bluuebunny/crossref_metadata_2025 huggingface dataset:
Genome Regulation by Long Non-Coding RNAs
Authors: Chang Howard Y.
Published: October 2019

The discovery of extensive transcription of long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation. Discussions will cover genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators, and both control and target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, long non-coding RNAs can function as modular scaffolds to specify higher order organization in RNP complexes and in chromatin states. More recently, lncRNAs have been found to control post-transcriptional processes and signal transduction as well. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs in developmental patterning and cancer.

Disclosures:

Chang: RaNA Therapeutics: Membership on an entity’s Board of Directors or advisory committees.
Monocyte Trafficking, Inflammation, and Atherosclerosis
Authors: Randolph Gwendalyn J.
Published: October 2019

Macrophages are central to the progression of atherosclerosis. An increased number of macrophages in plaque are associated with larger, more stenotic lesions. Furthermore, activated plaque macrophages promote rupture, the most significant clinical event affecting mortality. Plaque macrophages derive from monocytes that are recruited from blood. We have thus focused our efforts on understanding the mechanisms that regulate plaque macrophages, with emphasis on how macrophage-burden might be reduced to lower disease risk. We have developed techniques to discern whether macrophage contraction in plaques is due to emigration out of the plaque environment. Although this idea was our leading hypothesis, data obtained in a model of regression carried out in apoE-/- mice indicates that emigration of macrophages does not occur during regression. The idea was based on previous literature that monocyte-derived cells might be removed from sites of acute inflammation by emigrating to local lymph nodes. After finding that emigration of macrophages from resolving plaque in apoE-/- mice was poor, we revisited models of acute inflammation; in particular, thioglycollate-induced peritonitis, where emigration had been claimed to account for macrophage removal. New methodology to improve the quantification of macrophage loss from the peritoneum indicated that, like atherosclerosis, emigration of macrophages was a minor contributor to the contraction of macrophages associated with resolution. Instead, in both settings, macrophage loss was associated with a strong suppression of monocyte recruitment, coupled with ongoing macrophage apoptosis. These data strongly suggest that methods to block monocyte recruitment may provide a viable approach to reversing atherosclerosis. Current efforts focus on integrating the role of monocyte recruitment with local proliferation of macrophages in plaques, investigating macrophage motility in plaques during different disease states, and evaluating contraction of macrophages from plaques using other models of disease regression.

Disclosures:

No relevant conflicts of interest to declare.
The Potential to Enhance Hemostasis by Inhibiting Tissue Factor Pathway Inhibitor
Authors: Mast Alan E. , Maroney Susan A.
Published: October 2019

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factors (TF) X and VIIa. As such, it is a major regulator of the extrinsic clotting pathway. Compounds that block TFPI activity, or “inhibit the inhibitor,” effectively restore hemostasis in animal models of hemophilia. These compounds are thought to act by allowing unfettered TF-mediated thrombin generation through the extrinsic pathway in a manner that bypasses the need for the intrinsic pathway proteins, factor VIII and factor IX. Improved understanding of TFPI tissue expression and function is needed to better recognize how pharmaceutical agents targeting TFPI alter biochemical and physiological mechanisms of blood coagulation. Several advances have been made recently. TFPI is an alternatively spliced protein with two primary isoforms, TFPIα and TFPIβ, which have distinct cellular expression patterns and anticoagulant functions. TFPIα is produced by endothelial cells, and appears to localize within an intracellular granule with release following stimulation with thrombin or heparin. TFPIα is also produced by megakaryocytes and is released from activated platelets. Platelet TFPIα limits clot growth following vascular injury and alters bleeding in a murine hemophilia model. Interestingly, the anticoagulant activity produced by platelet TFPIα appears to be isoform specific and not mediated through inhibition of TF. TFPIβ is made by endothelial cells, localizes to endothelium, and is not present in platelets. TFPIβ is an effective inhibitor of TF-mediated cellular migration and dampens the effects of TF-induced intravascular coagulation in mice. This new knowledge of TFPI isoform expression patterns and anticoagulant activities has important implications for the development of TFPI-blocking pharmaceuticals to treat hemophilia. The recent findings also provide new insights into the biochemical mechanisms that activate blood coagulation pathways and regulate TF-mediated thrombotic and inflammatory disease.

Disclosures:

Mast: Novo Nordisk: Honoraria, Research Funding.
Genome-Wide Abnormality Patterns of B-Lineage Acute Lymphoblastic Leukemia in Adults in Comparison with Pediatric Cases
Authors: Wang Bai-Yan , Liu Yuan-Fang , Tang Jing-Yan , Gu Zhao-Hui , Zhang Wei-Na , Zhang Zi-Guan , Wang Qiang , Chen Bing , Wang Sheng-Yue , Zhu Yong-Mei , Li Jun-Min , Wang Jin , Bai Yun , Lu Gang , Yang Min-Jun , Zhang Jin-Li , Shi Jing-Yi , Wang Kan-Kan , Pan Chun-Ming , Lu Jing , Jiang Lu , Li Yang , Zhao Chun-Jun , Chen Jing , Gu Long-Jun , Mi Jian-Qing , Li Ben-Shang , Chen Zhu , Chen Sai-Juan
Published: October 2019

BACKGROUND

B-lineage acute lymphoblastic leukemia (B-ALL) represents the most common subtype in ALL. Genomic sequence information has been lacking in adult B-ALL, whose outcome remains dismal, while a comparison between genomic abnormalities in adult and pediatric groups is useful to further decipher disease mechanisms.

METHODS

We used whole exome sequencing (WES), copy number variation and molecular cytogenetics to catalog somatic mutations in 95 B-ALL patients (43 adults and 52 children).WES was conducted with appropriate depths for paired genomic DNA from bone marrow mononuclear cells at diagnosis and their matched peripheral blood samples during complete remission (CR) or saliva samples. Targeted deep sequencing (TDS) was performed in a validation cohort of 179 adult and 199 pediatric B-ALLs.

