bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Polymorphisms in the Folate Pathway and Risk of Childhood Acute Lymphoblastic Leukemia (ALL): MTHFR, MTHFD1, RFC1 and TS.
Authors: Yeoh Allen Eng-Juh , Kham Shirley Kow-Yin , Lu Yi , Foo Toon-Chai , Ariffin Hany , Quah Thuan-Chong
Published: October 2019

Folate is critical for DNA synthesis and methylation; dysfunction in methylating enzymes may result in genomic instability and DNA damage via the nucleotide synthesis pathways. We hypothesize that perturbation in the folate metabolism pathway may alter the risk of developing childhood leukaemia. We recently reported that methylenetetrahydrofolate reductase (MTHFR 677C>T) was associated with reduced risk in childhood ALL.

We aim to explore the value of a pathway approach, in this case exploring the impact of polymorphisms in multiple enzymes involved in the folate pathway, to determine its role in development of childhood ALL. As childhood ALL is highly heterogeneous and that some like TEL-AML1 and hyperdiploid subgroups have in utero origin, we postulated that the impact of folate metabolism may be greatest for in utero onset ALL. We analyzed the methylenetetrahydrofolate dehydrogenase (MTHFD1), reduced folate carrier (RFC1) and thymidylate synthase (TSER 5′-UTR 28bp repeat, TSER 3′-UTR 6bp deletion) in 360 children with ALL and 433 controls of Chinese and Malay origin (Ch=221, Mal=212). Genotyping was performed by RFLP-PCR, TEL-AML1 by RT-PCR, and hyperdiploidy either DNA index (≥1.16) or karyotyping. The latter was only available in some patients, especially in the Chinese.

Using logistic regression model analysis, MTHFR 677C>T and MTHFD1 1958G>A showed significant influence on leukemia susceptibility in the Chinese (n=235, p=0.005) and Malays (n=125, p=0.032) respectively.

In the Chinese cohort, MTHFR 677 CT and TT genotypes significantly decreased risk of childhood ALL as compared to CC wild type (p=0.004, OR=0.57, 95%CI=0.39–0.83), as well as in the hyperdiploidy subgroup (n=35, p=0.002, OR=0.28, 95%CI=0.12–0.65). The combined effect of RFC 80GA and AA genotypes with MTHFR 677CC wild type also decreased risk of childhood ALL in Chinese (p=0.05, OR=0.47, 95%CI=0.22–1.0). In the Malay cohort, the MTHFR 677 CT and TT genotypes played a protective role only in the TEL-AML1 subgroup (n=27) as compared to CC wild type (p=0.05, OR=0.24, 95%CI=0.06–1.0). However, interestingly MTHFD1 1958GG wild type decreased risk of childhood ALL as compared to MTHFD1 1958GA or AA genotypes (p=0.02, OR=0.54, 95%CI=1.11–3.05) when combined with MTHFR 677CC genotype. Haplotype analysis of TSER variants showed no significant effect in the 2 races.

Using a candidate pathway approach, we showed that polymorphisms in the folate pathway significantly modified the risk of developing childhood ALL especially the MTHFR, MTHFD1 and RFC1. This effect may arise from different genes of the same pathway and this may be different for different races. This supports a pathway way rather than a single-gene approach towards elucidation of cancer risk in childhood ALL.
Uniparental Disomy May Be Associated with NPM Mutations in AML with a Normal Karyotype.
Authors: Serrano Elena , Orantes Vanesa , Estivill Camino , Lasa Adriana , Brunet Salut , Aventin Anna M. , Sierra Jorge , Nomdedeu Josep F.
Published: October 2019

Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by an abnormal proliferation of the myeloid precursors and a maturation block. A large proportion of AML cases have either a normal karyotype or non-recurrent chromosomal alterations. Underlying genetic lesions of some of these cases have been characterized with the discovery of MLL-internal tandem duplications, activating FLT3 mutations and NPM mutations. Loss of heterozygosity (LOH) derives from the loss of one of the two alleles at a given locus and can be a sign of inactivation of tumor-suppressor genes. We performed a high-resolution genotype analysis on DNA obtained from 19 AML patients with a normal karyotype, both at diagnosis and in samples obtained in complete remission(assessed by multiparametric flow cytometry) using the 10K SNP Array (Affymetrix). Both LOH and copy number analysis, as well as visualization of these analysis were performed by means of the dChip software (M. Lin et al., Bioinformatics (2004), 20:1233–40). A mean call rate of 96.8%. SNP array-based LOH analysis revealed that 4 patients presented large regions of homozygosity at diagnosis which were absent from samples in complete remission. In all four patients copy number analysis indicated no gross chromosomal losses or gains, as was confirmed by conventional cytogenetic analysis. Therefore, it can concluded that the LOH observed in these four patients was due to the presence of uniparental disomy.

Simultaneous analysis of FLT-3 internal tandem duplications (FLT-3/ITD), FLT3- D835 mutations, NPM mutations and MLL rearrangements was performed using conventional molecular methods. Two of these patients (UPN2 and UPN12) had FLT-3/ITD in association with NPM mutations. UPN4 had a mutated form of NPM whereas in patient UPN16 FLT-3 and NPM genes were in the germ line configuration. All four cases were negative for MLL rearrangements and FLT-3-D835 mutations. These results suggest that NPM and FLT3 mutations may be associated with acquired somatic recombinations. It remains to be investigated whether there are loci preferentially involved by these events.

