ALBERT

Description: Biallelic mutations in the human breast cancer susceptibility gene, BRCA2, are associated with Fanconi anemia, implying that some persons who inherit 2 deleterious variants of BRCA2 are able to survive even though it is well established that BRCA2 is indispensable for viability in mice. One such variant, IVS7 + 2T 〉 G, results in premature protein truncation because of skipping of exon 7. Surprisingly, the persons who are either IVS7 + 2T 〉 G homozygous or compound heterozygous are born alive but die of malignancy associated with Fanconi anemia. Using a mouse embryonic stem cell–based functional assay, we found that the IVS7 + 2T 〉 G allele produces an alternatively spliced transcript lacking exons 4-7, encoding an in-frame BRCA2 protein with an internal deletion of 105 amino acids (BRCA2Δ105). We demonstrate that BRCA2Δ105 is proficient in homologous recombination-mediated DNA repair as measured by different functional assays. Evaluation of this transcript in normal and leukemia cells suggests that BRCA2Δ105 may contribute to the viability of persons inheriting this mutation. In this study, we have also characterized 5 other BRCA2 variants and found 3 of these (p.L2510P, p.R2336H, and p.W2626C) to be deleterious and 2 (p.I2490T and p.K2729N) probably neutral. Such studies are important to understand the functional significance of unclassified BRCA2 variants.

Description: Key Points In vitro analysis of VKORC1 mutations perfectly reflects patients’ warfarin resistance phenotypes. In silico docking of warfarin on a VKORC1 model reveals a putative docking site in agreement with the locations of OACR-associated mutations.

Description: Abstract 1125 Background and Objectives: Congenital Factor XIII (FXIII) deficiency is a rare, autosomal recessive bleeding disorder, with a significant majority of patients showing defects in the FXIII-A subunit. The disease is caused by a variety of F13A1 gene mutations resulting in a severe quantitative FXIII-A type I deficiency. Here, we report a wide spectrum of mutations identified in 41 severe Factor XIII-A deficiency patients (≥6 years of age, mean, 26.4; range, 7–60). Patients and Methods: A total of 41 patients were recruited in a multinational (23 centers, 11 countries), open-label, single-arm, phase 3, prophylaxis trial for the evaluation of the efficacy and safety of a novel recombinant FXIII (rFXIII). Eleven of the 41 patients (Israel [n=8], Switzerland [n=2] and Italy [n=1]) already had established genetic backgrounds and carried previously reported F13A1 mutations. Mutational screening in the remaining 30 patients who had unknown genetic status was done using direct sequencing on an ABI Prism 3130TL (Applied Biosystems, Weiterstadt, Germany). For two patients with splice-site mutations, cDNA analysis was done with RT-PCR (Quiagen One-step RT-PCR kit). The crystallographic model of the recombinant human cellular coagulation FXIIIA zymogen (EC: 2.3.2.13, resolution solved to 2.1Å) was downloaded from the Protein Data Bank (data file 1F13) for viewing, analysis and graphical rendering using YASARAver11.11.2. Classic molecular dynamic simulation approaches were used on the FXIII-A crystal structure to evaluate the effect of the novel missense mutations on protein structure. Results and Discussion: In total, 31 distinct mutations in 41 patients have been identified revealing 13 missense mutations, seven small deletions, six splice-site mutations, three nonsense mutations, one large deletion and one small insertion. Amongst this cohort of mutations, 16 mutations were novel. In one patient, a heterozygous missense mutation was detected in spite of severe deficiency symptoms shown by the patient. We assume that the other mutation could not be detected within the scope of our screening set up. The IVS5–1G〉A (c.691–1G〉A) splice-site mutation was the most commonly occurring (n=9 [21.9%]) mutation in this cohort. Two siblings carried a missense mutation in F13A1 gene (Ser295Arg) and in combination with a novel variant in F13B gene (Ser634Phe). This variant does not seem to significantly affect the B-subunit stability, since it is located in the terminal part of the molecule. Missense mutations are of special interest since they help to better understand the structure and function of FXIII A-subunit. The MD simulation of four novel missense mutations predicted a damaging effect on the protein structure for three of the missense