RESULTS

Eighty-four recurrent gene mutations were revealed by WES. Integrative analysis identified the involvement of 9 functional categories of genes: key fusions (KFs), epigenetic modifiers (EMs), signaling molecules (SMs), transcription factors (TFs), tumor suppressors (TSs), actin binding/cytoskeletons (ABCs), ion binding proteins (IBPs), trans-membrane proteins (TPs) and the others. Mutually cooperative or exclusive relationships were revealed among some of these categories. Genomic landscapes suggested two distinct mechanisms involved in the leukemogenesis: one is mainly driven by KFs together with mutations of TFs and TSs; the other results from the abnormalities of TFs and TSs, in cooperation with mutations of EMs, SMs, ABCs and IBPs recapitulating the role of KFs. A panel of histone/DNA methylation modifiers (HMMs) were revealed to bear potential value of relatively favorable prognosis in both adult and childhood patients.

CONCLUSIONS

We described the genome-wide abnormality patterns in adult B-ALL patients in comparison with those of pediatric cases, contributing to the understanding of leukemogenesis and the identification of potential new prognostic markers.

Disclosures

No relevant conflicts of interest to declare.
Outcomes of Combined Transplants of Cord Blood Plus Peripheral Blood from an HLA Matched Sibling in Thalassemia Patients
Authors: He Yuelin , Li Changgang , Wu Xuedong , Liu Sixi , Feng Xiaoqin , Li Chunfu
Published: October 2019

Background Cord blood from HLA matched sibling was widely used as stem source in transplants in patients with thalassemia major (TM), but failure of engraftment is frequent.

Aim To improve engraftment of CB transplant in patients TM

Patients and method A combined transplants of CB and peripheral blood (PB) from same HLA matched sibling was conducted (i.e. CB and PB were harvested and infused on day 0) from June, 2010 to February, 2014. Total 24 patients were involved in the study. Donor-recipient pairs were matched at HLA-A, B and DRB1 (antigen level) in 23 but one with HLA mismatched at one loci (DRB1). The conditioning protocol included intravenous busulfan, cyclophosphamide, fludarabine, and thiotepa. Prophylaxis of GVHD included Cyclosporine and short MTX. All donors (newborn) were sent into NICU for monitoring, and then, discharged after 3 days.

Results The median age at transplant was 4 years (range, 1.5-7 years), and the ratio of male-to-female patients was 16:8. The median follow-up time was 17 months (range, 5-49 months). The mean mononuclear cell (MNC) dose of CB was 5.67×107/kg body weight of the recipient (BWR; range, 2.1-8.75×107/kg BWR) while that of PB was 3.21×107/kg BWR (range, 0.47-7.31×107/kg BWR). The mean volume of infused CB was 91ml (range, 31-131ml) while that of PB was35ml (range, 10-71ml). The median ANC engraftment time was 28 days (range, 15-47days), while the median platelet engraftment time was 34.0 days (range, 0-128days). The estimated 4-year overall survival (OS) and TM-free survival (TFS) were both 100%. The cumulative incidences of graft rejection and grades II-IV acut egraft-versus-host disease (aGVHD) were both 0%. All donors are normal as peers with following up to date.

Conclusion These results suggest that patients with TM have excellent outcomes after CB plus PB combined transplant from an HLA matched sibling.

Disclosures

No relevant conflicts of interest to declare.
The Prevalence of Hereditary Hemoglobin Disorders and Its Implications for Newborn Screening in Germany
Authors: Grosse Regine , Schneppenheim Reinhard , Lukacs Zoltan , Oyen Florian , Muntau Birgit , Lukacs Paulina , Noack Bernd
Published: October 2019

Introduction: Hemoglobin disorders are among those hereditary diseases with evidence that an early diagnose and treatment improve the clinical outcome of affected children. So far hemoglobin disorders are not included in the German newborn screening program despite increased immigration from countries with populations at risk.

Methods: To determine the birth prevalence in a major German area we tested 16910 anonymized newborns from Hamburg. High pressure liquid chromatography (HPLC) and subsequent molecular-genetic testing were used for the detection and confirmation of sickle cell variant (HbS), of hemoglobin C (HbC) and of beta- and alpha-thalassemia variants.

Results: We found sickle cell disease (SCD) to be the most prevalent with a frequency of disease-consistent genotypes of 1 in 3382 newborns. The prevalence for sickle cell trait (SCT) was 1 in 301 births. Statistical analysis indicates with 96% probability a prevalence for SCD of at least 1 in 8455 births and for SCT of at least 1 in 393 births. Duffy-blood group typing showed evidence that affected children were likely of Sub-Saharan ancestry. More than 90% of the newborns found with SCD or SCT were of sub-Saharian ancestry, suggesting that in Hamburg mainly children from members of the African community are at risk for the hemoglobinopathy SCD. Our data indicate a 95% probability that the prevalence of SCD in Hamburg is at least 1,18/10.000 births.

Alpha Thalassemia trait had a prevalence of 1: 994, indicating with 97% probability a minimal prevalence of 1: 2114 births. In this study we did not find any child with beta-thalassemia major or HbH- disease, which indicates a birth prevalence of less than 1 in 16910 births during the study period.

32 chromatograms indicated heterozygosity for other hemoglobin variants and 1 pattern indicated homozygosity for HbCC.

