Jan B. Egan

Clonal competition with alternating dominance in multiple myeloma

Emerging evidence indicates that tumors can follow several evolutionary paths over a patients disease course. With the use of serial genomic analysis of samples collected at different points during the disease course of 28 patients with multiple myeloma, we found that the genomes of standard-risk patients show few changes over time, whereas those of cytogenetically high-risk patients show significantly more changes over time. The results indicate the existence of 3 temporal tumor types, which can either be genetically stable, linearly evolving, or heterogeneous clonal mixtures with shifting predominant clones. A detailed analysis of one high-risk patient sampled at 7 time points over the entire disease course identified 2 competing subclones that alternate in a back and forth manner for dominance with therapy until one clone underwent a dramatic linear evolution. With the use of the Vk*MYC genetically engineered mouse model of myeloma we modeled this competition between subclones for predominance occurring spontaneously and with therapeutic selection.

Whole-genome sequencing of multiple myeloma from diagnosis to plasma cell leukemia reveals genomic initiating events, evolution, and clonal tides

The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Genome-Wide Analysis Uncovers Novel Recurrent Alterations in Primary Central Nervous System Lymphomas

Purpose: Primary central nervous system lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma confined to the central nervous system. Whether there is a PCNSL-specific genomic signature and, if so, how it differs from systemic diffuse large B-cell lymphoma (DLBCL) is uncertain. Experimental Design: We performed a comprehensive genomic study of tumor samples from 19 immunocompetent PCNSL patients. Testing comprised array-comparative genomic hybridization and whole exome sequencing. Results: Biallelic inactivation of TOX and PRKCD was recurrently found in PCNSL but not in systemic DLBCL, suggesting a specific role in PCNSL pathogenesis. In addition, we found a high prevalence of MYD88 mutations (79%) and CDKN2A biallelic loss (60%). Several genes recurrently affected in PCNSL were common with systemic DLBCL, including loss of TNFAIP3, PRDM1, GNA13, TMEM30A, TBL1XR1, B2M, CD58, activating mutations of CD79B, CARD11, and translocations IgH-BCL6. Overall, B-cell receptor/Toll-like receptor/NF-κB pathways were altered in >90% of PNCSL, highlighting its value for targeted therapeutic approaches. Furthermore, integrated analysis showed enrichment of pathways associated with immune response, proliferation, apoptosis, and lymphocyte differentiation. Conclusions: In summary, genome-wide analysis uncovered novel recurrent alterations, including TOX and PRKCD, helping to differentiate PCNSL from systemic DLBCL and related lymphomas. Clin Cancer Res; 21(17); 3986–94. ©2015 AACR.

Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276

RNA interference screening identified XPO1 (exportin 1) among the 55 most vulnerable targets in multiple myeloma (MM). XPO1 encodes CRM1, a nuclear export protein. XPO1 expression increases with MM disease progression. Patients with MM have a higher expression of XPO1 compared with normal plasma cells (P<0.04) and to patients with monoclonal gammopathy of undetermined significance/smoldering MM (P<0.0001). The highest XPO1 level was found in human MM cell lines (HMCLs). A selective inhibitor of nuclear export compound KPT-276 specifically and irreversibly inhibits the nuclear export function of XPO1. The viability of 12 HMCLs treated with KTP-276 was significantly reduced. KPT-276 also actively induced apoptosis in primary MM patient samples. In gene expression analyses, two genes of probable relevance were dysregulated by KPT-276: cell division cycle 25 homolog A (CDC25A) and bromodomain-containing protein 4 (BRD4), both of which are associated with c-MYC pathway. Western blotting and reverse transcription-PCR confirm that c-MYC, CDC25A and BRD4 are all downregulated after treatment with KPT-276. KPT-276 reduced monoclonal spikes in the Vk*MYC transgenic MM mouse model, and inhibited tumor growth in a xenograft MM mouse model. A phase I clinical trial of an analog of KPT-276 is ongoing in hematological malignancies including MM.

Extramedullary myeloma whole genome sequencing reveals novel mutations in Cereblon, proteasome subunit G2 and the glucocorticoid receptor in multi drug resistant disease.