mutations (Glu229Arg, Leu275Phe, and Arg703Trp) based on changes in bonding patterns, free energy calculations, change in local secondary structure etc. The Leu588Gln located on the surface of barrel 1 domain was not observed to cause a significant change in local structure, nevertheless, owing to its surface presentation it might influence the interaction with FXIII B-subunit. The analysis for the two novel splice-site mutations (IVS7+1G〉A, IVS12+1G〉A) did not show any aberrant mRNA product. Inhibitor development is the most undesirable side-effect of treatment with plasma-derived or rFXIII products. Overall, the incidence of inhibitors in patients with congenital FXIII deficiency is unknown, but is expected to be much lower compared with other coagulopathies, e.g. hemophilia A. In the present study, none of the patients treated with rFXIII (a mean treatment period of 322 days) developed FXIII neutralizing antibodies. Four patients developed transient low-titer anti-rFXIII antibodies that had no neutralizing activity. Interestingly, two patients (male and female) were siblings carrying the same common splice-site mutation in intron 5 (IVS5-1 G〉A). The third patient was compound heterozygous for two missense mutations (Gln229Arg; Ser413Leu). The fourth patient was also found to be compound heterozygous for two missense mutations (Arg77His; Leu275Phe). Conclusion: To conclude, the identified mutations, including 16 novel mutations, are implicated in the causality of severe FXIII deficiency. However, none of these mutations were associated with the development of inhibitory antibodies in the context of treatment with rFXIII. Disclosures: Tehranchi: Novo Nordisk: Employment, Equity Ownership. Oldenburg:Biogen Idec, Swedish Orphan Biovitrum: Honoraria; Baxter, Bayer, Biotest, CSL Behring, Grifols, Inspiration, Novo Nordisk, Octapharma, Pfizer: Honoraria, Research Funding.

Description: Abstract 1736 EBV and KSHV are associated with a variety of lymphoid malignancies. Bortezomib, a proteasome inhibitor, is among the most potent activators of EBV and KSHV lytic infection and is active at nanomolar concentrations. Studying EBV Burkitt's cell lines, KSHV primary effusion lymphoma cell lines and EBV myeloma cell lines, we found that lytic gene expression is a class effect shared by other proteasome inhibitors, such as MG-132 and epoxomicin. Proteasome inhibition results in IKB stabilization and inhibition of NFKB activity and this effect has been implicated in many of the effects of bortezomib. We studied the promoters of EBV and KSHV immediate early viral protein reporters (EBV Zta and KSHV Rta) and found that IKB super-repressor did not activate promoter reporters suggesting that other pathways must be important in activation. Therefore, we sought to better understand the drug's impact on viral gene expression in virus-associated tumor cells. ER stress has been implicated in bortezomib's antitumor effects. Thapsigargin and tunicamycin are classic activators of ER stress that do not act through proteasomal inhibition. These agents were found to be potent activators of the EBV and KSHV lytic cycle. Previous studies have identified C/EBP family members (C/EBP alpha and beta) as activators of EBV and KSHV immediate early gene expression. In other studies, C/EBP family members (C/EBP beta, CHOP10) have been implicated in regulating the ER stress response. We found that bortezomib, thapsigargin and tunicamycin increased levels of the activating C/EBP beta LAP isoform as assessed by immunoblot and by real-time RT-PCR. Treatment also led to increase in ATF4, XBP1(s), CHOP10, and ATF6 cleavage, all consistent with induction of the ER stress response. Additionally, we showed that treatment with bortezomib increased C/EBP beta binding to previously characterized binding sites in the Zta promoter and expression of C/EBP beta LAP isoform was sufficient to activate EBV immediate early lytic promoters. Finally, we demonstrated that in tumor cell lines with C/EBP beta silenced by doxycycline regulated siRNA, induction of EBV lytic induction by bortezomib and thapsigargin was blocked. These results demonstrate that both human lymphoma associated herpesviruses (EBV and KSHV) are activated into lytic cycle by bortezomib and that these effects are mediated through ER stress pathways and specifically involve C/EBP beta. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1897 We have previously shown that haploidentical (HI) HSCT with low dose donor T-cell infusion provides a survival advantage in tumor bearing mice when compared to parent F1 or MHC-matched transplant models. We suggest that MHC difference in HI-HSCT generates early T-cell clonal activation against the unshared MHC haplotype, which eliminates residual tumor cells that express the unshared MHC haplotype. However, alteration in MHC antigen expression is a significant contributor to tumor escape from graft-versus-tumor (GVT) activity. Recent haploidentical transplant data revealed that uniparental disomy, the loss of the HLA haplotype, is a clinically relevant mechanism of tumor escape that leads to post-transplant leukemia relapse. Murine renal cell carcinoma, RENCA-TGL, cell line normally expresses only H2Kd as a MHC molecule. Therefore, in our haploidentical transplant model, T cell clonal activity is usually restricted against H2Kd molecule only. For circumventing the single haplotype expression of tumor model, we transfected this cell line with a H2Kb expressing vector, pAcGFP-NeoR-H2Kb, and generated stable clones with G418 selection. The clone that has more than 95% H2Kb expression used for in vivo experiments. Both tumor cell lines, i.e. parental and transfected clone, had similar in vivo tumor growth acceptance and growth rate. We then used two different haploidentical donors that were targeting different MHC haplotypes. Lethally irradiated B6D2F1 (H2Kb/d) mice were transplanted with T cell depleted bone marrow (TCD-BM) from either B6C3F1 (H-2Kb/k) (single haplo-1; SH1), or C3D2F1 (H2Kk/d) (single haplo 2; SH2) or both donor mice with low-dose (1×105) T-cells. In some experiments, animals were also injected either H2Kd or H2Kb/d expressing RENCA-TGL cells for the evaluation of GVT activity. Bone marrow (BM), spleens and thymi were harvested from recipients of single and double HI-HSCT at day 35 and showed similar cellularities. Interestingly, spleen and bone marrow had similar chimerism from both donors in DH-HSCT. There were no early transplant mortality, graft failure, weight loss and GVHD scoring difference among the double or single-haploidentical transplant recipients. In two other sets of experiments, we followed the tumor growth and the survival of tumor bearing mice after transplant. The recipients of DH-HSCT showed a better survival and GVT activity than the recipients of SH-HSCT in RENCA-TGL (H2Kb/d) bearing tumor model. These observations confirmed that MHC targeting plays a prominent role in tumor surveillance, and immune targeting the unshared MHC haplotype with haploidentical transplant induce remarkable survival advantage. Double HI-HSCT provides an unique anti-tumor activity that continues to exert GVT effect, even in case of MHC haplotype loss. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4027 The success of haploidentical (HI) hematopoietic stem cell transplantation (HSCT), suggests that graft-versus-leukemia (GVL) effect might have a substantial role in this transplant modality. Rigorous T-cell depletion (TCD) of the graft decreases the occurrence of graft-versus-host disease (GVHD) in HI-HSCT, however this results in immunodeficiency and high disease relapse rate, especially in patients with resistant or residual leukemia. Therefore, enhancing GVL activity of HSCT without increasing GVHD is crucial for improving the outcome of haploidentical transplant. Post-transplant IL-15 administration is shown to enhance immune reconstitution, particularly donor-derived NK and CD8+ T cell populations in murine models. We evaluated the efficacy of IL-15 for enhancing GVL effect in recipients of HI-HSCT. For developing clinically relevant haploidentical transplant models, different hybrid mice with B6 background that share the same haplotype (H2Kb) are used for our murine haploindentical transplant experiments. Lethally irradiated B6D2F1/J (H2Kb/d) mice are transplanted with B6CBAF1/J (H2Kb/k) TCD bone marrow (BM) and T cells at varying doses. Some animals were also given P815 tumor cells on the day of transplant. Administration of IL-15 significantly increased the numbers of CD8+ T and NK cells in the spleen and BM in the T cell depleted model at post-transplant day 28. Infusion of very low dose haploidentical T cells (1×104) with TCD-BM resulted in a conflicting effect on immune reconstitution, i.e. increased T cell numbers, and decreased NK cell population. Post-transplant IL-15 administration also