Conclusion: Our study sustains the notion that newborn screening in areas that see significant immigration is necessary to provide early intervention with the framework of a public health initiative. As long as a nationwide screening in Germany is not yet implicated all high-risk infants (those of African, Mediterranean, Middle Eastern, Indian, Caribbean, and South and Central American ancestry) should receive a neonatal screening for SCD. Any high-risk infant not screened at birth, or for whom neonatal screening results cannot be documented, should be screened for hemoglobinopathies prior to 2 months of age.

Disclosures

Grosse: Novartis Oncology Germany: Research Funding.
Minimal Renal Toxicity after Rituximab DHAP with a Modified Cisplatin Application Scheme in Patients with Relapsed and Refractory Diffuse Large B-Cell Lymphoma
Authors: Lisenko Katharina , McClanahan Fabienne , Schöning Tilman , Schwarzbich Mark-Alexander , Cremer Martin , Dittrich Tobias , Ho Anthony D. , Witzens-Harig Mathias
Published: October 2019

BACKGROUND: Rituximab (R) in combination with DHAP is a widely accepted salvage regimen in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). DHAP combines cisplatin (100 mg/m2) typically administered intravenously (i. v.) by continuous infusion over 24 hours, followed on day 2 by cytarabine (2 g/m2) in a 3-hour infusion repeated after 12 hours, and oral administration of dexamethasone (40 mg/d) for 4 consecutive days. A common adverse effect of this protocol consists of renal toxicity which may result in dose reduction or treatment discontinuation. Therefore novel approaches to overcome renal toxicity of R-DHAP are urgently warranted. Assuming that a lower single dose of cisplatin over several days would reduce renal toxicity, our institution has chosen to administer cisplatin at a dosage of 25 mg/m2 per day as a 3-hour infusion over 4 consecutive days. In this study we systematically examine efficacy and renal toxicity of this modified R-DHAP regimen.

METHODS: We retrospectively analyzed data of 122 patients with relapsed/refractory DLBCL who were treated at our institution from July 2002 to July 2013. Patients were grouped according to the number of R-DHAP courses applied and renal function was evaluated in each subgroup. Creatinine serum levels before each R-DHAP cycle and two to three weeks after the last R-DHAP were assessed and GFR was calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

RESULTS: Overall, 256 R-DHAP cycles were administered. 31 (25%), 61 (50%), 14 (12%) and 16 (13%) patients received one, two, three or four R-DHAP courses, respectively. Dose adjustments needed to be applied in only 5 (4%) patients who in total received 11 R-DHAP courses. A step-by-step evaluation of renal function after each R-DHAP course revealed that a GFR decrease can be observed after each chemotherapy cycle. However, in none of the subgroups GFR was lower than 60 ml/min/1.73 m2. In most patients, only renal impairment stage I and II was observed. Renal impairment stage III was observed in 12 patients (10%) and stage IV only in one patient (1%). The overall response rate of the modified R-DHAP protocol was 54% (CR 17%, CRu 5%, PR 32%, SD 15% and PD 29%, not known 4%).

CONCLUSION: A modified R-DHAP regimen with administration of cisplatin 25 mg/m2 over 4 consecutive days is effective and safe in relapsed/refractory DLBCL and leads only to minimal renal toxicity.

Disclosures

Witzens-Harig: Pfizer: Honoraria, Research Funding; Roche: Honoraria.
HLA-Mismatched Microtransplantation Vs HLA-Matched Nonmyeloablative Transplantation for Acute Myeloid Leukemia in Intermediate-Risk: Comparable Survival but Avoids of Gvhd
Authors: Guo Mei , Yu Chang-Lin , Dong Zheng , Hu Kaixun , SUN Qiyun , Qiao Jianhui , SUN Wanjun , Zuo Hongli , Huang Yajing , Qiao Junxiao , CAI Bo , LIU Zhiqing , Yao Bo , LIU Tieqiang , Zhao Hongxia , SUN Xuedong , Shen Xuliang , Zhan Xinrong , WANG Juan , Li Jianyong , HU Liangping , Zhou Qianqian , Ai Huisheng
Published: October 2019

The optimal therapy for intermediate-risk patients with acute myeloid leukemia (AML) in first complete remission (CR1) is uncertain. Recent studies shown that microtransplantation (MST) can improve survival in AML-CR1 patients. However, a comparison study between the MST and nonmyeloablative stem cell transplantation (NST) is lacking. 156 intermediate-risk AML-CR1 patients aged 9 to 59 years were enrolled in this study. 57 patients who had a HLA-identical donors were assigned to receive NST therapy with graft-versus-host disease (GVHD) prophylaxis. The other 99 who had no HLA-identical donors including 86 family-related, 9 distantly related and 4 unrelated donor were assigned to receive MST therapy but without GVHD prophylaxis. The probabilities of 10-year overall survival and leukemia-free survival was comparable in the MST-group and NST-group (70.7% vs. 61.4% and 59.6% vs. 57.9%). The NST-group exhibited a higher full donor chimerism (96.5%) and higher GVHD (33.3%), whereas the MST-group produced a higher donor microchimerism (75%), slightly higher relapse (32.3% vs. 22.8%) and significantly lower non-relapse mortality (6.9% vs. 19.3%, P=0.021) but without GVHD. In the MST-group, the patients with increase of WT1+ CD8+ T cells exhibited significantly higher leukemia-free survival and lower relapse than those without (92.0% vs. 40.0%, P=0.003; 8.0% vs. 50%, P=0.009). These results indicate that, compared to NST, MST produced a comparable survival, less transplantation-related mortality, avoidance of clinical GVHD and overcome limitations of HLA-barrier, suggesting a much safe and effective therapy for intermediate-risk AML-CR1, particularly for those without a HLA-identical donor.