Uniparental disomy and genetic lesions in normal karyotype AML Patient LOH FLT3 NPM D835 MLL UPN2 13q Mutated Mutated Germ line Germ line UPN4 6pter-p12.212q13.12-qter Germ line Mutated Germ line Germ line UPN12 2p Mutated Mutated Germ line Germ line UPN16 complex Germ line Germ line Germ line Germ line
Impact of Additional Cytogenetic Changes and FLT3 Mutations on the Outcome of Patients with Newly Diagnosed Acute Promyelocytic Leukaemia Treated with Single Agent Arsenic Trioxide.
Authors: Mathews Vikram , Thomas Maria , Srivastava Vivi M. , Chendamarai Ezhilarasi , George Biju , Lakshmi Kavitha M. , Viswabandya Auro , Sitaram Usha , Chandy Mammen , Srivastava Alok
Published: October 2019

The efficacy of single agent arsenic trioxide (ATO) in the management of relapsed and newly diagnosed patients with acute promyelocytic leukaemia (APL) has been established. The impact of FLT3 activating mutations and additional cytogenetic changes in newly diagnosed patients with APL treated with single agent ATO has never been reported. Between January 1998 and April 2005, 98 newly diagnosed cases of PML-RARα positive APL were treated with a regimen of single agent ATO at our centre. FLT3 activating mutations were seen in 33% (FLT3-ITD - 21.3%, D835V- 11.7%). Of the 69 patients evaluated for additional cytogenetic findings, 76.8% had an isolated t(15;17) while 23.2% had t(15;17) along with additional cytogenetic findings.

The presence of a FLT3 mutation was significantly associated with a higher mean white cell count (18.11±23.74 vs. 8.04±21.44, P=0.024) and a bcr3 PML-RARα isoform (48.4% vs. 20.6%, P=0.006). There was also a significant delay in achieving a molecular remission (MR) among patients with a FLT3 mutation as noted by the number of patients who were in MR prior to onset of consolidation therapy (60.9% vs. 85.7%, P=0.031). There was no significant difference in the hematological remission rate (CR), time to CR or early mortality between the group with and without a FLT3 mutation. At a median follow up of 20 months (range: 4 – 97), the 3 year Kaplan-Meier estimate of OS, EFS and DFS for patients with a FLT3 mutation was 82.51±7.24, 77.01±8.6, 88.82±7.48 percent while for those without a FLT3 mutation it was 92.06±3.4, 80.45±5.86, 89.02±5.31 percent. Statistical analysis of these survival curves by a log rank test did not reveal any significant difference between the two groups [Figure 1].

The presence of additional cytogenetic changes was not associated with any significant differences in the clinical or laboratory parameters at presentation. It also did not have an impact on the CR, MR, EFS, DFS or OS. Neither FLT3 activating mutations nor additional cytogenetic changes have a significant impact on the outcome of newly diagnosed patients with APL treated with single agent ATO in the short term. Whether either of these two parameters will affect long term outcome remains to be seen.

Figure 1: Kaplan-Meier product limit estimate of EFS based on FLT3 mutation status (n=94). Figure 1:. Kaplan-Meier product limit estimate of EFS based on FLT3 mutation status (n=94).
Bone Marrow Stroma (BMS) Prevents Apoptosis of Lymphoma Cells by Upregulation of BMS-Derived B-Cell Activating Factor (BAFF) and Activation of NF-kB.
Authors: Lwin Tint , Hazlehurst Lori A. , Moscinski Lynn C. , Dalton William S. , Tao Jianguo
Published: October 2019

Stromal cells are an essential component of the bone marrow microenvironment that regulate or supports tumor survival. The bone marrow stroma has long been recognized as a "sanctuary site" for lymphoma cells during traditional chemotherapy. In this study, we demonstrated that adhesion of the B-cell lymphoma cell lines SUDH-4 and 10 to bone marrow stroma inhibited mitoxantrone-induced apoptosis. This adhesion-dependent inhibition of mitoxantrone-induced apoptosis correlated with decreased activation of caspases- 8 and 9, and cleavage of caspase 3 and PARP. EMSA analysis demonstrated significantly increased NF-kB binding activity in cells adhered to stroma cells compared to lymphoma cells in suspension. This DNA binding activity could be attributed to cell adhesion-mediated proteolysis of p100 NF-kappaB2 resulted in generation of active p52, which translocates to the nucleus in complex with p65 and RelB. Furthermore, co-culture with stromal cells also induced expression of the NF-kB-regulated anti-apoptotic molecules, XIAP, cIAP1 and cIAP2. In addition, because of the knowledge that BAFF shown to be critical for maintenance of normal B cell homeostasis as well as the survival of malignant B cells, we characterized the functional significance of BAFF in BMS-mediated drug resistance and survival. BAFF was detected on BMS cell line HS-5 and BMS cells derived from lymphoma patients by flow cytometry. Concentrations of BAFF were 3- to 25-fold higher in bone marrow aspirate than in peripheral blood. Lymphoma cell adhesion to HS-5 cells significantly increased BAFF secretion evidenced by both ELISA and immunoblotting. Addition of BAFF counteracted mitoxantrone-induced apoptosis, and elicited a reduction in spontaneous apoptosis in primary lymphomas via activation of NF-kB activation. Finally neutralization of BAFF by TACI enhanced lymphoma cell response to chemotherapy and overcame cell-adhesion mediated drug resistance, suggesting that lymphoma cells utilize BAFF as survival factor. These data indicate that stromal cells prevent apoptosis of lymphoma cells by upregulation of BAFF and induction of NF-kB-regulated anti-apoptotic proteins. Bone marrow-derived BAFF plays a key role in lymphoma cell survival and drug resistance in bone marrow microenvironment.
Peroxisome Proliferator-Activated Receptor Gamma Promotes the Survival of Human T Lymphoma Cells through Its Regulation of Cellular Metabolism.
Authors: Yang Chunyan , Song Zibo , Jo Seung-Hee , Csernus Balazs , Chadburn Amy , Hyjek Elizabeth , Wang Y. Lynn
Published: October 2019

