Rational Targets in Systemic AL Amyloidosis

Introduction Dysproteinemia refers to a spectrum of gammopathies in which an aberrant clone(s) of late stage B lymphoid cells or plasma cells produces monoclonal immunoglobulins and/or fragments. The spectrum includes incidentally noted benign conditions like monoclonal gammopathy of undetermined significance (MGUS) to life threatening diseases such as multiple myeloma (MM) and systemic light chain (AL) amyloidosis. Exploitation of normal plasma cell biology forms the basis of treatments used in both MM and AL amyloidosis. In recent years, immunotherapeutic strategies targeting plasma cells have redefined the treatment landscape of MM and could be particularly useful in AL amyloidosis which is distinguished by a low burden of less proliferative disease and a paucity of high risk genetics. We sought to characterize the expression of potentially clinically relevant and/or immunotherapeutic targets in amyloidogenic plasma cells. Methods All patients diagnosed with systemic AL amyloidosis at Memorial Sloan Kettering Cancer Center, NY between January 1, 2012, and December 31, 2018, who had unstained bone marrow samples were identified. Decalcified formalin fixed paraffin embedded bone marrow biopsy sections were stained for G protein-coupled receptor, class C group 5 member D (GPRC5D), Cyclin D1 (CCND1), B-cell lymphoma 2 (BCL2), and B-cell maturation antigen (BCMA) expression using standard laboratory developed Immunohistochemistry (IHC) assays in a CLIA compliant setting. We scored the biopsies for percentage expression, and intensity of staining. We also obtained the demographic details, staging, and cytogenetic information for the patients from whom these samples were obtained. Results During the queried period, 32 unstained samples were available for testing from 27 unique patients. There were 27 diagnostic and 5 patients had additional samples from the time of relapse. The median age was 63 years (range 41-73). 64% of patients were male and 75% had lambda restricted plasma cells. Cytogenetic abnormalities using fluorescence in situ hybridization (FISH) were reviewed, t(11;14) was seen in 38% samples. The median clonal PC burden in BM at diagnosis was 10% (range 2-80%) and 36% had >10% plasma cells. In clonal PCs, the median BCL2 expression was 100% (range 80-100%) with a staining intensity of 2 (range 1-3). 84% (27) samples had BCL2 expression meeting threshold used in the Bellini study. CCND1 expression could be tested in 16 samples median expression 100% (range 20-100%) and median staining intensity 2 (range 1-3). Patients with CCND1 expression also had 100% BCL2 staining. GPRC5D expression was available in 18 samples and all samples tested expressed GPRC5D with median 80% (range 30-100%) with median intensity 1 (range 1-3). BCMA expression was available for 25 samples, with median expression 80% (range 50-100%) with a median staining intensity 2 (range 1-3). Among the five patients with samples from diagnosis and relapse, samples retained their expression of BCL2, BCMA, and GPRC5D. Among samples with t(11;14) by FISH, 92% expressed BCL2 (per Bellini study) and 58% expressed CCND1. Conclusion Cell surface expression of novel targets has enabled the development of several efficacious therapeutic strategies in MM. Amyloidogenic plasma cells express GPRC5D, BCMA and BCL2. Our data provides the rationale for careful investigation and development of targeted agents (BCL2 inhibitors, e.g.venetoclax) and immunotherapeutic strategies directed at GPRC5D and BCMA in AL amyloidosis. Figure 1 Figure 1. Disclosures Dogan: Roche: Consultancy, Research Funding; Peer View: Honoraria; Seattle Genetics: Consultancy; Takeda: Consultancy, Research Funding; Physicians' Education Resource: Honoraria; EUSA Pharma: Consultancy. Hassoun: Celgene, Takeda, Janssen: Research Funding. Mailankody: Fate Therapeutics: Research Funding; Allogene Therapeutics: Research Funding; Bristol Myers Squibb/Juno: Research Funding; Plexus Communications: Honoraria; Legend Biotech: Consultancy; Evicore: Consultancy; Jansen Oncology: Research Funding; Physician Education Resource: Honoraria; Takeda Oncology: Research Funding. Giralt: SANOFI: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Landau: Takeda: Research Funding; Genzyme: Honoraria; Takeda, Janssen, Caelum Biosciences, Celgene, Pfizer, Genzyme: Membership on an entity's Board of Directors or advisory committees.

