bluuebunny/crossref_metadata_2025 huggingface dataset:
Alternative Splicing Due to an Intronic SNP Defines a Novel HLA-B44-Restricted-Hematopoiesis-Restricted Minor Histocompatibility Antigen.
Authors: Kawase Takakazu , Akatsuka Yoshiki , Torikai Hiroki , Morishima Satoko , Kodera Yoshihisa , Morishima Yasuo , Kuzushima Kiyotaka , Takahashi Toshitada
Published: October 2019

Minor histocompatibility antigens (mHAgs) with expression limited to hematopoietic cells represent attractive targets for immunotherapy to induce selective graft-versus-leukemia (GVL) reactions. Here we report the identification of a novel mHAg which is recognized by an HLA-B*4403-restricted CTL clone. Microsatellite allele image analysis of two DNA pools generated from CTL-defined mHAg positive and mHAg negative groups was performed using microsatellite markers set at 100 kb intervals within the segment initially mapped by two-point genetic linkage analysis and detailed mapping of the chromosomal recombinant points. This approach defined a 0.53 Mbp region of chromosome 18q21–22 containing 12 candidate genes potentially encoding the mHAg, although the target gene could not be identified. Subsequently, cDNA expression cloning studies demonstrated that the CTL epitope of interest was encoded by a novel allelic splice variant of XM_209104, hereafter designated as XM_209104-av. Indeed, this gene was found to lie within the region predicted by microsatellite allele image analysis. The immunogenicity of the epitope was generated by differential protein expression due to alternative splicing, which was completely controlled by one intronic single nucleotide polymorphism (SNP) located in the consensus 5′ splice site adjacent to an exon. To our knowledge, this is the first example of a mHAg controlled by a SNP located in a region other than coding sequences. Because the CTL lysed also HLA-B*4402 positive, mHAg positive B-LCLs, this novel epitope peptide can bind to not only HLA-B*4403 but also HLA-B*4402 which is a relatively common HLA-B allele in Caucasian populations. Finally, the finding that the novel XM_209104-av showed low or no expression in normal tissues including resting hematopoietic cells, but significantly higher expression in primary acute leukemia cells, especially those of myeloid lineage, suggest that this novel epitope may be an attractive therapeutic target for immunotherapy not only as a minor H antigen but also as a leukemia-associated antigen.
Serum Cytokeratin-18 Fragments as Quantitative Marker of Epithelial Apoptosis in Liver and Intestinal Graft-Versus-Host Disease.
Authors: Luft Thomas , Conzelmann Michael , Rodionova Elena , Hess Michael , Strohhaecker Ulrich , Benner Axel , Görner Martin , Hegenbart Ute , Ho Anthony D. , Dreger Peter
Published: October 2019

Graft-versus-host disease (GvHD) is the main complication of allogeneic stem cell transplantation (SCT). However, diagnosis and differentiation of GvHD from infectious or toxic clinical conditions as well as evaluation of GvHD response to immunosuppressive treatment remains a challenge. Since apoptosis is a common histopathological finding in GvHD, we investigated whether the caspase-cleaved neo-epitope of cytokeratin 18 fragments (CK18F) might be a serum marker for ongoing GvHD-induced target organ inflammation.

Longitudinal monitoring of serum CK18F kinetics by M30 antibody-based ELISA in 49 patients who fulfilled histopathological and/or clinical criteria diagnostic for GvHD following SCT showed that CK18F were highly elevated in hepatic and intestinal GvHD (6.3±3.8 fold and 4.6±0.8 fold, respectively, over baseline). Responses of GvHD to immunosuppressive therapy were paralleled by CK18F decreases. The extent of CK18F increases tended to correlate with serological severity of acute (r=0.96) and chronic (r=0.76) liver GvHD. Differentially relevant clinical conditions, such as non-complicated infection-related diarrhea and toxic hyperbilirubinemia were not associated with significant CK18F rises. In conclusion, CK18F monitoring provides a simple serum marker for quantitative assessment of GvHD-associated apoptotic activity in intestinal and hepatic GvHD and for distinguishing active GvHD from irreversible end organ damage. Prospective studies are warranted to demonstrate the significance of CK18F for diagnosis, differentiation, prognosis, and treatment guidance of GvHD.
Costimulatory Molecule-Targeted Immunotherapy of Chronic Graft-Versus-Host Disease.
Authors: Kim Juyang , Kim Hye J. , Kim Jiyoung , Park Keunhee , Choi Hye-Jeong , Yagita Hideo , Cho Hong R. , Kwon Byungsuk
Published: October 2019

Chronic graft-versus-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Current therapies for cGVHD reduce symptoms but are not cures. Using the B10.D2àBalb/c (H-2d) minor histocompatibility antigen-mismatched model which reflects clinical and pathological symptoms of human cGVHD, we show that a single injection of an agonistic monoclonal antibody (mAb) to CD137, a member of tumor necrosis factor receptor superfamily, reverses skin fibrosis, ulceration and alopecia, ultimately leading to prolonged survival. The reversal is associated with markedly reduced helper type 2 CD4+ T cell (Th2) cytokines and increased production of the Th1 cytokine interferon-g (IFN-g). The Fas pathway is required for the therapeutic effect of anti-CD137 mAb on cGVHD. Taken together, these data indicate that anti-CD137 mAb has a therapeutic effect on cGVHD by removing Th2 cells mediating cGVHD. Our study demonstrate an agonistic mAb specific for a costimulatory molecule as a possible target for therapeutic intervention in cGVHD.
The MyD88 Adapter-Like (Mal) Protein Variant Leu 180, a Candidate Polymorphism for Protection Against Graft Versus Host Disease.
Authors: Holtick Udo , Middleton Peter G. , Harrold Jane L. , Holler Ernst , Gluckman Eliane , Hromadnikova Ilona , Dickinson Anne M. , Collin Matthew P.
Published: October 2019

The involvement of mediators of the innate immune response in immunological processes after allogeneic haematopoietic stem cell transplantation (HSCT) has been an important finding. Toll-like receptor (TLR) signalling plays a key role in the innate response to pathogens. The MyD88 adapter-like (Mal) protein is involved in TLR 2, 4 and 9 signalling. A polymorphism leading to a non functional variant (Ser → Leu 180) has been reported to be protective in cerebral malaria due to a reduced inflammatory response (O’Neill LA, ASH 2005). A dominant negative form of Mal inhibits NFκB activation by TLR-4 and LPS (Fitzgerald KA, Nature 2001). As bacterial products like LPS are believed to contribute to initiation and maintenance of graft versus host disease (GvHD), we hypothesised that one or two non functional Mal variants (Ser → Leu 180) present in donor or recipient cells might reduce the incidence of GvHD and improve survival after allogeneic HSCT.

