Real-Time Genomic Profiling Identifies Novel Mutations and Improved Therapy for Histiocytoses

Abstract BACKGROUND: The histiocytic disorders include several heterogeneous diseases including Langerhans cell histiocytosis (LCH,) juvenile xanthogranuloma, Erdheim-Chester disease (ECD,) and Rosai-Dorfman disease. These conditions have variable clinical courses and can be refractory to contemporary therapy, resulting in end-organ damage or even death. The etiopathogenesis of LCH and the other histiocytoses remained unclear for decades, until the identification of recurring BRAF V600E mutations, and more recently mutations in the downstream gene MAP2K1 (encoding the enzyme MEK) in LCH and ECD. Given the high risk of recurrence and the unpredictable response to therapy in some patients, we sought to characterize the genomic landscape of histiocytic lesions in patients in real-time. Our principal goal was to select alternative treatments for patients with inadequate response to standard therapies. As a secondary goal, we aimed to further characterize the biological effects of MAP2K1 mutations found in LCH and ECD, and determine their susceptibility to targeted therapies. METHODS AND RESULTS: We used a hybrid capture-based sequencing platform to molecularly profile eighty-five patient samples from patients with one of the above diagnoses. Fifteen patient samples (18%) harbored the BRAF V600E point mutation, and four LCH patients carried a novel 5 or 6 amino-acid in-frame deletion (indel) in BRAF (N486_P490del or N486_T491>K.) Eleven patient samples (13%) harbored activating mutations in MAP2K1. Additional recurrently altered genes included NRAS, KRAS and CDKN2A/B. Transcriptomic profiling also identified several patients with recurrent ALK gene fusions, previously described in other malignancies and recently also identified in histiocytosis not-otherwise-specified. One patient with multisystem LCH with CNS involvement was found to have a BRAF indel. She declined systemic chemotherapy, but agreed to treatment with a targeted agent. Based on the likelihood of resistance to BRAF V600E-specific inhibitors, we started treatment with the MEK inhibitor Trametinib resulting in resolution of disease-associated lymphadenopathy within days, and improvement of CNS symptoms as well. She remains in remission 4 months after the initiation of treatment. In two children, multi-system refractory LCH progressed to secondary HLH (hemophagocytic lymphohistiocytosis). Both demonstrated the presence of BRAF-V600E and their disease promptly responded to the BRAF inhibitor Dabrafenib. We then characterized the biological behavior of the MAP2K1 mutations using retroviral transduction in order to stably express these mutations in NIH/3T3 and BaF/3 cells. We demonstrate that these mutations all result in constitutive activation at baseline, as evidenced by increased phosphorylation of the target ERK. These mutant forms of MAP2K1 also express sustained activation of ERK in response to EGF stimulation. Additionally, we tested clinically available MEK inhibitors against mutant forms of MAP2K1, and show that all result in a dose-dependent decrease in phospho-ERK levels in vitro, supporting our hypothesis that MEK inhibition is a valid therapeutic approach in the histiocytic neoplasms. Finally, we demonstrate with an animal model that MAP2K1 is sufficient to induce disease. Using the cre-lox recombinase system in transgenic mice, we selectively express an activated form of MEK in CD11c-positive cells, which is largely restricted to the dendritic cell/macrophage lineage. These mice developed normally, but by a median of 17 weeks of age, mice became moribund and on necropsy exhibited hepatosplenomegaly with extensive infiltration of spleen, liver and lungs with CD68+ macrophages. DISCUSSION: Genomic profiling identified mutations in majority of patients, including a novel BRAF indel. We further show that these mutations result in activation of the MAP kinase pathway, and that activated MAP2K1 is capable of transforming hematopoietic cells resulting in a multisystem histiocytic disorder in mice. Finally, we demonstrate that available MEK inhibitors efficiently block disease-associated mutations. We propose that all patients with histiocytic neoplasms undergo comprehensive genomic profiling in order to identify potential causal mutations, and clinical trials for histiocytoses include MEK inhibitors in relapsed/refractory disease or even as upfront therapy. Disclosures Ali: Foundation Medicine: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Stevens:Foundation Medicine Inc.: Employment, Equity Ownership. Ross:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership.

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Molecular Analysis of RAS-RAF Tyrosine-Kinase Signaling Pathway Alterations in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.2876.2876 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2876-2876

Author(s):

Valentina Artusi ◽

Claudia Haferlach ◽

Alexander Kohlmann ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

...