Fish and chips: the origin of human gene families is a predictor of the location of GWAS signals

GWAS have identified thousands of loci associated with human complex diseases and traits. How these loci are distributed through the genome has not been systematically evaluated. We hypothesised that the location of GWAS loci differ between ancestral linkage groups (ALGs) related to the paralogy and function of genes. We used data from the NHGRI-EBI GWAS catalog to determine whether the density of GWAS loci relative to HapMap variants in each ALG differed, and whether ALG’s were enriched for experimental factor ontological (EFO) terms assigned to the GWAS traits. In a gene-level analyses we explored the characteristics of genes linked to GWAS loci and those mapping to the ALG’s. We find that GWAS loci were enriched or deficient in 9 and 7 of the 17 ALG’s respectively, while there was no difference in the number of GWAS loci in regions of the human genome unassigned to an ALG. All but 2 ALG’s were significantly enriched or deficient for one or more EFO terms. Lastly, we find that genes assigned to an ALG are under higher levels of selective constraint, have longer coding sequences and higher median expression in the tissue of highest expression than genes not mapping to an ALG. On the other hand, genes associated with GWAS loci have longer genomic length and exhibit higher levels of selective constraint relative to non-GWAS genes.Collectively, this suggests that understanding the location and ancestral origins of GWAS signals may be informative for the development of tools for variant prioritization and interpretation.

HELQ and EGR3 expression correlate with IGHV mutation status and prognosis in chronic lymphocytic leukemia

Background IGHV mutation status is a crucial prognostic biomarker for CLL. In the present study, we investigated the transcriptomic signatures associating with IGHV mutation status and CLL prognosis. Methods The co-expression modules and hub genes correlating with IGHV status, were identified using the GSE28654, by ‘WGCNA’ package and R software (version 4.0.2). The over-representation analysis was performed to reveal enriched cell pathways for genes of correlating modules. Then 9 external cohorts were used to validate the correlation of hub genes expression with IGHV status or clinical features (treatment response, transformation to Richter syndrome, etc.). Moreover, to elucidate the significance of hub genes on disease course and prognosis of CLL patients, the Kaplan–Meier analysis for the OS and TTFT of were performed between subgroups dichotomized by the median expression value of individual hub genes. Results 2 co-expression modules and 9 hub genes ((FCRL1/FCRL2/HELQ/EGR3LPL/LDOC1/ZNF667/SOWAHC/SEPTIN10) correlating with IGHV status were identified by WGCNA, and validated by external datasets. The modules were found to be enriched in NF-kappaB, HIF-1 and other important pathways, involving cell proliferation and apoptosis. The expression of hub genes was revealed to be significantly different, not only between CLL and normal B cell, but also between various types of lymphoid neoplasms. HELQ expression was found to be related with response of immunochemotherapy treatment significantly (p = 0.0413), while HELQ and ZNF667 were expressed differently between stable CLL and Richter syndrome patients (p < 0.0001 and p = 0.0278, respectively). By survival analysis of subgroups, EGR3 expression was indicated to be significantly associated with TTFT by 2 independent cohorts (GSE39671, p = 0.0311; GSE22762, p = 0.0135). While the expression of HELQ and EGR3 was found to be associated with OS (p = 0.0291 and 0.0114 respectively).The Kras, Hedgehog and IL6-JAK-STAT3 pathways were found to be associating with the expression of hub genes, resulting from GSEA. Conclusions The expression of HELQ and EGR3 were correlated with IGHV mutation status in CLL patients. Additionally, the expression of HELQ/EGR3 were prognostic markers for CLL associating with targetable cell signaling pathways.

Performance of Validated MicroRNA Biomarkers for Alzheimer’s Disease in Mild Cognitive Impairment

Background: Cerebrospinal fluid (CSF) microRNA (miRNA) biomarkers of Alzheimer’s disease (AD) have been identified, but have not been evaluated in prodromal AD, including mild cognitive impairment (MCI). Objective: To assess whether a set of validated AD miRNA biomarkers in CSF are also sensitive to early-stage pathology as exemplified by MCI diagnosis. Methods: We measured the expression of 17 miRNA biomarkers for AD in CSF samples from AD, MCI, and cognitively normal controls (NC). We then examined classification performance of the miRNAs individually and in combination. For each miRNA, we assessed median expression in each diagnostic group and classified markers as trending linearly, nonlinearly, or lacking any trend across the three groups. For trending miRNAs, we assessed multimarker classification performance alone and in combination with apolipoprotein E ɛ4 allele (APOE ɛ4) genotype and amyloid-β42 to total tau ratio (Aβ42:T-Tau). We identified predicted targets of trending miRNAs using pathway analysis. Results: Five miRNAs showed a linear trend of decreasing median expression across the ordered diagnoses (control to MCI to AD). The trending miRNAs jointly predicted AD with area under the curve (AUC) of 0.770, and MCI with AUC of 0.705. Aβ42:T-Tau alone predicted MCI with AUC of 0.758 and the AUC improved to 0.813 (p = 0.051) after adding the trending miRNAs. Multivariate correlation of the five trending miRNAs with Aβ42:T-Tau was weak. Conclusion: Selected miRNAs combined with Aβ42:T-Tau improved classification performance (relative to protein biomarkers alone) for MCI, despite a weak correlation with Aβ42:T-Tau. Together these data suggest that that these miRNAs carry novel information relevant to AD, even at the MCI stage. Preliminary target prediction analysis suggests novel roles for these biomarkers.

