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Background.Hepatocellular carcinoma (HCC) remains a major cause of cancer-related deaths worldwide, with limited treatment options. Long noncoding RNAs (lncRNAs) are essential regulators of HCC progressand are closely related to the pyroptotic cell death. However, the influence of pyroptosis-related lncRNAs on HCC remains indefinite. Methods. We systematically explored the expression profile of pyroptosis-related lncRNAs to establish a novel signature for predicting the outcome of HCC patients based on The Cancer Genome Atlas (TCGA) database by multiple analysis methods. Four lncRNAs with the highest hazard ratio in the above model were selected for external verification in the Gene Expression Omnibus (GEO) database. Finally, the expression of these lncRNAs was verified by real-time quantitative polymerase chain reaction (RT-qPCR) in different cell lines. Results. The results showed that 25 pyroptosis-related lncRNAs were evidently correlatedwith the prognosis of HCC patients. Cox regression analyses demonstrated the prognostic ability of the risk model established by the pyroptosis-related lncRNAs. And the high and low-risk groups were linked to different types of infiltrating immune cells and affected the physiological state of the tumor microenvironment in HCC. Conclusion.We constructed and validated an independent pyroptosis-related lncRNAs prognostic model and preliminarily described the potential immune correlations, providing novel prognostic factors and directions of immunotherapeutic strategies for HCC.

Chimeric antigen receptor (CAR) T cell therapy provides a potentially curative option for patients with relapsed or refractory large B-cell lymphoma (R/R LBCL). However, there are major limitations of this therapy which result in treatment resistance in B-cell malignancies, including the inadequate CAR T cell trafficking and tumor infiltration, frequent tumor antigen escape and poor CAR T cell persistence. Here we report the development of the CD19-specific CAR T cells capable of expressing interleukin (IL)-7 and chemokine (C-C motif) ligand (CCL)-19 upon CD19 engagement (referred to as 7 × 19 CAR T cells), the preclinical study and multicenter phase 1b clinical trial of 7 × 19 CAR T cell therapy in patients with R/R LBCL (NCT03258047). The clinical trial of 7 × 19 CAR T cells showed a favorable safety profile in a cohort of R/R LBCL patients (n = 39), with grade 3 cytokine release syndrome occurred in 5 (12.8%) patients and grade 3 or higher neurotoxicity in 4 (10.3%) patients. The overall response rate at 3 months post-single infusion was 79.5% (complete remission, 56.4%; partial response, 23.1%). With a median follow-up of 32 months, the median progression-free survival was 13 months, and the median overall survival was not reached, with an estimated rate of 53.8% (95% CI, 40.3–72.0%) at two years. Together, these long-term follow-up data from the multicenter clinical study suggest that 7 × 19 CAR T cells can induce durable responses with a median overall survival of greater than 2 years, and have a manageable safety profile in patients with R/R LBCL.

Relapsed and refractory (R/R) mantle cell lymphoma (MCL) remains an incurable lymphoma with a poor prognosis. Recently, there are a few studies demonstrating the efficacy of anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy in MCL, including ZUMA-2 study in which CD28-based CAR-T cells were used. However, long-term efficacy and safety associated with 4-1BB-based CAR-T therapy in MCL are not defined well. Here, we report three male patients with R/R classical MCL, who received CD19-directed 4-1BB CAR-T therapy and achieved complete remission, showed mild symptoms of cytokine-release syndrome (CRS) and had no neurological toxicity. During a follow-up of 24-35 months, all three patients remained in complete remission. Persistent B-cell depletion was observed in two patients. Recovery of CD19+ polyclonal B cells was detected in one patient at 6 months after CAR-T cell infusion. Recovery of serum immunoglobulin, including IgG, IgA and IgM, was not observed in two patients at the last follow-up. Only one patient developed herpes zoster, and the other two patients had no serious infection. This is the first report about the efficacy, long-term remission and safety of CD19-directed 4-1BB CAR-T therapy in R/R MCL.