163 donor recipient sibling pairs transplanted with full or reduced intensity conditioning without T depletion were identified from the Eurobank database. DNA was amplified using allele specific PCR primers: allele 1 (Ser)5′ CACCATCCCCCTGCTGTC; allele 2 (Leu) 5′ CACCATCCCCCTGCTGTT; reverse 5′ TGACGAAAGCCATCAG. The allele frequency was: allele 1 (Ser) 0.84; allele 2 (Leu) 0.16. There were no significant differences between the cohorts in age, gender mismatch, immunosuppression, conditioning or transplant center. The overall incidence of acute and chronic GvHD was reduced (83 vs 75% and 63 vs 52% respectively), when both donor and recipient possessed allele 2 (p=0.247 and p=0.155). A similar trend could be seen when the donor alone had at least one uncommon allele. The 5 year projected survival was not different for patient/donor pairs for any given combination of alleles.

To further test the functional relevance of the Mal polymorphism, PBMCs from 33 Donor/Recipient pairs were stimulated with LPS or PHA and TNFα levels were analysed after 24h and 72h respectively. After LPS stimulation the median TNFα levels were lower when one or two uncommon alleles were present (1871 vs 1774 vs 988 pg/ml; p=0.9 and p=0.178). PHA stimulated cells also showed lower median TNFα levels, approaching statistical significance for the combined group carrying at least one uncommon allele (6656 vs 5173 pg/ml, p=0.085) and being statistically significant when two uncommon alleles were present (6656 vs 1495 pg/ml, p=0.031).

We conclude that the presence of a non-functional Mal variant especially in donor cells may reduce the incidence of acute and chronic GvHD, although this did not reach statistical significance in this group of patients. There was no impact on survival. A larger cohort is currently being investigated.
High Alloreactive Potential of Central Memory T-Lymphocytes Expressing a Suicide Gene for the Cure of Hematologic Malignancies.
Authors: Kaneko Shin , Mastaglio Sara , Bondanza Attilio , Ponzoni Maurilio , Aldrighetti Luca , Toma Salvatore , Radrizzani Marina , La Seta Catamancio Simona , Ciceri Fabio , Nagasawa Toshiro , Bordignon Claudio , Bonini Chiara
Published: October 2019

Alloantigen targeting adoptive immunotherapy is a powerful therapeutic approach for the treatment of hematologic malignancies. A compelling example is represented by donor lymphocyte infusions after allogeneic hematopoietic stem cell transplantation. The clinical efficacy of T-cell based therapy relies not only on the ability of T cells to mediate a direct anti-tumor effect (GvT) and a protective immune response against pathogens, but also in their capacity to persist and expand in vivo, providing a long-term protection from disease relapse. Despite undeniable efficacy, the extensive exploitation of donor lymphocytes is limited by the risk of a severe and potentially life-threatening complication: Graft-versus-host disease (GvHD). To solve this double bind, we investigated the therapeutic potential of donor lymphocytes retrovirally transduced to express the suicide gene thymidine kinase of Herpes Simplex virus (TK) in patients affected by hematologic malignancies, and showed that the suicide machinery controls severe GvHD. To maximize the extent and persistence of GvT activity mediated by TK cells, we exploited the positive effects of IL-7 in maintaining the homeostasis of memory lymphocytes. We observed that while stimulation with anti-CD3 antibodies and culture in the presence of high doses of IL-2 generates mainly CD45RA−CD62L− effector memory (EM) TK cells with a limited ability to engraft and persist in vivo, stimulation with anti-CD3/CD28 conjugated cell sized beads and culture with low doses of IL-7 result in the generation of CD45RA−/CD62L+ TK cells with central memory (CM) functional phenotype, producing high levels of IL-2 upon stimulation, and expressing persistent high levels of IL-2R alpha and IL-7R alpha, a molecule associated to activation and long term survival memory T-cells, respectively. In mixed lymphocytes cultures (MLR), CM TK cells showed higher proliferative potential and lower sensitivity to apoptosis than EM TK cells, and such alloreactive CM TK cells kept CCR7 and IL-7R alpha expression even after 2ndary MLR. Accordingly, CM cells were more potent than EM cells in mediating allogeneic GvHD, after infusion in NOD/Scid mice, previously transplanted with allogeneic human skin. Newly developed homeostatic CM TK cells combine a high alloreactive potential with the selective sensitivity to GCV-mediated cell death, providing a tool for maximal anti-tumor activity with control of GvHD.
Pulmonary Embolism in Cancer Patients Is Associated with Lower Severity of Illness and Lower Mortality Rates with Increasing Use of CT Scans.
Authors: Villaruz Liza C. , Dang Qianyu , Ragni Margaret V.
Published: October 2019