Disclosures

No relevant conflicts of interest to declare.
Outcomes of Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia (CML) Who Switch to Imatinib Following Optimal Response to Second Generation Tyrosine Kinase Inhibitors
Authors: Kota Vamsi K , Winton Elliott F. , Arellano Martha , Jillella Anand , McLemore Morgan L. , El Rassi Fuad , Khoury H Jean
Published: October 2019

The second-generation TKIs (2G-TKIs) Dasatinib (DAS) and Nilotinib (NIL) yield faster responses in newly diagnosed chronic phase (CP) CML as compared to Imatinib (IM) however, long-term safety of these agents is a growing concern.

We identified 10 patients with CP CML diagnosed between 08/2013 and 06/2015 who initiated 2G-TKIs and were then switched after optimal response at 3 months to IM. DAS was administered to 8 patients at 100 mg/d and NIL in two patients at 300 mg twice a day. Response to TKI was assessed by quantitative reverse transcriptase polymerase chain reaction (qPCR) for BCR-ABL1. Response to 2G-TKIs after 3 months was as follows: CHR (n=10), 1 log (<10%, n=10), 2 log (<1%, n=7), and 3 log (<0.1%, n=2) reduction of BCR-ABL1 transcripts. Median qPCR at 3 months was 0.77% by IS (range 0-7%).

NIL was discontinued due to grades 2-3 non-hematological toxicities in both patients. DAS was discontinued due to patient or physicians' preference or drug availability. Median time to discontinuation of 2G-TKIs and initiation of IM was 103 days (range, 92 - 120) from diagnosis. IM was started at 400 mg/d and was well tolerated except in 2 patients who required dose-reduction and discontinuation due to grade 2 skin rash (1) and grade 2 anxiety (1). Both patients switched back to DAS at 54 and 228 days after initiation of IM respectively. 9/10 patients that switched to IM were evaluable with a follow up of at least 3 months. These patients have shown a continuous response with 9/9(100%) achieving a 2 log reduction at 6 months. With a median follow-up from initiation of IM of 8 months (range 2-20 months), 5/9 (55.5%) evaluable patients achieved MMR at 6 months and 3/6 MR5 at 9 months from diagnosis.

In conclusion, this retrospective analysis shows that IM can be safely and effectively administered following optimal response to 2G-TKIs. Longer follow-up is needed, but with up to 20 months follow-up, all patients showed continuous decrease in their transcripts (Figure 1) and no losses of major molecular response or progression to blast phase were observed. A strategy of induction with 2G-TKIs followed by maintenance with IM is worth evaluating in a prospective trial.

Figure 1. BCR-ABL1 qPCR (IS value) at diagnosis and follow-up after a switch to IM at 3 months from 2G-TKIs. Figure 1. BCR-ABL1 qPCR (IS value) at diagnosis and follow-up after a switch to IM at 3 months from 2G-TKIs.

Disclosures

Kota: Leukemia Lymphoma Society: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees.
A Primary Study of the Gene Mutations in Predicting Treatment Response to Decitabine in Patients with MDS
Authors: Chang Chunkang , Zhao Youshan , Xu Feng , Li Xiao
Published: October 2019

Background

To analyze whether some distinct gene mutations could predict treatment response to Hypomethylation Agent---Decitabine.

Patients and Methods

Targeted next-generation sequencing was performed on reported 30 recurrently mutated genes for 106 MDS patients before they accepted Decitabine therapy. For those who presented satisfactory response (CR) or poor response (NR or PD), their gene sequencing were repeated after Decitabine therapy to analyze the alteration of the gene mutation.

Results

The overall response rate was 67.0%, with 26.4% patients (28 cases) achieving CR. The median survival of patients who achieved CR was longer than those who just achieved NR/PD (22 months VS 14 months, p =0.021). Among the 14 TP53 mutant patients, ten achieved CR (71.4%). Moreover, two of 3 BCOR mutant patients (66.7%) and three of 5 KIF20B mutant patient (60.0%) also achieved CR. Zero of the patients who were with IDH1/2(n=11), GATA2 (n=5), SETBP1 (n=5), EZH2 (n=3) or CEBPa (n=1) mutations could get CR response (Figure1). On the other hand, total five of the 8 PTPRD mutant patients (62.5%) and five of 11 ASXL1mutant patients did not responded to Decitabine. In addition, 16 among the 28 CR patients accepted the target sequencing after the stop of decitabine therapy. Ten of the 16 patients showed decreased number of mutant genes (Table1). Besides, the TP53 mutation was completely fade away in five of 7 patients after Decitabine treatment (Table 2). Finally, the TP53 mutant and wild type of MDS patients achieved comparable overall survival, suggesting decitabine treatment could significantly alter the prognostic impact of adverse genetic alterations.

Conclusion

Some gene mutation (specially TP53) seemed predictive for Decitabine response. Further studies with large samples are needed to identify it.

Table 1. The number of mutant genes on CR patients before and after Decitabie therapy Sample Number of mutations response Before After Sample 586 3 2 CR Sample 967 3 2 CR Sample 1761 4 2 CR Sample 1619 2 3 CR Sample 1864 7 4 CR Sample 1854 3 1 CR Sample 1818 3 2 CR Sample 1522 2 4 CR Sample 2038 2 2 CR Sample 2055 2 1 CR Sample 702 3 1 CR Sample 828 2 0 CR Sample 1361 0 0 CR Sample 1002 2 1 CR Sample 1917 1 2 CR Sample 1154 0 0 CR

Table 2. Status of TP53 mutation after hypomethylating agents in CR patients. Sample Before After response Sample 586 TP53/8/D242H TP53-WT CR Sample 967 TP53/6/R174L TP53-WT CR Sample 1864 TP53/8/R244H TP53/8/R244H CR Sample 1854 TP53/4/W91 TP53-WT CR Sample 2038 TP53/8/F231S TP53/8/F231S CR Sample 2055 TP53/6/R196* TP53-WT CR Sample 1002 TP53/8/R234H TP53-WT CR

Disclosures

No relevant conflicts of interest to declare.
Favorable Immune Signature in CLL Patients, Defined By Antigen-Specific T-Cell Responses, Might Prevent Secondary Skin Cancers
Authors: Stickel Juliane S , Kowalewski Daniel Johannes , Schuster Heiko , Kahn Stefan , Backert Linus , Kanz Lothar , Salih Helmut R , Rammensee Hans-Georg , Stevanovic Stefan
Published: October 2019

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. The course of disease predisposes patients to the development of secondary malignancies, which might be due to a generalized immunosuppression induced by the disease and/or the therapies used for treatment. Skin cancer, including melanoma, is the most common secondary malignancy reported in these patients.

