bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulated TCR Zeta and Improved the IFN-γ Secretion and Cytotoxicity in T Cell from Patients with AML after IL-2, IL-7 and IL-12 Stimulation
Authors: Chen Shaohua , Shi Li , Yang Lijian , Lu Yuhong , Luo Gengxin , Zhu Kanger , Li Yangqiu
Published: October 2019

T cells from patients with acute myeloid leukemia (AML) display antigen receptor–mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain, a subunit of the TCR/CD3 complex. Up-regulate TCR signaling activation by TCR zeta recombinant vector transfection was showed to reverse IL-2 production and increased the activation in T cells from patients with chronic myeloid leukemia. This study was undertaken to explore the possibility that increased TCR zeta gene expression stimulated with immune cytokines may upregulate T cell receptor signaling activation in T cells from patients with AML. CD3+ T cells sorted from peripheral blood of 8 cases with AML were induced with different immune cytokines, including IL-2, IL-7, IL-12, anti-CD3 and anti-CD28 antibodies in vitro. The result showed that TCR zeta chain protein was significantly upregulated after stimulation with IL-2 + IL-7 at 72 hours (p<0.05) by western-blot analysis, the expression of Zap-70 which is TCR zeta-associated protein was increased apparently after stimulation by combined IL-7 and IL-12 at 72 hours (p<0.05). A higher level of interferon (IFN)-γ secretion was showed obviously with single stimulating factor or combination with different cytokines (p<0.05), especially in IL-12 + IL-7, CD3 + CD28 + IL-2 or CD3 + CD28 + IL-7 combination. In addition, the cytotoxicity of T cell from patients with AML was achieved after stimulation with different cytokines combinations at 7 days, especially in CD3 + CD28 + IL-2 group and CD3 + CD28 + IL-2 + IL-12 group. These result showed that downregulation of the TCR zeta chain in T cell from patients with AML is a reversible event. Combined different cytokines could restore TCR zeta chain deficiency and enhance IFN-γ production and cytotoxicity in T cells from AML patients. It is possible that upregulated TCR zeta chain expression in T cell from AML patients will effectively increase or recover their activation of antileukemia cytotoxicity.

Disclosures

Chen: National Natural Science Foundation of China (no. 81100353, 81270604), the Fundamental Research Funds for the Central Universities (No. 21611447, 21612116), And Medical Science Foundation of Guangdong Province(A2011325): Research Funding.
Differentiated Expression of microRNAs May be Regulated By the Vascular Endothelial Growth Factor in Acute Myeloid Leukemia
Authors: Ye Xiujin , Zhu Lixia
Published: October 2019

Background: Overexpression of the vascular endothelial growth factor (VEGF) is characterized in acute myeloid leukemia (AML), which stimulates endothelial cell mitogenesis and cell migration. MicroRNAs play a crucial role in the initiation and progression of malignancies. This study aimed to find the differential expression profiling of microRNAs that related with the expression level of VEGF in AML, and further explore the mechanism of VEGF in the course of AML.

Methods: The overexpression of VEGFa lentiviral vector LV-VEGFa-O or the knockdown of VEGFa lentiviral vector LV-VEGFa-shRNA or the empty lentiviral vector LV-NC was transfected into AML cells HL60 and U937. MicroRNAs was analysed by using a commercial microarray platform (miRCURY LNA™ v.18.0, Exiqon) for microRNAs. A set of selected microRNAs and VEGF mRNA were further analyzed by using quantitative real-time PCR in 45 bone marrow (BM) from primary AML patients.

Results: We have successfully constructed AML cell lines stably overexpressing VEGFa or knockdown VEGFa. Overexpression of VEGFa followed by a few changes of microRNAs profiling, that has-miR-1273f and has-miR-4679 were up-regulated, while eb-miR-BART10-3p and has-miR-3925-3p were down-regulated. However, knockdown of VEGFa significantly affected the expression profiling of microRNAs in AML cells. Thirty-three microRNAs were lower expression level than cells transfected with LV-NC (Figure 1). According to the expression level of VEGF, the AML patients were divided into two groups: VEGF-high group and VEGF-low group. We found five microRNAs (miR-20a, miR-93, miR-16-5p, miR-17 and miR-124-5p) were higher expression level in VEGF-high group than in VEGF-low group (p<0.05) (Figure 2).

Conclusion: Five microRNAs (miR-20a, miR-93, miR-16-5p, miR-17 and miR-124-5p) were positively correlated with VEGFa in AML. It is necessary to further explore the role of the selected microRNAs in the signal pathways of the regulation of VEGFa in the course of AML.

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
SKIP Is Underexpressed in AML Leading to Sphingosine Kinase Hypofunction
Authors: Ghazaly Essam , Smith Paul , Gnanaranjan Chathunissa , Young Bryan D , Joel Simon , Gribben John G. , Taussig David C
Published: October 2019

Background: Sphingosine kinase interacting protein (SKIP) has been shown to be mostly silenced by hypermethylation in AML [1]. SKIP interacts with and regulates the function of sphingosine kinase (SK) enzyme. SK activity results in phosphorylation of sphingosine (SPH) to form sphingosine 1 phosphate (S1P), which promotes cell survival and resistance to apoptosis. On the other hand, S1P precursors ceramide (CER) and SPH mediate antiproliferative and apoptotic responses. SKIP has been reported to negatively regulate SK1 activity in fibroblasts. Therefore, we investigated the consequences of SKIP silencing in primary AML cells. In addition, we studied the effects of SKIP re-expression in leukemic cell lines.