Peroxisome proliferator-activated receptor gamma is a metabolic regulator involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPAR gamma have been found to induce apoptosis in a variety of tumor cell lines including lymphomas. However, apoptosis induction may not depend on the receptor since high doses of PPAR gamma agonists are required for this process. Using cells containing or lacking PPAR gamma, we reported previously that PPAR gamma attenuates apoptosis induced by growth factor withdrawal in a murine lymphocytic cell line via a receptor dependent mechanism. PPAR gamma exerts this effect by enhancing ability of cells to maintain their mitochondrial membrane potential during growth factor deprivation. In the current study, we demonstrate that PPAR gamma is expressed in human primary T lymphoma tissues and activation of PPAR gamma protects cells from serum starvation-induced apoptosis in human T lymphoma cell lines. Further, we show that the survival effect of PPAR gamma is mediated through its actions on cellular metabolic activities. In serum-deprived cells, PPAR gamma attenuates the decline in cellular ATP and suppresses accumulation of reactive oxygen species (ROS) in favor of cell survival. Moreover, PPAR gamma regulates ROS through its coordinated transcriptional control of proteins and enzymes involved in ROS production and scavenge. Introduction of PPAR gamma into a PPAR gamma-null T lymphoma cell line leads to increased cell survival. Meanwhile, knocking down the receptor in a PPAR gamma-positive lymphoma cell line reduces cell survival rate. Our studies identify cell survival promotion as a novel activity of PPAR gamma and suggest that high expression of PPAR gamma in lymphoma cells confers on them a survival advantage that renders cells resistant to growth factor and nutrient deprivation. These findings highlight the need for further investigation into the role of PPAR gamma in lymphoma and other types of cancer prior to widespread use of its agonists as anticancer therapeutics.
Constitutive Expression of SALL4B in Mice Confers Properties of Leukemic Stem Cells to Committed Hematopoietic Progenitors.
Authors: Chai Li , Cui Wei , Ma Yupo , Yang Jianchang , Kong Nikki , Kao Grace , Silberstein Leslie
Published: October 2019

The homeotic gene SALL4 is a transcription factor involved in multi-organ developments. Recently, our group and others have shown that murine Sall4 plays an important role in maintaining the properties of embryonic stem (ES) cells by interacting with Oct4 and Nonag. Knock down of Sall4 expression in mouse ES cells led to their spontaneous differentiation and loss of self-renewal ability. Parallel to these findings, using quantitative RT-PCR approach, we found that human SALL4 was preferentially expressed in the CD34+CD38− hematopoietic stem cell (HSC) population, and was dramatically down regulated in the CD34+CD38+ progenitor (HPC) population, suggesting that SALL4 functions similarly at the HSC level as it does in ES cells. In contrast to normal hematopoiesis, we found that SALL4 was constitutively expressed in the leukemia initiation stem cell (CD34+CD38− and CD34+CD38+), and its expression in human acute myeloid leukemia (AML) was deregulated. To investigate the effect of constitutive expression of SALL4, we have generated a transgenic mouse model with constitutive expression of SALL4B, one of the SALL4 isoforms. The SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequent AML that was transplantable. Further analysis on the pre-leukemic and leukemic SALL4B transgenic mice showed constitutive expression of SALL4B in the HSC and HPC subpopulations (GMP), which resulted in the expansion of the GMP population and eventually led to myeloid leukemia development. Activation of Wnt/beta catenin pathway and up-regulation of Bmi-1, the two key elements that are known to be involved in the self-renewal of HSC and leukemia stem cell (LSC), were found in the SALL4B transgenic mice. Taken together, our data suggests that constitutive expression of SALL4B contributes to leukemogenesis by conferring leukemia stem cell properties to committed murine hematopoietic progenitors. To our best knowledge, SALL4 is one the few genes, if not the only gene, that play an important role in the self-renewal properties of ES cell, HSC and LSC.
Blinded Mid-Treatment FDG-PET in Newly Diagnosed Aggressive Non-Hodgkin Lymphoma (NHL): First Results of a Prospective Multicenter Study.
Authors: Schot Bart W. , Zijlstra Josée , Hoekstra Otto S. , Pruim Jan , van Imhoff Gustaaf W. , Sluiter Wim , Raemaekers John M.M. , Oyen Wim , Van Gelder Michel , Eriks Olga , Huijgens P.C. , Vellenga Edo
Published: October 2019

In aggressive B-cell NHL, determination of early response to induction therapy by FDG-PET may improve risk stratification. In a multicenter prospective trial in patients with aggressive NHL (stage II–IV) treated with 2 or 3 weekly (R)CHOP chemotherapy, FDG-PET was performed after the third cycle of chemotherapy. Both clinicians and nuclear medicine physicians were blinded for PET and clinical outcome, respectively. Clinical decisions were based on conventional diagnostic methods (CDM) only. In total 114 patients were included of which 95 were evaluable. 19 patients were excluded because of patient refusal (n=7), early progression (n=7) or logistical reasons (n=5). Median age was 55 yrs (range 20–82), 72% had DLBC-NHL, 76% IPI<3. The majority (85%) of patients completed chemotherapy according to protocol. 14(15%) went off protocol because of less than PR after 3 cycles of (R)CHOP or progression on treatment based on CDM or excess toxicity. CR(u) was reached in 60% of the patients. 34 (36%) patients relapsed and 2 patients died of NHL after a median follow-up of 26 months. PET was scored qualitatively using a Likert scale. PET after 3 courses was negative in 45 patients and positive in 50. 24/50 (48%) PET positive patients progressed or relapsed as compared to 10/45 (22%) PET negative patients. Median progression free survival (PFS) for PET positive versus PET negative patients was 19.8 months versus not yet reached (p=.0087). In a multivariate analysis, IPI and PET were independent predictors for PFS.