In a recent study we reported on spontaneous memory T cell responses against CLL-associated antigens defined by HLA ligandome analysis in CLL patients. Moreover, retrospective analysis suggests a significant survival benefit for CLL patients showing immune reactions to more than one of the tested antigens (Kowalewski et. al., PNAS 2015).

Here we analyzed our CLL patient cohort for the incidence of secondary malignancies, and in particular for skin cancers. A total of 15 CLL-associated HLA-peptides (6 A*02, 3 A*03, 6 B*07) were implemented for stimulation of HLA-matched PBMC obtained from CLL patients. 45 CLL patients (Binet A n=15, Binet B, n=15, Binet C n=15, median age, 33.3% (15/45) with prior CLL specific therapy) were analyzed for antigen-specific memory T-cell responses in IFNγ-ELISPOT assays. 13 patients (28.9%, response cohort) showed specific T cell response against > 1 antigen, while 32 patients (71.1%, negative cohort) showed none or only one immune response. The median age was 71 in the response cohort and 73 in the negative cohort. 40.0% (6/15) of CLL patients with Binet stage A and 33.3% (5/15) with stage B were classified as responders whereas only 13.3% (2/15) of patients with stage C showed >1 immune responses. Of the included CLL patients with prior therapy only one patient (8%) showed > 1 immune responses. In the analyzed CLL patient cohort 7 skin cancers (melanoma: n=2, squamous cell carcinoma: n=2, basal cell carcinoma: n=3), 3 precancerous skin lesions and 9 other secondary malignancies including renal cell carcinoma (n=2), colon (n=3), breast (n=2) and prostate cancer (n=2) were observed. In the negative cohort 34.4% (11/32) of the patients developed a secondary malignancy, while only 15.4% (2/13) in the immune response cohort presented with secondary neoplasms. Strikingly all skin cancers and precancerous skin lesions were observed in the non-immune responder cohort. Furthermore multiple secondary malignancies (e.g. squamous cell carcinoma and colon cancer) were only observed in the cohort that showed no immune response.

Taken together our data suggest that a favorable immune signature in CLL patients, i.e. the ability to mount T-cell responses against CLL-associated antigens not only is associated with improved overall survival but also might protect these patients from the development of secondary skin cancers.

Disclosures

No relevant conflicts of interest to declare.
Macrofocal Multple Myeloma Is a Particular Subgroup of Multiple Myeloma
Authors: Fan Jianling , Hou Jian , Du Juan , Jin Lina , Peng Lihui , Chen Baoan , Xi Hao , Zhang Hui , Jiang Hua , Zhou Fan , Fu Weijun
Published: October 2019

Some young multiple myeloma (MM) patients, coined macrofocal MM, have been reported to present with multiple lytic bone lesions and less than 10% bone marrow plasma cells (BMPCs). They usually have low tumor burden and favorable outcome. To further understand the clinical and laboratory features of macrofocal MM, we retrospectively analyzed about 1000 symptomatic MM patients of two centers and found that 49 (4.9%) patients fulfilled the criteria of macrofocal MM. Median age was 54 years; 39 (80%) were male. The immunoglobulin subtype included IgG (13), IgA (5), IgD (4), light chain only (4) and non-secretory type (23). International Staging System (ISS) stage 1 in 45 (92%), and no stage 3 patient. Durie-Salmon (DS) Staging System stage 3A in 48 (98%), and 2A in 1. Extramedullary plasmacytomas (EMPs) in 39 (80%) patients. The mean percentage of BMPCs was 2.1%, and 44 (90%) patients had less than 5% BMPCs. No patients had hypercalcemia and renal impairment. FISH cytogenetics, available in 11 patients, showed IgH translocation in 7, 13q del in 2, t(4;14) in 1, 1q21 amp in 6 and p53 del in 2 patients. Serum free light chain (sFLC), available in 14 patients, showed abnormal sFLC in 7 of 14 patients and 4 in 7 non-secretory type patients. Thirty eight (77.6%) patients received new drug-based induction therapies and 14 patients received autologous stem cell transplantation (ASCT) as consolidation therapy. After a median follow-up of 36 months,the estimated median progression-free survival (PFS) was 41 months and the median overall survival time (OS) had not been reached. Forty four macrofocal MM with ISS stage 1 and DS stage 3A were compared to all (93) typical symptomatic MM patients who were diagnosed at the same period with the same stages according to both ISS and DS Staging System. For macrofocal MM, there were significantly more male patients and EMPs, higher hemoglobin levels and lowerb2-microglobulin (b2M) levels than that of typical MM. There were no significant differences in the proportions of patients received novel agents-based chemotherapy and ASCT between two groups, however, the estimated median PFS of macrofocal MM was significant superior to typical MM (41 vs 23 months, respectively; P= 0.001) (Figure 1), and the estimated median OS was also better in macrofocal MM than typical MM (not reached vs 61 months; P= 0.004) (Figure 2). Multivariate analysis showed that only the type of disease was the independent prognostic factor of both PFS and OS. In summary, Macrofocal MM is a particular subgroup of MM with multiple bone lesions and lower tumor burden, higher frequency of EMPs, and better survival than typical symptomatic MM.

