scholarly journals Lack of a Spleen Signal on Diffusion Weighted MRI Is Associated with High Tumor Burden and Poor Prognosis in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4471-4471
Author(s):  
Leo Rasche ◽  
Manoj Kumar ◽  
Grant Gershner ◽  
James E McDonald ◽  
Samrat Roy Choudhury ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Differential Gene Expression ◽  
Research Funding ◽  
Imaging Techniques ◽  
Tumor Burden ◽  
Diffusion Weighted Mri ◽  
Medical Sciences ◽  
High Tumor Burden ◽  
Differential Gene

Abstract Background: Whole body medical imaging is an integral component of Multiple Myeloma (MM) evaluation as it reveals bone disease and/or focal lesions. While established imaging techniques, such as PET-CT, are powerful in delineating focal pathologies, they suffer from low specificity when it comes to assessment of diffuse bone marrow (BM) signals. Especially in patients treated with G-CSF, discrimination between malignant and non-malignant BM signals is barely possible. Diffusion-weighted MRI with background suppression (DWIBS) is a novel functional MRI technique which measures water diffusion in vivo. Basically, diffusion of water is more restricted in tissues with high cellularity, making DWIBS a promising tool to investigate malignant diffuse signals. Initially, we searched for an internal reference to classify the level of BM infiltration. We selected the spleen, since it is the abdominal organ with the highest restriction on DWIBS. Unexpectedly, we observed significant heterogeneity in the spleen signal itself including a subgroup of MM patients with total loss of the signal on DWIBS. Perplexingly, these patients suffered from high tumor burden and poor outcome. Here, we report on our strategy to elucidate this phenomenon and to evaluate its clinical value. Methods: We investigated 295 transplant-eligible newly diagnosed MM patients and 72 cases with monoclonal gammopathy of undetermined significance (MGUS). This study was approved by the institutional review board (#205415). DWIBS was performed on a 1.5 Tesla Philips Achieva scanner. The spleen signal was assessed on DWI and ADC maps by two experienced investigators in consensus read. The Kaplan-Meier method was used for survival analyses. Molecular characterization included fluorescence in situ hybridization, and gene expression profiling of CD138-enriched plasma cells (PCs). Differential gene expression was performed using a threshold of 2-fold expression difference. Wilcoxon or Fisher's exact tests were used to compare the median of a continuous variable or the distribution of discrete variables across groups, respectively. Correlation coefficients were determined using Spearman's rank correlation. Results: Absence of the spleen signal on DWIBS was a frequent phenomenon in MM, seen in 71/295 (24%) patients. In all of these patients asplenia was excluded using alternative imaging techniques. Lack of a signal was highly positively associated with tumor-load parameters, such as the degree of BMPC infiltration (P=1x10-10) or International Staging System (ISS) 3 (P=0.0001). In contrast, it was not associated with age, gender, or the tumor progression markers gain(1q) and del(17p). Patients with absence of spleen signal experienced unfavorable outcome (hazard ratio of 1.8 for PFS and OS). In order to further investigate the biological underpinnings of this phenomenon we performed a differential gene expression analysis of purified CD138 MM cells. No differentially expressed genes were found between patients with and without spleen signal, suggesting that the absence of the spleen signal mainly reflected increased tumor burden rather than specific tumor features. As a proof of concept, we addressed the spleen signal in individuals with MGUS, and longitudinally in MM patients who presented with absence of the spleen signal at diagnoses. Indeed, in all 78 individuals with MGUS the signal was preserved, and the majority of MM patients showed re-appearance of the spleen on DWIBS during treatment as the tumor burden declined. Interestingly, re-appearance of the spleen was helpful to distinguish between malignant and non-malignant hyperintensities in the BM, making the spleen signal a promising parameter for MM follow-up investigations. Conclusions: Due to the low frequency of abnormalities affecting the spleen, this organ is often overlooked during abdominal examinations using cross-sectional imaging techniques. Here we show that the spleen signal on DWIBS provides clinically useful information on tumor burden in MM. Moreover, it opens the avenue to distinguish between malignant and reactive BM hypercellularity on imaging in patients receiving treatment. Our observation strongly supports the hypothesis, that the spleen signal is suppressed by a high BMPC involvement. Yet, the reasons for this phenomenon are still elusive. Figure. Figure. Disclosures Roy Choudhury: University of Arkansas for Medical Sciences: Employment, Research Funding. Epstein:University of Arkansas for Medical Sciences: Employment. Barlogie:Dana Farber Cancer Institute: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; Celgene: Consultancy, Research Funding; Millenium: Consultancy, Research Funding; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; Multiple Myeloma Research Foundation: Other: travel stipend. Davies:Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

