Yuka Aoki

Molecular diagnostics of a single drug-resistant multiple myeloma case using targeted next-generation sequencing

A 69-year-old man was diagnosed with IgG λ-type multiple myeloma (MM), Stage II in October 2010. He was treated with one cycle of high-dose dexamethasone. After three cycles of bortezomib, the patient exhibited slow elevations in the free light-chain levels and developed a significant new increase of serum M protein. Bone marrow cytogenetic analysis revealed a complex karyotype characteristic of malignant plasma cells. To better understand the molecular pathogenesis of this patient, we sequenced for mutations in the entire coding regions of 409 cancer-related genes using a semiconductor-based sequencing platform. Sequencing analysis revealed eight nonsynonymous somatic mutations in addition to several copy number variants, including CCND1 and RB1. These alterations may play roles in the pathobiology of this disease. This targeted next-generation sequencing can allow for the prediction of drug resistance and facilitate improvements in the treatment of MM patients.

Elevation of Plasmin-α2-plasmin Inhibitor Complex Predicts the Diagnosis of Systemic AL Amyloidosis in Patients with Monoclonal Protein

Objective The complication of systemic immunoglobulin light chain (AL) amyloidosis in patients with monoclonal immunoglobulin affects the prognosis, but amyloid deposition in tissues is sometimes difficult to detect due to bleeding tendencies and preferential distributions. However, fibrinolysis is known to be exacerbated in patients with systemic AL amyloidosis specifically. We therefore explored new biomarkers for predicting a diagnosis of systemic AL amyloidosis focusing on coagulation and fibrinolysis markers. Methods We reviewed the clinical features and treatment outcomes of patients with serum monoclonal protein, including primary systemic AL amyloidosis and multiple myeloma (MM), treated at our hospital between January 2008 and December 2014. Results Among several biomarkers, only the serum level of plasmin-α2-plasmin inhibitor complex (PIC) in patients with systemic AL amyloidosis (n=26) at the diagnosis was significantly higher than in patients with MM without AL amyloidosis (n=26) (mean±standard deviation, 3.69±2.82 μg/mL vs. 1.23±0.97 μg/mL, p<0.01). The cut-off for predicting a diagnosis of systemic AL amyloidosis in patients with serum monoclonal protein was 1.72 μg/mL with 84.6% sensitivity and 80.8% specificity. Hepatic involvement resulted in a significantly higher PIC level than no involvement in patients with systemic AL amyloidosis. The serum PIC level was also associated with the hematological response of systemic AL amyloidosis. Conclusion PIC is a useful biomarker for the diagnosis and management of patients with systemic AL amyloidosis.

Other Iatrogenic Immunodeficiency-associated Lymphoproliferative Disorder Presenting as Primary Bone Lymphoma in a Patient with Rheumatoid Arthritis.

Primary bone lymphoma (PBL) is a rare disorder. We herein present a case of other iatrogenic immunodeficiency-associated lymphoproliferative disorder (OIIA-LPD) presenting as PBL. A 63-year-old woman was diagnosed with rheumatoid arthritis and had been treated with methotrexate for seven years. Two months before admission, she suffered from pain in the limbs. Magnetic resonance imaging revealed multiple irregular lesions in the bones of the limbs, which showed an uptake of (18)F-FDG on positron emission tomography. A biopsy of the right radius revealed diffuse large B-cell lymphoma, leading to the diagnosis of OIIA-LPD. She received rituximab-containing regimens resulting in a complete response.

Abstract 3188: Targeted semiconductor sequencing of 409 cancer-related genes for somatic mutations and copy number variations in multiple myeloma

Background: 20169s NCCN guideline recommended that induction therapy used conventional chemotherapy such as proteasome inhibitors and Imids in MM. Overall survival is improving by these Novel agents. However, we don9t know how to choose these new agents. In the future, we can use next generation sequencer as a tool of drug choosing system. So we reviewed newly diagnosed 11 patients with MM who had received novel agents in our institution.The comprehensive analysis of genetic alterations in tumor by next generation sequencing can allow for the prediction of drug resistance and facilitate improvements in the treatment of MM patients. Method: DNA was extracted from magnetic bead-enriched bone marrow CD138-positive malignant plasma cells from 11 cases of MM, and CD138-negative cells were used as matched non-tumor cells. Forty nanograms of DNA were used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel that offers targeted coverage of all exons in 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers. (covered regions: 95.4% of total). We sequenced 15,992 regions which obtained more than 1.5 megabases of target sequence. Result: Each sample underwent on average 8.3 million sequencing reads after quality filtering. The mean read depths were 539x, and >95% of targeted bases were represented by at least 20 reads. The average number of non-synonymous mutations detected per patient was 5.8 (range 0-11). We also detected copy number variations in which segments of the genome can be duplicated or deleted from sequencing data. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of MM patients. Pathway assessment has shown that somatic aberrations within MM genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB. Conclusion: We performed targeted next-generation sequencing for rapid (2 days), standardized, and cost-effective gene analysis of malignant plasma cells from patients with MM. We can use targeted next-generation sequencing as tool of drug choosing system. Citation Format: Hiroshi Ikeda, Yasushi Sasaki, Kazuya Ishiguro, Tadao Ishida, Yuka Aoki, Takashi Tokino. Targeted semiconductor sequencing of 409 cancer-related genes for somatic mutations and copy number variations in multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3188.