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

Disclosures

Fan: The National Natural Science Foundation of China: Research Funding. Hou:Novartis: Membership on an entity's Board of Directors or advisory committees.
Th17/Th22 Cells Mediate Breakthrough Graft-Versus-Host-Disease
Authors: Furlan Scott N. , Tkachev Victor , Watkins Ben , Betz Kayla , Panoskaltsis-Mortari Angela , Blazar Bruce R. , Kean Leslie
Published: October 2019

Acute graft-versus-host disease (aGVHD) develops in more than half of patients after allogeneic hematopoietic cell transplantation (allo-HCT) despite poly-pharmacy immunoprophylaxis. Importantly, the dysregulated pathways responsible for this breakthrough disease remain largely unidentified. Thus, the discovery of these pathways represents one of the critical challenges for the field of allo-HCT. To address these needs, we have developed a model of aGVHD in rhesus macaques, which allows us to study the mechanisms of aGVHD both in its untreated state and in a variety of immunoprophylactic settings. Using a systems-based approach, we have created both a multiparameter flow cytometric and transcriptomic map of the immune landscape of aGVHD in allo-HCT recipients, in comparison to two critical control groups: (1) healthy untransplanted controls, and (2) those receiving autologous transplantation.

We find that recipients of allo-HCT receiving 1) no immunoprophylaxis 2) monotherapy with CTLA4Ig or 3) monotherapy with sirolimus develop early fulminant aGVHD with multi-organ disease (Figure 1a-grouped as 'Primary GVHD'). The immunophenotype of T cells from the Primary GVHD cohort exhibits an effector/memory phenotype with robust proliferation and acquisition of cytotoxic function. Transcriptomic analysis reveals enrichment of Th1-associated transcripts (IL12RB2, CCR5, CXCR3) as well as programs of proliferation early in the post transplant period (Figure 1b). Flow cytometric data confirms an increase in the number of CD4 and CD8 T cells producing the Th1 cytokine, IFN-g at this time-point (Figure 1c).

In contrast, standard-of-care Tacrolimus/Methotrexate (Tac/Mtx) as well as novel CTLA4Ig/sirolimus combination immunoprophylaxis (CoBS) both significantly improved survival of animals after allo-HCT. However, similar to human patients undergoing allo-HCT, these recipients often developed clinical signs of breakthrough aGVHD (starting around day 30 post-transplant) characterized by both gastrointestinal and skin pathology. This cohort was thus termed the "Breakthrough GVHD" cohort (Figure 1a). Unexpectedly, despite the presence of breakthrough clinical aGVHD, the Tac/Mtx and CoBS cohorts were still able to control programs of T cell proliferation, effector phenotype acquisition and Th1 cytokine skewing. However, both transcriptional and flow cytometric profiles demonstrated enrichment for molecules that reflect Th17/Th22 skewing (RORC, IL17A, AHR, and IL22) (Figure 2a) and production of IL17a (Figure 2b).

These results suggest that while current methods of immunoprophylaxis are able to limit both T cell proliferation and Th1 polarization, breakthrough Th17/Th22 pathway activation occurs despite these therapies. These data suggest that emphasis should be placed on exploration of pharmacologic inhibitors of IL17/IL22 for the prevention/treatment of breakthrough aGVHD.

Disclosures

No relevant conflicts of interest to declare.
Post-Thrombotic Syndrome and Quality-of-Life after Venous Thromboembolism in Young and Middle-Aged Women
Authors: Ljungqvist Maria , Holmstrom Margareta , Kieler Helle , Larfars Gerd
Published: October 2019

Introduction: Post-thrombotic syndrome (PTS) is the most common complication after a venous thromboembolism (VTE). PTS is a chronic condition affecting health-related quality-of-life (QoL). In this study we aimed to determine the risk of PTS and how it affects QoL after a first episode of VTE in young and middle-aged women.

Methods: We conducted a cohort study, including 1438 women with a first episode of VTE. Patients were recruited from 'Thrombo Embolism Hormonal Study' (TEHS), a Swedish nation-wide case-control study on risk factors for VTE in women 18-65 years of age. Consecutive patients with a first episode of deep vein thrombosis (DVT) in the lower leg or pulmonary embolism (PE) were included between 2002 and 2009. In 2011 all women still living in Sweden were followed up through a questionnaire. PTS was measured using self-reported Villalta score and Veins-QoL was used to measure QoL.

Results: After a median follow-up time of 6 years 1049 patients accepted participation in the follow-up study. The reported prevalence of PTS was 20 % for all patients, 28 % among women with a previous episode of a proximal DVT, 19 % among women with a previous distal DVT and 12 % among women with PE. Women with a history of leg symptoms before the first VTE-event had a higher risk of PTS (OR 3.5 (95% CI 2.5 - 4.8), with a prevalence of 32% compared to 12% in women with no history of leg symptoms. Obese women were at increased risk of PTS (OR 1.9, 95% CI 1.4 - 2.7) compared to non-obese. Similar women with proximal DVT (OR 1.6, 95% CI 1.1 - 2.3) and ipsilateral recurrence (OR 3.8, 95% CI 1.9 - 7.7) had increased risk of PTS. Patients with PTS scored lower on Veins-QoL (44 vs. 52, p < 0.01).

Conclusions: PTS is a common complication of VTE. Women with a history of leg-symptoms before time of VTE-diagnosis have more than 3-fold increased risk of PTS. Occurrence of PTS significantly reduces QoL.