Extramedullary disease (EMD) in Multiple Myeloma (MM) is characterized by the detection of monoclonal plasma cells outside the bone marrow niche, and is frequently associated with poor prognosis. Here we describe novel genomic events leading to drug refractory disease in a heavily pretreated 37-year-old IgG-kappa MM patient presenting with progressive, multi-drug refractory EMD. For the first time we report an acquired truncating mutation of Cereblon (CRBN) as well as point mutations in proteasome subunit G2 and the glucocorticoid receptor as an explanation for drug resistance. Initial myeloma treatment for the patient occurred over multiple years and included the immunomodulatory drugs (IMiDs) thalidomide and lenalidomide, the proteasome inhibitor bortezomib, cortiosteroids, radiation, one autologous and two allogeneic transplantations. She experienced extramedullary relapse, presenting as an extensive neck mass and smaller soft tissue nodules in the upper left triceps. The most recent therapy immediately prior to genomic sequencing was hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone) incorporating alkylating agent cyclophosphamide with transient minor response. The patient was then enrolled in a pilot study utilizing next generation sequencing (NGS) to identify novel markers and potential therapeutic targets. Samples were acquired with patient consent in compliance with Mayo Clinic Institutional Review Board. For this study we completed array comparative genomic hybridization, whole exome, whole genome (insert = 1 kb) and RNA sequencing (RNASeq) of a biopsy taken from the neck mass to thoroughly interrogate the tumour genome of this patient. The presence of mutations of interest was evaluated by capillary sequencing in an expanded cohort of 25 CD138+ MM samples with low CRBN expression. The neck mass pathology confirmed sheets of atypical plasma cells, kappa light chain restriction, CD138+, CD20− and CD45−. Array comparative genomic hybridization revealed multiple copy number abnormalities, most notably del1(p13.2–34.2), monosomy 13 and monosomy X. Therapy subsequent to biopsy for genome sequencing was with pomalidomide and dexamethasone without response. Unfortunately, the patient succumbed to her disease in less than the 12 weeks required at the time for sequencing and data analysis. Sequencing revealed a highly disturbed genome (Figure 1) consisting of 4 somatic insertions/deletions, 38 intra-chromosomal rearrangements, and 35 translocations, including the high risk marker and initiating tumour event t(14;16). Furthermore, 271 nonsynonymous, somatic point mutations were detected in genes including KRAS, PIK3CA, ATM, and NFKB2 (Table I). Importantly, a Q99* truncating mutation as well as a R283K point mutation were observed in CRBN, that we recently demonstrated as essential for the anti-MM action of IMiDs (Zhu, et al 2011). To our knowledge this is the first documented mutation of Cereblon in a primary myeloma sample. Additional sequencing of CRBN in the expanded cohort of 25 patients revealed a synonymous mutation in only one sample. Figure 1 Circos plot depicting genome wide somatic variants, rearrangements and copy number changes derived from next generation sequencing. Numbers with circles around them indicate the following: 1) somatic single nucleotide variation (SNV), 2) location of SNV ... Table I Summary of clinically relevant single nucleotide variations We also observed in the patient biopsy a potentially clinically relevant nonsynonymous point mutation in proteasome assembly chaperone 2, PSMG2 (E171K). PSMG2 is a proteasome assembly protein involved in mammalian 20S proteasome maturation (Hirano, et al 2005). Mutations in proteasome assembly components contribute to proteasome inhibitor resistance (Keats, et al 2007), possibly explaining this patient’s bortezomib-refractory disease. Capillary sequencing of PSMG2 in our expanded cohort revealed no mutations, although exonic deletion of PSMG2 has also been reported in myeloma (Walker, et al 2012). The last nonsynonymous point mutation associated with drug resistance was identified in NR3C1 (G369A), a glucocorticoid receptor. Mutation of NR3C1 has been associated with resistance to steroid therapy (Bray and Cotton 2003), which this patient received and proved refractory. No NR3C1 mutations were identified in our expanded cohort and none have been previously reported in other myeloma genomes (Chapman, et al 2011, Walker, et al 2012). Mutations in NR3C1 have however been described in the glucocorticoid resistant MM.1R cell line (Moalli, et al 1992). Patients with low NR3C1 expression levels who received thalidomide demonstrated better progression-free survival and overall survival than those with low NR3C1 who did not receive thalidomide (Heuck, et al 2012). While these mutations suggest causality of drug-refractory disease, they do not identify pathways that can be exploited with targeted therapies. Additional mutations were observed in pathways for which targeted therapies are available. This patient had mutations in KRAS (G12C) and in ATM (T1985I), both of which affect the signalling of MEK downstream, thus making MEK a therapeutic target of interest in this patient. While there are no approved MEK inhibitors available for MM treatment, more than 100 trials are currently investigating MEK inhibitors, of which three of these trials are being conducted in MM patients (www.clinicaltrials.gov). The patient tumour also contained canonical, activating mutations in PIK3CA (E542K). Interestingly, one study demonstrated that 64% of PIK3CA mutations occur in exon 9, where codon 542 is located. Moreover, 19% of patients with PIK3CA mutations also presented with KRAS mutations, of which 9% are G12C (Janku, et al 2012), found in our patient. The PI3K pathway is vitally important as it regulates downstream targets, such as AKT and MTOR, which are responsible for cell proliferation, growth, survival and metastasis (Bartholomeusz and Gonzalez-Angulo 2012). In addition, a number of clinical trials are currently investigating PIK3 inhibitors (www.clinicaltrials.gov). In summary, this is the first description of CRBN mutations in a primary myeloma sample and furthermore of a “triple negative” MM patient possessing mutations probably contributing to resistance to all three major drug classes utilized in MM therapy. These mutations were not replicated in our validation cohort of 25 patients with low level CRBN expression and functional data have not yet been obtained, thus further investigation is necessary to better understand the mutation frequency and the functional significance of mutation in these genes. In summary, our approach utilizing comprehensive next generation sequencing not only identified mutations suggestive of the patient’s refractory disease, but also revealed unforeseen therapeutic options highlighting the importance of this technology in advancing individualized medicine.