These results corroborate and extend the evidence that mid-treatment FDG-PET is highly predictive for PFS and may be a useful tool for risk-adapted management of aggressive NHL.
Intensifying Methotrexate (MTX) Dosage Reduces Treatment Failure in Adults with Burkitt or Burkitt-Like Leukaemia/Lymphoma (BL) Treated with an Adapted BFM Protocol.
Authors: Tauro Sudhir , Begum Gulnaz , Lauritzsen Grete Fossum , Delabie Jan , Roberts Claudia , Mahendra Premini , Holte Harald
Published: October 2019

The use of short-duration intensive combination chemotherapy protocols has improved survival in adults with Burkitt/Burkitt-like leukaemia and lymphoma (BL). Systemic methotrexate (MTX) is an integral component of these regimens, but the dosage varies between treatment schedules, and the precise dose required to optimise tumour-kill without causing severe toxicity is not known. In this study of 66 adults with sporadic BL, we have investigated whether the dosing intensity of MTX can influence treatment failure (defined as disease relapse or resistance to treatment, or death due to therapy). There were 49 males and 17 females in the cohort (median age 36 years, range 16–69y), including 9 with HIV disease and 2 organ-transplant recipients. Majority of patients (66%) had St Jude stage III/IV disease. The median increase in serum LDH level relative to normal (adjusted LDH) was 1.4 (range 1–65). Patients were treated with a combination of CNS-directed and systemic chemotherapy comprising of a pre-phase [fractionated (Fr) cyclophosphamide and prednisolone], followed by a possible total of 6 cycles of alternating Fr ifosfamide, dexamethasone, vincristine, cytarabine and etoposide [Cycle A], with Fr cyclophosphamide, dexamethasone, vincristine and adriamycin [Cycle B] as outlined by the German BFM paediatric protocols. Patients received a 24h intravenous infusion of MTX on d1 of each cycle of treatment. Based on the mean MTX dose administered per cycle, patients were stratified into three dosage groups: low (<1gm/m2, n=4), intermediate (1–1.5g/m2, n=24) or high (>1.5–3g/m2, n=34). There were 3 toxic deaths, disease was refractory in 8 patients and 9 experienced disease relapse. Durable complete responses following BFM were observed in 46/66 (70%) patients. A significantly lower proportion of patients receiving high-dose MTX (17%) experienced treatment failure compared to 45% and 50% in the intermediate and low-dose groups respectively (Fisher’s Exact p=0.01). Risk stratification on the basis of pre-treatment stage and bulk of disease, adjusted LDH and ECOG score was however unable to identify patients who may benefit from intensifying MTX dosage. These data thus uniquely highlight the impact of MTX dose in influencing outcomes in adult BL as well as the need for novel biological markers to identify patients requiring additional therapeutic strategies.
Enteropathy-Type T-Cell Lymphoma: Clinical Features of a Series of 62 Cases. Jan Delabie, Harald Holte, Andrew Lister and Julie Vose for the International T-Cell Lymphoma Project.
Authors: Delabie Jan , Holte Harald , Lister Thomas A. , Vose Julie M.
Published: October 2019

In an international retrospective clinical study of 1159 adult, non-cutaneous mature T-cell lymphomas, verified by an expert pathology panel, 62 cases of enteropathy-type T-cell lymphoma (ETTCL) were identified, representing 5.3%. Patients in this study were diagnosed at one of the participating centers in North America, Europe, and Asia between 1990 and 2002. ETTCL represented the 6th most common T-cell lymphoma, affecting predominantly the small bowel. Of interest, about a third of the cases of ETTCL were obtained from Norway, as the only participating center from Scandinavia. Twenty out of 52 cases (38%) were associated with celiac disease. As expected, abdominal pain was the most frequently recorded symptom, followed by general symptoms such as fatigue, weakness and anorexia. Overall as well as failure-free 5-year survival were poor, 20% and 4%, respectively. Univariate statistical analysis revealed that high T-IPI score (Gallamini, et al: Blood 103: 2474–2479, 2004) but not high IPI score, high CRP and LDH levels, and tumor size >5cm were adverse prognostic factors at p values <0.05. Most patients (51/60) were treated with anthracycline-containing chemotherapy regimens, whereas 9 received other or no treatment and showed a worse survival, probably due to a negative patient selection. Multivariate analysis controlling for T-IPI score, revealed that only high CRP levels and tumor size >5cm were independent prognostic factors. In conclusion, ETTCL is a clinically aggressive and fatal disease for which, currently, no effective treatment exists. T-IPI score, CRP levels and tumor size can be used as prognostic factors.
Hodgkin Lymphoma in Very Young Children Can Be Treated Successfully without Radiation Therapy.
Authors: Belgaumi Asim F. , Al-Kofide Amani A. , Joseph Nicey , Khafaga Yasser , Malik Rubina J. , Mourad Walid , Mohamed Gamal-Eldin H. , Sabbah Rajih S.
Published: October 2019

Hodgkin Lymphoma (HL) is rarely seen in <5-year olds in developed nations. Even in developing countries, where a tendency towards younger age of presentation has been shown, this represents a minority of cases. Little is known about the biology and behavior of these very young patients with HL as compared to older children.

Methods: We retrospectively reviewed HL cases diagnosed and treated at our institution between 1975 and 2003. HL was diagnosed histopathologically and staged clinically. The pediatric age group ranged from 0–14 years. Treatment strategy for these very young children was focused on the elimination of radiation therapy (XRT).

Results: 69/368 (18.75%) patients were less than 5 years at diagnosis. When compared to older patients, there was a trend towards male predominance (M:F 4.31 v. 2.65; p=0.2), but no difference in the incidence of B-symptoms (26.1% v. 32.9%; p=1.0) and stage distribution (p=1.0). There was less mediastinal involvement (p=0.025) or bulky disease (p=0.01) in the younger patients. These patients had more mixed cellularity and less nodular sclerosis subtype (p=0.025). Fifty-five were treated with chemotherapy (CTX) alone, 12 with combined modality therapy (CMT) and 2 with XRT only. 35/55 CTX patients were treated with ABVD (20 per standard schedule, 13 modified and 2 unknown), 12 MOPP and 8 with hybrid or combination CTX (4 MOPP/ABVD, 3 COPP/ABVD and 1 unspecified). All CMT patients received ABVD (9 standard and 3 modified) and XRT (1500cGy/IF for 5, 1500cGy/EF for 4 and 2400cGy/EF, 2720cGy/IF and 3060cGy/IF for one each). The two XRT alone patients had stage I cervical disease and received 3900cGy and 3250cGy IFXRT. At ten years the EFS and OS for these patients under 5-years of age was 81.5% and 90.4%, respectively, compared to 75.5% and 90.5% for the children between 5 and 14 years of age (p>0.5 for both comparisons). OS (86.4% v. 100%; p=0.3, Log Rank test) and EFS (81.0% v. 90.9%; p=0.4, Log Rank test) for CTX v. CMT groups were not statistically significantly different. The CTX group had more B-symptoms (29.1% v. 16.1%) and higher stage disease (stage III/IV 47.3% v. 25%; stage IV 12.73% v. 0%).