Methods: CTS and K562 cells were transfected with SKIP gene using standard techniques. SKIP is normally silenced in both cell lines. Intracellular and extracellular S1P, SPH and CER were measured by UPLC-MS/MS. In addition, intracellular SK activity was determined based on C17 S1P production from C17 SPH substrate. Chemosensitivity to doxorubicin, Imatinib and Ara-C in transfected cells was also studied.

Results: In Primary AML cells, intracellular S1P and CER concentrations were reduced compared to G-CSF-mobilized peripheral blood mononuclear cells. In addition, SK activity was found to be downregulated in AML primary cells. When we transfected leukemic cell lines with SKIP gene, S1P and CER showed at least 2 fold increase in intracellular and extracellular basal levels compared to vector alone control (Figure 1). Further studies confirmed a significant increase in intracellular SK activity in SKIP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase in S1P and CER was associated with increasing apoptotic signals as evidenced by Annexin V staining and cleaved PARP expression. Moreover, chemosensitivity to Imatinib and AraC was significantly increased in SKIP transfected cell lines. These experiments confirm the regulation of SK1 function by SKIP.

Conclusion: These data indicate that SKIP is downregulated in AML leading to reduced SK activity, which ultimately inhibits the apoptosis response.

Figure 1. Effect of SKIP transfection on intracellular concentrations of S1P. Figure 1. Effect of SKIP transfection on intracellular concentrations of S1P.

1. Saied, M.H., et al., Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats. PLoS One, 2012. 7(3): p. e33213.

EAG and PS contributed equally

JG and DT contributed equally

Disclosures

No relevant conflicts of interest to declare.
Genomic Analisys of Notch Mutations in a Case of Alagille Syndrome with Acute Lymphoblastic Leukemia
Authors: Bernardi Simona , Tononi Paola , Marin Vargas Sergio , Bassi Giulio , Takam Kamga Paul , Perbellini Omar , Ambrosetti Achille , Meneghini Vittorio , Scaffidi Luigi , Martinelli Giovanni , Iacobucci Ilaria , Pizzolo Giovanni , Delledonne Massimo , Krampera Mauro
Published: October 2019

Alagille syndrome (ALGS), or arteriohepatic dysplasia, is a congenital multisystem disease due to Notch signalling pathway mutations, most commonly affecting JAGGED-1 (ALGS type 1), and more rarely NOTCH-2 (ALGS type 2), leading to hepatic, lung, renal and ocular dysfunction (chronic cholestasis, peripheral pulmonary artery stenosis, dysplastic kidneys pigmentary retinopathy), and skeletal abnormalies (minor vertebral segmentation, characteristic facies, posterior embryotoxon/anterior segment defects). ALGS is an autosomal dominant disease, but it is characterized also by variable penetrance and clinical expression and somatic/germline mosaicism.

A 20-year-old man with ALGS was admitted to the University Hospital of Verona because of pancytopenia. Following analyses led to the diagnosis of Philadelphia chromosome/bcr-abl-negative, CD10-positive, B-lineage acute lymphoblastic leukemia (common B-ALL).

In order to identify the genetic components involved in this complex phenotype, we sequenced the exome of a bone marrow sample collected from the patient. By genome interpretation with Knome pipeline applied to the reference genome UCSC hg19, we found missense variants both in NOTCH-2 (E38K) and JAGGED-1 (P871R) genes that are mainly involved in the syndrome, although their effect on protein function was predicted not to be deleterious. However, we detected putative damaging mutations in genes such as PAX5 (R38H) and NOTCH-1 (K1821N), which might be strongly related to the observed disease. In fact, PAX5 is a member of PAX protein family of transcription factors implicated into regulation of early development that binds NOTCH-2 and likely altering its functionality. On the other hand, NOTCH-1 is involved in cell growth and proliferation and thus the predicted alteration of function of the corresponding protein may have an important role in neoplastic transformation.

Disclosures

Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.
Synthesis and Preliminary Assessment of 99m tc-HYNIC-Fab´s (Rituximab) for Non-Hodgkin Lymphoma Diagnosis
Authors: Camacho Ximena , Machado Camila , Banchero Agustina , García Maria Fernanda , Calzada Victoria , Fernández Marcelo , Moreno María , Gambini Juan Pablo , Alonso Omar , Chammas Roger , Cabral Pablo , Riva Eloísa
Published: October 2019

Introduction: Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy; more than 90% are B cell lymphomas and express CD-20 antigen. Rituximab® (RTX) is an IgG1κ chimeric anti-CD20 mAb that binds specifically to the CD20-antigen on B lymphocytes. Our group previously reported that 99mTc-RTX represents a promising molecular imaging agent for NHL [1]. When used for tumor imaging, intact IgG exhibits high liver uptake. Antibody fragments (Fab´s) are quickly eliminated from blood and normal tissues (except kidneys), achieving high tumor/blood and tumor/normal tissue ratios with renal clearance. The development of radiolabeled Fab´s directed against specific targets may become a new strategy for NHL staging and surveillance.

Objective: To radiolabel Fab´s (RTX) with 99mTc and to perform its chemical and biological evaluation.

Methodology: We performed antibody fragmentation with papain and, once purified, fragments were identified by MaldiTOF/TOF and derivatized with Suc-HYNIC as a bifunctional coupling agent. A mixture of Tricine/SnCl2.2H2O was added to Fab´s (RTX)-HYNIC and radiolabeled with 99mTcO4-. Radiochemical purity was determined by HPLC. The in-vitro radiochemical stability of the radiolabeled Fab´s were analyzed in saline and serum up to 4 h. In-vitrobinding and competition assays were performed using Ramos and Raji cell lines up to 90 min. Biodistribution studies were evaluated in normal Balb/c mice and in Raji tumor-bearing Nude mice at 0.5 and 1 h.