Purpose Diffuse large B cell lymphoma (DLBCL) is an aggressive B-cell malignancy with clinical and molecular heterogeneity whose genetics may have clinical implications for patient stratification and treatment. The circulating tumor DNA (ctDNA) is a novel noninvasive, real-time, and tumor-specific biomarker harboring tumor-derived genetic alterations that are identical to those of tumor cells, thus showing great promise in individualized medicine, including precise diagnosis, prediction of prognosis, response monitoring, and relapse detection for DLBCL. Patients and methods In this study, we applied NGS analysis to tumor biopsies and ctDNA samples from 16 DLBCL subjects. Then, we compared the genomic alterations from 41 newly diagnosed patients and 56 relapsed/refractory (R/R) patients. Results Our results show that ctDNA can function as a liquid biopsy for tracking recurrently mutated genes in DLBCL (sensitivity: 87.50%). The mutational profiles of newly diagnosed and R/R DLBCL groups largely overlapped, but the frequencies of some gene mutations differ between the two cohorts. The distribution of mutations also revealed different frequencies in the two cohorts due to different signaling pathways. Genes from apoptosis pathway, immune response and BCR pathway suffered more mutations in R/R patients. Conclusion Overall, this study establishes ctDNA as an easily accessible source of tumor DNA for DLBCL genotyping and provides a deeper understanding of the somatic alteration spectrum for both newly diagnosed and R/R DLBCL patients.

Different strategies were taken to make virotherapy more effective at killing cancer cells. Among them, oncolytic virus which arms the therapeutic gene to enhance antitumor activity is a prevalent approach. In this study, a newly developed oncolytic vaccinia virus (OVV) that expresses Beclin-1 (OVV-BECN1) was tested for its in vitro and in vivo oncolytic activity in blood cancer. Results showed that the OVV exhibited higher infectivity for leukemia cells. OVV-BECN1 induced significant apoptosis-independent cell death either in wild-type leukemia and multiple myeloma (MM) cell lines or caspase-3 shRNA leukemia cell lines, and had a superior antitumor activity compared to the parent OVV. Autophagic cell death induced by OVV-BECN1 was demonstrated in vitro and in vivo experiments. Finally, upregulation of SIRT-1, a member of class III histone deacetylases, by OVV-BECN1 resulted in the deacetylation of LC3 and its distribution from the nucleus toward the cytoplasm, which might contribute to induction of autophagy. Overall, our data showed a favorable therapeutic effect of the oncolytic vaccinia virus on blood cancers through oncolytic and autophagic mechanisms, and may therefore constitute a promising and effective therapeutic strategy for treating human leukemia and MM. However, further studies are warranted for its reliable clinical translation.

During imatinib therapy, many patients with chronic myeloid leukemia (CML) develop severe neutropenia, leading to treatment interruptions, and potentially compromising response to imatinib. Berbamine (a bisbenzylisoquinoline alkaloid) has been widely used in Asian countries for managing leukopenia associated with chemotherapy. To investigate whether berbamine shows clinical benefit in reversing imatinib-associated neutropenia, we analyzed 63 chronic-phase CML patients who had developed grade ≥2 neutropenia and were treated with (n = 34, berbamine group) or without (n = 29, control group) berbamine. Among those patients with grade 2 neutropenia, five of 13 (38.5%) progressed to grade 3 neutropenia without berbamine support, while in the berbamine group, the rate decreased to 3/20 (15%) (p = 0.213). Although the rate of recovery from grade ≥3 neutropenia was similar in the two groups (94.1 vs. 90.5%, p = 0.559), berbamine markedly shortened the recovery time (median, 11 vs. 24 days, p = 0.006), and prevented recurrence of grade ≥3 neutropenia (18.8 vs. 52.6%, p = 0.039). Moreover, with berbamine support, the time to achieve complete cytogenetic response was significantly shorter (median, 6.5 vs. 10 months, p = 0.007). There were no severe adverse events associated with berbamine treatment. In conclusion, the present study reveals the potential clinical value of berbamine in the treatment of CML with imatinib-induced neutropenia. The use of berbamine may improve response to imatinib by stimulating normal hematopoiesis and faster neutropenia recovery.