Conclusions: HL patients <5years old do not present with higher risk disease than older children. They can successfully be treated without XRT using CTX alone. XRT can be reserved for treating the few who relapse. This may result in reduction in XRT related toxicity, which can be significant in these very young children.
Multivalent Anti-CD20/Anti-CD22 Bispecific Antibody Fusion Proteins Made by the DNL Method Show Potent Lymphoma Cytotoxicity.
Authors: Rossi Edmund A. , Losman Michele J. , Nordstrom Diane L. , Cardillo Thomas , Loo Meiyu , Kopinski John , Qu Zhengxing , Goldenberg David M. , Chang Chien-Hsing
Published: October 2019

Purpose: Multivalent and either monospecific or bispecific antibodies have shown the potential for improved efficacy in a number of preclinical studies. A panel of such agents based on hLL2 (epratuzumab, anti-CD22) and hA20 (humanized anti-CD20) was made and evaluated for cytotoxicity in vitro and anti-tumor activity in vivo.

Methods: The Dock and Lock (DNL) method (Rossi et al., Proc. Natl. Acad. Sci. USA, 2006,103:6841) is a novel platform technology for the site-specific and covalent assembly of modular components. DNL was utilized to generate two hexavalent monospecific (hex-hA20 and hex-hLL2), and two hexavalent bispecific (DNL-1 and DNL-2) stably tethered complexes, each composed of four Fab fragments conjugated to an IgG at the latter’s carboxyl termini of the heavy chain. DNL-1 consists of four hA20 Fab fragments tethered to an hLL2 IgG. DNL-2 consists of four hLL2 Fab fragments tethered to an hA20 IgG.

Results: For each construct, the DNL method produced a single, defined, homogeneous structure that is stable in serum. All of the constituent Fab fragments are functional, with binding affinities similar to the parental MAbs. In vitro analyses using human Burkitt lymphoma cell lines demonstrate that both the monospecific and bispecific constructs induce strong cell adhesion and potently inhibit cell growth without the requirements for further cross-linking. DNL-1, DNL-2 and hex-hA20 each completely inhibited the growth of cultured Daudi cells at concentrations as low as 1 nM and exhibited two to three logs greater potency than that of hA20 IgG or rituximab under the same experimental conditions. Anti-tumor efficacy was demonstrated in SCID mice bearing disseminated Daudi lymphoma. Treatment with 4 μg of hex-hA20 or DNL-2 at 1, 4 and 7 days post intravenous injection of 1 × 107 Daudi cells resulted in significantly increased survival of tumor-bearing mice compared to controls treated with saline.

Conclusion: These findings suggest that the DNL method can be used to make multivalent proteins with potent anti-tumor activity, as shown here with anti-CD20/anti-CD22 bispecific antibody fusion proteins.
The Humanized Anti-CD40 Antibody, SGN-40, Promotes Apoptosis Signaling and Is Effective in Combination with Standard Therapies in Lymphoma Xenograft Models.
Authors: Lewis Timothy S. , Sutherland May S.K. , Jonas Mechthild , Cerveny Charles G. , McCormick Renee , Wahl Alan F. , Drachman Jonathan G. , Grewal Iqbal S. , Law Che-Leung
Published: October 2019

CD40 is a TNF receptor family member that is expressed on B cells and by a wide variety of transformed cells including non-Hodgkin’s lymphoma (NHL), multiple myeloma, and various solid tumors. The humanized anti-CD40 antibody, SGN-40, is a partial agonist that induces apoptosis as well as mediates ADCC against CD40+ NHL B cell lines, contributing to in vivo antitumor activity observed in human lymphoma xenograft models. The current study demonstrates the ability of SGN-40 to initiate multiple signaling cascades upon ligation of CD40 on NHL cell lines. SGN-40 was shown to activate the stress-induced p38 MAP kinase and pro-survival pathways including NF-κB, p42/44 MAP kinase and, to a lesser extent, AKT. Consistent with the apoptosis-inducing activity of SGN-40, cleavage of caspase-3 and its downstream substrate poly (ADP-ribose) polymerase was detected in NHL cell lines. SGN-40 signaling was qualitatively similar to that mediated by trimeric recombinant human CD40 ligand (rhCD40L). However, the overall magnitude of signaling was lower with SGN-40 compared to rhCD40L, consistent with the partial agonistic properties of SGN-40. In addition, constitutive phospho-AKT levels, a key pro-survival signal, were found to be very low in most high-grade lymphoma cell lines and primary NHL specimens, in contrast to the high levels reported in carcinomas. Low AKT activity may bias lymphoma cells toward apoptosis in response to SGN-40 signaling. To augment SGN-40-induced cell killing, in vitro combination studies with chemotherapeutics have been performed using the Ramos, RL, and HT NHL lines. We now report that SGN-40 has additive activity when combined with cisplatin, melphalan, or mitoxantrone and is synergistic with bleomycin. Furthermore, in vivo activity of combination therapy has been demonstrated in a subcutaneous Ramos lymphoma xenograft model. While tumor growth was delayed by either CHOP (cyclophosphamide, adriamycin, vincristine, prednisone) or SGN-40 alone, the combination of SGN-40 and CHOP was significantly more active. Our results suggest that SGN-40 can be combined with standard lymphoma therapies resulting in improved therapeutic efficacy, and provide a rationale for combination clinical trials involving SGN-40. The molecular mechanisms through which standard chemotherapeutic agents enhance SGN-40-mediated cell killing in target lymphoma B cells are currently being investigated.
HCD122, an Antagonist Human Anti-CD40 Monoclonal Antibody, Inhibits Tumor Growth in Xenograft Models of Human Diffuse Large B-Cell Lymphoma, a Subset of Non-Hodgkins Lymphoma.
Authors: Hsu Ssucheng J. , Esposito Lin A. , Aukerman Sharon L. , Kantak Seema , Mirza Amer M.
Published: October 2019