scholarly journals The SF3b Splicing Complex Regulates DNA Damage Response in Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1237-1237 ◽  
Author(s):  
Shalini Sankar ◽  
Miriam Guillen Navarro ◽  
Frida Ponthan ◽  
Simon Bomken ◽  
Sirintra Nakjang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Differential Expression ◽  
Differential Gene Expression ◽  
Research Funding ◽  
Gene Set Enrichment Analysis ◽  
Rna Seq ◽  
Self Renewal ◽  
Strand Breaks ◽  
Differential Gene

To identify potential regulators of propagation and self-renewal of Acute Lymphoblastic Leukaemia (ALL), we performed an explorative genome-wide RNAi screen followed by CRISPR ex vivo and in vivo validation screens in the t(4;11)-positive ALL cell line SEM. These screens identified the splicing factor PHF5A as a crucial component of the leukemic program. PHF5A is a subunit of the SF3b protein complex, which directs alternative splicing by binding to the branchpoint of pre-mRNA. Mutations in members of this complex including SF3B1 have been implicated in several haematological malignancies. Functional perturbation experiments demonstrated that PHF5A depletion impairs proliferation, viability and clonogenicity in a range of ALL and AML cell lines strongly suggesting that PHF5A is required for leukemic propagation and self-renewal. To identify genetic programs affected by PHF5A inhibition, we performed RNA-seq followed by analysis of differential gene expression and splicing events. We identified 473 genes with differential expression upon PHF5A knockdown. In addition, we performed in-depth analysis of splicing patterns by examining both differential exon/intron usage and exon junction formation. These analyses demonstrated that loss of PHF5A affects splicing of more than 2500 genes with exon skipping and intron retention being the most frequent splicing events. In order to identify processes and pathways affected by PHF5A, we performed gene set enrichment analysis (GSEA) on both differential expression and splicing. While gene sets associated with RNA processing including splicing, turnover and translation were enriched in both data sets, the differential gene expression signature was also linked to DNA repair processes including base excision, mismatch and homologous recombination repair. In line with these findings, knockdown of either PHF5A or its partner protein SF3B1 induced DNA strand breaks as indicated by comet assay and increased y-H2AX levels. Furthermore, both PHF5A and SF3B1 depletion sensitized ALL cells towards the DNA crosslinking agent mitomycin C. Closer inspection of RNA-seq datasets revealed reduced FANCD2 expression and skipping of exon 22 associated with impaired mono-ubiquitination of the FANCD2 protein as a consequence of PHF5A and SF3B1 knockdown. Furthermore, expression of RAD51, a key component of double strand break repair, also decreased upon PHF5A and SF3B1 knockdown. Notably, in vitro pharmacological inhibition of SF3b complex activity using H3B-8800 (or Pladienolide B) showed a very similar effect on FANCD2 expression, and ubiquitination as well as decrease of RAD51 and an increase in y-H2AX levels on a dose and time-dependent manner. This strongly suggests a mechanistic link between impaired RNA splicing and the repair of DNA double-strand breaks. These combined data show that leukemic cells are highly dependent on a functional SF3b splicing complex. Interference with its function results in DNA damage and also sensitizes towards DNA damaging agents pointing towards a possible benefit of the combined application of inhibitors targeting the SF3b complex with more conventional chemotherapy. Disclosures Ponthan: Epistem Ltd: Employment. Zwaan:Sanofi: Consultancy; Incyte: Consultancy; BMS: Research Funding; Roche: Consultancy; Janssen: Consultancy; Daiichi Sankyo: Consultancy; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Pfizer: Research Funding; Celgene: Consultancy, Research Funding. Vormoor:Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.

scholarly journals Single Cell RNA Sequencing Identifies Potential Molecular Indicators of Response to Melflufen in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1194-1194
Author(s):  
Philipp Sergeev ◽  
Sadiksha Adhikari ◽  
Juho J. Miettinen ◽  
Maiju-Emilia Huppunen ◽  
Minna Suvela ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Single Cell ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Cell Populations ◽  
Drug Efflux ◽  
Single Cell Rna Sequencing ◽  
Differential Gene