Identification of LncRNA Prognostic Biomarkers Associated with Copy Number Variants in Gastric Cancer

Backgrounds: Gastric cancer is one of the most common gastrointestinal carcinomas worldwide, with a poor prognosis. Prognosis prediction is very important in the treatment of gastric cancer .This study aimed to explore the prognostic value of lncRNA deregulated by copy number variants (CNVs) in gastric cancer. Methods: Multi-omics cluster analysis was performed to identify subtypes on the prognosis associated coding genes, CNVs and methylation sites. We conducted survival analyses on median expression level for all identified lncRNAs. Finally, we constructed and validated a prognostic model on lncRNAs with data of public and our center. Results: As a result, we identified six subtypes of gastric cancer with different prognosis (P=0.00446) and a total of 83 disease associated lncRNAs. We finally obtained five prognostic lncRNA biomarkers. Survival analyses showed that the high expression of all identified lncRNAs positive associated with worse prognosis. We also found that the prognostic model on five identified lncRNAs could predict survival with high 5-years AUC 0.69. And the differences of survival between high risk and low risk groups were significant in both of our and public database. In the multivariable analyses, we found that the prognostic model was an independent prognostic factor (p = 0.0104).Conclusions: We concluded that the prognostic model on five identified lncRNAs was closely related to the overall survival of gastric cancer and may serve as promising prognostic biomarkers of gastric cancer.

Effect of Remote Ischemic Postconditioning on miRNA-145 and Troponin I levels in STEMI patients undergoing primary percutaneous coronary intervention

Background: Primary Percutaneous Coronary Intervention Procedure (PPCI) results in reperfusion injury which will result in more extensive infarction. Remote Ischemic Postconditioning (RIPC) is a protective strategy to reduce the increase in the area of ​​infarction. miRNA-145 also plays a role in the protective effect of IPC and RIPC. Research Methods: This study uses a pre and post test approach only with control group design with experimental research designs. Data is taken at the Integrated Heart Services Installation RSUP Dr. M. Djamil Padang from July to November 2019, 40 patients with ST-segment elevation myocardial infarction (STEMI) performed RIPC. Bivariate analysis was performed to determine differences in levels of miRNA-145 and troponin I in STEMI patients underwent PPCI with and without RIPC using the Wilcoxon test and the Mann Whitney test. Results:A total of 40 patients who underwent the PPCI procedure were divided into two groups PPCI + RIPC (n = 20) and PPCI without RIPC (n = 20). There were no significant differences in the basic characteristics between the two groups. There were no significant difference in escalation of median expression of miRNA-145 in PPCI+ RIPC [pre test 36.33 (27.44-52.39), post test 34.83 (27.65-65.26), p = 0.765] compared to PPCI without RIPC [pre test 31.66 (26.31-43.28), post test 33.43 (26.83-64.97), p = 0.765]. There were an increase in median troponin I levels in both groups, PPCI+ RIPC [pretest 4,104.70 (67.30-40,000.00), post test 30,448.50 (120.00-16.3192.20), p = 0.001] and PPCI without RIPC [pretest 826.50 (17.00-48.259.00), post test 42.784.50 (2,119.00-162.897.00), p = <0.001]. Conclusion:There were no significant difference in median expression of miRNA-145 in STEMI patients before and after (48 hours) underwent PPCI+RIPC and PPCI without RIPC. There were a significant difference in median levels of troponin I in STEMI patients before and after (48 hours) underwent PPCI+RIPC and PPCI without RIPC. Keywords: Remote Ischemic Postconditioning, miRNA-145, troponin I

The Effect of Remote Ischemic Postconditioning on miRNA-145 and Troponin I levels in STEMI patients undergoing primary percutaneous coronary intervention

