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

Introduction

Multiple myeloma (MM) is characterized by malignant plasma cell proliferation in the bone marrow (BM) associated with monoclonal protein in the serum and/or urine.^1,2 Hematopoietic stem cell transplantation and novel agents such as bortezomib, thalidomide, and lenalidomide have improved the survival of MM patients.^3,4 However, most patients eventually relapse even after the achievement of a complete therapeutic response. Improvements in molecular profiling technologies have provided new insight into the basic molecular events underlying the development of MM as well as the mechanisms of anticancer drug resistance. The transition from the long-established one-size-fits-all approach to new strategies based on individual genetic profiles provides an opportunity to transform current diagnostics into individual prognostic and even predictive classifications.

In MM, there are two distinct genetic subtypes based on copy number alterations and translocations. Approximately half of all MM cases are hyperdiploid, which is characterized by multiple trisomies of chromosomes 3, 5, 7, 9 11, 15, 19, and 21 and a lower prevalence of primary translocations involving the immunoglobulin heavy chain (IgH) locus at 14q32.^5,6 The remaining cases form the nonhyperdiploid group, and chromosomes 8, 13, 14, and 16 are frequently lost. Nonhyperdiploid myeloma is strongly associated with translocations of IgH alleles with various partner chromosomes. Copy number alterations in chromosomal regions, such as 1q, 6q, 8p, and 16q, occur in both subtypes. Overall, nonhyperdiploid MM is associated with worse survival compared with hyperdiploid MM.

Methods for determining DNA content and, ultimately, ploidy in MM include conventional cytogenetics, fluorescence in situ hybridization (FISH), comparative genomic hybridization, and, recently, massively parallel whole genome sequencing. Because unique mutations have been observed in individual human cancer samples, the identification and characterization of the molecular alterations of individual cancer patients is a critical step toward the development of more effective personalized therapies. For example, next-generation sequencing (NGS) technologies have revolutionized cancer genomics research by providing a comprehensive method of detecting genomic alterations associated with somatic cancer.^7–9 In this study, we sequenced all exons of 409 cancer-related genes in matched tumor and normal DNA samples from a multidrug-resistant myeloma patient using a next-generation semiconductor sequencing protocol.

Case report

A 69-year-old male presented in October 2010 with back pain. Physical examination and magnetic resonance imaging revealed large focal lesions in the fourth thoracic vertebra (Figure 1A) and first lumbar vertebra (Figure 1B). Laboratory evaluation revealed a white blood cell count of 2.5×10³/μL with no atypical cells, a red blood cell count of 3.67×10⁶/μL, a hemoglobin level of 11.6 g/dL, and a platelet count of 122×10⁶/μL. The serum total protein level was 10.7 g/dL, the albumin level was 3.4 g/dL, the serum β2 microglobulin level was 4.2 mg/dL, and the serum calcium level was 8.9 mg/dL. The concentrations of IgG, IgA, and IgM were 6,284, 34, and 25 mg/dL, respectively. The monoclonal protein IgG was increased, and serum immunofixation revealed the production of IgG with λ light-chain restriction (data not shown). The proliferation of plasma cells (more than 10% among all nucleated cells) was also detected in BM aspirates. When BM biopsy was performed, the infiltration of plasma cells expressing IgG λ monoclonal protein was identified by pathological investigation, and the patient was diagnosed with MM (Stage II according to the International Staging System). Chromosome analysis at this time using conventional Giemsa banding of BM-derived metaphase spreads revealed a normal karyotype (46, XX) in all analyzed cells. The patient was then treated with one cycle of high-dose dexamethasone, followed by three cycles of bortezomib plus dexamethasone. He achieved complete response according to the International Myeloma Working Group uniform response criteria. His symptoms were also significantly improved.

Figure 1 Sagittal T1-weighted magnetic resonance images depict focuses of plasma cell infiltration and pathologic fractures in the T4 (A) and L1 (B) vertebrae. Note: Red arrows indicate large focal lesions in the vertebrae.