changed this immune reconstitution pattern and significantly increased both T and NK cell numbers in recipients of HI-HSCT. In P815 challenged mice that were transplanted with very low dose T cell added TCD-BM, IL-15 administration significantly increased anti-tumor activity of the graft and improved survival (Figure 1) without increasing GVHD. This effect was observed when IL-15 administration was given at a later time point rather than immediately following transplantation, possibly allowing for more donor cell engraftment and T cell proliferation to take place. IL-15 administration without T cell infusion did not result in any survival improvement. We conclude that in our experimental HI transplant models, IL-15 administration augments anti-tumor effect of the HI-HSCT without increasing GVHD risk, and this effect requires presence of donor derived T cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2583 Background: Management of acute lymphoblastic leukemia (ALL) requires use of immunosuppressive agents like high-dose steroids and antimetabolites for prolonged periods which can predispose these patients for CMV reactivation and disease. As opposed to hematopoietic stem cell transplant there has been a real paucity of literature regarding clinical manifestations and management of CMV reactivation in ALL. In countries like India with a background of high CMV seropositivity (〉90%), reactivation is a serious concern in ALL patients while receiving chemotherapy. Timely recognition and treatment can avoid the morbidity and mortality as well as help maintaining dose intensity which is the key to achieve cure in ALL patients. Methods: This retrospective case series included adult ALL patients (〉14 years) who were being treated with chemotherapy between July 2009 to July 2011 at a tertiary care centre and detected to have CMV viraemia (Real time quantitative PCR with Roche CMV DNA QuantKit). PCR was done in patients with possibility of CMV infection based on clinical suspicion. Case records were analyzed for demography, chemotherapy details, clinical features, laboratory parameters, viral load, antiviral therapy and response. Results: Among 203 adult ALL patients, 23 (males-18, females-5) were detected to have CMV viremia. Median age was 23 years (range, 16–44 years). Occurrence of CMV reactivation was most common during later part of induction or re-induction phase of therapy which includes high dose of steroids (14/23) followed by maintenance therapy with 6-mercaptopurine and methotrexate (5/23) and high dose cytarabine based treatment (4/23). Presenting features were: fever (19/23), fever alone (2/23) respiratory symptoms (9/23), anorexia (10/23), loose stools (8/23), abdominal pain (7/23) and splenomegaly (1/23). Abnormal laboratory parameters were: leukopenia or thrombocytopenia (14/23), deranged liver function tests (12/23). CT thorax was abnormal in 3 patients. Bacterial and fungal co-infection was seen in 5/23 patients. Median CMV viral load was 3.0 ×103 copy numbers (range, 708–1.38×106). Eighteen of these patients were treated with intravenous gancyclovir for a period of 14 days. In remaining 5 patients abnormal clinical and lab parameters improved either with antibiotic therapy or spontaneously. Median time to fever defervescence was 4 days (range, 2–5 days). Blood counts recovered after median period of 5 days (range 3–9 days). Gancyclovir related neutropenia and transaminitis developed in 1 patient. CMV titre became undetectable after a period of 2–4 weeks. Conclusion: Awareness of diverse clinical manifestations of CMV infection and high index of suspicion is important for timely diagnosis. Early diagnosis and treatment with gancyclovir reduces the morbidity, empirical use of other antimicrobials and avoids delays in administration of chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1453 The use of haploidentical donors extends the potential clinical application of HSCT. However, relapse of resistant malignancy contributes to low success rates in high risk patients. Relapse may be due to the ability of leukemic cells to immunologically escape a single donor's GVL effects. We hypothesized that the use of two haploidentical donors, each targeting a different recipient haplotype, will increase anti- leukemia activity after double haploidentical SCT. We performed murine studies to establish new single haploidentical (SH) and double-haploidentical (DH) murine models that