Results: Radiochemical purity of radiolabeled Fab´s were ≥90%. The in-vitro radiochemical stability studies showed that the radioconjugate was stable and no significant transchelation was detected. In-vitro binding and competition assays confirm that after its derivatization and radiolabeling, Fab´s (RTX) retained its specificity of binding to CD-20 antigen. This results confirm that Fab´s (RTX) affinity for CD20+ NHL cells remained unaffected after its derivatization. In-vivobiodistribution studies show that radiolabeled Fab´s has renal uptake with neglectable uptake in other organs, indicating that the primary route of clearance is renal. Lymph-node/muscle ratios of 4.00 and 2.55 at 0.5 and 1 h post injection, respectively.

Conclusions: Fab´s (RTX) were easily and rapidly labeled demonstrating good stability and radiochemical purity. Based on lymph-node uptake and lymph-node/muscle ratios, 99mTc-HYNIC-Fab´s (RTX) may be useful for tumor molecular imaging agent for NHL.

Disclosures

No relevant conflicts of interest to declare.
Preventive Effects of Cardotoxicities By Liposomal Anthracyclines non Pegilated in 95 Perspective Patients Affected By Lymphoma and Monitored By Biomarkers and Ecocardiography
Authors: Gini Guido , Olivieri Jacopo , Bocci Caterina , Sampaolo Michela , Trappolini Silvia , Olivieri Attilio , Offidani Massimo , Rupoli Serena , Poloni Antonella , Scortechini Anna Rita , Capelli Debora , Montanari Mauro , Scortechini Ilaria , Perna Gian Piero , Leoni Pietro
Published: October 2019

Anthracyclines (AC) still constitute the mainstay of the 1st line treatment in lymphoma: their use, however, is limited by the occurrence of Cardiac Toxicity (CT). The exact prevalence of AC CT occurring after widely used regimens such as R-CHOP or ABVD is unknown and there is uncertainty about the best monitoring method and possible prophylactic or therapeutic interventions.

METHODS: We started a prospective observational trial in lymphoma patients undergoing treatment with conventional or liposomal AC. We used a comprehensive approach to monitor for AC CT, using a telemedicine(TM) system integrating echocardiography, ECG and biomarkers (Troponin I - TnI).

RESULTS: In this final analysis, 95 patients completed the planned treatment (52 males and 43 females). Median age was 56.03 years (range 19.1 to 78.5 years), and 36 patients were > 65 years. 23 were HL and 72 NHL (DLBCL was the most represented subtype with 47 cases). Liposomal AC was used in 31 patients and classical AC in 64, with mean cumulative doses of 283.33 and 272.76 mg/sqm, respectively.

10/95 patients (11%) developed a TnI rise above 0.08 ng/ml and 39/95 (41%) above 0.03 ng/ml. With both cut-offs, the rises occurred more frequently at cumulative doses >200 mg/sqm. The major arising occurred in the group underwent classical AC, while in the group underwent liposomal AC although the value before first infusion was in 10% more than 0.03 at the cumulative dose of 300 mg /sqm there's a plateau of troponin value.

Thanks to this monitoring system we noticed 2 Acute cardiac toxicity events with resolution in 100% of cases. Furthermore in those cases where has registered a subclinical CT has begun a prompt Cardiological therapy by ACE inhibitors and Beta Blocker to reduce the relative risk of Cardiological events.

CONCLUSIONS: Even with low cumulative doses, with a median follow up of 13 months, subclinical signs of AC CT were found in at least 11% of patients. The use of liposomal AC allow the safe treatment of patients with a previous heart disease diagnosis rather it seem protective in the higher cumulative doses.

A longer follow up will be able to clarify the impact of arising of TnI more than 0.03 and 0.08 on the developing of AC CT in all our series.

On the basis of our experience a multicentric trial has begun on behalf Italian Lymphoma Foundation (FIL)

In a low-risk setting for AC CT, a monitoring strategy combining clinical, imaging, instrumental and biomarker data seems to enhance the sensitivity of separate methods. This strategy is feasible and resource-saving thanks to the integration in a TM system.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Langerhans Cell Histiocytosis: A Single Institution Retrospective Analysis of Eleven Patients
Authors: Stefoni Vittorio , Broccoli Alessandro , Casadei Beatrice , Derenzini Enrico , Gandolfi Letizia , Pellegrini Cinzia , Quirini Federica , Argnani Lisa , Zinzani Pier Luigi
Published: October 2019

Langerhans cell histiocytosis (LCH), is a rare disorder which has a substantially unknown etiology, pathophysiology, and may manifest through a variety of clinical presentations ranging from solitary eosinophilic granuloma to severe multisystem disease. LCH is more common in children, although it can affect any age; the most common sites of involvement are bone, skin, and lung.

From a histological point of view LCH derives from accumulation of proliferating cells with surface markers and ultrastructural features similar to cutaneous Langerhans cells, intermixed with inflammatory cells, particularly eosinophils.

Below, a retrospective analysis of LCH patients treated at our institution.

Between 1997 and 2013 we have treated 11 LCH patients, including 6 females and 5 males with a median age at time of diagnosis of 42.9 years (range 22.2-62.3). All diagnoses were reviewed by our pathologist.