Background: Primary Percutaneous Coronary Intervention Procedure (PPCI) results in reperfusion injury which will result in more extensive infarction. Remote Ischemic Postconditioning (RIPC) is a protective strategy to reduce the increase in the area of ​​infarction. miRNA-145 also plays a role in the protective effect of IPC and RIPC. Research Methods: This study uses a pre and post test approach only with control group design with experimental research designs. Data is taken at the Integrated Heart Services Installation RSUP Dr. M. Djamil Padang from July to November 2019, 40 patients with ST-segment elevation myocardial infarction (STEMI) performed RIPC. Bivariate analysis was performed to determine differences in levels of miRNA-145 and troponin I in STEMI patients underwent PPCI with and without RIPC using the Wilcoxon test and the Mann Whitney test. Results:A total of 40 patients who underwent the PPCI procedure were divided into two groups PPCI + RIPC (n = 20) and PPCI without RIPC (n = 20). There were no significant differences in the basic characteristics between the two groups. There were no significant difference in escalation of median expression of miRNA-145 in PPCI+ RIPC [pre test 36.33 (27.44-52.39), post test 34.83 (27.65-65.26), p = 0.765] compared to PPCI without RIPC [pre test 31.66 (26.31-43.28), post test 33.43 (26.83-64.97), p = 0.765]. There were an increase in median troponin I levels in both groups, PPCI+ RIPC [pretest 4,104.70 (67.30-40,000.00), post test 30,448.50 (120.00-16.3192.20), p = 0.001] and PPCI without RIPC [pretest 826.50 (17.00-48.259.00), post test 42.784.50 (2,119.00-162.897.00), p = <0.001]. Conclusion:There were no significant difference in median expression of miRNA-145 in STEMI patients before and after (48 hours) underwent PPCI+RIPC and PPCI without RIPC. There were a significant difference in median levels of troponin I in STEMI patients before and after (48 hours) underwent PPCI+RIPC and PPCI without RIPC. Keywords: Remote Ischemic Postconditioning, miRNA-145, troponin I

Toll-like receptor 9 negatively related to clinical outcome of AML patients

Background Acute myeloid leukemia (AML) can modulate toll-like receptor-9 (TLR9) expression and activation. This study was conducted to elucidate the expression of TLR9 in AML patients and its relation to the prognosis of the disease. Results The study included 40 newly diagnosed AML patients managed in the hospital in addition to 20 sex and age matched normal volunteers as control. TLR9 expression assay was conducted on peripheral blood samples of AML cases before the start of treatment as well as the controls by immunophenotyping. TLR9 expression was ranging from 0.10 to 2.40% in AML patients with higher expression among the control, ranging from 0.94 to 8.25%. The median TLR9 expression in AML patients was significantly lower with advanced cytogenetic risk score. It is not significantly differing in relation to patients’ sex, age group, and FAB type of AML. However, significant lower median expression was found in relation to clinical outcome. TLR9 expression ≤ 1% showed lower median overall survival time when compared to those with > 1% expression. Conclusion This study concluded that AML patients express TLR9 in leukemic cells with very low percentage. This expression was negatively related to the clinical outcome.

The role and robustness of the Gini coefficient as an unbiased tool for the selection of Gini genes for normalising expression profiling data

We recently introduced the Gini coefficient (GC) for assessing the expression variation of a particular gene in a dataset, as a means of selecting improved reference genes over the cohort (‘housekeeping genes’) typically used for normalisation in expression profiling studies. Those genes (transcripts) that we determined to be useable as reference genes differed greatly from previous suggestions based on hypothesis-driven approaches. A limitation of this initial study is that a single (albeit large) dataset was employed for both tissues and cell lines. We here extend this analysis to encompass seven other large datasets. Although their absolute values differ a little, the Gini values and median expression levels of the various genes are well correlated with each other between the various cell line datasets, implying that our original choice of the more ubiquitously expressed low-Gini-coefficient genes was indeed sound. In tissues, the Gini values and median expression levels of genes showed a greater variation, with the GC of genes changing with the number and types of tissues in the data sets. In all data sets, regardless of whether this was derived from tissues or cell lines, we also show that the GC is a robust measure of gene expression stability. Using the GC as a measure of expression stability we illustrate its utility to find tissue- and cell line-optimised housekeeping genes without any prior bias, that again include only a small number of previously reported housekeeping genes. We also independently confirmed this experimentally using RT-qPCR with 40 candidate GC genes in a panel of 10 cell lines. These were termed the Gini Genes. In many cases, the variation in the expression levels of classical reference genes is really quite huge (e.g. 44 fold for GAPDH in one data set), suggesting that the cure (of using them as normalising genes) may in some cases be worse than the disease (of not doing so). We recommend the present data-driven approach for the selection of reference genes by using the easy-to-calculate and robust GC.