Abstract 139: Interaction between monocytes and bone marrow microenvironment in pathogenesis of multiple myeloma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Multiple myeloma (MM) is an incurable haematological malignancy characterized by the clonal proliferation of malignant plasma cells within the bone marrow. The current data on MM disease progression indicate that bone marrow microenvironment plays crucial role in pathogenesis of MM. Myeloma cells contacts with bone marrow stromal cells (BMSCs), which secrete factors/cytokines, promoting tumor cell growth and survival. Paracrine secretion of cytokines, such as IL-6, insulin-like growth factor-1, inflammatory protein-1a in BMSCs promotes MM cell proliferation and protects against drug-induced cytotoxicity. MM lytic bone disease is caused by osteoclast activation and osteoblast inhibition. Disease-related bone complications result in significant morbidity due to pain, pathologic fractures and spinal cord compression. The bone microenvironment creates a supportive niche for MM progression. Osteoclasts and BMSCs, along with extracellular matrix and cytokines stimulate myeloma cell proliferation and confer chemoresistance. Therefore, the reciprocal interactions among tumor cells, osteoclasts, osteoblasts, and bone marrow stromal cells impact both the establishment and progression of MM. In current study, monocyte can directly promote osteogenic differentiation of mesenchymal stem cells through cell contact interactions and production of osteogenic factors. This mechanism is mediated by the activation of STAT3 signaling pathway in the mesechymal stem cells that leads to the upregulation of osteoblasts-associated genes such as Runx2 and alkaline phosphatase (ALP), and the downregulation of osteoblast inhibitors such as DKK1 to drive the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocyte, a component of bone marrow microenvironment, in the MM progression. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BMSCs and/or monocytes of MM patients. Consistently, we observed increased proliferation of MM cell lines in the presence of either BMSCs or monocytes compared to cell line-only control. Furthermore, the co-culture of BMSCs plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the bone marrow microenvironment. Citation Format: Hiroshi Ikeda, Yuka Aoki, Toshiaki Hyayashi, Yumiko Maruyama, Tadao Ishida, Takashi Tokino, Yasuhisa Shinomura, Yasushi Sasaki. Interaction between monocytes and bone marrow microenvironment in pathogenesis of multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 139. doi:10.1158/1538-7445.AM2014-139

Abstract LB-175: Methylome analysis by the combination of methylated DNA enrichment and next-generation sequencing in multiple myeloma

[Aim] To attempt methylome analysis in multiple myeloma (MM) cells using high-throuput next-generation sequencer, and assess the association between the methylome status and aneuploidy status detected by array CGH analysis in MM cells. [Methods] After the extraction of DNA from 2 MM cell lines and 9 primary MM samples, we enriched sheared fragments of methylated DNA using MethylminerTM Methylated DNA Enrichment Kit (invitrogen). Fragment libraries were then prepared followed by emulsion PCR and templated beads preparation. Sequencing analysis using SOLiDTM3 system (life technologies) was performed for these fragment libraries from enriched methylated DNA. In this study, using adoptors with individual barcodes when library preration, we could analyse at 12 samples (including input samples) at one run. Analyses after sequencing were the following. We counted tag numbers in promoter region of each gene (determined by sequences between +/− 100 bp from transcription start site (TSS)) read by sequencing. The number of the tags quantitatively reflects methylation levels of corresponding DNA sequences. We also performed array CGH analysis in the 82 primary MM samples. Combining these methods, we can assess the association between DNA methylation and chromosomal instability. [Results and comments] Of all RefSeq genes which have CpG islands in their promoter region (18592/30638 genes, determined by unique accession number in NCBI36/hg18), the number of genes which showed at least one tag was 6549 (35.2%). Referring to the results of mRNA expression analysis using cDNA microarray (Agilent, matched by the accession number), we found a significant reduction of expression levels according to the tag number increase. Also, a very high correlation between tag numbers and mRNA expression levels (Rs = −0.562. We also analysed the genome-wide association between expression up-regulation by decitabine (DAC) and DNA methylation. A correlation between tag numbers and fold-up regulation by DAC was significant, although correlation coefficient was not that high (Rs=0.239). This result means DAC could affect gene expression by a mechanism not depending on demethylation effect. We have also performed the same analysis with genes in which there are no CpG islands. Without CpG islands, only almost no correlations were found between mRNA expression levels (Rs=−0.087), fold up-regulation by DAC (Rs=0.067) and tag numbers. Interestingly, in genes without promoter methylation, gene ontology (GO) analysis demonstrated that genes related to immune response were significantly up-regulated by DAC. This could reflect the direct cytotoxic effect of DAC or some other mechanism. We are now performing the array CGH analysis in primary MM samples to assess the association between methylome status and aneuploidy status in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-175. doi:10.1158/1538-7445.AM2011-LB-175