Pulmonary embolism (PE) is a major cause of morbidity and mortality in patients with cancer (CA). However, predicting which CA patients will develop PE is not possible and thromboprophylaxis is infrequently used. In order to define the incidence and clinical characteristics of CA patients with PE, we analyzed discharge data from all patients with PE collected by the Pennsylvania Health Care Cost Containment Council (PHC4) from 1997–2001. The analyses included all PE discharges with ICD-9 codes 415.11 and 415.19, excluding pregnant patients and all second or later PE admissions. Cancer diagnoses were enumerated by ICD-9 codes 140–239 and diagnostic procedures by revenue codes for computerized tomographic (CT) (including spiral CT) scans. Of 33,036 PE patients identified in this period, one-quarter had cancer, among whom PE incidence increased marginally, from 1,589 (24.0%) in 1997 to 2,262 (25.4%) in 2001, p = 0.072. CA patients were more likely to be Caucasian, OR 1.22, p<0.001, greater than 70 years of age, OR 1.16, p<0.001, and male, OR 1.19, p<0.001. CA of the lung was the most common CA type associated with PE, 1150 (17.7%), followed by CA of the digestive tract, 1150 (17.4%); genitourinary tract, 832 (12.6%); and lymphatic/hematopoetic tissues, 771 (11.7%). CT scans were performed with increasing frequency in CA PE patients, from 27.7% in 1997 to 50.0% in 2001, p < 0.001, and more frequently in Caucasians than in blacks, OR =1.61, p<0.001, and in those 70 years or younger, OR =1.91, p<0.001. At the same time, MediQual Atlas severity of illness scores decreased over time, from a mean 2.6 in 1997 to 2.3 in 2001, p<0.001, with a significant decrease in the two higher categories, 65.8% vs. 38.1%, and an increase in the three lowest categories, 34.2% to 61.9%, p<0.001. CA patients were less likely than non-CA patients to have other risk factors for PE, i.e. trauma, 3.7% vs. 8.1%, p< 0.001, or congestive heart failure, 12.9% vs. 20.8%, p<0.001. Although CA patients had higher mortality rates than non-CA PE patients, OR 1.86, p<0.001, these rates decreased from 23.3% in 1997 to 18.6% in 2001, p<0.001. In conclusion, patients with cancer account for one fourth of all PE, and they are typically elderly Caucasian males with cancer of the lung or digestive tract. The increasing use of CT scans in this group is associated with lower severity of illness scores and decreasing mortality rates, which suggest PE may be diagnosed earlier. Consideration of early thromboprophylaxis among CA patients at risk for PE may improve health outcomes.
Inflammatory Disease Associated Gene Polymorphisms Detected in Patients with Chronic Graft-vs-Host Disease.
Authors: Shimada Masako , Onizuka Makoto , Oba Taku , Ogawa Yosiaki , Kawada Hiroshi , Miyamura Koichi , Hotta Tomomitsu , Kodera Yosihisa , Inoko Hidetoshi , Ando Kiyoshi
Published: October 2019

Chronic graft-vs-host disease (cGVHD) is the most serious and common long-term complication of allogeneic hematopoietic stem cell transplantation(HSCT). Incidence of cGVHD is 20% to 70% of recipient surviving more than 100days after HSCT. cGVHD most commonly affects the skin, liver, eyes, lung, or the mouth, while multiple other sites may also be affected. Similarities of clinical features of cGVHD and several inflammatory diseases and auto-immune diseases have been commonly observed. Autoantibody formation has also been noted in experimental models of cGVHD, although the pathogenetic role of antibodies in cGVHD is still poorly defined. Both donor and host factors contribute to the pathogenesis of GVHD. While the most important determinant is the compatibility of HLA, non HLA genetic associations with cGVHD have not been extensively studied. Therefore we investigated a relationship between cGVHD and well known inflammatory disease associated gene polymorphisms. We analyzed 100 pairs of Japanese HLA-matched sibling recipients and donors who underwent HSCT in Japanese Red-cross Nagoya First hospital(1990–2001). We examined SNPs, Fc receptor-like3 (FCRL3), programmed cell death 1(PDCD1), peptidyl arginine deiminase(PADI4), solute carrier family 22 memberA4(Slc22A4), and DLG5 by PCR-SSO methods or TaqMan assays, and analyzed correlation between these gene polymorphisms and clinical feature of cGVHD. Statistical analysis were done by using Fisher’s exact probability test.

So far FCRL3-169T allele has been correlated with high incidence of cGVHD(p=0.017). Especially, 18 patients out of 37 who has been occurred extensive cGVHD have -169T/T genotype. On the other hand, there is no relationship between cGVHD and FCRL3 SNP in donors. Our result is FCRL3 -169T allele has been correlated with incidence of cGVHD. FCRL3 is one of a new family genes of FCγ receptor II/III, and is located at chromosome 1q21-23 which is the regions implicated in susceptibility to multiple autoimmune disease. FCRL3 has high structural homology with classical FCγreceptor, although it’s ligands and function are not yet known. Contrary to our result, FCRL3 -169C allele is disease-susceptible allele to inflammatory disease. Since the expression of FCRL3 are detected in the germinal center in spleen and tonsils, residual host B cells or other types of cells expressing the gene might be involved in pathogenesis of cGVHD. This study is the first to report the relationship between cGVHD and inflammatory disease associated gene polymorphisms in hosts.
Bronchiolitis Obliterans Syndrome after Hematopoietic Stem Cell Transplantation: A Retrospective Multicenter Survey of the Late Effect Working Party EBMT.
Authors: Arat Mutlu , Socié Gérard , Passweg Jacob R. , Caballero Dolores , Ljungman Per T. , Bonifazi Francesca , Kumbasar Ozlem O. , Fernandez A.O. , Broeters D. , Guidi Stefano , Greil J. , Zander Axel R. , Tichelli Andre , Bekassy Albert N.
Published: October 2019

Aim and Scope: Substantial morbidity and mortality is caused by respiratory complications in 40–60% of patients after HSCT. Bronchiolitis obliterans syndrome (BOS) is reported in 2–13% of patients, occurring within 6–12 months post transplant. The LEWP launched a retrospective survey in 2003 to document the incidence, current practice for the diagnosis, clinical work-up and treatment of BOS after allogeneic HSCT.

