bluuebunny/crossref_metadata_2025 huggingface dataset:
Faster Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation of Younger Children Compared with Older Children and Adults.
Authors: Fehse Natalja , Kabisch Hartmut , Erttmann Rudolf , Kroeger Nicolaus , Zabelina Tatjana , Freiberger Petra , Zander Axel R.
Published: April 2020

Allogeneic stem cell transplantation (SCT) is a potentially curative treatment of otherwise incurable malignant hematological disorders and severe immunodeficiency diseases. The post-SCT immunoincompetence can last for up to several years and might cause significant morbidity and mortality. Immune reconstitution after SCT has been studied extensively in adults, but only a few reports have been published on a paediatric population or have compared paediatric and adult population. This study was carried out in order to determine the influence of age on quantitative reconstitution of peripheral blood lymphoid subsets. A cohort of 69 adult and 109 paediatric patients received allogeneic SCT from related or unrelated fully HLA matched or 1 locus -mismatched donors for different malignant and non-malignant diseases. Analysis of lymphocyte subsets was performed at various time points: until day 30, between days 31–60, 61–90, 91–180, 181–365, between 1–2 years, between 2–5 years and more than 5 years post transplant (at the last time point only paediatric patients were analysed).

Comparing the paediatric with adult patients, we observed statistically significant faster recovery of CD19+, CD19+CD5+, CD4+CD45RA+ cells during the whole period of observation, and of total lymphocyte count (TLC) (< 60d and 1-2y), CD3+ (< 60d and 90d-2y), CD3+HLA-DR+ (31-60d), CD3+CD4+ (< 60d and > 90d), CD4+CD45R0+ (< 60d), CD3+CD8+ (< 60d and 90d - 2 y) and CD56+CD3−cells (< 31d) in the paediatric group. Interestingly, the recovery of CD56+CD3− cells was faster from day 61-365 in the adult group (significant faster on day 91–180).

The paediatric patients were divided in two subgroups: 1) aged < 10 years; 2) aged > 10 years. The following lymphocyte subsets recovered significant faster in the younger group: TLC (31-60d, 91-365d and > 2y), CD3+ (91-180d and > 1y), CD3+HLA-DR+ (31-60d), CD3+CD4+ (31-60d and > day 90), CD4+CD45RA+ (61-90d and > day 180), CD4+CD45R0+ (91-180d and 2-5y), CD3+CD8+ (91-180d and > 2y), CD19+ (31-60d, 91-180d and 2-5y), CD19+CD5+ (31-60d, 181-365d and 2-5y) and CD56+CD3− cells (61-180d).

The thymopoietic pathway of T helper cells is important throughout childhood; however, the thymus has a diminished capacity for the regeneration of these cells in adulthood. This can be the explanation of the faster recovery of T helper cells in the younger patients in this study. Besides, we found, that paediatric patients have a faster reconstitution of B cells and cytotoxic T cells. This might be because of the higher number of stem cells in the paediatric transplantation.
Therapeutic Plasma Levels of OKT3 Muromonab Are Effective as Second-Line and Subsequent Treatment in Recipients of Stem Cell Allografts with Steroid-Resistant Acute Graft-Versus-Host Disease.
Authors: Knop Stefan , Hebart Holger , Gscheidle Heidi , Holler Ernst , Kolb Hans J. , Niederwieser Dietger W. , Einsele Hermann
Published: April 2020

Acute graft-versus-host disease (aGvHD) remains a major complication in recipients of hematopoietic cell allografts and is associated with poor outcome. Salvage treatment is difficult after failure of steroids. OKT3 muromonab is a monoclonal murine IgG2a antibody directed against the CD3 antigen on T-lymphocytes that has proven effective in transplant settings both of solid organs and bone marrow. Here we report on the results of OKT3 treatment in 43 patients who had received stem-cell or bone marrow allografts for hematologic malignancies and subsequently been diagnosed with steroid-resistant aGvHD. Overall, 48 treatment cycles of OKT3 (5 mg intravenous injection once daily) were administered for acute GvHD of grades II (n = 12), III (n = 27) and IV (n = 9). Mean duration of OKT3 therapy was 9 (range, 1 –16) days. Twenty cycles of OKT3 were administered as second line and 28 as third-plus line (third line, n = 14; fourth line, n = 14) treatment regimens. Side effects were mild to moderate, comprising fever, dyspnea, tachycardia, and vomiting. Thirty of 42 evaluable patients responded to therapy for an overall response rate of 69 %. Complete remissions were seen in 6 (11.9 %) and partial remissions in 24 patients (57.1 %). Response to treatment was 88.8 % for skin GvHD; 53.3 % for gut aGvHD; and 8.7 % for liver involvement. Recurrent or progressive acute GvHD was observed in 23 patients (54.8 %) after a median of 9 (range, 1 – 58) days. Pharmacokinetic studies of monoclonal antibody OKT3 were performed in 17 patients, 16 of whom achieved at least a partial remission. Treatment duration was > 5 days in 16 patients. Adequate plasma levels (> 1000 ng/ml) were observed in 13 patients after a median of 6 (1 – 11) days on treatment. OKT3 concentration became undetectable at a median of merely 4 (3 – 11) days after discontinuation of therapy. Survival at 100 days from transplantation was 58.1 % and at one year, 25.5 % with the most important causes of death being infectious complications (44.7%) and GvHD (33.2%). Patients treated with OKT3 earlier in the course of aGVHD showed a trend to better survival: 35% of patients with 2nd-line therapy were alive at one year from transplantation, compared to 17.4% with 3rd- plus line treatment (p=0.21). There was also a trend to a better one-year survival for patients with grade II (33% of patients alive) when compared to grade III/IV aGvHD (16% of patients alive; p=0.24). Thus, a significant proportion of patients with corticosteroid-resistant acute GvHD showed a response to salvage treatment with OKT3. More effective antimicrobial prophylaxis and treatment are mandatory to overcome considerable treatment-related mortality in those high-risk patients.
Treatment of Steroid-Refractory Acute Graft Versus Host Disease with Low Dose Combination Monoclonal Antibody Therapy: A Pilot Study.
Authors: Moore John J. , Johnston Amanda , David Ma D.F. , Sam Milliken , Keith Fay , Tony Dodds
Published: April 2020