Keyword(s):

Multiple Myeloma ◽

Signaling Pathway ◽

Mutation Rate ◽

Plasma Cell ◽

Deep Sequencing ◽

Response To Therapy ◽

Braf V600e ◽

Employment Equity ◽

Available N ◽

Equity Ownership

Abstract Abstract 2876 Multiple myeloma (MM) is a malignancy of abnormal plasma cells and a correlation with poor outcome has been described for immunoglobulin heavy-chain (IgH) translocations, deletions of 13q or 17p. Thus far, a convincing relationship between specific mutations, disease onset and progression has not been fully established. Aberrant impairment of important signaling pathways can drive oncogenesis and contribute to MM development. We therefore chose to study NRAS, KRAS and BRAF, three members of the RAS-RAF signaling pathway, as well as TP53 and CCND1, two fundamental genes in cell cycle control. We here investigated 41 MM cases to further elucidate molecular mechanisms underlying this disease. Bone marrow (n=35) or, in case of plasma cell leukemia, peripheral blood (n=6) specimens were collected between 12/2006 and 6/2011 and molecular analyses using a deep-sequencing assay (454, Branford, CT) in combination with the 48.48 Access Array technology (Fluidigm, South San Francisco, CA) were performed on mononuclear cells after Ficoll enrichment or magnetic activated plasma cell sorting using anti-CD138 beads (RoboSep, STEMCELL Technologies SARL, France). The cohort included 16 female and 25 male patients at first diagnosis, with a median age of 63 years (range: 33–84 years). Based on fluorescence in situ hybridization (FISH), the cohort was characterized as follows: IgH rearrangements were detected in 54.3% of patients (19/35: n=6 with t(4;14), n=9 with t(11;14), n=3 with t(14;16), n=1 other; data not available: n=5). A deletion 13q14 was present in 64.9% of patients (24/37; data not available: n=4). Trisomy 3 was detected in 48.0% of patients (12/25; data not available: n=16), trisomy 9 was detected in 50.0% of patients (12/24; data not available: n=17), trisomy 11 was detected in 46.4% of patients (13/28; data not available: n=13), and trisomy 15 was detected in 56.2% of patients (9/16; data not available: n=25), respectively. Interestingly, in all cases where FISH data was available (n=36), at least 1 aberration was detectable. Further, we studied the occurrence of somatic mutations in NRAS, KRAS, BRAF, TP53 and CCND1. In our cohort, we detected an overall mutation rate within the RAS pathway of 41.4% (17/41), in line with a recent report (Chapman et al., Nature, 2011). KRAS was the most frequently mutated gene with 21.9% of cases with mutations (9/41 patients), followed by NRAS (19.5%; 8/41 patients). Recently, BRAF V600E mutations have gained clinical interest since they became manageable by targeted treatment in melanoma. Interestingly, Chapman et al. discovered a mutational rate of 4% by sequencing of 161 MM patients (Nature, 2011). Even if BRAF is not a frequently mutated gene in MM, it justifies upfront diagnostic screening since these patients may benefit from new treatments. In our cohort, 2/41 patients harbored BRAF V600E mutations. Moreover, because of their involvement in the same signaling pathway, we also noticed that mutations affecting NRAS, KRAS or BRAF were predominantly mutually exclusive, except for one patient who concomitantly harbored a BRAF and a NRAS mutation. Additionally, we observed an overall molecular TP53 mutation rate of 12.2% (5/41 patients). In these 5 patients, in total 7 mutations (5 missense substitutions; 2 frame-shift mutations) were detected. 1/4 cases concomitantly harbored a deletion of the TP53 gene, as detected by FISH. Finally, we were interested in the analysis of CCND1, which is located on 11q13, a region frequently involved in chromosomal translocations (9/20 IgH translocated cases in our cohort). Here, we were able to detect 2/41 (4.8%) CCND1 mutated cases. Concerning the correlation between IgH rearrangements and molecular aberrations we observed that 21.9% (9/35; n=5 IgH status not available) of patients that were IgH rearranged, concomitantly carried a TP53 or RAS-RAF mutation. In more detail, 2/5 TP53 mutated patients and 50.0% (8/16) RAS-RAF mutated cases concomitantly harbored an IgH rearrangement. Taken together, MM patients are currently stratified in part based on cytogenetic/FISH classification. We demonstrated that deep-sequencing analyses support an additional molecular characterization. In our cohort, all patients carried mutations detected by FISH and 23/41 (56.1%) carried a molecular mutation. Future clinical studies need to confirm the frequencies of these mutations as well as their association with response to therapy and outcome. Disclosures: Artusi: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment.