Patients&Method: Between 2003 – 2005, 31 centers reported 226 patients with BOS. Adjusted BOS criteria were accepted (normal spirometry pretransplant, respiratory symptoms in the absence of an infection and combination of 3/4 items at onset: FEV1 <80% predicted/measured; FEV1/FVC <80% and MMFR <50% predicted/measured and residual volume >120%; HR CAT scan of the thorax. FOB and BAL were used in 60% of centers, but open lung bx.was not recommended by 75%.

Results: The median age of the patients was 30.9 years (1–62) and 19% was pediatric. The donors were 64.6% siblings. HSCT indication was hematological malignancy in 92.3% (37% AL, 21% CL). The graft source was BM in 60% and PBSC in 39%. The preparative regimen was myeloablation in 85.8%. The majority of the patients had antecedent GVHD (acute 66%, chronic 89%). The median interval from transplant to onset of BOS was 11.7 mo.s (1–260). Two third of the patients are still alive (22% in remission and 70% still receiving therapy). Median survival is 39.5 mo.s (1–277). The main reasons for high mortality rate are BOS (69%), relapse (15%) and GVHD (14%). The results of the univariate analysis on KM curves are summarized in Table 1.

Conclusion: Univariate data showed a negative impact of male gender, early onset (<11.7 mo.s) and MUD transplants. BOS is still an important cause of non-relapse late morbidity and mortality. Diagnosis and treatment are difficult and heterogeneous. The final analysis with multivariate methods should validate current results.

Variables tested in univariate analyses Variables Med. OS, months (95% CI) P NR: not reached Sibling/MUD NR 75.9 (28.1–123.6) 043 Male/Female 72.5 (47.6–97.4) NR 0.038 Onset: Early/Late 59 (33.3–84.8) NR 0.00001 Ablative/Non Ablative 102.8 (67.7–137.9) NR 0.191
AKT Kinase Addiction in Multiple Myeloma.
Authors: Bardeleben Carolyne , Yan Huajun , Frost Patrick , Hoang Bao , Shi Yijiang , Sharma Sanjai , Lichtenstein Alan
Published: October 2019

Cancer cells with activating kinase mutations are singularly hypersensitive to drugs that target these kinases, a phenomenon referred to as kinase or oncogene addiction. Although the AKT kinase is not known to be mutated in multiple myeloma (MM), it is frequently hyperactivated due to mutations in upstream signaling proteins. Previous work indicated that MM cells with heightened AKT activity were hypersensitive to drugs targeted to mTOR, a downstream substrate of AKT, suggesting an addiction to this pathway. “Kinase addiction” would theortically render the cell dependent on the kinase for growth. This dependency implies that other cascades of viability and/or proliferation may become downregulated subsequent to kinase activation. To test for AKT kinase addiction in MM we stably transfected the U266 MM cell line that is known to be protected against apoptosis by an autocrine Il-6/IL6R pathway (Caitlett-Falcone et al., Immunity 10, 105–115, 1999) with a constitutively active AKT construct (U266-AKT). AKT-transfected MM cells were protected against fas-induced apoptosis even when the autocrine IL-6 protective pathway was paralyzed by anti-IL6 and anti-IL6 receptor antibodies. However, as expected for an AKT addicted cell line, the U266-AKT cell line was more sensitive to agents that target the PI3/AKT/mTOR pathway, including two AKT inhibitors and the PI3K inhibitors LY294002 and wortmanin in terms of increased level of apoptosis and/or growth inhibition in comparison to the control U266 cell line. This “kinase-addicted” state was associated with down regulation of several components of the autocrine anti-apoptotic pathway of U266 cells. Expression of IL6, the IL6 receptor subunits gp80 and gp130 and STAT3 were all markedly silenced following AKT transfection. This down regulation of the survival pathway was relatively specific as expression of the fas receptor and VEGF receptors were relatively unaffected. The decrease in protein expression was also reflected in the decrease of mRNA for the receptor subunits, suggesting a transcriptional mechanism for down regulation. As expected from the decrease in autocrine IL-6 signaling, AKT-transfected cells also demonstrated a marked inhibition of Jak1 and MAPK 42/44 phosphorylation and enzymatic activity. In summary, over expression of activated AKT results in an AKT-addicted state whereby the MM cell becomes dependent on AKT for survival as other growth-promoting and anti-apoptotic cascades become paralyzed. These data support AKT as an important target for therapeutic intervention of MM and suggest such therapy would be most successful in patients whose MM cells contain the highest amount of activated AKT.
Biological Pathways and In Vivo Anti-Tumor Activity Induced by Atiprimod in Multiple Myeloma (MM).
Authors: Neri Paola , Tassone Pierfrancesco , Shammas Masood , Yasui Hiroshi , Schipani Ernestina , Batchu Ramesh B. , Blotta Simona , Prabhala Rao , Catley Laurence , Hamasaki Makoto , Hideshima Teru , Chauhan Dharminder , Jacob Gary S. , Picker Donald , Venuta Salvatore , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