CD40, a member of the tumor necrosis factor receptor family, is expressed in all human B-cell malignancies and engagement by the CD40 ligand (CD40L) is important for both cell proliferation and cell survival. CD40L has been shown to be co-expressed with CD40 in neoplastic B-cells from Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkins Lymphoma (NHL), suggesting the importance of an autocrine CD40/CD40L loop in these malignancies. HCD122 (formerly known as CHIR-12.12) is a fully human, highly potent, IgG1 antagonist anti-CD40 monoclonal antibody (mAb) that blocks CD40/CD40L interactions in vitro and also mediates ADCC. Previous studies showed that HCD122 can mediate ADCC in vitro and has anti-proliferative and anti-tumor activities as a single agent in CLL, MM, and Burkitts Lymphoma in vitro and in vivo. In this study, the activity of HCD122 on a subtype of NHL, Diffuse Large B-Cell Lymphoma (DLBCL) was examined. The DLBCL derived cell lines, RL and SU-DHL-4, were selected for this study based upon in vivo characterization as well as their sensitivity to Rituximab as reported in the literature. These cell lines were subsequently confirmed for the expression of CD40 and CD20 by flow cytometry. The in vivo anti-tumor effects of HCD122 as single agent was demonstrated in these two xenograft models and was compared to Rituximab, an anti-CD20 antibody therapeutic currently approved for the treatment of relapsed or refractory, low-grade or follicular, NHL. HCD122 when administered intraperitoneally weekly at 1 mg/kg significantly reduced tumor growth with a tumor growth inhibition (TGI) of 85.5% (P<0.01) in the RL model. At the same dose and schedule in the RL model, TGI achieved with Rituximab was 31.7% (P>0.05). In the SU-DHL-4 model, an 85% TGI (P<0.01) was observed at the 1 mg/kg dose of HCD122. In comparison, Rituximab at this dose elicited a 57.6% TGI (P<0.05). Additionally, the downstream CD40/CD40L signal transduction pathways were also examined in order to elucidate the molecular mechanism underlying the HCD122-mediated effects in DLBCL. Taken together, these results support the clinical development of HCD122 for the treatment of DLBCL. Currently HCD122 is in Phase I trials for treatment of CLL and MM.
In Vitro Activity of SYK and BCR-ABL Inhibitors in Aggressive Lymphomas.
Authors: Bertoni Francesco , Rinaldi Andrea , Sasso Anna , Gaidano Gianluca , Zucca Emanuele , Uccella Silvia , Pruneri Giancarlo , Ponzoni Maurilio , Tibiletti Maria Grazia , Catapano Carlo , Cavalli Franco
Published: October 2019

The B cell receptor tyrosine kinase SYK is a critical component of the B-cell receptor signalling pathway in normal B cells. We have recently reported that SYK is amplified and over-expressed in mantle cell lymphoma (MCL) and that the growth of MCL and diffuse large B cell lymphoma (DLBCL) cell lines over-expressing SYK is inhibited by piceatannol, a known SYK inhibitor (Bertoni et al, ASH 2005; Rinaldi et al, BJH 2006). Others have reported important SYK expression in splenic marginal zone B cell lymphomas, in DLBCL and peripheral T cell lymphomas (PTCL) (Ruiz-Ballesteros et al, 2005; Mahadevan et al, w Streubel et al, 2006), suggesting SYK targeting agents could be useful for the treatment of various lymphoma subtypes. SYK inhibitors are already in clinical development for treatment of asthma. Here, we report on the activity on lymphoma cell lines and primary cells of the SYK/ZAP-70 inhibitor #1 (Novartis) and of the BCR-ABL inhibitors imatinib (Novartis) and nilotinib (Novartis), which could act as cross-selecting SYK inhibitors (Atwell et al, 2004).

We treated four human MCL and three DLBCL established cell lines with increasing doses of the SYK/ZAP-70 inhibitor #1, imatinib and nilotinib for 72 h. Cell viability was measured with the MTT assay. The two cell lines expressing high levels of SYK, JeKo-1 and SUD-HL-6, were sensitive to the compounds (IC50: Syk/ZAP-70 inhibitor #1, 1–5 μM; imatinib, 15–20 μM; nilotinib, 10 μM). Cells with lower SYK expression were generally less sensitive to all three compounds. To obtain further data on the relevance of SYK inhibition in lymphoma, we treated nine lymphoma primary cells with the piceatannol (Sigma), the SYK/ZAP-70 inhibitor #1 and nilotinib. Responses, defined as <50% decrease in viable cell number, were observed with piceatannol (4/9 samples), the Syk/ZAP-70 inhibitor #1 (3/9 samples) and nilotinib (3/9 samples). Immunoblotting experiments aimed to elucidate the mechanism of action are under-way and data will be presented at the meeting.