Severe (Grade III–IV) acute graft versus host disease (aGVHD) is a serious complication of allogeneic haemopoietic stem cell transplantation. Patients not responding to high dose corticosteroids have a very poor prognosis (60–90% mortality). Previous second line agents, such as anti-thymocyte globulin, have had limited success in controlling steroid-refractory aGVHD. Recently monoclonal antibodies that target cytokines (TNF and IL-2) have been trialled, with several case series reporting responses to high dose infliximab (10mg/kg, x1-9 doses) and daclizumab (1mg/kg, x5-8 doses), alone or in combination. Here we report the results of a pilot study using reduced-dose infliximab and daclizumab, along with strict antimicrobial prophylaxis, for refractory aGVHD. The aim was to maintain the efficacy of monoclonal antibody therapy whilst reducing the infectious complications seen in previous series.

Since May 2003 we have treated 10 allogeneic transplant patients with one or both of the monoclonal antibodies. All had failed a minimum of 3 days methylprednisolone 2mg/kg/day, started a median of 35 days post-transplant. The first four patients received infliximab 10mg/kg, x2-3 doses. Two of four had a complete response with one patient alive 15 months after antibody treatment. The remaining three died of opportunistic infections. Following this study, a second cohort of patients (n=6) were given low dose, combination therapy (infliximab 5mg/kg, x2 doses and daclizumab 1mg/kg, x4 doses). 5/6 patients had complete resolution of aGVHD and 5/6 are alive at a median of 130 days post-treatment. One patient died of MRSA pneumonia despite resolution of GVHD. The remaining 5 patients have had minimal and manageable infectious complications whilst on strict prophylactic regimens. In conclusion, low dose combination monoclonal antibody therapy appears to be a promising and safe treatment for refractory aGVHD.
ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.
Authors: Zhang Weirui , Motto David , Ginsburg David
Published: April 2020

Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.
Calpain I Null Mice Display Lymphoid Hyperplasia.
Authors: Kuchay Shafi , Nunez Rafael , Bartholomew Amelia M. , Chishti Athar H.
Published: April 2020

Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, homeostasis, cell proliferation, cell motility, and apoptosis. The conventional calpains include mu-calpain (calpain I) that requires micromolar calcium for half maximal activation and m-calpain (calpain II) that functions at millimolar calcium concentration in vitro. To evaluate the physiological function(s) of mu-calpain, we utilized genetically altered mu-calpain null mice as the model system. Spleen biopsy of mu-calpain null adult mice showed consistent expansion of white pulp (lymphoid hyperplasia) due to an increase in number and size of follicles and PALS (periarteriolar lymphatic sheets), relative to matched wild type mice. Also, cervical lymph nodes of mu-calpain null mice showed lymphoid hyperplasia and marked plasmacytosis. The majority of mu-calpain null mice displayed multi-organ lymphoid infiltration. Using immunofluorescence labeling and multiparameter flow cytometry, the cellular composition of the hematopoietic compartment was quantified in both bone marrow and spleen of mu-calpain null mice. The mu-calpain null mice exhibited an overall increase in the number of B-cell lineage; showing ~40% increase of B220+ cells in the bone marrow and ~19% increase of B220+ cells in the spleen. Interestingly, T-cell compartment (CD3+, CD4, CD8+) and NK lineage (NK1.1+) cells were significantly reduced. Myeloid specific GR1+, CD11b+ granulocytic cell lineage, and megakaryocytic CD41+ cells were also noticeably increased in the bone marrow of mu-calpain null mice. Together, these results reveal a central role of mu-calpain in the maintenance of lymphoid homeostasis, contributing to the regulation of B-cell lineage. Ongoing experiments involve investigating quantitative effects of mu-calpain deletion on the survival and apoptotic cell death of B220+ B-cell lineage in response to various insults such as Fas ligand, cycloheximide, irradiation, 9-AC, and staurosporine. The outcome of these studies will elucidate the precise biochemical function(s) of mu-calpain by identifying key substrates that regulate apoptosis in a physiologically relevant environment.
HSP27 Protects Malignant Hematopoietic Cells Against VP-16-Induced Apoptosis through Modulation of P38 and C-JUN.
Authors: Schepers Hein , Geugien Marjan , van der Toorn Marco , Bryantsev Anton L. , Kampinga Harm H. , Vellenga Edo
Published: April 2020