Atiprimod (N-N-diethl-8, 8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) is a novel orally bio-available agent with anti-inflammatory properties. Although its in vitro anti-MM activity have been previously reported, here we have investigated molecular changes induced by Atiprimod as well as its in vivo activity in murine models of MM. Atiprimod inhibits in vitro growth and survival of IL-6 dependent as well as independent MM cell-lines in a time- and dose-dependent manner. Evaluation of changes in gene expression profile following treatment with Atiprimod identified down-regulation of genes involved in adhesion, cell-signalling, cell-cycle and BMP pathways and up-regulation of genes implicated in apoptosis and bone metabolism. The signalling pathway analysis identified the integrin, TGF-beta and FGF signaling as well as Wnt/b-catenin, IGF1 and cell cycle regulation networks as being most modulated by Atiprimod treatment. Next, we evaluated its in vivo activity in three different murine models of MM. A xenograft model bearing subcutaneous MM cells confirmed in vivo the anti-MM activity of Atiprimod and established its dose-response activity; a model based on engraftment of human fetal bone chip implanted in SCID mice (SCID-hu) with INA-6 cells, confirmed its ability to overcome the protective effects of the bone marrow milieu on MM cell growth, survival and drug resistance; and a SCID-hu model engrafted with primary patient MM cells confirmed its activity in the context of primary human disease recapitulating the clinical condition. Finally, we observed reduced number of osteoclasts, following Atiprimod treatment, compared to control bone samples confirming its beneficial effects on bone remodelling. Taken together, these data demonstrate the in vitro and in vivo anti-tumor activity of Atiprimod and delineate potential molecular targets triggered by this agent, providing a preclinical rational for its clinical evaluation in MM.
Eradication of Tumors in Pre-Clinical Models of Multiple Myeloma by Anti-CS1 Monoclonal Antibody HuLuc63: Mechanism of Action Studies.
Authors: Rice Audie , Dillon Myles , van Abbema Anne , Jesaitis Lynne , Wong Melanie , Lawson Stacey , Liu Gao , Zhang Yin , Powers David , Rhodes Susan , Caras Ingrid , Law Debbie , Afar Daniel
Published: October 2019

Introduction: We have recently shown that CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family, is uniformly expressed on myeloma cells from multiple myeloma (MM) patients. Based on its high expression in MM and limited expression in normal cells, we propose CS1 as a novel and specific antibody target for the treatment of MM.

Methods: A panel of monoclonal anti-CS1 antibodies (mAbs) was generated to identify a potential therapeutic candidate. MAb clones MuLuc63 and MuLuc90 were selected for testing in CS1 positive MM xenograft models in vivo in severe combined immunodeficient mice. HuLuc63, a humanized IgG1 version of MuLuc63, was generated as the potential therapeutic candidate for the treatment of MM. HuLuc63 and Fc-modified versions of HuLuc63 were tested for anti-tumor activity in mouse models vivo. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays were performed to define the potential mechanism of action for HuLuc63.

Results: Both MuLuc63 and MuLuc90 exhibited significant in vivo anti-tumor activity compared to isotype control antibodies in the L363 MM xenograft model. MuLuc63 was significantly more potent, resulting in rapid tumor eradication in most of the animals for the length of the study (~4 months). Based on these results, MuLuc63 was humanized to generate HuLuc63, which exhibited similar affinity for CS1 when compared to the mouse parent antibody. In two different MM xenograft models, L363 and OPM2, HuLuc63 exhibited significant anti-tumor activity resulting in tumor eradication in a high proportion of animals. To investigate the mechanism of action, two modified versions of HuLuc63 were tested in xenograft models. One version, HuLuc63-Ala,Ala, exhibits a mutation in the Fc region that decreases the ability to interact with the Fc receptor on natural killer (NK) cells. The second version, HuLuc63-LF, exhibits low levels of fucosylation in the Fc region that would result in increased binding to the Fc receptor. Compared to HuLuc63, the LF version exhibited significantly better in vivo anti-tumor activity towards, while the Ala,Ala mutant exhibited no anti-tumor activity. These data indicate that the Fc region of HuLuc63 is critical for its anti-tumor activity, and suggest ADCC as a possible mechanism of action. In vitro, HuLuc63 exhibits substantial ADCC towards L363 and OPM2 cells. The activity was dose-dependent, with increasing cytotoxicity being observed with concentrations ranging from 0.01μg/mL to10 μg/mL.

Conclusions: These pre-clinical data support HuLuc63 as a new therapeutic for the treatment of MM and suggest that ADCC is part of the mechanism of action. HuLuc63 will be entering a phase I clinical study for multiple myeloma.
The Humanized Anti-CD40 Antibody SGN-40 Inhibits Tumor Growth in LAGκ-1A, a CD40+ Mouse Model of Human Multiple Myeloma.
Authors: Campbell Richard A. , Gordon Melinda S. , Sanchez Eric , Chen Haiming , Turker Lauren , Trac Olivia , Li Mingjie , Pang Shen , Bonavida Benjamin , Said Jonathan , Drachman Jonathan G. , Berenson James R.
Published: October 2019

CD40 is a TNF receptor found on the cell surface of mature B cells (B lymphocytes) and most B-cell malignancies including multiple myeloma (MM). SGN-40 is a high-affinity, humanized monoclonal antibody that targets the CD40 antigen. Recently, it has been shown that SGN-40 decreases the proliferation of malignant B cells by partial agonistic signaling and effector functions in vitro. In this study, we examined the anti-MM effects of SGN-40 in vivo using a CD40+ SCID-hu murine model of human myeloma, LAGκ-1A. Each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGκ-1A tumor fragment into the left hind limb muscle. The tumor was allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum. Mice were then randomly assigned to one of four SGN-40 treatment groups (6 mice per treatment group). SGN-40 was administered via intraperitoneal injection twice per week at doses of 0.1, 0.3, 1, and 3 mg/kg. Control mice were given a control IgG antibody (3 mg/kg) using the same schedule. Mice receiving the higher doses of SGN-40 showed marked inhibition of tumor growth (0.3 mg/kg, P < 0.02; 1 mg/kg, P < 0.03; and 3 mg/kg, P < 0.04) and reduction of paraprotein levels (1 mg/kg, P < 0.05; and 3 mg/kg, P < 0.03) compared to mice receiving control antibody. At the lowest dose of SGN-40 evaluated (0.1 mg/kg) a slight inhibition of tumor growth was observable, but there was no effect on human paraprotein. Treatment with SGN-40 was not associated with any observed toxicity. Based on these data with SGN-40 monotherapy, we are currently investigating the antitumor activity of SGN-40 plus bortezomib as well as other available anti-MM agents using our in vivo SCID-hu myeloma murine model. These data for single-agent SGN-40 are encouraging and support testing SGN-40 both alone and in combination regimens to treat MM patients.
Pharmacokinetic (PK) and Pharmacodynamics (PD) Study of Two Doses of Bortezomib (Btz) in Patients with Relapsed Multiple Myeloma (MM).
Authors: Stewart A. Keith , Sullivan Dan , Lonial Sagar , Mohrbacher Ann F. , Chatta Gurkamal , Shustik Chaim , Burris Howard , Karol Michael , Zannikos Peter , Reece Donna E.
Published: October 2019