Abstract Introduction Melphalan flufenamide (melflufen), is a novel peptide-drug conjugate that targets aminopeptidases and selectively delivers alkylating agents in tumors. Melflufen was recently FDA approved for the treatment of relapsed/refractory multiple myeloma (MM) patients. Considering the challenges in treating this group of patients, and the availability of several new drugs for MM, information that can support treatment selection is urgently needed. To identify potential indicators of response and mechanism of resistance to melflufen, we applied a multiparametric drug sensitivity assay to MM patient samples ex vivo and analyzed the samples by single cell RNA sequencing (scRNAseq). Ex vivo drug testing identified MM samples that were distinctly sensitive or resistant to melflufen, while differential gene expression analysis revealed pathways associated with response. Methods Bone marrow (BM) aspirates from 24 MM patients were obtained after written informed consent following approved protocols in compliance with the Declaration of Helsinki. BM mononuclear cells from 12 newly diagnosed (ND) and 12 relapsed/refractory (RR) patients were used for multi-parametric flow cytometry-based drug sensitivity and resistance testing (DSRT) evaluation to melflufen and melphalan, and for scRNAseq. Based on the results from the DSRT tests and drug sensitivity scores (DSS), we divided the samples into three groups - high sensitivity (HS, DSS > 40 (melflufen) or DSS > 16 (melphalan)), intermediate sensitivity (IS, 31 ≤ DSS ≤ 40 (melflufen) or 10 ≤ DSS ≤ 16 (melphalan)), and low sensitivity (LS, DSS < 31 (melflufen) or DSS < 10 (melphalan)). To identify genes, responsible for the general sensitivity to melphalan-based drugs we conducted differential gene expression (DGE) analyses separately for melphalan and melflufen focusing on the plasma cell populations, comparing gene expression between HS and LS samples for both drugs ("HS vs. LS melphalan" and "HS vs. LS for melflufen", respectively). In addition, to explain the increased sensitivity of RR samples, we conducted the DGE analysis for ND vs. RR samples and searched for similarities between these three datasets. Results DSRT data indicated that samples from RRMM patients were significantly more sensitive to melflufen compared to samples from NDMM (Fig. 1A). In addition, we observed that samples with a gain of 1q (+1q) were more sensitive to melflufen while those with deletion of 13q (del13q) appeared to be less sensitive, although these results lacked significance (Fig. 1A). After separating the samples into different drug sensitivity groups (HS, IS, LS), DGE analysis showed significant downregulation of the drug efflux and multidrug resistance protein family member ABCB9 in the melflufen HS group opposed to the LS group (2.2-fold, p < 0.001). A similar pattern was detected for the melphalan HS vs. LS comparison suggesting that this alteration might be a common indicator of sensitivity to melphalan-based drugs. Furthermore, in the melflufen HS group we observed downregulation of the matrix metallopeptidase inhibitors TIMP1 and TIMP2 (3-fold and 1.6-fold, p < 0.001, respectively), and cathepsin inhibitors CST3 and CSTB (3.2-fold and 1.3-fold, p < 0.001, respectively) (Fig. 1B). This effect was observed in both "ND vs. RR" and "HS vs. LS for melflufen" comparisons, but not for melphalan, suggesting that these changes are associated with disease progression and specific indicators of sensitivity to melflufen. Moreover, gene set enrichment analysis (GSEA) showed activation of pathways related to protein synthesis, as well as amino acid starvation for malignant and normal cell populations in the HS group. Conclusion In summary, our results indicate that melflufen is more active in RRMM compared to NDMM. In addition, samples from MM patients with +1q, which is considered an indicator of high-risk disease, tended to be more sensitive to melflufen. Based on differential GSEA and pathway enrichment, several synergizing mechanisms could potentially explain the higher sensitivity to melflufen, such as decreased drug efflux and increased drug uptake. Although these results indicate potential indicators of response and mechanisms of drug efficacy, further validation of these findings is required using data from melflufen treated patients. Figure 1 Figure 1. Disclosures Slipicevic: Oncopeptides AB: Current Employment. Nupponen: Oncopeptides AB: Consultancy. Lehmann: Oncopeptides AB: Current Employment. Heckman: Orion Pharma: Research Funding; Oncopeptides: Consultancy, Research Funding; Novartis: Research Funding; Celgene/BMS: Research Funding; Kronos Bio, Inc.: Research Funding.

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