In conclusion, our data indicate that pharmacological inhibition of SYK is a therapeutic approach to be further investigated in subsets of aggressive lymphomas.
An Fc-Optimized Monoclonal Antibody Against CD32B, the Human Inhibitory Fc-γ Receptor IIB, in the Immunotherapy of B-Cell Malignanacy.
Authors: Stavenhagen Jeffrey B. , Gorlatov Sergey , Tuaillon Nadine , Rankin Christopher T. , Veri Maria-Concetta , Inzunza David , Burke Stephen , Huang Ling , Li Hua , Thomas Shannon , Johnson Syd , Ravetch Jeffrey , Koenig Scott , Bonvini Ezio
Published: October 2019

Monoclonal antibodies (mAbs) are widely used in the treatment of lymphoma and autoimmune diseases. The activity of several mAbs depends on the binding of the Fcγ regions to low-affinity Fcγ receptors (FcγR) expressed on effector cells, chiefly mononuclear phagocytes, cells that express both activating and inhibitory FcγRs. FcγRIIB (CD32B), the inhibitory FcγR, is expressed by normal and malignant B lymphocytes and is itself an immunotherapeutic target. A recently characterized anti-human CD32B-specific mAb (2B6) was Fc engineered to customize the FcγR binding profile for increased binding to the activating receptor FcγRIIIA (CD16A), resulting in an improved activating-to-inhibitory binding ratio. Fc-optimized versions of 2B6 showed enhanced cytotoxicity in vitro and increased potency in animal tumor models, including transgenic mice harboring the human low-affinity allele of the activating receptor, CD16A-158phe. The enhancement in tumor depletion correlated with an increased binding to FcγRIV, a mouse functional homolog to human CD16A. The expression of FcγRIV is limited to monocyte and macrophages, consistent with the role of these cells in tumor cell killing in vivo. These data support CD32B as a target for immunotherapy of B cell lymphoproliferative disorders and Fc optimization to increase the activating-to-inhibitory FcγR-binding ratio as a means to designing improved antibodies.
MLL-AF9 Initiates Leukemia from a Single Committed Hematopoietic Progenitor.
Authors: Krivtsov Andrei V. , Stubbs Matthew C. , Wright Renee , Feng Zhaohui , Kung Andrew L. , Armstrong Scott A.
Published: October 2019

Recent experiments have demonstrated that MLL-translocation associated fusion proteins can transform either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP) into leukemia stem cells. However, it may be that leukemogenic process differs when HSC are the cell of origin as compared to myeloid progenitors. We transduced either HSC or GMP with a retrovirus expressing MLL-AF9 and GFP followed by single cell sorting of transduced cells. Approximately 80% of singly sorted MLL-AF9 transduced GMP (MLL-AF9-GMP) and about 25% of MLL-AF9 transduced HSC (MLL-AF9-HSC) could be serially re-plated over 9 passages. Upon transplantation into syngeneic mice, 83% (n=12) of MLL-AF9-HSC single cell derived clones induced AML with a median latency 70 days. Approximately 30% (n=20) of MLL-AF9-GMP single cell derived clones induced AML, with median latency 112 days. When MLL-AF9-GMP single cell derived clones were co-infected with an empty retrovirus (to provide additional oncogenic events as a result of retroviral integration) before transplantation into recipient mice, 93% of the transplanted mice (n=15) developed AML with mean latency 65 days, similar to leukemia initiated from HSC. This suggests that either single GMP or HSC can be transformed into leukemia initiating cells. However, extra mutations appear to be required to induce leukemia from committed progenitors. Consistent with this hypothesis, southern blot analysis performed on leukemias initiated from 5,000 and 15,000 MLL-AF9 transduced HSC or GMP demonstrated polyclonal AML arising from HSC compared to oligoclonal AML arising from GMP. Next, we used bioluminescent imaging to follow disease kinetics. When 15,000 MLL-AF9 transduced HSC were injected into recipient mice, the disease accumulated in a linear fashion over 42 days. However, when 15,000 MLL-AF9 transduced GMP were injected the disease developed more slowly over 75 days. Immunophenotypic analysis of the resultant leukemias demonstrated that the HSC-derived and GMP-derived leukemias were quite similar, with a GMP-like population containing LSC in both cases. Globally, the two cell types were also very similar with their gene expression profile being more similar to GMP than any other progenitor or stem cell population. However, we found that in addition to the previously reported 363-gene “self-renewal associated signature” LSC derived from HSC also possessed high-level expression of genes such as Flt3, Mcl1, and Notch-1. Preliminary analysis also suggests that gene expression differences between HSC and GMP-derived leukemia stem cells may have prognostic significance in human AML. These data suggest that AML derived from different cells of origin, while globally quite similar, require a different number of genetic events, and have gene expression differences that may influence drug response.
CD34+CD19−, CD34+CD19+ and CD34−CD19+ Cells May All Have Leukemia-Initiating Potential in NOD/Scid Mice.
Authors: Le Viseur Christoph , Hotfilder Marc , Rosemann Annegret , Stam Ronald , Schrauder Andre , Shultz Leonard , Schrappe Martin , Pieters Rob , Vormoor Josef
Published: October 2019