The interaction between killer-cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) molecules expressed on target cells is known to modulate the cytolytic activity of natural killer (NK) cells. To date, a wide range of KIR genotypes has been observed, which varies among different ethnic populations. We report here comparison of the KIR gene content and genotypic structure of KIRs in 106 individuals from Eastern mainland Chinese Han and 97 from Taiwanese Han. All 17 KIR genes were observed in the two populations. Framework genes 2DL4, 3DL2, 3DL3 and 3DP1 were present in all individuals. The two populations had very similar frequencies in most loci, however, significant differences were noted in the frequencies of KIR3DS1 and KIR2DS4D (KIR2DS4 deletant variant). A total of 35 and 29 genotypes were identified in the individuals from the Eastern mainland Chinese and the Taiwanese Hans, respectively. Some pairs of KIRs showed significant positive and negative linkage disequilibrium (LD). Our data showed that there were minor distinctions in KIR gene frequencies, genotypes and LD between the two populations, which shed light on a possible geographic genetic demarcation among different Chinese communities.

Reduced-intensity allogeneic transplantation combined with imatinib mesylate for chronic myeloid leukemia in first chronic phase

There is limited data from developing countries on the current status of imatinib treatment for chronic myeloid leukemia (CML), thus we retrospectively analyzed 116 Chinese CML patients who received imatinib between 2003 and 2008. The response rates for 102 patients in chronic phase were: complete hematologic, 94.1%; complete cytogenetic, 69.6%; and complete molecular response, 54.9%. For 14 patients in the accelerated phase, the respective response rates were 85.7, 35.7 and 28.6%. The 3-year progression-free survival and 5-year overall survival were 73.3 and 74.8%. Although skin hypopigmentation occurs as the most common side effect (77.6%), imatinib is still well tolerated. In addition to the known pretreatment characteristics of spleen size, leukocyte and platelet counts, disease phase and Sokal scores, we found that delayed therapy, variant Philadelphia chromosome translocations and IM-related grade 3/4 leucopenia were associated with an inferior cytogenetic response. Four factors emerged as predictors of disease progression: molecular response, cytogenetic response, disease phase and disease duration prior to imatinib treatment, but only the latter three remained significant after multivariate analysis. The results indicate that the suboptimal outcome in Chinese patients is associated with delayed imatinib therapy, so the importance of the optimal treatment opportunity for CML should be emphasized.

Abstract 1159 Poster Board I-181 Background In allogeneic SCT, genetic factors such as donor and recipient gene polymorphisms of pro- and anti-inflammatory cytokines have been associated with the incidence and severity of GVHD. However, the influence of such donor and recipient gene polymorphisms on other outcomes, such as early infection after SCT, has seldom been described. TNFαa, TNF receptorII (TNFRII), IL-10 and TGF gene contain multiple single nucleotide polymorphisms (SNPs) in the promoter and codon region. We thus studied the association of this panel of candidate gene polymorphisms with the incidence of the first episodes of early bacterial infections within 100 days after allo-HSCT. Methods A total of 138 unrelated donor/recipient pairs, who had undergone HLA-matched allo-HSCT from January 2001 to March 2009 at the First Affiliated Hospital of Zhejiang University School of Medicine, were tested for TNFαa(TNFαa-857 C>T, TNFαa-863 C>A, TNFαa-1031 T>C), TNFRII (codon196 T>G), IL-10 (IL10-1082 A>G, IL10-819 T>C, IL10-592 A>C), TGF (TGF-509 T>C, TGF+869 C>T) polymorphism allele frequencies and genotype. SNPs were analysed by Multiplex SnaPshot. We considered the first episodes of early bacterial infections within 100 days after allo-HSCT have developed when sepsis, pneumonia, or septic shock was diagnosed according to previously published criteria. Results (1) 133 patients achieved complete donor chimerism in the peripheral blood and 5 patients had graft failure. All patients achieved an absolute neutrophil count (ANC) greater than 0.5×109/L at day 13 (7∼22) and platelet recovery at day 15 (7∼64). The cumulative incidence of at least one bacterial infection was 47.8% (pneumonia and intestinal infection are the most popular) within 100 days after allo-HSCT. There is no significant difference in the time to neutrophil recovery in patients who experienced early bacterial infections with those who did not (13.6 vs 12.8,P=0.115). (2) The TNFαa-857 C/C genotype and TNFRII 196 T/T genotype of the donor were significantly associated with a higher risk of early bacterial infection ( for TNFαa-857 C/C genotype: 53.3% vs 29.0%, P=0.024 ; TNFRII 196 T/T genotype: 53.5% vs 33.3%, P=0.038); (3) The TGF-509 T/T genotype of the donor was significantly associated with a higher risk of early bacterial infection (62.5% vs 41.8, P=0.038); (4) Transplantation from donors with IL10-819 C/C genotype or IL10-592 C/C genotype were significantly associated with a higher risk of early bacterial infection (for IL10-819 C/C genotype: 71.4.1% vs 45.3%, P=0.034; IL10-592 C/C genotype: 70.0.1% vs 45.8%, P=0.055); (5) The genotypes of TNFαa-863, TNFαa-1031, IL10-1082 and TGF+869 were not found to be associated with the risk of early bacterial infection. Conclusions Recent studies have shown that the generation potential of IL-10 is influenced by the polymorphism of the IL-10 gene. The IL10-819*C allele and IL10-592 *C allele are associated with higher secretion of IL-10 than IL10-819*T allele and IL10-592*A allele. In our data, a higher risk of early bacterial infection with IL10-819 C/C and IL10-592 C/C genotype was postulated to be associated with a higher IL-10 production, which suppressed reactive T-cell response. These results, which is the first report of TNFαa, TNFRII, IL-10and TGF polymorphic features of Chinese population with the risk of early bacterial infection after HLA-matched unrelated allo-HSCT, suggest an interaction of the donor TNFαa-857C/C, TNFRII 196 T/T, IL10-819 C/C, IL10-592 C/C and TGF-509 T/T genotypes on risk of early infection. These results are helpful for predict allo-HSCT outcome, identify more suitable donors and clarify therapy on an individual patient basis. Disclosures No relevant conflicts of interest to declare.