With regard to the site at onset, 9 patients had bone involvment, among these, four patients had only bone involvment, the other five patients also lung, oral cavity and lymph nodes.

At time of onset 4 patients showed no symptoms, while the remaining 7 showed a variety of symptoms ranging from B symptoms to tinnitus, dizziness, and other neurological symptoms such as diplopia. Among the study group 6 patients had multisystemic involvement.

All patients except one had CT scan performed before, during, and at follow-up, the remaining patient was studied and followed through follow-up with PET scan.

As first-line therapy 8 patients underwent chemotherapy, 2 patients radiation therapy, 1 patient required only steroid therapy. The most frequently used chemotherapy regimen for these 8 patients was MACOP-B, a third generation, CHOP-like regimen.

Responses to first-line therapy were as follows: 7 complete remissions (CR), resulting with chemotherapy (5), radiation therapy and steroid therapy, two partial remissions (both obtained with chemotherapy) and two stable diseases (1 with chemotherapy and 1 with radiation therapy).

Two patients relapsed, of whom one has ran several lines of chemotherapy, including autologous stem cell transplantation. Both are alive at the time of the last follow-up.

To date all patients are alive but one, who died of pulmonary embolism while he was in stable disease. Six patients are in CR (60%), two in SD (20%) and two in PD (20%).

In conclusion, our monocentric experience of 11 LCH patients confirms what reported in the literature in terms of heterogeneity of presentation, age, sites of involvement, symptomatology and treatment demanded. Coming to the the results our retrospective analisys shows that ten of the eleven study population patients (90.9%) are to date still alive after a significant median time of follow-up; six out of these ten patients (60%) are in CR.

Disclosures

No relevant conflicts of interest to declare.
The Assessment of Risk Factors for Thrombosis in Children Treated for Non- Hodgkin Lymphoma
Authors: Smalisz-Skrzypczyk Katarzyna , Klukowska Anna , Malinowska Iwona , Sobocinska-Mirska Agata , Romiszewski Michal , Matysiak Michal
Published: October 2019

Thrombosis occurs quite frequently in patients with NHL, it is

a complication resulting from the presence of neoplastic disease and vascular catheter

and a side effect of the treatment (corticosteroids, L-asparaginase).

The aim of the study was to assess the factors predisposing to thrombosis in children

treated for non-Hodgkin's lymphomas.

6 cases of thrombosis in 31 children (19%) treated in the Department in 2007-2013 due to the NHL were analyzed. All patients with thrombosis were assesed: age of onset, location, cause of the thrombosis and the anticoagulant treatment. A analyzed patients underwent testing

for congenital thrombophilia.

Age at onset of thrombosis ranged from 2 to 17.5 years, median 9.5 years.

Arterial thrombosis occurred in 1 (17%), venous thrombosis in 5 (83%) patients. Thrombosis

occurred in 4 children with lymphoblastic lymphoma (LBL), 1 child with large cell anaplastic lymphoma (LCAL) and 1 child with primary mediastinal B-cell lymphoma (PMBCL). Most of the cases were concerned to central catheter (n = 5, 83%). Massive thrombosis

occurred in 2 children (33%). There was no recurrence of thrombosis. The most common

causes of thrombosis were: the presence of the central catheter (n = 5, 83%) and treatment with L-asparaginase (n = 4, 66%). The presence of mutation on the factor

V Leiden was discovered in 1 patient. 2 patients were treated with a recombinant tissue plasminogen activator in systemic infusion (16%), low molecular weight heparin was used

in 5 patients (83%), then changedd to warfarin in 3 (50%) children.

The bleeding complications of the treatment were not observed. All the children showed total

resolution of thrombi.

Conclusions:

1 The predominant location and cause of thrombosis in children with NHL is a central catheter

.2 Thrombosis occurs the most often in children with LBL.

3 The risk factor of thrombosis in children is the treatment with L-asparaginase

4 It was not found that congenital thrombophilia iss an additional risk factor for thrombosis

in children with NHL.

5 Thrombosis in children with NHL responds well to treatment with anticoagulants.

Disclosures

Klukowska: Octapharma AG: Investigator Other.
Outcome of Lymphocyte Predominant Hodgkin Lymphoma
Authors: Khalid Arifa , Zafar Huma , Wali Rabia , Riaz Shazia , Badar Farhana
Published: October 2019

BACKGROUND: Lymphocyte predominant Hodgkin Lymphoma (LPHL) , also known as Hodgkin paragranuloma or lymphocytic and histiocytic predominant Hodgkin disease. It accounts for 5% of the lymphoma in the western world Since LPHL is a rare type of lymphoma different treatment modalities used to treat it including radiotherapy alone, combined chemotherapy and radiotherapy, chemotherapy alone and recently the use of Rituximab anti CD20 monoclonal antibody as LPHL expresses CD20 marker . The results are variable. Patients of LPHL also have frequent relapses beyond 5 years of treatment as compared to cHL and high rates of transformation to DLBCL .

OBJECTIVE:

To review outcome of LPHL patients treated with various chemotherapy regimens ( ChlVpp/ABVD) and (ABVD/COPDac) alone or in combination with radiotherapy.

METHODS AND MATERIALS: Data of total 301 patients reviewed . Out of which 15 were of LP Hodgkin (4.98%). Retrospective review of 15 patients of LP Hodgkin Lymphoma patients enrolled between January 2006 to December 2013 was done. Data regarding age, sex, stage of disease , treatment and outcome was analyzed.