In August 2012, the serum concentrations of IgG and free light chain (FLC) gradually increased, suggesting the worsening of his MM. His complete blood count was as follows: 1.8×10³/μL white blood cells, 3.61×10⁶/μL red blood cells, 11.6 g/dL hemoglobin, and 55×10⁶/μL platelets. BM analysis showed complex aberrations often observed in this patient (Figures 2A and 2B) and an elevated plasma cell percentage (59.8%). His karyotype was 39, XY, del(1)(p22p36), −3, −6, der(8)t(6:8)(p11.1:p23), −10, t(11:14)(q13:q32), −12, add(13)(q22), add(16)(q22), −17, add(18)(p11), −19, −20, +mar [4]/46, XY [20]. The chromosomal translocation t(11:14)(q13:q32), which generates the IgH/CCND1 fusion gene, was also identified in our case by FISH (Figure 2C).

Figure 2 Evaluation of a bone marrow aspirate. Notes: (A) Conventional karyotyping of metaphase cells from BM aspirate was performed using the G-banding technique. Complex cytogenetic aberrancies including loss of chromosomes, and additional uncharacterized materials at chromosome 8 (1) and 13 (2) are shown here. In addition, a dicentric translocation involving chromosome 11 and 14 (3) were also observed. (B) Visualization of CNVs over the entire genome in the karyotype view. The decreased copy number is indicated in red with increased copy number indicated in blue. (C) Interphase FISH studies were performed on BM aspirates using IgH/CCND1 dual color dual fusion probe (Vysis Inc., Des Plaines, IL, USA). The cell showed one orange (normal CCND1), one green (normal IgH), and two yellow signals (arrows), indicating typical t(11;14) rearrangement. Abbreviations: CNV, copy number variant; FISH, fluorescence in situ hybridization; BM, bone marrow.

The patient was started on a bortezomib, cyclophosphamide, and dexamethasone regimen. No serious complications occurred during the course of the treatment, and a partial response was observed with a decrease in the serum FLC value. After seven cycles of this regimen, however, his condition progressively deteriorated, with increases in serum lambda immunoglobulin light chain and LDH, a deterioration of renal function, and the appearance of circulating plasma cells in the peripheral blood (up to 5% of the total peripheral leukocyte population). He was admitted for combination chemotherapy with combination chemotherapy with bortezomib, cyclophosphamide, lenalidomide, and dexamethasone, but his response was poor (Figure 3), and an increase in myeloma cells was detected by BM biopsy. Unfortunately, the patient has since passed away, and his family did not choose to perform postmortem examination.

Figure 3 Clinical course of the patient. Abbreviations: HD, high dose; DEX, dexamethsone; Cy, cyclophosphamide; FLC, free light chain; IgG, Immunoglobulin G.

The patient provided consent for use of his medical record and samples for clinical and research purposes, and the examination was performed in accordance with the Declaration of Helsinki. The sequence study was approved by the Institutional Review Boards of Sapporo Medical University. Retrospectively, to better understand the molecular pathogenesis in this patient, we sequenced 409 cancer-related genes in matched tumor and nontumor DNA samples at relapse in August 2012 using an Ion Torrent PGM (Life Technologies, Carlsbad, CA, USA). DNA was extracted from magnetic bead–enriched BM CD138 positive tumor cells from the patient, and CD138 negative cells were used as matched nontumor cells. DNA (40 ng) was used for multiplex polymerase chain reaction (PCR) amplification with an Ion Ampliseq Comprehensive Cancer Panel (Life Technologies), enabling the targeted coverage of all exons of 409 cancer-related genes frequently cited and mutated (covered regions =95.4% of total). The 15,992 amplicons obtained represented more than 1.2 Mb of target sequence. Library preparation and sequencing with an Ion Torrent PGM was performed as previously described.⁹ The mean read depths were 125× (tumor) and 152× (normal). Alignment to the human genome build 19 and variant calling were performed by Ion Reporter Software 4.0. Mutations were also validated by conventional Sanger sequencing. We identified eight nonsynonymous somatic mutations (6.49 mutations/Mb; Table 1). We included missense mutations in seven genes (SYNE1, IKBKB, ERBB3, MYH11, CYLD, TP53, and CDH2) and a frameshift mutation in EGFR. Changes in relative copy number were also assessed from the sequencing data, and we identified 133 copy number variant (CNV) regions, including 87 gain and 46 loss regions (Figure 2B and Table S1). Importantly, we found a gain in the copy number of CCND1, a gene encoding cyclin D1. To check the contamination of tumor cells in the CD138 negative subset, we compared CD138 negative DNA of this patient and peripheral blood DNA from two healthy donors. We found single nucleotide variants in the CD138 negative DNA of this patient; however, all variants had previously been reported in the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) (Tables 2 and 3). Therefore, we can rule out tumor cell contamination in the CD138 negative subset.