Abstract 1158 Poster Board I-180 Background The importance of theTumor Necrosis Factor (TNF), including TNF-αa and TNF-β, in both the initial preconditioning and effector phases of aGVHD is well established. TNF and TNF receptor II (TNFRII) gene contains multiple single nucleotide polymorphisms (SNPs) in the promoter and transcription start site. There are conflicting datas regarding the cytokines gene polymorphisms and the risk of aGVHD in several studies and no data about Chinese population. This present study was designed to test association of TNFA, TNFB and TNFRII genotype for gene polymorphisms of both donors and recipients with incidence and severity of aGVHD in HLA-matched unrelated allo-HSCT within Chinese population. Methods A total of 138 unrelated donor/recipient pairs, who had undergone HLA-matched allo-HSCT from January 2001 to March 2009 at the First Affiliated Hospital of Zhejiang University School of Medicine, were tested for TNFA (TNFαa-857 C>T,TNFαa-863 C>A,TNFαa-1031 T>C), TNFB (TNFβ+252 A>G) and TNFRII (codon 196 T>G) polymorphism allele frequencies and genotype. SNPs were analyzed by Multiplex Snapshot. Results (1) The TNFαa-857 C/C genotype of the donor or recipient was significantly associated with a higher risk of aGVHD (for donor type:75.7% vs 41.9%, P=0.001; for recipient type: 72.7% vs 50.0%, P=0.039) and a higher incidence of grade II-IV aGVHD( for donor type:50.5% vs 19.4%, P=0.002; for recipient type:48.2% vs 25.0%, P=0.033). (2) The TNFβ+252*G allele of the donor or recipient was significantly associated with a higher incidence of aGVHD (for donor type:74.5% vs 46.9%, P=0.005; for recipient type: 75.0% vs 47.1%, P=0.005); (3) The TNFRII196 T/T genotype of the donor or recipient was significantly associated with a higher incidence of aGVHD (for donor type:73.7% vs 53.8%, P=0.028; for recipient type: 73.3% vs 58.3%, P=0.086); (4) TNF and TNFRII geng polymorphic features, together with other clinical and biological factor (patient's age, donor-recipient gender, diagnosis, conditioning regimen, transplant material and GVHD prophylaxis), were subjected to multivariate analysis for aGVHD manifestation in order to exclude indirect association of gene polymorphic features. In multivariate analysis, donor-recipient gender (female to male) (RR=1.602,95%CI: 1.035-2.479, P=0.034), the TNFαa-857 C/C genotype of donor (RR=2.177, 95%CI: 1.204-3.938, P=0.01) and the TNFβ+252*G allele of recipient (RR=1.920, 95%CI: 1.116-3.304, P=0.018) were found to significantly contribute to the development of aGVHD. The TNFαa-857C/C genotype of donor (RR=3.211, 95%CI: 1.373-7.509, P=0.007) and the TNFβ+252*G allele of recipient (RR=2.174, 95%CI: 1.063-4.443, P=0.033) were also associated with a higher incidence of grade II-IV aGVHD; (5) The genotypes of TNFαa-863 and TNFαa-1031 were not found to be associated with the risk of aGVHD. Conclusions These results, which is the first report of TNF and TNF receptor polymorphic features of Chinese population with the risk of aGVHD, suggest an interaction of the donor and recipient TNFαa-857, TNFβ+252 and TNFRII196 genotypes on risk of aGVHD. These results are helpful for predict allo-HSCT outcome, identify more suitable donors and clarify therapy on an individual patient basis. Disclosures No relevant conflicts of interest to declare.