RESULTS: Total 15 LP HD patients with age range from 2 to 18 years were included from 2006 to 2013. Mean age of patients was 10 years. Median was 9. Out of these 13 (86.7% ) were males and 2 (13.3%) females, most of them are of stage III (40%). Majority , 9 patients (60 %). were treated with ChlVpp/ABVD protocol. XRT was given to 5 patients (33.3%). Total 12 (80%) are still on follow up and 3 (20%) relapsed.

CONCLUSION: Patients of Lymphocyte predominant Hodgkin Lymphoma treated with chemotherapy had a better outcome .

Disclosures

No relevant conflicts of interest to declare.
a Pediatric Primary Renal Lymphoma Case Presenting with Acute Renal Failure Findings
Authors: Akici Fahrinnisa Ferhan , Tugcu Deniz , Aydogan Gonul , Salcioglu Zafer , Sayilan Sen Hulya , Akcay Arzu , Kiyak Aysel , Dogan Oner , Kilinc Yurdanur -
Published: October 2019

Lymphomatous involvement of the kidneys is a common manifestation of systemic non-Hodgkin’s lymphoma (NHL). In contrast, lymphoma originating within the kidneys is a rare event in children. In this study we report a case of primary renal lymphoma presenting with acute renal failure in a 14-years-old boy. According to histopathological and immunohistochemical examinations, the diagnosis of B-cell renal lymphoma was established.

A fourteen years old boy presented to our Pediatric Hematology - Oncology Unit, with 4-month history of weakness and anorexia. He had mild abdominal pain. Physical examination revealed no significant abdominal or systemic. The laboratory data were white blood cell count 4100/mm3; hemoglobin level 10.8 g/dL, mean corpuscular volume 107 fL; mean corpuscular hemoglobin concentration 33.6 g/dL); platelet count 179,000/mm3;erytrocyte sedimentation rate 50mm/h; blood urea nitrogen level 142 mg/dL; creatinine level 4.85 mg/dL; lactate dehydrogenase level 465 IU/L. There were no pyuria or hematuria, and urinary culture and stain results were negative. An abdominal ultrasonography revealed an increase in bilateral kidney sizes. Abdomen computed tomography scan showed an increase in thickness of renal parenchyma with hypodense appearance and with bilateral mild ectasia in pelvicalyceal system.

An ultrasonography guided renal biopsy demonstrated precursor B-cell lymphoblastic lymphoma. Hematoxylin and eosin section of these renal masses revealed neoplastic lymphoma cells with increased nuclear-cytoplasmic ratio, prominent nucleoli and mitotic figures.The lymphoma cells stained positivie for terminal deoxynucleotydyl transferase and B-lymphocyte marker CD10 and CD20 immuno-histochemical stain. Extrarenal abdominal involvement was excluded by imaging techniques. Bone marrow examination was normal. He was diagnosed as non-Hodgkin’s lymphoma, pre B-cell type and was treated with multidrug chemotherapy (induction regimen of prednisolone, vincristine, daunorubicin and L-asparaginase) and prophlactic intratecal methotrexate according to British-French-Munster (BFM) protocol. After the initial therapy, renal function test were improved rapidly and during consolidation and maintenance chemotherapy, they continud as normal. The patient is still in complete remission and was followed normal renal function with no sign of disease recurrence 5 years later diagnosis..In conclusion, diagnose of PRL is difficult. Percutaneous kidney biopsy is the most expeditious method towards the establishment of an early diagnosis for appropriate chemotheraph. Early diagnosis and urgent therapy, with supporting therapy are necessary for good prognosis.

Disclosures

No relevant conflicts of interest to declare.
Intravenous Administration of Rituximab in the Treatment of Primary Cutaneous B-Cell Lymphomas (PCBCLs): A Retrospective Study
Authors: Paterno Giovangiacinto , Zizzari Annagiulia , Nasso Daniela , Tonialini Lorenzo , Angeloni Cecilia , Vaccarini Sara , Postorino Massimiliano , Pupo Livio , Franceschini Luca , Provenzano Ida , Rizzo Manuela , Giannì Laura , Anemona Lucia , Mauriello Alessandro , Lombardo Giuseppe Alfonso , Scala Enrico , Cantonetti Maria
Published: October 2019

Introduction. Rituximab has been demonstrated to be effective either as intralesional or as systemic therapy in PCBCL, We report our experience in the treatment of PCBCL with intravenous Rituximab.

P atients and Methods From February 1999 to February 2014 we treated 75 patients: 47 Follicle Center Lymphoma, 23 Marginal Zone Lymphoma, 5 Diffuse Large Lymphoma Leg type. The stage was T1 in 38 pts, T2 in 22 pts and T3 in 15 pts. In 24 patient prior treatment included: CHT (11), Radiotherapy (4), Surgery (4) or alpha2Interferon (IFN) (5). Rituximab at dosage of 375 mg/m2 for a minimum of 4 cycles, was administered alone (51 patients) or in association with CHT (13). RT (2) or IFN (3).

Results. No patient presented adverse effects during the Rituximab infusion. A reduction of circulating B lymphocytes was observed for 11 months, on the average, without an increased risk of infections. No added toxicity was observed in patients treated with Rituximab plus CHT. Overall response rate was 97,3% (CR 82,6 %, PR 14,6 %). Five–years Overall Survival (OS) was 86,9% with Disease Free Survival of 57%. According to stage OS was in T1 94,3%, in T2 90,5%, in T3 73,6%. (T1+T2 vs T3: p<0,05).