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Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms

Blood ◽

10.1182/blood.v126.23.481.481 ◽

2015 ◽

Vol 126 (23) ◽

pp. 481-481

Author(s):

Benjamin Heath Durham ◽

Eli L. Diamond ◽

Julien Haroche ◽

Zhan Yao ◽

Jing Ma ◽

...

Keyword(s):

Mapk Pathway ◽

Single Agent ◽

Braf V600e ◽

Rna Seq ◽

Wild Type ◽

Employment Equity ◽

Mek Inhibitors ◽

Whole Exome ◽

Recurrent Mutations ◽

Equity Ownership

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of the BRAF V600E mutation in ~50% of patients with LCH and the non-LCH Erdheim-Chester Disease (ECD) provided the first molecular target in these patients and novel insights into the pathogenesis of these disorders. However, recurrent mutations in the majority of the ~50% of BRAF V600E-wild type patients with non-LCH are unknown. Moreover, recurrent mutations outside of the MAP kinase pathway are undefined throughout histiocytic neoplasms. To address these issues, we performed whole exome sequencing (WES) of frozen biopsies from 24 patients with LCH (n=10) or ECD (n=14) paired with peripheral blood mononuclear cells. 13/24 patients also underwent RNA sequencing (RNA-seq). All mutations in activating kinases were validated by droplet-digital PCR, while targeted-capture next-generation sequencing validated all others. Both adult (n=18; n=2 with LCH) and pediatric cases (n=9; n=8 with LCH) were included. Using combined WES/RNA-seq, activating kinase alterations were identified in 100% of patients. In LCH, 60% and 40% had BRAF V600E and MAP2K1 mutations, respectively. In non-LCH 51%, 14%, 14%, and 7% were BRAFV600E, ARAF, MAP2K1, and NRAS mutant (Fig1A). Overall, a mean of 7 non-synonymous mutations per adult patient was identified (range 1-22) compared with 5 mutations per pediatric patient (range 4-9; p =ns). Mutations affecting diverse cellular processes were found to co-exist with kinase mutations including mutations in epigenetic modifiers and the p38/MAPK pathway. In addition to kinase point mutations, RNA-seq identified recurrent, in-frame kinase fusions-a first for these disorders. All identified fusions were validated using FISH and RT-PCR. This includes novel fusions in BRAF (RNF11-BRAF and CLIP2-BRAF), as well as therapeutically important fusions in ALK (2 separate KIF5B-ALK fusions) and NTRK1 (LMNA-NTRK1;Fig1B). Expression of each fusion in Ba/F3 cells conferred cytokine-independent growth. Importantly, the BRAF fusions were found to be sensitive to MEK inhibition but resistant to vemurafenib while the ALK fusions conferred sensitivity to the ALK inhibitors crizotinib or alectinib. We next interrogated a validation cohort of 37 BRAF V600E-wild type, non-LCH, formalin-fixed, paraffin-embedded tissue samples using targeted mutational profiling for MAP2K1, ARAF, NRAS, KRAS, and PIK3CA. This revealed activating mutations in MAP2K1 (32%; n=12), NRAS (16%; n=6), KRAS (11%; n=4), PIK3CA (8%; n=3), and ARAF (3%; n=1). Three of the investigated non-LCH patients with refractory disease and progressive organ dysfunction were treated with targeted therapies based on the discovery of novel kinase alterations described above. Treatment of 2 refractory MAP2K1- mutant, non-LCH patients with MEK inhibitors (trametinib or cobimetinib) resulted in dramatic clinical improvement (Fig1C). Both patients have been maintained on MEK inhibitor single-agent therapy with a sustained clinical response for >100 days. Further evidence of effective targeted inhibition was found in a refractory ECD patient carrying an ARAF S214A mutation. This patient failed to respond to 3 lines of prior therapies and suffered near blindness due to disease infiltration in the retina and optic nerves. Given a recent report of complete response to sorafenib in a lung cancer patient with an ARAF S214C mutation, we initiated sorafenib. Within 12 weeks, there was improvement in the patientÕs eyesight and decreased infiltrative disease, coinciding with >50% decrease in mutant ARAF DNA in plasma cell-free DNA. Whole exome and transcriptome sequencing identified activating kinase mutations or translocations in all patients with the common downstream effect of activating the MAPK pathway. The preliminary, dramatic, clinical efficacy observed with use of MEK and RAF inhibitors in MAP2K1 - and ARAF-mutated, non-LCH patients further supports the central role of targeting the MAPK pathway in these tumors. The discovery of the discussed mutations and fusions in diverse kinases provides critical new insights into the genetic events central to a spectrum of adult and pediatric histiocytic neoplasms. Figure 1. Figure 1. Disclosures Off Label Use: This abstract describes use of MEK inhibitors (both tremetinib and cobimetinib) as well as sorafenib for MEK1 and ARAF mutant histiocytosis. . Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc.: Employment. Ali:Foundation Medicine Inc.: Employment. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria.