Current data on the leukemic stem cell (LSC) compartment in childhood acute lymphoblastic leukemia (ALL) are conflicting. The traditional hypothesis supposed that childhood ALL originates in a lymphoid progenitor cell and this is assumed to be consistent with the overall good treatment responses in pediatric patients. In accordance with this hypothesis, our previous studies failed to detect involvement of immature CD34+CD19− progenitor cells in ALL/t(12;21) (Hotfilder et al., Blood 2002) while high-risk ALL/t(9;22) and t(4;11) appears to originate in a more primitive CD34+CD19− cell (Hotfilder et al., Cancer Res 2005). In order to characterize the leukemia-initiating cell in vivo, we established a mouse xenograft model by serial intrafemoral transplantation of NOD/scid mice with flow sorted subpopulations from childhood ALL. Samples were taken from the bone marrow of children with ALL/t(12;21) (n=1), t(4;11) (n=3) and t(11;19) (n=1) and B-cell precursor ALL without a marker translocation (n=2). Primary transplantations were performed with freshly thawed unsorted cells, followed by secondary, tertiary and quaternary transplantations with flow sorted populations. Human leukemic engraftment was defined by a proportion of >5% human CD45+ cells in the murine bone marrow that simultaneously express CD34 and/or CD19. From the bone marrow of leukemic mice, we isolated different leukemic populations and successfully re-transplanted 2×103 − 1×105 CD34+CD19− cells, 2×104 − 6×106 CD34+CD19+ lymphoid progenitors and 3×104 − 2×106 more differentiated CD34−CD19+ blasts onto secondary, tertiary and quaternary mice (average purity after flow sorting: >96%). So far, we detected leukemic engraftment in 60 of 161 (37%) transplanted mice (with many mice - having only recently been transplanted - still alive). These include 7 of 36 (19%) mice engrafted with CD34+CD19− cells, 33 of 72 (46%) mice engrafted with CD34+CD19+ cells and 20 of 53 (38%) mice engrafted with CD34−CD19+ cells. With as few as 2 × 103 CD34+CD19− cells being sufficient to re-initiate the leukemia, this intrafemoral ALL-NOD/scid mouse model represents a very sensitive functional assay for candidate LSC in childhood ALL. We have initiated limiting dilution experiments with the different subpopulations to quantify LSC frequency in the different compartments and to exclude that low levels of contaminating blasts with an immunophenotype different from the main transplanted cell population blurred the results. We are also currently investigating whether there is heterogeneity in the CD34+CD19− compartment in respect to standard and high-risk ALL. Altogether, our data indicate that all three subpopulations, CD34+CD19−, CD34+CD19+ and CD34−CD19+ cells, may have the capacity to transfer the leukemia onto NOD/scid mice and that lymphatic LSC may not loose their self-renewal potential with differentiation.
Immortalization of Human HSPC by a Self-Renewal Signal Coupled with Telomere Maintenance.
Authors: Wei Junping , Wunderlich Mark , Krejci Ondrej , Alvarez Sara , Cigudosa Juan , Mulloy James C.
Published: October 2019

Expression of the leukemia-associated fusion protein AML1-ETO (AE) in human hematopoietic stem and progenitor cells (HSPC) results in long-term cytokine-dependent cells that maintain CD34 expression, multipotential differentiation and the ability to engraft NOD/SCID mice. These cells gradually lose telomere length and are not immortal. To determine whether expression of telomerase would cooperate with AE in immortalization of human HSPC, we transduced AE-expressing CD34+ cells with the telomerase gene, hTERT. In four independent AE cell cultures, hTERT expression allowed the cells to proliferate beyond the crisis point. Cells retained B lymphoid, myeloid and erythroid differentiation potential, displayed continuous replating ability in methylcellulose assays and engrafted in NOD/SCID mice. The hTERT-expressing cells had a significant increase in progenitor cell numbers, more cells in S phase, and increased resistance to gamma radiation-induced apoptosis compared to control cells. No genetic changes were identified by G-banding or comparative genomic hybridization during the passage through crisis, implying that these signals are sufficient for the immortalization. Similar results were obtained using a long-term culture established with the related core binding factor (CBF) fusion gene CBFβ-MYH11 (CM). By microarray analysis we identified a number of genes implicated in stem cell self-renewal, including HOXB4, GATA3 and STAT3, that are upregulated in CBF-expressing cells when compared to control CD34+ cells. Ectopic hTERT expression did not affect the level of expression of these self-renewal genes. We hypothesize that the continual rescue of progenitor cells during culture, as a result of telomerase maintenance of telomeres, is responsible for the proliferation and survival phenotype observed in hTERT expressing AE and CM cells. This is supported by our findings that control cultures contained significantly more DNA damage foci as detected by immunofluorescence staining for phosphorylated H2AX. In addition, signal-free chromosome ends were significantly more abundant in control cultures as demonstrated by a telomeric FISH probe when compared to the hTERT cultures.

Senescence-associated β-gal staining was prominent in control cells as they slowed proliferation after 7 months of growth, but hTERT-expressing cells did not display SA-β-gal staining and continued proliferating indefinitely, for over two years at this point in time. We conclude that the self-renewal signal supplied by a CBF oncogene cooperates with telomere maintenance initiated by hTERT for the immortalization of human HSPC. This system permits a molecular dissection of the signals involved in HSPC self-renewal and leukemogenesis and in the contribution of hTERT expression to this process.
Mesenchymal Stem Cells Produce Nitric Oxide, a Key Molecule for T Cell Suppression, upon Interaction with Activated T Cells.
Authors: Sato Kazuya , Ozaki Katsutoshi , Oh Iekuni , Meguro Akiko , Tatara Reine , Hatanaka Keiko , Nagai Tadashi , Muroi Kazuo , Ozawa Keiya
Published: October 2019

The molecular mechanisms by which mesenchymal stem cells (MSCs) suppress T cell proliferation are poorly understood, and whether a soluble factor plays a major role remains controversial. We demonstrated that nitric oxide (NO) is important for T cell suppression by MSCs (ASH 2005, 403a). Here we further demonstrate that the production of NO in the presence of MSCs is dependent on CD4 or CD8 T cells but not on CD19 B cells. MSCs inhibits B cell proliferation induced by LPS, suggesting that mechanisms of supppression by MSCs are different between T cells and B cells. Inducible NO synthase was exclusively detected in MSCs co-cultured with activated T cells, indicating that the producer of NO is MSC. Experiments with transwell system revealed that separation by transwell membrane reduces the induction of NO and T cell suppression. RAW246.7 macrophage cell line showed a similar transwell-mediated inhibition, suggesting that the inhibition by transwell is a common feature of NO and that direct contact is critical for efficient NO production and T cell suppression. Furthermore, inhibitors of prostaglandin synthase or NO synthase restored the proliferation of T cells, whereas an inhibitor of indoleamine 2,3-dioxygenase and a transforming growth factor-β-neutralizing antibody had no effect. In the view of that NO is upstream of PGE2, NO may be a key regulator of T cell suppression induced by MSCs. Finally, we used inducible NO synthase knockout mice to reconfirm all results here. MSCs from knockout mice did not produce NO even in the presence of activated wild type T cells and had a reduced ability to suppress T cell proliferation. Meanwhile, proliferation of splenocytes from knockout mice was suppressed in the presence of wild type MSCs and NO production was readily detected, confirming that NO produced by MSCs plays a critical role in T cell suppression.