In the present study, expression and function of Heat Shock Protein 27 (HSP27) was analyzed in acute myeloid leukemia (AML), since HSP27 expression is linked to unfavourable prognosis. HSP27 protein was predominantly expressed in monocytic blasts (M4-M5, 91%, N = 11) and absent in myeloid leukemic blasts (M1-M2, N = 5). A similar lineage restricted expression was observed in normal hematopoietic cells: high expression in normal CD34+ cells and monocytes, and absent in granulocytes. To study the functional role of HSP27, RNA interference (RNAi) studies were performed in the leukemic TF-1 cell line. These experiments demonstrated a twofold increase in VP-16-induced apoptosis after HSP27 siRNA. In contrast, CD95 Fas-induced apoptosis remained the same, as a result of CD95 Fas-mediated upregulation of HSP27. Additional investigations demonstrated that the increased VP-16-induced apoptosis after HSP27 RNAi, was associated with an enhanced phosphorylation of p38 and c-Jun. This VP-16-induced phosphorylation was subsequently followed by the release of cytochrome c into the cytoplasm, which increased twofold after siRNA treatment. These results indicate that HSP27 plays an important role in the protection against VP-16-induced apoptosis through the modulation of p38 and JNK activation, probably through interference with DAXX-mediated ASK1 activation. This was further underscored by co-immunoprecipitation studies, demonstrating complex formation of DAXX and HSP27 in an ASK1-dependent manner. However, in the investigated AML samples, VP-16-mediated apoptosis correlated moderately with HSP27 expression, although HSP27 was highly expressed and phosphorylated and activated in primitive monocytic AML blasts. This is likely due to the co-expression of p21Waf1/Cip1, which is in the majority of the monocytic AML M4-M5 blasts constitutively localised in the cytoplasm and interferes with apoptosis via the DAXX-ASK1-dependent pathway. Preliminary data indicate that overexpression of a cytoplasmic form of p21 is able to reduce the VP-16-induced apoptosis after RNAi for HSP27 as compared to controls, suggesting a predominant anti-apoptotic role of p21 over HSP27. In summary, we demonstrate a role for HSP27 in the survival of leukemic cells by modulation of the DAXX/p38/JNK apoptosis pathway. This survival advantage can further be promoted by the co-expression of cytoplasmic localised p21Waf1/Cip1 protein, indicating that strategies in AML treatment should be focused on targeting multiple signal transduction pathways.
Leukemia Inhibitory Factor: A Newly Identified Chemoattractant and Regulator of Metastasis of Rhabdomyosarcomas and Neuroblastomas to Bone Marrow.
Authors: Wysoczynski Marcin , Jankowski Kacper , Miekus Kasia , Kucia Magda , Janowska-Wieczorek Anna , Ratajczak Janina , Ratajczak Mariusz Z.
Published: April 2020

Rhabdomyosarcomas (RMS) and neuroblastomas (NBS) are tumors that originate in skeletal muscle and neural tissue respectively, and frequently metastasize/infiltrate bone marrow (BM). We have recently demonstrated a crucial role for the stromal-derived factor (SDF)-1- CXCR4 axis in RMS and NBS metastases to BM (Blood2002:100, 2597). However, our recent studies with the CXCR4 antagonist T140 suggest involvement of other factor(s) besides SDF-1 that direct the motility/metastasis of these cells. Based on the fact that the gp-130 receptor signaling cytokine Leukemia Inhibitory Factor (LIF) plays an important role in trafficking of normal muscle and neural cells and the development of muscle and neural tissue we hypothesized that LIF could be this missing chemoattractant that is involved in directing metastasis of RMS and NBS cells to the BM environment. Supporting this notion we found that LIF is secreted by BM stroma cells and LIF-R (a gp130 receptor family member) is expressed at the protein level on several human RMS and NBS cell lines as determined by FACS analysis. Next, we found that LIF-R is functional on RMS and NBS cells, mediates phosphorylation of MAPKp42/44, PI-3K-AKT, and JAK-STAT pathways and activation of small GTP-ase - Rac-1, and both RMS and NBS cells are chemoattracted by LIF. Furthermore, LIF increases adhesion of RMS and NBS cells to stroma and endothelium and upregulated expression of angiopoietic factors such as VEGF and IL-8. Finally we found that LIF-mediated responses were specifically inhibited by anti-LIF-R blocking antibodies. In conclusion, we present evidence for first time that LIF-LIF-R (cytokine-gp130 signaling receptor axis) may direct metastasis and enhance the invasive potential of human solid tumor cells. Hence molecular targeting of LIF or LIF-R may become a new antimetastatic strategy to improve therapeutic outcome from pediatric sarcomas. Molecular strategies aimed at inhibiting the LIF-LIF-R axis, and the SDF-1-CXCR4 axis, for example, by the use of small-molecule inhibitors, could lead to the development of new anti-metastatic therapies that could complement the conventional radio- or chemotherapy in preventing the dissemination of RMS and NBS cells into bone marrow and lymph nodes. This is currently being tested in animal models in our laboratory.
Crosstalk between Breast Cancer Cells and Bone Marrow Endothelium Require the Engagement of PI3K/AKT Signaling.
Authors: Brandao Joana G. , Barata Joao T. , Nunes Raquel , Nadler Lee M. , Cardoso Angelo A.
Published: April 2020