Btz (VELCADE®) is approved for patients (pts) with relapsed MM. This study characterized PK/PD of btz at 2 doses (1.0, 1.3mg/m2) after single- and multiple-dose administration, and evaluated their relationship. 24 pts with relapsed MM and creatinine clearance ≥50mL/min were randomized to btz 1.0 (n=12) or 1.3mg/m2 (n=12) on D 1, 4, 8 and 11 of a 21-D cycle (C). Pts could continue therapy for ≥8 Cs if beneficial. PK/PD samples were drawn at multiple time points on D1 and D11 of C1 and C3 over 48h. Plasma concentrations of btz were similar following administration of 1.0 and 1.3mg/m2; differences in mean plasma Cmax and AUC values were within the degree of intersubject variability (Table). There was a decrease in clearance and an increase in terminal half-life of btz on D11 vs D1 of C1.The volume of distribution of btz suggests extensive peripheral tissue distribution. After single or repeat administration, mean maximum percent inhibition of 20S proteasome activity (vs baseline) in whole blood was 70–84% and 73–83% for the 1.0 and 1.3mg/m2 doses, respectively. The percentage of pts who had >90% inhibition was greater in subsequent doses vs first dose. Reversibility of 20S proteasome inhibition in whole blood was demonstrated. PK/PD modeling revealed an exposure-response relationship, with Emax and EC50 values consistent across study days. These results confirm btz is a potent inhibitor of the 20S proteasome. The efficacy of single-agent btz has been explored and validated in the pivotal phase 2 SUMMIT and phase 3 APEX trials at 1.3mg/m2, and explored in the phase 2 CREST trial at 1.0mg/m2. Complete safety and efficacy data by dose will be presented.

Mean±SD PK parameters of btz in plasma 1.0mg/m2 1.3mg/m2 1.0mg/m2 1.3mg/m2 D1/C1 (n=11) D11/C1 (n=12) an=5;bn=11;cn=10;dn=7;en=11;fn=9 Tmax (h) 0.13±0.13 0.11±0.06 0.07±0.03 0.20±0.29 Cmax (ng/mL) 56.7±36.3 112±122 106.2±46.7 88.6±47.6 AUC48h(ng·h/mL) 26.5±12.4 34.6±19.8 82.8±35.9b 82.4±28.6b AUClast (ng·h/mL) 22.6±12.6 31.5±18.6 137±106 122.2±67.4 CL (L/h) 102.1±48.1 111.6±73.6 23.2±17.8 28.0±19.8 Vβ (L) 1976±2498a 2015±2974d 1659±752c 2415±1711 t1/2λz(h) 30.7±44.8a 11.5±12.7d 78.9±50.9c 75.6±49.9 D1/C3 (n=12) D11/C3 (n=12) Tmax (h) 0.12±0.12 0.10±0.05 0.16±0.17 0.10±0.05 Cmax (ng/mL) 66.5±42.6 120.3±70.7 83.9±69.3 114.9±98.3 AUC48h(ng·h/mL) 66.4±24.1 79.4±24.5b 101.6±58.2b 85.2±18.7f AUClast (ng·h/mL) 74.8±35.8 86.0±27.6 227±181 160.7±67.1 CL (L/h) 32.2±19.0 32.1±15.4 15.1±13.9 18.2±9.2 Vβ (L) 1852±951d 2059±1231 3294±2993e 2505±1641 t1/2λz (h) 39.9±14.4d 49.1±34.6 193±169e 108.6±64.8
Final Results of a Phase II Study of Bortezomib (Velcade) in Combination with Liposomal Doxorubicin (Doxil) and Thalidomide (VDT) Demonstrate a Sustained High Response Rates in Patients (pts) with Relapsed (rel) or Refactory (ref) Multiple Myeloma.
Authors: Chanan-khan Asher Alban , Padmanabhan Swaminathan , Miller Kena C. , Musiel Laurie , Yu Jihnhee , Bernstein Zale P. , Manochakian Rami , Czuczman Myron S.
Published: October 2019

Background: Tumor microenvironment (ME) plays an important role in MM. It is associated with disease progression, metastasis, and resistance to therapy. Therefore, targeting the ME and the tumor cell simultaneously may be an effective way to overcome resistance in pts with rel/ref MM.

Aim: Orlowski et al reported improved anti-tumor responses when bortezomib (V) was combined with doxil (D) in pts with hematologic malignancies. We investigated clinically, this approach i.e., targeting the MM cell as well as its ME, using a combination of V, D and low-dose thalidomide (T) as salvage therapy for pts with rel/ref MM. Here we report the final results along with survival data of this phase II study.

Methods: All pts with rel/ref disease were eligible for this study. V was given at 1.3mg/m2 (D1,4,15,18) and D at 20mg/m2 (D1,15) every 4 weeks with daily T (200 mg) for 4–6 cycles. SWOG criterion was used to assess response. Low-dose coumadin (1–2 mg po qd) was used for prevention of venous thromboembolism (VTE).

Results: Twenty three pts (9M, 14F; median age −57 , range 43–79 yrs; 21MM, 2 WM) have been enrolled. All pts had Stage III disease, median b2M was 4.2 and median number of prior therapies were 5(range 1–6). Prior therapies included stem cell transplant(SCT) in (41%), T (41%), adriamycin(A) (65%) steroids (82%) and velcade (12%). 74% had refractory disease. Seventeen pts have completed at least 1 cyc and are available for toxicity and response evaluation. One pt died of sepsis prior to completing 1 cyc and 1 pt with PR was taken off study for non-compliance after 1 cyc. ORR was with 65%(CR+PR) with 23% CR all of whom were IFE negative. The Median Progression Free survival was 10.9 months with an median overall survival of 15.7 months. Toxicity: 2 pts developed Gr. II plantar-palmar erythrodysthesia (PPE) and 1 had Gr. III cellulitis. No VTE was noted. No significant non-hematologic Gr. III/IV toxicity were seen. Despite prior exposure to anthracycle, we did not noted any cardiotoxicity with D.No significant neuropathy was noted.