Disclosures

No relevant conflicts of interest to declare.
IRF8 Regulates Cell Cycle of Hematopoietic Stem Cells
Authors: Qiu Qingsong , Liu Ping , Zhao Xuemei , Zhang Chun , Li Donghe , Zhang Ruihong , Ren Ruibao
Published: October 2019

IRF8 is expressed predominately in hematopoietic cells as a transcription factor and regulator of innate immune receptors signaling. It plays a critical role in the development of innate immune and adaptive immune cells, including dendritic cells, monocytes, eosinophils, basophils, B and T lymphocytes. It also functions as a tumor suppressor, as IRF8 deficient mice manifest a chronic myelogenous leukemia (CML)-like syndrome. In addition to various lineages of hematopoietic cells, we have found that IRF8 is expressed in hematopoietic stem cells (HSCs). However, the function of IRF8 in HSCs was unknown. In this study we investigated the role of IRF8 in regulating HSCs. We found that the number of long-term (LT)-HSCs (Lin- Sca1+ c-Kit+ CD48- CD150+) is significantly reduced in IRF8 knockout mice (IRF8-/-), comparing to the wild-type (WT) controls. Long-term reconstitution assays showed that IRF8-/- LT-HSC's repopulation capability is severely impaired compared to equal amount of WT mouse LT-HSCs. The effect of IRF8 depletion on HSC's self-renewal capacity is unlikely due to the influence of the CML-like syndrome, since the disease is not transplantable and only seen in the primary mice. In addition, the number of LT-HSCs is also decreased in E14.5 fetal liver of IRF8-/- mice, when the myeloproliferative disorder has not been manifested. A cell cycle analysis showed that the number of LT-HSCs in S, G2 or M phase is greatly reduced in IRF8-/- mice comparing to that in WT mice. Transcription profiling analysis of LT-HSCs revealed that the expression of key regulators of cytokine/growth factor signaling and factors controlling HSC self-renewal are downregulated in IRF8-/- mice comparing to that in WT mice. These results indicate that IRF8 plays a critical role in regulating cell cycle entry of HSCs. This function of IRF8 may play an important role in activating HSCs to enhance immunity and innate immunity.

Disclosures

No relevant conflicts of interest to declare.
Single-Cell Analysis of Lymphoid-Primed Multipotent Progenitors (LMPPs) Reveal Alterations in Lineage Commitment during Aging
Authors: Borikar Sneha , Philip Vivek , Trowbridge Jennifer J.
Published: October 2019

During aging, the hematopoietic compartment undergoes lineage skewing, biased toward myeloid differentiation at the expense of lymphoid differentiation. This skewing clinically presents as impaired adaptive immunity and an increased risk of myeloproliferative disorders. However, little is known of the regulatory mechanisms underlying these changes in differentiation potential due in part to the inadequacy of current analytic techniques to evaluate lineage potency of individual progenitor cells. Recent demonstration that long-lived hematopoietic progenitor cells drive steady-state hematopoiesis has shifted focus onto the progenitor cell compartment to understand clonal dynamics of native hematopoiesis. Here, we critically assess the functional and molecular alterations in the multipotent progenitor cell pool with aging at the single-cell level. We developed novel in vitro and in vivo assays to define the heterogeneity of the LMPP population and test cell-fate potential from single cells. Our results demonstrate, for the first time, distinct, intrinsic lineage potential of single in vitro LMPPs at the cellular and molecular level. We find that clonal alterations in the lymphoid-primed multipotent progenitor (LMPP) compartment contributes to the functional alterations in hematopoiesis observed during aging. Unbiased single-cell transcriptome analysis reveals that true multipotential clones and lymphoid-restricted clones are reduced with aging, while bipotential and myeloid-restricted clones are modestly expanded. Furthermore, myeloid-restricted clones gain myc driver signatures, molecularly identifying clones emerging during aging that are susceptible to transformation. Our study reveals that aging alters the clonal composition of multipotential progenitor cells, directly contributing to the global loss of the lymphoid compartment and increased susceptibility to myeloid transformation.

Disclosures

No relevant conflicts of interest to declare.
A Double Blind Randomised Clinical Trial to Prevent Serious Side Effects of Dexamethasone during Pediatric Acute Lymphoblastic Leukemia Treatment
Authors: Warris Lidewij T , van den Heuvel-Eibrink Marry M , Aarsen Femke K , Pluijm Saskia MF , Bierings Marc B , van den Bos Cor , Zwaan Christian M. , Tissing Wim JE , Veening Margreet A , Pieters Rob , van den Akker Erica LT
Published: October 2019

Background/Objectives: Dexamethasone is highly effective in the treatment of pediatric acute lymphoblastic leukemia (ALL), but can induce serious metabolic and neuropsychological side effects. Recent studies have led to the hypothesis that neuropsychological side effects could be due to cortisol depletion of the cerebral mineralocorticoid receptor. We therefore studied whether adding a physiological dose of hydrocortisone to dexamethasone treatment reduces neuropsychological and metabolic side effects in children with ALL.

Design/Methods: We performed a multicentre double-blind, randomised controlled trial with a crossover design. Patients (3-16 years) treated with dexamethasone pulses according to the DCOG ALL protocols were included. Patients received hydrocortisone 10 mg/m2/day in a circadian rhythm during one dexamethasone course and placebo during another dexamethasone course in a randomised order. The primary outcome measures were mood and behavior (parent-reported Strenght and Difficulties Questionnaire- Dut (SDQ)). Secondary outcome measures included sleep (Sleep Disturbance Scale for Children (SDSC)), neurocognitive function, and metabolic parameters.

Results: 50 subjects were enrolled, of which 48 patients completed both courses. For the total group no significant effects were found. However, in the subgroup of children with clinically significant behavioral side effects, adding hydrocortisone resulted in a significant reduction of side effects on overall stress (-4.9 (=0.9 SD), P =0.00), mood (-1.8 (=0.9 SD), P =0.03), behavior (-1.0 (=0.7 SD), P =0.01), and impact (P <0.05). The subgroup with dexamethasone-related sleeping problems did benefit on total sleeping problems (-4.3, P< 0.05). In contrast, hydrocortisone addition did not affect metabolic parameters.