Conclusions. Rituximab is effective and safe in the treatment of PCBCL even in heavily-treated or elderly patients. In our patients only the stage of disease was significant for the prognosis. A higher number of patients are necessary to indicate Rituximab in biological and clinical subsets of patients as a front-line therapy.

Disclosures

No relevant conflicts of interest to declare.
Turning a Tumor into a Vaccine Factory: In Situ Vaccination for Low-Grade Lymphoma
Authors: Marron Thomas , Bhardwaj Nina , Crowley Elizabeth , Keler Tibor , Davis Thomas A. , Brody Joshua
Published: October 2019

BACKGROUND: Lymphomas are the 5th most common cancer in the U.S. and most are incurable with standard therapy. Previously, we completed three trials of ‘in situ vaccination’ - combining low-dose radiotherapy (XRT) with intratumoral administration of TLR9 agonist (CpG). We demonstrated induction of anti-tumor CD8 T cell responses and clinical remissions of patients’ non-irradiated sites of disease, lasting up to 4+ years. One limitation may have been the paucity of intratumoral dendritic cells (DC). DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells.

METHODS: Flt3L– the predominant DC differentiation factor– induces tumor leukocyte infiltration and regression of lymphoma tumors pre-clinically and a new formulation of this cytokine -CDX-301- was shown to mobilize BDCA-1 and BDCA-3 DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DC (the CpG-responsive DC subset). We initiated a phase I/II study of a new iteration of the in situ vaccine, adding Flt3L-priming and replacing the prior TLR9 agonist with the TLR3 agonist poly-ICLC (Fig 1A). The vaccine consists of:

-intratumoral Flt3L administration to increase DC within the tumor

-low-dose XRT to induce immunogenic tumor cell death and release tumor-associated antigens, and

-intratumoral poly-ICLC administration to activate tumor antigen-loaded DC.

RESULTS: Six patients have been enrolled, two patients have completed therapy. Treated patients had 2-200-fold increases in BDCA1 and BDCA3 intratumoral DC after Flt3L administration and marked DC activation after XRT and poly-ICLC. Both treated patients have had partial remissions of untreated sites per Cheson criteria, persisting or improving for >6 months after vaccination. These include regressions of bulky lymph nodes (Fig 1B), as well as peripheral blood (Fig1C) and bone marrow disease. A patient with significant peripheral blood tumor burden experienced >10-fold decrease in malignant B cells with concurrent increase in non-tumor B cells, suggesting a degree of cell specificity in the tumor-killing mechanism. Adverse effects have been mild.

CONCLUSIONS: Preliminary results suggest that the Flt3L-primed in situ vaccine is feasible, safe and immunologically and clinically effective, warranting further study.

Figure 1 Figure 1.

Disclosures

Off Label Use: CDX-301 - the purpose of which is to mobilize dendritic cells to an injected tumor site.. Crowley:Celldex Therapeutics Inc: Employment. Keler:Celldex Therapeutics, Inc.: Employment. Davis:Celldex Therapeutics Inc: Employment.
TLR9 and Dendritic Cells Are Required for CD8+ T Cell Responses to the AAV Capsid
Authors: Rogers Geoffrey L. , Herzog Roland W
Published: October 2019

CD8+ T cell responses to the adeno-associated virus (AAV) capsid have posed a significant barrier to transduction in clinical trials of AAV-mediated gene therapy for hemophilia B, as reactivation of a memory CTL response to the capsid is capable of eliminating transduced hepatocytes in the absence of immunosuppression. Recently, it has been suggested that innate immune responses induced by the toll-like receptor (TLR) pathway can influence the development of adaptive immune responses to AAV-mediated gene transfer. In particular, reports have implicated TLR2 (AAV capsid), TLR9 (AAV genome), and MyD88 (downstream signaling adaptor of both these TLRs). Herein, we have used a modified AAV2 with an insertion of the immunodominant MHC class I epitope of ovalbumin into the capsid (AAV2-SIINFEKL) to study the mechanism of CD8+ T cell responses to the AAV capsid. Using an H2-Kb-SIINFEKL tetramer reagent, we determined that anti-capsid CD8+ T cell responses depended on the TLR9-MyD88 pathway. While the frequency of circulating capsid-specific CD8+ T cells peaked around 7-10 days post-injection and subsided after about 21 days in wild type (WT) mice, tetramer-positive cells were not detected in TLR9-/- or MyD88-/- mice. The kinetics and magnitude of the response was unaltered in TLR2-/- mice. Mice deficient in STING, a downstream adaptor of multiple cytoplasmic DNA sensing pathways, also developed comparable capsid-specific CD8+ T cell frequencies to WT mice, suggesting that this is not a general effect of pattern recognition of DNA. Interestingly, the frequency of capsid-specific CD8+ T cells was not reduced in AP3-/- mice, which are deficient in type I IFN signaling downstream of TLR9. Adoptively transferred OVA-specific OT-1 T cells proliferated in WT but not TLR9-/- mice that received AAV2-SIINFEKL, confirming the importance of TLR9. The effect was antigen-specific, as OT-1 cells in WT mice that received AAV2 lacking SIINFEKL showed minimal proliferation comparable to TLR9-/- mice. In addition to pattern-recognition receptors, we also assessed the role of antigen-presenting cells in the CD8+ T cell response to capsid. The formation of capsid-specific CD8+ T cells was unaltered in mice that received gadolinium chloride to inactivate macrophages, or in B cell-deficient μMT mice. Depletion of B cells in WT mice prior to vector administration also failed to affect the anti-capsid CD8+ T cell response. However, transient depletion of dendritic cells (DCs) in CD11c-DTR mice resulted in a delayed development of capsid-specific CD8+ T cells. Seven days post-injection, DC-depleted mice had a significantly reduced frequency of tetramer-positive CD8+ T cells which recovered to normal by 10 days, likely due to the repopulation of DCs before the input capsid was completely cleared. Overall, our results show that TLR9 signaling, most likely in DCs, is required for the formation of de novo anti-capsid CD8+ T cell responses.