Conclusions: Pt with rel/ref MM usually have aggressive disease with paucity of effective regimens. VDT is an effective salvage regimen. Final results show high response rates with 22% of the patients achieving complete (IFE−) remissions. Responses were noted regardless of type of prior therapy. VDT could be safely given in patients with renal failure/insufficiency. Venous Thromboembolism (VTE) does not appear to be a problem with low dose coumadin prophylaxis. Final results of this phase II study will be presented at the 48 th annual ASH meeting.
Treatment with Bendamustine, Thalidomide and Prednisolone (BPT) in Patients with Refractory or Relapsed Multiple Myeloma after Autologous Stem Cell Transplantation or Conventional Chemotherapy: Results of a Phase I Clinical Trial.
Authors: Poenisch Wolfram , Rozanski Marta , Goldschmidt Hartmut , Hoffmann Franz-Albert , Boldt Thomas , Schwarzer Andreas , Ruffert Ute , Rohrberg Robert , Schwalbe Elke , Uhlig Jens , Zehrfeld Thomas , Schirmer Veronika , Haas Antje , Kreibich Ute , Niederwieser Dietger
Published: October 2019

Thalidomide is an active single agent in advanced relapsed or refractory multiple myeloma (MM). The combination of low dose thalidomide with bendamustine and prednisolone might maintain or increase efficacy of the drug while avoiding dose limiting toxicity (DLT).

Patients and Methods: The treatment consists of a fixed dose of bendamustine (60mg/qm) day 1, 8, and 15 with prednisolone (100 mg) day 1, 8, 15, and 22. In addition, thalidomide is given in three escalating doses, starting with 50 mg to a maximum of 200 mg daily. At each dose level 8 to 12 relapsed or refractory patients, half of them after conventional chemotherapy and half after high dose therapy with stem cell support, were enrolled. Cycles were repeated every 28 days for a minimum of 2 and a maximum of 10 cycles either until a maximal response was achieved, or until a DLT or a disease progression were observed. Between March 2004 and May 2006, 28 patients were enrolled: 8 in the first dose level with 50 mg thalidomide; 8 in the second dose level with 100 mg and 12 in the third dose level with 200 mg. All patients had received a minimum of 2 prior treatment regimens. Seven patients had been refractory to the last treatment. Median age was 67 years (range: 40 – 78). All patients completed 2 cycles of BPT-treatment and are therefore evaluable. Response was assessed using EBMT criteria modified to include near complete remission (nCR) and very good partial remission (VGPR).

Results: Twentyfive of 28 patients responded after at least 2 cycles of chemotherapy with 3 CR, 1 nCR, 5 VGPR, 15 PR and 1 MR. Two patients had stable disease and one patient was refractory. With a median follow up of 15 months, EFS and OS at twelve months were 34 % and 92 %, respectively. The most common side effects were constipation (11 patients WHO grade 1, 8 patients WHO grade 2), polyneuropathy (15 patients WHO grade 1, 3 patients WHO grade 2) and somnolence (4 patients WHO grade 1). None of the 28 patients developed dose-limiting hematoxicity as defined by an ANC < 1,0 Gpt/l for > 7 days or an ANC < 0,5 Gpt/l for > 3 days or platelet count < 25 Gpt/l. Transient neutropenia was reported in 9 patients (WHO grade 3 and 4) but no thrombocytopenia was observed.

Conclusion: BPT with a dose between 50 and 200 mg thalidomide daily is well tolerated in patients with relapsed or refractory MM.
Rituximab in CD20 Positive Multiple Myeloma: A Prospective Study from the IFM Group.
Authors: Moreau Philippe , Voillat Laurent , Benboubker Lotfi , Dumontet Charles , Mathiot Claire , Hulin Cyrille , Robillard Nelly , Loiseau Herve Avet , Herault Olivier , Garnache Francine , Varoqueaux Nathalie , Harousseau Jean-luc , Bataille Regis
Published: October 2019

Multiple myeloma (MM) is a heterogenous disease. A strong association between small mature plasma cell morphology, t(11;14) and CD20 expression has been described in approximately 10% of the patients with MM (Robillard et al, Blood 2003). In this subgroup of patients with MM expressing CD20, rituximab (anti-CD20 chimeric monoclonal antibody) could target the antigen and could have a clinical impact. Thus we conducted a prospective phase II trial of 4 weekly IV infusions of 375 mg/m2 rituximab in patients with MM expressing CD20 on at least 33% of tumor cells. From 07/2004 to 02/2006, 14 consecutive patients, median age 65 years, with either stage I MM never pretreated (n = 7) or stage III MM in relapse or refractory after a median of 2 lines of therapy (n= 7) were treated. Immunophenotype using flow cytometry revealed that a median of 75% of tumor cells were expressing CD20 (range, 33–100%) at the onset of therapy. Responses were evaluated 3 months after therapy according to EBMT criteria. At the reference date of June 1st, 2006, a single patient, who had relapsed 8 months after a double autologous SCT, experienced a minor response, ongoing 18 months after rituximab therapy. At the time of rituximab therapy, 100% of its plasma cells were expressing CD20, and 3 months after treatment, bone marrow examination showed 0.6% of plasma cells, none of them expressing CD20. Five patients (1 with relapsed MM and 4 with stage I MM) experienced stable disease, ongoing for 3, 4, 4, 11 and 12 months, respectively. Three patients with stage I MM had stable disease but subsequently progressed 10, 11, and 15 months after therapy, respectively. The last 5 patients with relapsed MM did not respond to anti-CD20 therapy, despite partial clearance of CD20+ plasma cells in the bone marrow in 2 cases. Conversely in the 3 latter cases, the percentage of CD20+ plasma cells did not decrease despite rituximab therapy.