Conclusion: This randomised controlled trial provides evidence that addition of a physiological dose of hydrocortisone in circadian rhythm during dexamethasone treatment in pediatric ALL patients reduces side effects on behavior, mood and sleep. This novel and simple intervention may be valuable for all patients on high-dose dexamethasone treatment.

Disclosures

No relevant conflicts of interest to declare.
SETD2 Heterozygous Loss in Leukemia Leads to Chemotherapy Resistance through Attenuation of the DNA Damage Response
Authors: Mar Brenton G. , Kahn Josephine , Zon Rebecca L , Grauman Peter V , Boettger Kevin , Chu Scott Haihua , Armstrong Scott A. , Ebert Benjamin L.
Published: October 2019

Relapsed leukemia is chemotherapy resistant and survival is poor. We recently found that mutations in epigenetic regulators are enriched in relapsed pediatric B-cell acute lymphoblastic leukemia (B-ALL), suggesting that they are associated with clonal survival and chemotherapy resistance. The mutations included loss of function mutations in the epigenetic regulator SETD2, which had not before been described in B-ALL. SETD2 is the only known mammalian Histone 3 Lysine 36 (H3K36) trimethyltransferase and one or two copy loss leads to a global decrease or elimination, respectively, of its chromatin mark, H3K36me3.

Using CRISPR/Cas9 to engineer isogenic human and murine leukemia cells, we have demonstrated that SETD2 loss leads to resistance to DNA damaging chemotherapy used in standard ALL therapy, consistent with the gain of SETD2 mutations at relapse. Leukemia cells with SETD2 heterozygous deletion demonstrated a significant increase in IC50 to the DNA damaging anti-metabolites 6-thioguanine (6TG) and cytarabine (AraC), but not the protein synthesis inhibitor L-Asparaginase (Asp). To model the enrichment of SETD2 mutations at relapse, we competed a small population of SETD2 heterozygous cells with isogenic control leukemia cells. Within 3 weeks, treatment with 6TG and AraC, but not Asp or vehicle, led to a dramatic selection of SETD2 heterozygous cells, mimicking the selective pressure of SETD2 clones in patients.

Recent reports have shown that H3K36me3is important in the DNA damage response (DDR). Consistent with this, we find that heterozygous SETD2 loss impairs the DDR signaling pathway with attenuated phosphorylation of multiple DDR components, including Chk1 and Chk2, in response to chemotherapy. Consequently, SETD2 heterozygous cells showed an abrogation of the apoptotic response to the DNA damaging chemotherapy agents 6TG and AraC, despite a normal apoptotic response to Asp.

In summary, we have demonstrated that SETD2 loss leads to resistance to DNA damaging chemotherapy. The resistance is caused by a failure to trigger DDR signaling and apoptosis after chemotherapy induced DNA damage. These findings identify heterozygous SETD2 lossas a novel mechanism of chemotherapy resistance in leukemia and have implications for therapy selection and novel therapeutic interventions.

Disclosures

No relevant conflicts of interest to declare.
Quantitative Multiplexed Immunohistochemistry Assays for Exploring CAR Modified T Cells and Checkpoint Inhibitors in Lymphoma Trials
Authors: Tran Thai Hong , Scott Krupa , Lee Shyun-Shyun , Singh Reshma , Cogan Shawn , Bordeaux Jennifer , Jennifer Hummel , Lameh Jelveh , Tribouley Catherine , Kassim Sadik , Tangri Shabnam , Dakappagari Naveen
Published: October 2019

Although there have been compelling advances in the cancer immunotherapy space recently in the form of chimeric antigen receptor (CAR) modified T-cells and checkpoint inhibitors, advanced tools to explore the therapeutic mechanisms of their combination are not adequately developed or widely available. To address this growing need, we developed a robust quantitative fluorescent immunohistochemistry platform using multiplex AQUA (Automated Quantitative Analysis) technology to evaluate checkpoint inhibitor expression, enumerate CAR T cells and determine the interaction between tumor cells and immune cells via novel co-localization algorithms. We explored utility of this method both in preclinical- and clinical model systems. In an immunodeficient mouse model of B-cell lymphoma, we evaluated homing of CAR T cells to malignant B-cells in primary lymphoid organs. We determined the phenotype and functional status of the CAR T cells via multiplex analyses of CD4, CD8, PD1 and FOXP3 expression. Additionally, to enable combination immunotherapies in Diffuse Large B-Cell Lymphoma (DLBCL) setting, we explored prevalence of adaptive immune resistance mechanisms in the form of PD1 and PD-L1 expression in immune- and tumor cell compartments via landmarks created by cytoplasmic and nuclear stains in both primary and secondary biopsies from DLBCL patients (n = 63). To support patient selection for CAR T trials, we quantified expression and prevalence of relevant tumor antigens that could not be scored reproducibly by traditional methods to yield objective cut points. We anticipate utilization of these quantitative multiplexed IHC methods for optimal selection of patients into upcoming novel combination immunotherapy trials

Disclosures

Tran: Genoptix: Employment. Scott:Genoptix: Employment. Lee:Genoptix: Employment. Singh:Novartis: Employment. Cogan:Novartis: Employment. Bordeaux:Genoptix: Employment. Jennifer:Genoptix: Employment. Lameh:Genoptix: Employment. Tribouley:Novartis: Employment. Kassim:Novartis: Employment. Tangri:Genoptix Inc., a Novartis company: Employment. Dakappagari:Genoptix Inc., a Novartis company: Employment.