The presence of breast cancer cells in the patient’s bone marrow (BM) at diagnosis is associated with resistance to treatment, disease relapse and poor prognosis. Identification of the factors implicated in the homing, survival and latency of breast cancer cells in the BM should contribute to the design of more efficient therapeutic strategies for breast cancer. There is evidence that breast cancer can recruit endothelial progenitors from the BM. Also, other epithelial tumors seem to preferentially adhere to BM endothelial cells. Therefore, we hypothesized that BM endothelium may play a significant role in the biology of breast cancer cells residing in the BM. Co-cultures in Matrigel showed that breast cancer cells interact with BM endothelium to form heterotypic multicellular networks. Moreover, breast cancer cells migrate towards BM endothelium assembled as capillary-like structures, but not to structures of BM mesenchymal stem cells or BM stroma. This migration was abrogated by pertussis toxin-mediated blockade of chemokine receptor signaling, suggesting the involvement of endothelium-secreted chemokine(s). We then evaluated the impact of breast cancer cells in the survival and proliferation of BM endothelium. All breast cancer lines tested (n=4) promoted the proliferation of BM-derived endothelial cells. This effect is mediated through the engagement of the PI3K/Akt pathway (phosphorylation of Akt at Ser437 and Thr308, and activation of its downstream substrates GSK3β, PRAS-40 and FKHRL1) since its specific blockade abrogated the stimulatory effects of breast cancer on BM endothelium. We next determined whether, reciprocally, BM endothelium impacts on breast cancer cell survival. These experiments were performed in serum-free media to enhance dependency of breast cancer cells from microenvironmental stimuli. In all cases tested, BM endothelium promoted survival/proliferation of breast cancer cells. This stimulation was accompanied by the engagement of the PI3K/Akt pathway in breast cancer cells and, in three of the four lines, the phosphorylation of Erk1/2. These effects were also observed for breast cancer cells that showed constitutive activation of Akt (MCF-7 and ZR-75-1 cells). Specific blockade of PI3K/Akt abrogated the BM endothelium-promoted survival of breast cancer cells, thus demonstrating the critical role of this pathway. These studies show that crosstalk between BM endothelial cells and breast cancer cells may impact on the survival of both cell types. These findings provide new light on the mechanisms that may facilitate the development of a tumor-permissive BM microenvironment in breast cancer, and the creation of breast cancer-supporting BM niches. Importantly, this study implicates BM endothelium as a therapeutic target in breast cancer and suggest that blockade of PI3K/Akt may impact the outcome of patients with metastatic breast cancer.
Risk-Adapted Therapy in Adults Previously Untreated Hodgkin’s Lymphoma (HL) with ABVD Followed by Involved Field Radiotherapy (IFRT).
Authors: Pavlovsky Santiago , Corrado Claudia S. , Pavlovsky Miguel A. , Giunta Mario , Zoppegno Lucia , Prates Virginia , Lastiri Francisco , Cerutti Ider , Narbaitz Maria C. , Rudoy Silvia , Cavagnaro Fernando , Kurchan Alejandra , Palomino Elsa , Campo Cesar
Published: April 2020

Background: The present goal for the treatment of HL is to develop a combined modality therapy with high response rate, disease-free survival (DFS) and overall survival (OSV) with minimal toxicity.

Methods: On December 1996, the GATLA started a non randomized protocol for 15 to 75 years old (median 28) patients previously untreated. Patients with clinical stage I, II, IIIA without bulky tumor (< 10 cm mass or < 1/3 thoraxic diameter) (low risk) received 3 cycles of ABVD followed by IFRT 25 Gy to all node areas of more than 2 cm at diagnosis. A total of 46 out of 218 patients (21%) with low risk who failed to achieve complete remission (CR) after 3 cycles of ABVD were included as high risk completing 6 cycles of ABVD. Patients with clinical stage IIIB and IV or all other stages with bulky disease or persistance lymph nodes areas after 3rd cycle of ABVD (high risk) received 6 cycles of ABVD followed by IFRT 30 Gy to residual areas after the third cycle of ABVD or bulky areas at diagnosis. The dose of ABVD was the standard; Adriamycin 25 mg/m2, Bleomycin 10 IU/m2, Vinblastine 6 mg/m2 and Dacarbacine 375 mg/m2 all IV on day 1 and 15 of each 28 days cycles. Patients who achieved partial remission (PR) were salvage with other regimen mainly ESHAP x 3 cycles followed by high dose therapy with autograft rescue.

Results: A total of 171 (99%) out of 172 patients with low risk achieved CR. One 74 year old patient died of pneumonia after the third ABVD. A total of 187 (85%) of 219 patients with high-risk achieved CR, 25 PR, and 7 failed to respond (FR) (P<0,001). The estimate DFS at 60 months was 92% and 67% (P< 0.001), while the OS was 98% and 89% (P=0.004) for low and high risk respectivelly. Of the 25 patients with PR, all received second line therapy followed in 15 by an autograft. Twelve patients are in CR, 5 are in PR, 2 in progressive disease and 6 died. Of the seven who FR, four died of PD an three are alive with the disease. One patient developed a MDS/AML after relapsing from an autograft and 8 months after having been rescued with BEACOPP. Five other second cancer (1 ovary, 2 NHL, 1 lung, 1 colon) appeared after treatment, two died and 3 remain in CR of their HL. Using the IPI HL 165 patients (48%) have scored 0–1, 145 (43%) scored 2–3, and 30 (9%) scored 3–4. The rate of CR was 96%, 88% and 87% respectively (P<0.016). The EFS at 60 months was 89%, 72% and 55% respectively (P=0.004). The OSV was 98%, 89% and 86% (P=0.037).