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Single Molecule Real Time (SMRT™) Sequencing Sensitively Detects Polyclonal and Compound BCR-ABL in Patients Who Relapse on Kinase Inhibitor Therapy,

Blood ◽

10.1182/blood.v118.21.3752.3752 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3752-3752 ◽

Cited By ~ 1

Author(s):

Catherine C. Smith ◽

Michael Brown ◽

Jason Chin ◽

Corynn Kasap ◽

Sara Salerno ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Kinase Inhibitor ◽

Drug Resistant ◽

Smrt Sequencing ◽

Sequencing Technology ◽

Employment Equity ◽

Pacific Biosciences ◽

Equity Ownership ◽

Tki Treatment

Abstract Abstract 3752 Background: Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD where TKI-resistant mutations are detected, next-generation platforms are unable to generate reads of sufficient length to determine if two mutations separated by 500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) circular consensus sequencing technology is a novel third generation deep sequencing technology capable of rapidly and reliably achieving average read lengths of ∼1000bp (Travers et al, 2010) and frequently beyond 3000bp, allowing sequencing of the entire ABL KD on single strand of DNA. We sought to address the ability of SMRT sequencing technology to distinguish polyclonal from compound mutations using clinical samples obtained from patients who have relapsed on BCR-ABL TKI treatment. Results: We analyzed an 863bp area of the BCR-ABL KD in 6 patients who had clinically relapsed on ABL kinase inhibitor therapy. SMRT sequencing detected mutations at a sensitivity of ∼1–2% of the total sequenced population, and successfully distinguished polyclonal from compound BCR-ABL KD mutations in several patient samples. Results were largely consistent with those obtained by PCR subcloning and sequencing, although SMRT sequencing detected additional mutations and/or mutation combinations. In the most complex case, 7 distinct mutation-bearing alleles were detected in an individual patient after sequential relapse on imatinib and dasatinib. Mutant clones contained single and compound mutations combining distinct mutations (Y253H, T315F, T315A, T315I, T319A, E355G). Three distinct substitutions at residue T315 were detected: T315A, T315I and T315F. Notably, these findings are clinically important as the T315A mutation confers resistance to dasatinib but not imatinib, while the T315F and T315I mutations are resistant to all three clinically approved BCR/ABL inhibitors (imatinib, dasatinib, and nilotinib). Phospho-flow analysis for p-Crkl, a direct substrate of BCR-ABL, was conducted following ex vivo exposure of patient cells from the same time point to all three BCR-ABL inhibitors, and demonstrated the existence of distinct populations of cells with varying sensitivity to each drug (i.e. polyclonal drug sensitivity), underscoring the potential clinical importance of distinguishing polyclonal from compound mutations. Additionally, SMRT sequencing routinely detected alleles harboring compound mutations not detectable by conventional direct sequencing. Data analysis of samples from additional patients is ongoing and will be presented. Conclusions: Pacific Biosciences RS SMRT sequencing sensitively detects KD mutations in patient samples and can distinguish TKI-resistant clones containing compound mutations to reveal a complex mutational landscape in an individual patient not detectable by conventional sequencing. SMRT sequencing of the BCR-ABL KD can feasibly be developed into a rapid and economical clinical test with the additional advantages of increased sensitivity and reliability over current methods. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles promises to further facilitate a personalized approach to patient management. Disclosures: Brown: Pacific Biosciences: Employment. Chin:Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.

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Targeted MEK Inhibition in Patients with Previously Treated Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.4775.4775 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4775-4775 ◽

Cited By ~ 4

Author(s):

Christoph Heuck ◽

Yogesh Jethava ◽

Rashid Z Khan ◽

Scott Miller ◽

Alan Mitchell ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Mapk Pathway ◽