Several factors have been described to explain resistance against rituximab in a variety of B-cell malignancies such as the level of CD20 expression, dissociated action of complement-dependent cytotoxicity and antibody-dependant cellular cytotoxicity, polymorphism in FcgammaRIIIA receptor, and may be inadequate dose schedule. These mechanisms could explain the marginal activity of rituximab as single-agent in CD20+ MM.
The CALM-AF10 Fusion Gene Expressed in Transgenic Mice Produces Acute Leukemia.
Authors: Caudell David L. , Zhang Zhenhau , Chung Yangjo , Aplan Peter D.
Published: October 2019

The rare but recurring translocation [t(10;11)(p13;q21)] occurs in patients with both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML); a more recent analysis of clinical cases showed that of the T-ALL cases, t(10;11) are restricted to the TCRγδ lineage. This translocation was initially described in immature T-cells; subsequently the molecular features were characterized in the U937 cell line and shown to result in the production of a CALM-AF10 fusion gene. In order to study the leukemic effects of a CALM-AF10 fusion in mice as a model of human disease, a CALM-AF10 fusion cDNA was cloned from the U937 cell line and inserted into an expression vector that utilized Vav 5′ and 3′ regulatory elements. The Vav gene is expressed in all hematopoietic tissues and is essential for lymphocyte development; Vav regulatory elements have been used by a number of groups to direct transgene expression to the hematopoietic compartment. We generated transgenic Vav-CALM-AF10 mice by pronuclear injection, and followed offspring from two founder mice that were shown to express the CALM-AF10 transgene in hematopoietic tissues for 18 months. Mice were clinically healthy for the first 9 months of life, but had T and B cell developmental abnormalities, including decreased CD4+/CD8+ cells and increased CD4−/CD8− cells in the thymus. In addition, CALM-AF10 mice had decreased B220+/IgM+ cells in the spleen, as well as an unusual Mac1+/B220+ population of cells in the spleen. Eight of 20 offspring from founder C10, and 7 of 16 from founder E6 developed acute leukemia between 9 and 18 months-of-age. Additionally, 5 of 20 mice from the C10 founder and 3 of 16 mice from the E6 founder were found dead unexpectedly but were too necrotic for analysis. Animals that developed acute leukemia were either found dead without premonitory symptoms or presented with ruffled fur, hunched posture and dyspnea. Gross findings included splenomegaly and hepatomegaly. CBC analysis indicated a leukocytosis and anemia and peripheral blood and bone marrow contained increased numbers of blasts and immature forms. Spleens from leukemic mice were effaced by leukemic blasts, which also invaded peripheral tissues including liver, lung, and kidney. Tissue sections from most of the leukemias were myeloperoxidase (MPO) positive and negative for CD3, F4/80, and B220, and immunophenotype was typically Mac1+, Gr+/−, CD4−, CD8−, and B220−. Spleen and/or liver from animals with leukemia were evaluated for clonal T-cell receptor (TCR) β or δ rearrangements. Two of 11 had clonal TCR β rearrangements, one of which also had a clonal TCR δ rearrangement. These findings suggest that CALM-AF10 produces a predominately myeloid leukemia in mice, some of which also have T-cell features. The long latency period and incomplete penetrance suggests that collaborative events may be needed to initiate leukemogenesis.
AML1/MDS1/EVI1 Induces Essential Thrombocythemia in Mice Independently of Jak2 Mutations.
Authors: Senyuk Vitalyi , Li Donglan , Fazzina Raffaella , Cattaneo Francesca , Rinaldi Ciro R. , Dickstein Jerome , Nucifora Giuseppina
Published: October 2019

The molecular etiology of subgroups of essential thrombocythemia (ET), as well as some other myeloproliferative disorders (MPDs) has been recently better understood by the identification of an activating somatic point mutation (Valine 617 to Phenylalanine) in Jak2 detected in patients with MPDs. Approximately 23%–57% of ET patients harbor this mutation, which is thought to be one of the dominant factors contributing to the disease. However, the pathways disrupted in at least half of ET patients who have wild type Jak2 are not known. The fusion gene AML1/MDS1/EVI1 (AME), a product of the t(3;21)(q26;q22) translocation, is associated with several hematopoietic disorders, primarily chronic myelogenous leukemia and acute myeloid leukemia, and to a lesser extent with ET. We have investigated the role of AME by generating C57BL/6J mice that express this fusion oncogene in their bone marrow after bone marrow infection and transplantation. After a latency of 12 ± 1.8 months, all the reconstituted mice invariably developed an ET-like disease. The disease was fatal and the animals died of anemia probably caused by bleeding. Until 1–2 weeks before death, at which time the mice were severely cytopenic, the peripheral blood hemoglobin and leukocyte counts of the reconstituted AME mice were normal. The only striking difference was the platelet counts, which were consistently above the normal range (1456–3153 and 940–1608 K/ml for AME and control mice, respectively, p=0.00029). Platelets appeared dysplastic with anisocytosis, various degrees of degranulation, and giant size. The bone core biopsies of AME mice were especially remarkable for large and giant megakaryocytes with a tendency for clustering. Touch preparations of the biopsies showed progressive maturation in the granulocytic and erythroid precursor cell lines, a normal myeloid to erythroid ratio and no evidence of dyshematopoiesis. These finding coupled with markedly increased levels of TPO but normal concentrations of c-Mpl and Epo-R are concordant with the morphologic and laboratory features of patients with ET. The sequence analysis of exon 12 of Jak2 demonstrated no guanine-to-thymine transversion at nucleotide 1849 in all AME mice tested. These data are valuable because they provide a novel mouse model for understanding the deregulated pathways in ET patients with wild-type Jak2.