Targeting Bcl-2 and CD38 As Part of Personalized HSCT Conditioning Regimen in Chemorefractory Acute Leukemia in Children

Introduction The outcome HSCT in a cohort of children with chemorefractory acute leukemia (AL) is poor. The incidence of relapse exceeds 50% and survival varies from 10 to 40%. Additional attempts at remission induction with various combinations of chemotherapy are unlikely to improve the outcome and may contribute to toxicity. We hypothesized that personalized targeted therapy combined with high-dose preparative regimen may improve the outcome of alloHSCT in a cohort of pediatric patients with refractory AL. Bcl-2 and CD38 were chosen as potential targets due to frequent expression in pediatric acute leukemia, availability of marketed targeted therapies, venetoclax and daratumumab, and expected non-overlapping toxicity profile of these agents and the conditioning regimen. Materials and methods A total of 26 pts with chemorefractory acute leukemia (AML-18, T-ALL-6, ABL-2, primary refractory - 9, refractory relapse - 17, 18 male, 8 female, median age 11.3 years), underwent HSCT between November 2017 and May 2019. All patients were transplanted from haploidentical donors, all patients had active disease (AD) at the moment of SCT. For 19 (73%) pts it was the first alloHSCT, for 7 (27%) pts it was the second HSCT. Median bone marrow leukemia burden before cytoreduction was 11% (3-90). Bcl-2 expression on the tumor cells was detected in 23 pts with the median expression of 58% (1.5-99), CD38 expression was detected in 24 pts with the median expression of 88% (11-100). Thirteen pts received treosulfan-based conditioning and 13 - TBI-based (12 Gy). TBI was used among patients with second HSCT (n=7), patients with T-ALL (n=4) and patients with massive extramedullary disease (n=2). GvHD prophylaxis included tocilizumab at 8 mg/kg on day -1 and abatacept at 10 mg/kg on day -1, +7, +14, +28. According to the expression of Bcl-2 and CD38 on tumor cells, 24pts (92%) received daratumumab (anti-CD38 monoclonal antibody) on day -7 at 10-16 mg/kg, 21 pts (81%) received venetoclax at 300 mg/m2/day on days -7 to -2 and 12 pts additionally received plerixafor at 240 mcg/kg x 3 on days -7 to -5. TCRαβ+/CD19+ depletion of PBSC with CliniMACS technology was implemented in all cases. The median dose of CD34+ cells in the graft was 9.5 x106/kg (range 4.9-13.3), αβ T cells - 26.5x103/kg (range 5.6- 84). Modified (CD45RA-depleted) donor lymphocyte infusions (DLI) were administered prophylactically in all pts. Results Two patients died before engraftment due to septic event. Primary engraftment was achieved in 24 of 24 evaluable pts (100%), the median time to neutrophil and platelet recovery was 12 and 13 days. All pts achieved complete remission on day +30. There were no significant toxic effects after targeted therapy, 4 pts (21%) had daratumumab-related infusion reactions, one patient with T-ALL died after engraftment due to septic event. aGVHD grade I-II developed in 5 pts (20%), two of them required systemic immunosuppressive therapy. There were no cases of aGVHD &gt; grade II, neither cases of cGVHD. The median NK- cells count by the day +30 was 0.07 x 106/ml (range 0.01- 0.4), the median levels of αβ T cells and gd T cells were 0.018 x 106 /ml (range 0 - 1.1) and 0.015 x 106 /ml (range 0,01 - 1.3), respectively. Nine (37.5%) pts (5 with T-ALL, 1 with ABL, 3 with AML) relapsed, median interval to relapse was 103 days (55-345). At the moment of reporting 14 (53%) patients are alive in complete remission with a median follow up of 12.7 months (1.9-23m). Event-free and overall survival at 1 year for AML are 76% (SE13) and 76% (SE14), all patients with T-ALL and one with ABL died of disease progression. Conclusion We suggest that addition of venetoclax and daratumomab to the backbone of myeloablative haploidentical HSCT with αβ T cell depletion is not associated with increased toxicity and may lead to improved outcome in a cohort of pediatric patients with chemorefractory AML. In a subgroup of patients with T-ALL this approach does not seem to produce visible benefit. This approach can be further tested in a prospective trial with the goal to increase the anti-leukemic efficacy of HSCT. Figure Disclosures Maschan: Miltenyi Biotec: Other: lecture fee. Off Label Disclosure: venetoclax daratumomab plerixafor