Lessons from next-generation sequencing analysis in hematological malignancies

Next-generation sequencing has led to a revolution in the study of hematological malignancies with a substantial number of publications and discoveries in the last few years. Significant discoveries associated with disease diagnosis, risk stratification, clonal evolution and therapeutic intervention have been generated by this powerful technology. As part of the post-genomic era, sequencing analysis will likely become part of routine clinical testing and the challenge will ultimately be successfully transitioning from gene discovery to preventive and therapeutic intervention as part of individualized medicine strategies. In this report, we review recent advances in the understanding of hematological malignancies derived through genome-wide sequence analysis.

Targeted sequencing using a 47 gene multiple myeloma mutation panel (M3P) in -17p high risk disease

We constructed a multiple myeloma (MM)‐specific gene panel for targeted sequencing and investigated 72 untreated high‐risk (del17p) MM patients. Mutations were identified in 78% of the patients. While the majority of studied genes were mutated at similar frequency to published literature, the prevalence of TP53 mutation was increased (28%) and no mutations were found in FAM46C. This study provides a comprehensive insight into the mutational landscape of del17p high‐risk MM. Additionally, our work demonstrates the practical use of a customized sequencing panel, as an easy, cheap and fast approach to characterize the mutational profile of MM.

Clinical Implementation of Integrated Genomic Profiling in Patients with Advanced Cancers

DNA focused panel sequencing has been rapidly adopted to assess therapeutic targets in advanced/refractory cancer. Integrated Genomic Profiling (IGP) utilising DNA/RNA with tumour/normal comparisons in a Clinical Laboratory Improvement Amendments (CLIA) compliant setting enables a single assay to provide: therapeutic target prioritisation, novel target discovery/application and comprehensive germline assessment. A prospective study in 35 advanced/refractory cancer patients was conducted using CLIA-compliant IGP. Feasibility was assessed by estimating time to results (TTR), prioritising/assigning putative therapeutic targets, assessing drug access, ascertaining germline alterations, and assessing patient preferences/perspectives on data use/reporting. Therapeutic targets were identified using biointelligence/pathway analyses and interpreted by a Genomic Tumour Board. Seventy-five percent of cases harboured 1–3 therapeutically targetable mutations/case (median 79 mutations of potential functional significance/case). Median time to CLIA-validated results was 116 days with CLIA-validation of targets achieved in 21/22 patients. IGP directed treatment was instituted in 13 patients utilising on/off label FDA approved drugs (n = 9), clinical trials (n = 3) and single patient IND (n = 1). Preliminary clinical efficacy was noted in five patients (two partial response, three stable disease). Although barriers to broader application exist, including the need for wider availability of therapies, IGP in a CLIA-framework is feasible and valuable in selection/prioritisation of anti-cancer therapeutic targets.

Simultaneous characterization of somatic events and HPV-18 integration in a metastatic cervical carcinoma patient using DNA and RNA sequencing.

Objective Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient’s cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. Materials and Methods We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient’s lung metastasis. Results We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient’s tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. Conclusions We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis.