated by use of a comparative Ct method, revealed that GL, his mother, and aunt each contained a single intact P2Y12 allele. Thus, GL, his mother, and aunt suffer from haploinsuffiency in their P2Y12 alleles. However, GL’s remaining allele is normal, while his mother and aunt each have a remaining mutant allele. This explains the differences in clinical phenotypes between GL and his mother and aunt. The quantitative PCR result, supported by Southern blotting results, supports our hypothesis and illustrates the platelet phenotype associated with P2Y12 haploinsufficiency.