Abstract 3396 Poster Board III-284 OBJECTIVE: Allogeneic hematopoietic stem cell transplantation (HSCT) is a successful curative therapy for a variety of hematological malignancies. The success of allogeneic HSCT partly depends on the graft-versus-leukemia (GVL) effect caused by donor lymphocytes. Apart from alloreactive T cells, alloreactive natural killer (NK) cells also play an important role in the induction of GVL effect after HSCT. However, the effect of NK cell alloreactivity on the outcome of unrelated hematopoietic stem cell transplantation is controversial. NK cell alloreactivity after allogeneic HSCT is regulated by killer cell immunoglobulin-like receptors (KIRs). So, our study is to investigate the impact of KIRs on the outcome after unrelated hematopoietic stem cell transplantation. METHODS: 116 cases of unrelated transplants between Jan. 2001 to May. 2008 were involved in the study. The inhibitory KIRs identified with HLA ligands are as follows: KIR2DL2/KIR2DL3 bind HLA-C1; KIR2DL1 recognizes HLA-C2; and KIR3DL1 recognizes HLA-Bw4. KIR-ligand mismatch was defined as the absence of one or more recipient HLA epitopes for the corresponding donor-inhibitory KIRs. Samples containing either one or two group B haplotypes were assigned the genotype designation B/x. Samples that lacked all KIR B loci were assigned the genotype A/A. RESULTS: We found that KIR-ligand mismatch was significantly associated with a decreased leukemic relapse risk (p=0.019; HR=0.329, 95% CI 0.131-0.830), mainly in myeloid disease (p=0.003; HR=0.193, 95% CI 0.066-0.563). In myeloid disease, the cumulative risk of relapse was lower in the patients with KIR-ligand mismatch than KIR-ligand match (10.7% vs. 41.5%, p=0.002). This effect of the KIR-ligand mismatch on relapse was comparably significant between AML/MDS subgroup (12.6% vs. 40%, p=0.03) and CML subgroup (4% vs. 41.7%, p=0.008). For standard-risk myeloid leukemia, KIR-ligand mismatch resulted in a lower relapse rate than KIR-ligand match, although no statistical significance was reached. (11.3% vs. 26%, p=0.356). For high-risk myeloid leukemia, the risk of relapse in patients with KIR-ligand mismatch was significantly lower than that in patients with KIR-ligand match (9.1% vs. 71.4%, p=0.002). Furthermore, the impact of KIR-ligand mismatch was found mainly in the HLA-match group (13.9% vs. 62.5%, p= 0.001) and KIR haplotype, Bx group (11.5% vs. 55%, p=0.01). In myeloid disease, KIR-ligand mismatch also improved 5-year overall survival (OS) (p=0.034; HR=0.430, 95% CI 0.194-0.924) and disease-free survival (DFS) (p=0.024; HR=0.445, 95% CI 0.221-0.897). No effect was seen in patients with lymphoid disease. AA in the donor significantly decreased the cumulative incidence of relapse compared with Bx (12.9% vs. 28.6%, p=0.024). We further found that more activating KIR genes in the donor resulted in an increased relapse risk (p=0.005; HR=1.463, 95% CI 1.123-1.906). Meanwhile, the presence of donor activating KIR2DS3 gene was associated with increased relapse risk (p=0.003; HR=5.046, 95% CI 1.746-14.575) and decreased OS (p=0.004; HR=3.181, 95% CI 1.432–7.064) and DFS (p=0.003; HR=2.919, 95% CI 1.430-5.959) in myeloid disease. CONCLUSION: Our study indicated that, in unrelated HSCT for myeloid leukemia, selection of donors with KIR-ligand mismatch offered a lower relapse risk and a long-term survival advantage. The presence of KIR2DS3 in the donor was an important risk factor for myeloid leukemia. If donors carry low number of activating KIR genes, the risk of relapse is reduced. Disclosures: No relevant conflicts of interest to declare.