mimic the possible scenarios which might be encountered clinically rather that using more traditional Parent → F1 models. We first established a haploidentical transplant model using two different hybrid mouse strains as donor and recipient in the experiments. Lethally irradiated B6CBAF1 (H2Kb/k) recipients were transplanted with T cell depleted (TCD) bone marrow (BM) from B6D2F1 (H2Kb/d) donors. Recipient mice harvested at days 28, 42 and 56, showed more than 90% donor cell engraftment, including donor derived lymphopoiesis and myelopoiesis, without evidence of graft versus host disease. Subsequently, lethally irradiated B6CBAF1 (H2Kb/k) recipients were transplanted with TCD-BM from two haploidentical donors (DH model) including B6SJF1 (H2Kb/s) (donor 1 - D1) and B6D2F1 (H2Kb/d) (donor 2 - D2). We observed recipients for 90 days and all mice survived without evidence of GVHD or weight loss. Analyses of blood collected retro-orbitally at day 90 revealed that recipients of DH transplants had significantly higher WBC and neutrophil counts than recipients of SH HSCT from either D1 or D2 respectively. DH recipients consistently showed successful engraftment with mixed chimerism in both bone marrow and spleen. There was no difference in thymopoiesis, B cell and myeloid cell reconstitution compared to SH transplants. In contrast, the number of splenic T cells was higher in SH recipients of D1 marrow (B6SJF1). We then explored the effects of low dose T cell infusions (1×105) on chimerism of donor cells. Low dose T cell infusion from either D1 or D2 did not affect the BM cellularity, but did increase the degree of dominance of that donor's cells in the BM and spleen. A similar outcome was observed when this study was extended to other models such as B6C3 + C3D2F1 → B6D2F1 and B6CBAF1 + B6SJF1 → CB6F1 models where all recipients of DH transplants survived without evidence of GVHD. Recipients of TCD DH transplants were challenged with P815 tumor cells. We used B6SJF1 (D1) + B6CBAF1 (D2) → B6D2F1 model in tumor experiments. Interestingly, recipients of TCD-DH transplants exhibited a significantly better survival than recipients of D1 SH or D2 SH transplants. However, after a low dose T cell infusion (1 ×105), recipients of D2 BM survived significantly better than recipients of D1 BM. In contrast to the TCD model, recipients of DH BM + DH T cells show similar probability of survival with recipients of D2 SH BM + D2 SH T cells. We conclude that TCD DH HSCT results in successful engraftment of both types of BM cells. Additionally, infusion of low dose haploidentical T cells improves the anti-tumor effect without stimulating GVHD. Double haploidentical HSCT may be an ideal platform to enhance GVL effects after transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: Myelomonocytic cells play a key role in the progression of many solid tumors. However, very little is known about their contribution to the progression of hematopoietic cancers. We investigated the role of monocytes in the progression of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We demonstrated that coculturing human monocytes in vitro with CD19+ BCP-ALL blasts from patients “conditioned” them to an inflammatory phenotype characterized by significant up-regulation of the chemokine, CXCL10. This phenotype was also observable ex vivo in monocytes isolated from BCP-ALL patients, which show elevated CXCL10 production compared with monocytes from healthy donors. Functionally, the “conditioned” monocytes promoted migration and invasive capacity of BCP-ALL cells. Increased invasion was mediated by matrix metalloproteinase 9 expression and activity in the BCP-ALL cells induced by the monocyte-derived CXCL10. However, neither the “conditioned” monocytes nor the CXCL10 produced by these cells had any effect on the proliferation/viability of BCP-ALL cells and angiogenesis. Collectively, our results strongly suggest a protumoral role for human monocytes in BCP-ALL, orchestrated by CXCL10 and its effect on tumor cell migration and invasion. These observations highlight the importance of the CXCL10/CXCR3 chemokine circuit in BCP-ALL progression.

Description: Key Points VKORC1:p.Arg98Trp disrupts a di-arginine ER retention motif, resulting in mislocalization and degradation of the mutant VKORC1 protein. A second low-efficiency di-lysine ER localization and retention motif contributes to the partially deficient phenotype of VKCFD2 patients.