Table 1 Somatic mutations identified in our case Notes: List of total coverage, variant read coverage, and variant frequencies of somatic mutations identified in DNA isolated from BM aspirates of this case. BM mononuclear cells were separated using Ficoll–Paque density sedimentation, and plasma cells were purified by positive selection with anti-CD138 magnetic-activated cell separation microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Somatic mutations were detected using statistical approaches in tumor (CD138 positive) and normal (CD138 negative) samples from the Ion Reporter software 4.0 tumor-normal workflow. A sequencing coverage of 25× and a minimum variant frequency of 15% of the total number of distinct tags were used as cutoffs. Mutations were called if they occurred in <1% of reads in the normal control, and were absent from dbSNP and the 1,000 Genomes Project database. Abbreviation: BM, bone marrow.

Table 2 Nucleotide variants identified in CD138-negative bone marrow aspirates from our case-1 Notes: DNA was extracted from CD138-negative BM aspirates of this case and peripheral blood of healthy donor-1 (TT) using the QIAamp DNA Mini kit (Qiagen GmbH, Hilden, Germany) following manufacturer’s instructions. DNA (40 ng) was used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel (Life Technologies, Carlsbad, CA, USA), enabling the targeted coverage of all exons of 409 cancer-related genes in a 4-tube reaction (covered regions =95.4% of total). Nucleotide variants on the CD138-negative BM aspirates of this case were detected using the peripheral blood of healthy donor-1 as a normal control. A sequencing coverage of 25× and a minimum variant frequency of 15% of the total number of distinct tags were used as cutoffs. adbSNP ID number. Abbreviations: BM, bone marrow; PCR, polymerase chain reaction.

Table 3 Nucleotide variants identified in CD138 negative bone marrow aspirates from our case-2 Notes: DNA was extracted from CD138 negative BM aspirates of this case and peripheral blood of healthy donor 2 (Y.S.) using the QIAamp DNA Mini kit (Qiagen GmbH, Hilden, Germany) following manufacturer’s instructions. DNA (40 ng) was used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel (Life Technologies, Carlsbad, CA, USA), enabling the targeted coverage of all exons of 409 cancer-related genes in a four tube reaction (covered regions =95.4% of total). Nucleotide variants on the CD138-negative BM aspirates of this case were detected using the peripheral blood of healthy donor-2 as a normal control. A sequencing coverage of 25× and a minimum variant frequency of 15% of the total number of distinct tags were used as cutoffs. adbSNP ID number. Abbreviations: BM, bone marrow; PCR, polymerase chain reaction.

Discussion

MM is a plasma cell malignancy characterized by a heterogeneous clinical course. Treatments for MM have remarkably improved in recent years, due in part to the introduction of novel therapies such as bortezomib, thalidomide, and lenalidomide. Despite these advancements, the prognosis of patients with relapse and refractory MM remains poor, and novel therapies are needed. Alternatively, the identification of novel targets or signaling pathways regulating myeloma cell proliferation would improve the clinical outcome and survival of refractory MM patients. Several pathways related to drug resistance and cell survival, such as Notch1, Akt, and NF-κB, are activated to protect MM cells from death.^10–12 Here, we describe the characterization of genetic abnormalities found in myeloma cells in a patient with refractory MM.