Killer cell immunoglobulin-like receptors (KIR) are a family of inhibitory and activatory receptors and are expressed by most NK cells. The interaction between KIR and human leukocyte antigen (HLA) molecules expressed on target cells is known to modulate the cytolytic activity of NK cells. At present, seventeen KIR genes have been identified, and the number of KIR gene loci has been reported to vary among individuals, resulting in a heterogeneous array of KIR genes in different populations. KIR haplotypes are divided into two distinctive groups based on their gene contents. Ruggeri, et al, first reported that KIR-ligand mismatch can alleviate aGVHD, reduce relapse and improve disease-free survival in mismatched hematopoietic stem cell transplants, while some studies show that this kind of mismatch is deleterious. Now the effect of NK cells alloreactivity on outcome of hematopoietic stem cell transplantation remains controversial. Our study is to analyze the KIR gene contents and investigate the impact of KIR-ligand mismatch on outcome following hematopoietic stem cell transplantation in Chinese population. METHODS: 203 cases of allogeneic hematopoietic stem cell transplantation between Jan. 2001 to May. 2008 were involved in the study. KIR genes were typed by using PCR-SSP, and HLA-A, -B and -C loci genes were used by PCR-SSP or PCR-SSO technology. KIR-ligand incompatibility were assessed based on HLA-Cw (divided into C1 and C2 group) and three major inhibiting KIR genotypes (KIR2DL1, KIR2DL2 and KIR2DL3) of 203 donor/recipient pairs. These patients received myeloablative (n=180) or nonmyeloablative (n=23, with ATG) conditioning followed by hematopoietic stem cell transplantation from HLA matched (n=164) or mismatched (n=39) donors. All patients received mycophenolate mofetil (MMF) combined with CsA and short course MTX regimen as prophylaxis for aGVHD. RESULTS: All seventeen KIR genes were observed in the Chinese population. Framework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were present in all individuals. The most frequent non-framework KIR genes were: KIR2DL1 (99.5%), KIR3DL1 (97.0%), KIR 2DS4 (97.5%), KIR2DL3 (99.5%) and KIR2DP1 (99.5%). The other gene frequencies were KIR2DS2 (25.1%), KIR2DL2 (25.6%), KIR2DL5A (32.5%), KIR2DL5B (30.0%), KIR2DS5 (23.6%), KIR2DS1 (43.8%), KIR2DS3 (21.2%) and KIR3DS1 (34.0%). The most prevalent haplotype group found in the population was A haplotype. Group A haplotypes outnumbered group B haplotypes in frequency by approximately 3:1, with individuals having two group A haplotypes accounting for 48.8% (99/203). HLA genotyping showed that 156 out of 203 (76.8%) donor-recipient pairs could be characterized by lack of recipient HLA-Cw ligand for donor KIR2DL1, KIR2DL2/2DL3. KIR-ligand mismatch was not associated with any deleterious or beneficial influence on relapse or overall survival (OS) in Chinese population with hematopoietic stem cell transplantation. But KIR-ligand mismatch could lead to a decreased incidence of aGVHD (36.1% vs 56.3%, P=0.018). When donor NK cells had KIR2DS2 gene, the recipient acquired a decreased incidence of aGVHD (13.2% vs 49.3%, P CONCLUSION: Our data demonstrated that Chinese population was distinct in KIR gene frequencies and putative KIR haplotypes in comparison to some other populations. KIR-HLA mismatch was not any better effect on relapse, OS, but it had a better effect on aGVHD. Donors with gene KIR2DS2 or KIR2DS5 were associated with lower incidence of aGVHD in allogeneic hematopoietic stem cell transplantation.