Disclosures

Herzog: Genzyme: AAV-FIX technology Patents & Royalties.
Long Term Trends in Chronic Myeloid Leukemia (CML) Following the FDA Approval of Tyrosine Kinase Inhibitors (TKIs), on Bone Marrow Transplant Rates and Clinical Outcomes - a Study of the Nationwide Inpatient Sample Between 1998-2011
Authors: Patel Parshva , Varadarajan Indumathy , Varma Gaurav , Christina Trotta , Ward Kristine , Jain Maneesh , Styler Michael
Published: October 2019

Background: Single-institution and international, retrospective studies have demonstrated a decrease in the number of allogeneic stem cell transplants (alloSCTs) in patients with CML in the first few years after the introduction of TKIs. However alloSCT remains the only curative therapy in patients who develop resistance to multiple TKIs, and in those who present with blast crises. The long-term impact of TKIs on the trend of SCTs in the United States in CML remains unexplored. Therefore, we sought to examine the trend in the number of SCTs, as well as the mortality and clinical complications for CML over the decade following the FDA approval of Imatinib.

Methods: We queried the Healthcare Cost and Utilization Project's Nationwide Inpatient Sample (NIS) between 1998 and 2011 using the ICD-9 code 205.1 for CML in the primary and secondary diagnosis fields. Only patients 18 years or older were included in the analysis. We monitored the trends in allo-SCT, mortality and clinical complications related to CML. A p value of <0.05 was considered significant. We have also attempted to analyze the trends in cost and length of hospitalization during this decade. Cost of hospitalization was adjusted for inflation in reference to the year 2011 and cost to charge ratio.

Results: A total of 53254 (weighted n=262964) hospitalizations for CML (Male 54.6% and average age 65.9+/-0.8 yrs) occurred from 1998-2011. There has been a 408% decrease in the rate of transplants and 79.9% decrease in in-hospital mortality since the introduction of TKIs (p<0.0001). There has also been a 48.3% reduction in the average length of hospital stay, despite which there has been a 17% increase in cost of hospitalizations for CML (p<0.0001). There have also been marked reductions in the complications related to CML with the advent of TKI therapy. There has been almost 200% reduction in blast crises (p<0.0001), a 120.8% decrease in splenic infarct/splenic vein thrombosis (p<0.0001), 56.1% reduction in epistaxis (p <0.0004).

Conclusion: This analysis supports the widely held belief that the introduction of TKIs has not only reduced the reliance on allogeneic transplants, but has also led to remarkable improvement in mortality and CML related complications. Increase in cost of hospitalization could be related to the overall increase in health care cost, 4.5% yearly from 1998-2000, 3.9% from 2000-2007 and 1.8% 2007-2010.

Disclosures

No relevant conflicts of interest to declare.
Characterisation of Novel Hypomorphic and Null Mutations in Klf1 Derived from a Genetic Screen for Modifiers of a-Globin Transgene Variegation
Authors: Huang Stephen , Gillinder Kevin R. , Ilsley Melissa , Magor Graham , Tallack Michael , Sorolla Anabel , Oey Harald , Harten Sarah , Clemens-Daxinger Lucia , Whitelaw Emma , Perkins Andrew C
Published: October 2019

Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated a-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for Modifiers of murine metastable epi-alleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation; i.e. an increased percentage of GFP+ circulating red blood cells. Both strains harbour point mutations in the erythroid specific transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain. D11 homozygous mice die in utero of anaemia at 14.5DPC. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have mild compensated microcytic anaemia which models the phenotype in a recently described human family. Mice Carrying the H350R mutation were interbred with Klf1+/- mice. Klf1H350R/- mice have severe perinatal haemolytic anaemia and marked splenomegaly. Furthermore blood haemoglobin content, haematocrit and red blood cell size (MCV) were significantly reduced in Klf1H350R/- mice compared to wildtype and D45 homozygous offspring of the same age. Analysis of Klf1H350R/- by flow cytometry showed an increase in circulating immature red blood cells. In the bone marrow, a lack of mature red blood cells was observed. Flow cytometric analysis of the spleen from Klf1H350R/- animals revealed an expansion of erythroid cells and a relative reduction in B and T Cells. Gel shifts assays of a recombinant Klf1 zinc finger protein with the H350R mutation showed normal binding to the b -globin promoter sequence but weak binding to the Alas2 intronic enhancer site. Furthermore b -globin gene expression was near normal whereas expression of other known Klf1 target genes was decreased. We will discuss how H350R disrupts function from ChIP-seq and RNA-seq in primary fetal liver tissue. Previous studies of the linkers in C2H2 zinc finger transcription factors have revealed their necessity as structural and regulatory components for the C2H2 class of transcription factors. Our results thus far show that the second linker of Klf1 has a role in maintaining the integrity of Klf1 function (at a subset of Klf1-occupied sites,) and does not act just as a spacer for the zinc fingers.