In nonhyperdiploid MM, the IgH gene (14q32) commonly fuses with FGFR3 (4p16), MMSET (4p16.3), CCND3 (6p21), CCND1 (11q13), and MAF (16q23), resulting in the direct or indirect dysregulation of cyclin D.¹³ Among the nonhyperdiploid myelomas, the hypodiploid subtype (≦44 chromosomes) has the most aggressive clinical phenotype, but the genetic differences between the groups have not been completely defined. Cytogenetic analysis revealed that this patient had a hypodiploid karyotype with 39 chromosomes and complex chromosomal abnormalities, including t(11:14)(q13:q32). CCND1 expression is generally related to copy number aberrations. Although we did not analyze CCND1 mRNA expression, CNV analysis revealed a gain of 11q13–q21, suggesting the presence of cyclin D1 dysregulation.

Several NGS platforms are available for the sequencing of targeted genomic regions to analyze a variety of disease-associated changes, such as point mutations, insertions, deletions, and CNVs. CNV analysis of the sequencing data revealed that this patient had diverse DNA copy number alterations, including large and regional gains and losses (Figure 2B and Table S1). Additionally, we detected eight somatic mutations among 409 cancer-related genes (Table 1). We considered gene sets based on existing insights into the biology of this MM patient. It has been proposed that activation of the NF-kB pathway is important in the pathogenesis of MM, as well as in resistance to chemotherapy. We observed two point mutations and three CNVs affecting NF-κB pathway genes, including IKBKB, CYLD, IKBKE, CD79B, and SYK (Tables 1 and S1). Although additional experiments are required to establish the functional significance of genetic alterations of these genes in MM cells, NF-κB pathway activation may be involved in the molecular pathogenesis of this patient’s disease.

Alterations in the tumor suppressor retinoblastoma (RB) and p53 or their respective pathways are frequently observed in human cancers.¹⁴ In MM, a deletion or mutation of p53 (17p13) or RB1 (13q14.2) is considered to be predictive of poor prognosis. We found a monoallelic chromosome 17 deletion and a missense mutation in the DNA-binding domain of p53 (Arg158Gly), suggesting the abrogation of p53 transcription. Moreover, the deletion of the CNV in 13q14.2 was detected in this patient, resulting in the inactivation of two key regulators of the cell cycle, RB1 and p53.

A recent study has described an increased rate of mutations in receptor tyrosine kinases (RTKs) and their associated signaling effectors, pointing to a more potent role of this pathway in MM than was previously appreciated.¹⁵ We found two mutations in RTKs, including a missense mutation in ERBB3 and a truncating mutation in EGFR, and have suggested a role of aberrant RTK signaling in the development or progression of MM in this patient. In addition, few studies have addressed the functional roles of the remaining three mutated genes (SYNE1, MYH11, and CDH2) in myeloma, and further investigations are required. SYNE1 is frequently silenced by DNA methylation in primary cancers of the colon and lung,^16,17 suggesting that a loss of SYNE1 function may be a genetic event that promotes tumor progression. MYH11 is a member of the myosin family, and inversion at the MYH11 locus is found in acute myeloid leukemia.¹⁸ Additionally, MYH11 mutations have been shown to occur in human colorectal cancers with microsatellite instability.¹⁹ N-cadherin, encoded by the CDH2 gene, is a transmembrane protein and plays an important role in cell adhesion. Recently, circulating N-cadherin levels was reported to be a negative prognostic factor in patients with MM.²⁰ In this study, we performed comprehensive genomic analyses using PCR target enrichment and semiconductor-based sequencing of matched tumor and normal DNA samples obtained from an individual with refractory MM. We detected several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of this patient. Although its value should be further confirmed in larger samples, targeted NGS is considered a valuable tool for high-throughput genetic testing in clinical research.

Acknowledgments

This work was supported in part by the Scientific Support Programs for Cancer Research Grant-in-Aid Scientific Research on Innovative Areas Ministry of Education, Culture, Sports, Science, and Technology. This is the same research abstract number 4424 AACR Annual Meeting Philadelphia 2015.

Disclosure

The authors report no conflicts of interest in this work.

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Supplementary material

Table S1