Conclusions: We can conclude by saying that with risk-adapted therapy, 92% of the 391 patients achieved CR, with a CR + PR response of 98%. A total of 78% are event-free and 93% are alive at 60 months with remarkable low-toxicity.
G-CSF Administration Increases Pulmonary Toxicity for Hodgkin’s Disease Patients Treated with Bleomycin-Containing Chemotherapy.
Authors: Martin William G. , Habermann Thomas M. , Colgan Joseph P. , Ristow Kay M. , Ansell Stephen M.
Published: April 2020

Background: In Hodgkin’s Disease (HD), G-CSF has been used to maintain maximum dose intensity by avoiding neutropenia and treatment delays. Pre-clinical data and anecdotal reports have shown an association between G-CSF administration and bleomycin pulmonary toxicity (BPT). This complication may cause access morbidity and have a detrimental impact on survival in this otherwise good prognosis population. We embarked on a retrospective analysis to determine if G-CSF increased the incidence of this complication or had an impact on outcomes.

Methods: We reviewed all patients managed for HD at Mayo Clinic, Rochester, from January 1986 to February 2003. BPT was defined by the presence of pulmonary symptoms, bilateral interstitial infiltrates, and no evidence for an infectious etiology. Patients who received G-CSF support with one or more of their bleomycin-containing chemotherapy cycles were considered as being treated with G-CSF.

Results: A retrospective review identified 141 patients with HD treated with a bleomycin-containing regimen. The mean age of patients at diagnosis was 34 years. Fifty four percent were males. The histology was nodular sclerosing in 85%. The Hasenclever index was 0 in 15%, 1 in 35%, 2 in 24%, 3 in 16%, 4 in 9%, and ≥ 5 in 1%. The G-CSF and non G-CSF groups were balanced for poor prognosis risk factors including Hasenclever index. Frontline chemotherapy included ABVD in 57%, MOPP-ABV(D) in 33%, COPP-ABV in 8%, BEACOPP in 3%, and Stanford V in 2%. Forty one percent of patients received radiation. The 5-year overall survival (OS) and progression free survival (PFS) for all patients were 87% and 79% respectively. G-CSF was administered to 52% (74/141) of patients. BPT occurred in 18% of patients (25/141). A higher rate of BPT was observed in patients receiving G-CSF, 26% (19/74) versus 9% (6/67) in non G-CSF patients (p=0.0141). In this population, G-CSF administration was the only factor associated with an increased risk of BPT. There was no difference between the two groups for BPT associated risk factors including radiation, bleomycin dose, renal insufficiency, smoking history, and underlying lung disease. Additionally these risk factors showed no correlation with the development of BPT. The mortality from BPT was 4.2% (6/141) in all patients and 24% (6/25) in patients who developed the pulmonary syndrome. Of the 6 patients who died acutely, 83% (5/6) received G-CSF. CR rates were equal between the G-CSF and non G-CSF groups at 92% and 94% respectively. Five year OS and PFS were equal between the G-CSF and non G-CSF groups at 82% versus 89% (p=0.473) and 74% versus 84% (p=0.152) respectively.

Conclusion: G-CSF administration increases the incidence of pulmonary toxicity in HD patients treated with bleomycin based chemotherapy. The major impact was on treatment related morbidity as 5-year OS and PFS were equal between the G-CSF and non G-CSF groups.
New Animal Models Reveal Stage-Specific Hematopoietic Functions for the BMP Signaling Pathway.
Authors: Evans Todd , Gupta Sunny , McReynolds Lisa M.
Published: April 2020

Genetic analyses in mouse and zebrafish demonstrate that a conserved BMP signaling pathway is essential for normal development of the embryonic vascular and hematopoietic systems. However, it has not been clear which aspects of hemato-vascular development are regulated by the pathway beyond the initial specification of embryonic mesoderm. We present new animal models for studying BMP signaling at specific stages of both embryonic and adult hematopoiesis. First, we developed approaches to modulate BMP signaling conditionally at specific stages of development in transgenic zebrafish. For this purpose we adapted the Crelox system to express a dominant-negative BMP receptor in cells derived from specific progenitor populations, with subsequent analysis of hematopoietic and vascular development using GFP reporter strains of fish. For example, we find that BMP signaling is required in LMO2+ cells for the normal development of both hematopoietic and vascular systems. Yolk sac vasculature and trunk angiogenesis are both disrupted, and primitive erythropoiesis is also impaired, when these cells express the mutant receptor. In contrast, BMP signaling is apparently no longer required in primitive erythroid cells once they have activated expression of the GATA-1 gene. In a second approach, we studied hematopoiesis in zebrafish mutants for Smad5, which is an intracellular mediator of BMP signaling. For example, homozygote piggytail (pgy) mutant embryos live until day 3–5, yet the fish have little to no blood and no circulation. Surprisingly, we find that key hematopoietic regulatory genes including GATA-1, GATA-2, SCL, and LMO2 are expressed in the pgy mutant fish, but that their patterns of expression are altered. Thus, the progenitor cells initiate a hematopoietic program but fail to differentiate. We also established adult pgy fish that carry a GATA1::GFP transgene, to facilitate the analysis of adult-stage blood cell profiles using flow cytometry. Blood cell populations were monitored under normal conditions and following phenylhydrazine-induced hemolytic anemia in wild-type and pgy/+ fish. Preliminary results indicate that pgy/+ adult fish are able to mount an erythropoietic response to hemolytic anemia. However, the kinetics are altered, specifically in the transition from the proerythroblast to the polychromatophilic erythroblast stage. In summary, we present data using novel animal models disrupted for normal BMP signaling, and demonstrate roles for this pathway at specific stages of differentiation for both embryonic and adult hematopoiesis.
Antibody Response to Pneumococcal Polysaccharide Vaccination Predicts Pneumococcal Disease in Splenectomized Patients with Hematological Diseases.
Authors: Cherif Honar , Bjorkholm Magnus , Kalin Mats , Konradsen Helle B. , Landgren Ola
Published: April 2020