Disclosures

Perkins: Novartis Oncology: Honoraria.
Hematopoeitic Stem Cells and Their Progenitors Critically Require Autophagy to Promote Early Engraftment Following Allogeneic Stem Cell Transplantation
Authors: Lineburg Katie E , Leveque-El Mouttie Lucie , Le Texier Laetitia , Teal Bianca , Kuns Rachel D , Hill Geoffrey R , MacDonald Kelli P A
Published: October 2019

Stem cell transplantation (SCT) is the only curative therapy for the majority of hematological malignancies and relies on successful engraftment of donor hematopoietic stem cells (HSC) to reconstitute the patient's hematopoiesis and immunity. Graft-versus-host disease (GVHD) and graft failure are complications of SCT that contribute to significant morbidity and mortality. Recent studies have established a role for autophagy in the long-term survival and function of HSCs and as a regulator of hematopoiesis. We investigated the contribution of autophagy to HSC in the setting of allogeneic transplantation. Using LC3-GFP bone marrow as a graft source in the well-established B6 into F1 model of GVHD, together with imaging flow cytometry, we demonstrated increased autophagy (LC3-GFP punctae) within donor HSC and progenitor cells in GVHD compared to non-GVHD recipients. To assess the contribution of autophagy to HSC development and function we used mice deficient in Atg5, an essential protein for the autophagy pathway. In primary transplants, ATG5 KO foetal liver (FL) had reduced ability to engraft lethally irradiated congenic recipient mice compared to WT FL. Furthermore, competitive transplantation of 50% ATG5 KO FL cells together with 50% WT FL cells demonstrated that ATG5 KO cells had a reduced capacity to reconstitute compared to congenic WT FL cells. Finally, to address the role of autophagy in HSC in the setting of GVHD, we transplanted WT or ATG5 KO FL ± T cells into lethally irradiated B6D2F1 recipients. At D10 post-transplant ATG5 KO recipients failed to establish tri-lineage engraftment in peripheral blood and lacked expansion of myeloid precursor cells in the BM, leading to engraftment failure and significantly reduced survival compared to WT FL recipients (50% WT vs 10% Atg5 KO p<0.0001). This early engraftment failure was confirmed to be autophagy specific using VAVcre x Atg7fl/fl donor mice, in which Atg7 deficiency is restricted to hematopoietic cells. Furthermore, the essential requirement for autophagy in early progenitors and HSC was confirmed using LysMcre Atg7fl/fl mice, which ablate Atg7 from the granulocyte-macrophage progenitor population onwards. Survival, or engraftment, in mice receiving the LysMcre Atg7fl/fl donor graft did not differ significantly from those receiving a WT graft. We demonstrate that autophagy is increased in the GVHD setting and that without autophagy early myeloid precursors fail to provide short term reconstitution leading to primary graft failure and mortality. Thus, intervention to increase autophagy in these cells post-transplant may improve engraftment in the clinic.

Disclosures

No relevant conflicts of interest to declare.
Force-Induced Cooperative Unfolding of Two Distinctive Domains in a Single Gpibalpha Molecule
Authors: Chen Yunfeng , Ju Lining , Lou Jizhong , Zhu Cheng
Published: October 2019

GPIbα, a major member of the GPIb-IX-V complex, initiates a mechano-signaling pathway that leads to platelet intracellular calcium flux when binding to VWF at the A1 domain1. Exactly how this signal is transduced across the membrane is unknown. A recent work identifying the unfolding of a jextamembrane mechanosensitive domain (MSD) (Fig.1 A,C) suggested that this unfolding might play a role in the signal transduction of GPIbα2. Using molecular dynamics (MD) simulations to pull the GPIbα leucine-rich repeat domain (LRRD) from the VWF A1 domain, we observed unfolding of the LRRD (Fig.1 A) (manuscript under review).

Here we used a biomembrane force probe (BFP) to study single GPIbα unfolding (Fig. 1E,F). Platelet GPIbα was pulled by A1 coated on the probe through a ramping phase (pink, Fig. 1G) to a clamping phase to wait for bond dissociation under a 25-pN force (red, Fig. 1G). Two unfolding signatures were identified: i) ramped unfolding, featured by a sudden force kink in the ramping phase (dashed circle, Fig. 1G); ii) clamped unfolding, featured by an abrupt force drop in the clamping phase (dashed ellipse, Fig. 1G). Unfolding lengths from both signatures were measured from the force vs. time data (Fig. 1G), based on which the unfolding events of MSD (7-25 nm), LRRD (25-56 nm) or both together (56-80 nm) were differentiated. Intriguingly, LRRD unfolding was only observed in the ramping phase, while MSD could unfold in both ramping and clamping phases.

The frequency of observing both LRRD and MSD unfolding in the same cycle was much higher than the product of the frequencies for LRRD and MSD to unfold (the joint probability for both to unfold if they were independent), suggesting that the two GPIbα domains unfolded cooperatively (Fig 2A). Chi-square tests also rejected the hypothesis that MSD and LRRD unfolded independently (p =5.24×10-5). Separating the ramped and clamped unfolding of MSD and evaluating their respective cooperativity with LRRD unfolding demonstrated similar results (Fig 2A). Agreeing with these, both the ramped and clamped unfolding of MSD occurred much more frequently in the presence of LRRD unfolding (Fig 2B, left), and vice versa for the unfolding of LRRD (Fig 2B, right). Cooperative unfolding of LRRD and MSD may play a key role in transducing signals across platelet membrane.

References

1 Nesbitt, W. S. et al. The Journal of biological chemistry277, 2965-2972, doi:10.1074/jbc.M110070200 (2002).

2 Zhang, W. et al. Blood125, 562-569, doi:10.1182/blood-2014-07-589507 (2015).

Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures

No relevant conflicts of interest to declare.