Patients with hematological diseases undergoing diagnostic or therapeutic splenectomy are at increased risk of acquiring fulminant pneumococcal infections. Vaccination is a straightforward option in reducing these infections. Certain patient subgroups showing inadequate immunological response to pneumococcal vaccination may be candidates for alternative prophylactic measures. We prospectively studied the antibody response to vaccination with 23-valent pneumococcal capsular polysaccharide vaccine (Pneumovax N) in splenectomized patients with hematological disorders in relation to clinical characteristics and pneumococcal disease. A total of 76 splenectomized patients (Hodgkin s lymphoma 26, indolent lymphoma 10, aggressive lymphoma 8, immune hemolytic anemia 6, immune thrombocytopenic purpura 22, and others 4) with a median age of 52 years (range 18–82) were included. Antibody titers were determined using an enzyme-linked immunosorbent assay before and 12 months after vaccination. A weak immunological response was observed in 27 (35%) patients (poor responders) and an adequate response in 49 (65%) (good responders). During the follow-up period of 5–9 years after vaccination and despite repeated revaccination in many cases, a total of 5 episodes of microbiologically verified pneumococcal infections were reported in poor responders, while only one episode was noted among good responders (p=.01). Underlying malignant hematological diseases were more frequent among poor responders than among good responders (p=.002). The distribution of patients according to age, gender, immunoglobulin levels, time between splenectomy and vaccination (<1 year>), time between preceding chemotherapy/radiotherapy and vaccination (<6 months>) and/or previous radiotherapy did not differ between poor and good responders. In conclusion, a significant number of splenectomized patients with hematological diseases respond poorly to pneumococcal vaccination and have a significantly increased risk of post-splenectomy pneumococcal infections despite vaccination. In the absence of clinical parameters that can reliably predict a poor antibody response, measurement of antibody titers 12 months after vaccination seems to be the most adequate method for identification of patients in this subgroup. Poor responders may be offered other prophylactic measures such as antibiotic prophylaxis and/or immunization with pneumococcal conjugate vaccines. Studies to further elucidate the last alternative are ongoing.
Phase I/II Trial of Dendritic Cells Pulsed with Telomerase.
Authors: Marten Angela , Sievers Elisabeth , Albers Peter , Muller Stefan , Franchy Christian , von Rucker Alexander , Strunk Holger , Schild Hans H. , Sauerbruch Tilman , Schmidt-Wolf Ingo G.H.
Published: April 2020

Human telomerase reverse transcriptase (hTERT) could be a potential target for immunotherapy in cancer patients because it is detectable in more than 85% of human tumors including renal cell carcinoma. Here, we present results of a clinical phase I/II trial using dendritic cells (DC) pulsed with hTERT-peptides in patients with progressive metastatic renal cell carcinoma. Patients received injections of hTert-peptides pulsed autologous DC intradermally and intratumorally. DC were generated from leukapheresis products and matured by addition of a cytokine cocktail. Beside toxicity and feasibility aspects, a complex immune monitoring was a major point of interest in this study. All ten patients were in progressive disease when entering the protocol. The dendritic cell vaccine was well tolerated, no toxicity was observed. Two patients showed a mixed response (MR) and one patient a stable disease (SD). Seven out of ten patients remained in progression. Clinical outcome was based on a comparison of CT scans before and after treatment, i.e. three months after entering the protocol. 2/8 patients showed DTH reactivity after vaccination. Interestingly, there was a significant increase in cytotoxic activity of effector cells from all responders (SD and MR patients) after the first vaccination. In most cases, effector cells of responders showed cytotoxic activity in contrast to those of non-responders. Most interestingly, all non-responders had shown no cytotoxic activity before treatment. In contrast, responders showed cytotoxic activity already before start of therapy. Therefore, cytotoxic activity might be used as a predictive marker in the future. With tetramer staining, we could detect higher amounts of tumor specific cytotoxic cells in responding patients compared to non-responders. Responders possessed increasing amounts of IFN-gamma producing immunological effector cells, tested by ELISpot assay. In summary, telomerase-pulsed DC could induce a tumor-specific immune response against renal cell carcinoma.
Endosomal Targeting Sequences from Non-Classical Antigen Presenting Molecules Can Direct Antigens into the MIIC and Other Antigen Processing Compartments.
Authors: Gansert Jennifer L. , Niazi Kayvon R. , Ochoa Maria T. , Sieling Peter A. , Modlin Robert L.
Published: April 2020

The ultimate goal of these studies is to enhance the development of MHC class II-restricted helper T cell responses that are required for effective and durable immunotherapy. In order to optimize the processing and presentation of MHC class II-restricted epitopes, we are using a differential targeting strategy that relies upon the unique endosomal targeting sequences from the individual isoforms of the CD1 family of non-classical antigen presenting molecules. Distinct endosomal compartments differ with respect to pH and resident proteases. MHC class II molecules can load antigen in each of the different endosomal compartments. This is in addition to the traditional antigen loading compartment, MIIC. Therefore, by forcing the expression of a tumor antigen across different endosomal compartments, we hope to maximize the number of successfully processed epitopes. To achieve this, we have developed a PCR-based strategy to link the leader, transmembrane, and endosomal targeting sequences from the four different human CD1 isoforms, CD1a-d, to DNA encoding for a surrogate antigen, green fluorescent protein (GFP). Using this strategy, we have demonstrated that fusion of individual CD1 tails to the sequence for GFP resulted in four distinct expression patterns in HELA cells transfected with these constructs. Using immunofluorescent staining and confocal laser microscopy, we determined the pattern of antigen expression in various endosomal compartments. GFP variably colocalized with recycling endosomes (ARF-6), early endosomes (CD71, transferrin receptor), late endosomes (CD63), and late endosomes/lysosmes/MIIC (CD107a, LAMP-1). Three of the four CD1 tails also resulted in co-expression of GFP with the MHC class II molecule, HLA-DR. The expression pattern for each CD1 tail is shown in Table 1. These experiments validate the feasibility of the CD1 targeting approach. Current studies are underway to apply this strategy to the leukemia antigen, WT1, using recently completed and sequenced WT1/CD1 fusion constructs.

ARF-6 CD71 CD63 CD107a CD1a −/+ − − − CD1b −/+ − + ++ CD1c + −/+ + ++ CD1d + −/+ − +

Table 1. Pattern of GFP/CD1 fusion expression in different endosomal compartments. Fusion of the endosomal targeting sequences from human CD1 molecules to the model antigen, GFP, results in different patterns of antigen expression within distinct endosomal compartments. The degree of colocalization between GFP and the indicated markers of endosomal compartments is represented as follows: (−), no colocalization; (−/+), infrequent colocalization; (+), frequent colocalization; (++), extensive colocalization.
Glutathione-S-Transferase π Expression Helps for Differential Diagnosis between Mantle Cell Lymphoma and Marginal Zone Lymphoma with t(11;14).
Authors: Rolland D. , Gazzo S. , Felman P. , Callet-Bauchu E. , Traverse-Glehen A. , Baseggio L. , Paret A. , Chapuis F. , Salles G. , Coiffier B. , Berger F. , Thieblemont C.
Published: April 2020

Glutathione-S-transferase π (GSTπ), whose gene is located in 11q13, is belonging to a complex multigene family of enzymes that may detoxify electrophilic xenobiotics. Recent gene expression profiling analysis showed that GSTπ transcript is belonging to the mantle cell lymphoma (MCL) signature and constitutes one of the most expressed mRNA in MCL*. Furthermore it has been reported by immunohistochemistry that overexpression of cyclin D1 in MCL was associated with a high level of GSTπ protein expression. We then looked for the specificity of a high level of GSTπ protein expression in MCL, and particularly when comparing to marginal zone lymphoma (MZL) with t(11;14). Material and Methods: Immunochemistry analysis of GSTπ and cyclinD1 expression was performed 111 non Hodgkin’s lymphoma including MCL, MZL, small lymphocytic lymphoma (SLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL) cases with or without t(11;14). Results: A strong GSTπ expression in more than 50% of the cells was observed in 98% of the MCL. None of the MZL cases with t(11;14) expressed GSTπ, except one case that expressed a weak signal in less than 20% of the cells. None of the MZL without t(11;14) expressed GSTpi. Only two (13%) FL graded 3 according to OMS classification expressed GSTπ in less than 50% of the cells. A high GSTπ expression (>50% cells stained) was detected in 29% of the DLBCL and 43% of the SLL.

* C. Thieblemont et al. Non-germinal B-cell lymphoma exhibit distinct genomic profiles allowing molecular diagnosis. Blood2004;103:2727–2737

n GST π expression >50% GST π expression 5–50% π GST expression <5% MCL with t(11;14) 25 23 0 2 MZL with t(11;14) 5 0 1(weak) 4 MZL without t(11;14) 31 0 1 (weak) 30 FL without t(11;14) 15 0 2 13 DLBCL without t(11;14) 14 4 1 9 SLL without t(11;14) 21 6 3 12

In conclusion, 98% of the MCL with t(11;14) expressed high level of GSTπ whereas none of our MZL with t(11;14) and high transcriptional cyclinD1 expression expressed GSTπ. This results may help for differential diagnosis between MCL and MZL with t(11;14) and suggest different transcriptional regulation of GSTπ in lymphoma subtypes with t(11;14), contributing possibly to the chemoresistant phenotype of MCL. Finally, this confirmed our previous work on MZL with t(11;14).
Campylobacter jejuni Infection in Intestinal Lymphoma: A Strong Association with Immunoproliferative Small Intestinal Disease.
Authors: Baginsky Lysa A. , Diss Tim C. , Ye Hongtao , Wren Brendan , Du Ming Qing , Isaacson Peter G. , Dogan Ahmet
Published: April 2020

Immunoproliferative small intestinal disease (IPSID) is a lymphoma of the mucosa-associated lymphoid tissue (MALT) that produces a characteristic truncated immunoglobulin α-heavy chain. Bacterial involvement is suspected in this disease, as some tumours resolve after antibiotic treatment and other MALT lymphomas have proven associations with infectious agents. A recent report implicated Campylobacter jejuni in the development of IPSID. In this study, we used a PCR based method to identify C. jejuni in paraffin embedded archival material of intestinal lymphomas. The cases studied included IPSID, other non-IPSID MALT lymphomas, mantle cell lymphomas (MCL), diffuse large B-cell lymphomas (DLBCL), follicular lymphomas (FL) and enteropathy-type T-cell lymphomas (ETTL) as well as inflammatory diseases such as Crohn’s disease and coeliac disease. Campylobacter specific 16S ribosomal DNA (16S rDNA) was amplified by PCR and the products were sequenced to confirm Campylobacter sequences and to classify Campylobacter species. The results are summarised in Table 1. Campylobacter 16S rDNA was amplified from 11/22 IPSID samples and from 12/121 other samples including 6/11 DLBCL. The sequence analysis showed that, in IPSID, the sequences were mostly specific for C. Jejuni whereas generic sequences consistent with several other Campylobacter species were obtained in other lymphomas. The IPSID cases originated from a very wide range of geographical locations suggesting that the association with C.jejuni was disease specific rather than environmental. Our results confirm a close association between C.jejuni infection and IPSID but also suggest that Campylobacter sequences can be found in other intestinal lymphomas, in particular DLBCL.

Table 1

Diagnosis 16S rDNA (VC6) C jejuni C jejuni or related Not sequenced IPSID 11/22 7 2 2 MCL 2/8 0 2 0 DLBCL 6/11 0 4 2 MALT 1/15 0 0 1 FL 0/13 0 0 0 ETTL 0/5 0 0 0 Crohn’s 0/12 0 0 0 Coeliac 1/13 0 0 1 Others 2/44 2 0 0     TOTAL 23/143 9 8 6
The Role of Sprouty Family Members in Hematopiesis in Zebrafish and Mammals.
Authors: Mendenhall Eric M. , Eckfeldt Craig E. , Ekker Stephen C. , Verfaillie Catherine M.
Published: April 2020

In an effort to identify novel regulators of hematopoiesis, we identified a Sprouty family member, SPRY1, significantly enriched (>1.5 fold, p-value < 0.03) in human hematopoietic stem cells (HSC) compared to more committed hematopoietic progenitor cells (HPC) using Affymetrix-based global gene expression profiling (see Eckfeldt et al. abstract). Predominantly FGF, but also VEGF, and EGF signaling induces expression of Sprouty family members (SPRY1-4). SPRY proteins form heterodimers with other Sprouty members along with homodimers. They and are intracellular antagonists and agonists of receptor tyrosine kinase signaling via the RAS/MAPK pathway. SPRY proteins are best characterized as antagonists of the FGF, EGF, and VEGF pathways. As part of a high-throughput functional analysis of the genes identified by human gene array in zebrafish, we evaluated the role of the only identified zebrafish homologue for Sprouty, SPRY4, in zebrafish hematopoiesis. Using morpholino (MO) antisense technology we knocked-down expression of zebrafish SPRY4 during early zebrafish development in a double transgenic GATA1:dsRed/Fli1:eGFP zebrafish line. The SPRY4 morpholino injected fish (SPRY4[superscript]MO[/superscript]) showed an almost complete loss of blood with normal vasculature, which was confirmed by injection of a second unique morpholino designed against SPRY4. [italics] In situ [/italics]hybridization of the hematopoietic markers SCL, GATA1 and Ikaros were all significantly reduced or absent at multiple developmental stages in the SPRY4[superscript]MO[/superscript] embryo. Together with the normal development of the vasculature, this data indicates SPRY4 acts early in hematopoiesis and after the specification of the hemangioblast. [italics] In situ[/italics] hybridization for SPRY4 demonstrated expression in the otic vesicle, tail bud and lateral plate mesoderm, the first site of hematopoietic stem cells. Coinjection of SPRY morpholino and low doses of VEGF morpholino did not rescue the SPRY blood phenotype, indicating that the blood phenotype is not likely through the VEGF pathway. Previous gain-of-function experiments injecting SPRY4 mRNA showed an expansion of hematopoietic progenitors (Furthauer et al. Development 2001), a phenotype we confirmed with overexpression of SPRY4 cDNA. Experiments are ongoing to overexpress human SPRY1 or zebrafish SPRY4 cDNAs in mammalian cells to determine if the hematopoietic phenotype correlates with the zebrafish data. Additional genes differentially expressed in human HSC and HPC evaluated via the high throughput zebrafish screen will also be presented. This is the first description of functional validation of a gene from a human microarray in zebrafish, an essential step in obtaining meaningful in vivo data from global gene profiling studies.