Objective Response ◽

Genomic Profiling ◽

Employment Equity ◽

Advisory Committees ◽

Comprehensive Genomic Profiling ◽

Equity Ownership

Abstract Background: Diagnostic and therapeutic advances have significantly improved the outcomes for multiple myeloma (MM) patients. However, pts who are refractory to or relapse after therapy with immune modulatory drugs and proteasome inhibitors remain a therapeutic challenge. Comprehensive genomic profiling via clinical next generation sequencing (NGS)-based assays studies of MM cases have revealed multiple targetable mutations that were previously unexploited in MM. Methods: Between June 2013 and May 2014 we performed genomic profiling of 351 patients who had progressed after initial therapy to assist physicians in therapy planning. Comprehensive genomic profiling was performed using the FoundationOne¨ or FoundationOne Heme¨ assays. FoundationOne assays 374 cancer-related and 24 frequently rearranged genes via DNA-seq, and FoundationOneHeme assays 405 cancer-related and 31 frequently rearranged genes via DNA-seq as well as 265 frequently rearranged genes by RNA-seq. All samples were sequenced in a CLIA-certified CAP-accredited laboratory to an average depth >500x . Patients with activating alterations of KRAS, NRAS or BRAF were considered for therapy with the targeted agent trametinib (TMTB) as were patients who had a gene expression signature suggesting activation of the MAPK pathway. Retrospective review of this case series was approved by the UAMS institutional review board. Results: We identified 63 patients who underwent treatment with Trametinib. 60 were treated based on activating mutations of KRAS, NRAS or BRAF and 3 were treated based on a GEP signature. The median age was 65 and patients had a median of 5 lines of prior therapy (range 1-20). 38 of 63 patients had prior treatment with Total Therapy. 43 underwent salvage with chemotherapy prior to initiation of TMTB, 15 had salvage transplants, 33 patients were exposed to novel agents (Pomalidomide, Carfilzomib) and 33 had Metronomic therapy before TMTB. 25% of patients were ISS stage 3 and 37% had GEP70 defined high risk. 13 had PET defined extra medullary disease (EMD). 41 patients were administered TMTB monotherapy and 22 received TMTB treatment in combination with other agents. In general the treatment was well tolerated. 10 patients discontinued therapy because of toxicities, 29 discontinued because of disease progression or death. None of the deaths were attributed to TMTB, Best treatment responses were SD in 30, PR in 8, VGPR in 2 and CR in 3 of the 63 pts. For 25 patients with evaluable PET data, treatment resulted in complete resolution of FDG avid lesions in 9 patients and a better than 50% reduction in 15 (Figure 1). We will present updated data on clinical responses as well as toxicities. Conclusions: Treatment with targeted therapy guided by prospective comprehensive genomic profiling across all classes of genomic alterations in this heavily pretreated population of MM patients resulted in an unexpectedly high objective response rate. Observation of CR with TMTB monotherapy further supports continued investigation of this individualized approach to MM management. Disclosures Van Laar: Signal Genetics: Employment, Equity Ownership. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Zangari:Norvartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. van Rhee:Millenium: Speakers Bureau; Sanofi: Speakers Bureau; Celgene: Speakers Bureau; Janssen: Speakers Bureau. Morgan:Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees.

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A Subcutaneously Administered Investigational RNAi Therapeutic (ALN-CC5) Targeting Complement C5 for Treatment of PNH and Complement-Mediated Diseases: Preliminary Phase 1/2 Study Results in Patients with PNH

Blood ◽

10.1182/blood.v128.22.3891.3891 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3891-3891 ◽

Cited By ~ 14

Author(s):

Anita Hill ◽

Anna Gaya Valls ◽

Morag Griffin ◽

Talha Munir ◽

Anna Borodovsky ◽

...

Keyword(s):

Phase 1 ◽

Clinical Activity ◽

Inadequate Response ◽

Employment Equity ◽

Complement Activity ◽

Part C ◽

Activity Inhibition ◽

Treatment Naïve ◽

Equity Ownership ◽

Ongoing Phase

Abstract Background:Uncontrolled complement activation plays a pivotal role in a variety of disorders such as paroxysmal nocturnal hemoglobinuria (PNH). Challenges to be addressed with eculizumab therapy include inter-individual variation in clearance of eculizumab, economic burden, and improvements in the current biweekly intravenous infusion maintenance schedule. ALN-CC5 is a subcutaneous (SC) investigational RNA interference (RNAi) therapeutic targeting hepatic complement C5 (C5) synthesis. Previously presented data from our ongoing Phase 1/2 study showed that ALN-CC5 was generally well tolerated and exhibited a clamped C5 knockdown and complement activity inhibition in healthy volunteers (Hill et al. Haematologica 2016; 101, Suppl 1). The aim of this abstract is to report updated tolerability and clinical activity of ALN-CC5 in patients with PNH. Methods: A phase 1/2 single-ascending dose (Part A) and multiple-ascending dose study (Part B) of ALN-CC5 was conducted in healthy adult volunteers and in patients with PNH (Part C). In Part C, patients with PNH received weekly doses of 200 mg or 400 mg of ALN-CC5 for 2 to 16 weeks; ALN-CC5 is administered subcutaneously at a concentration of 200 mg/mL. The primary endpoints are safety and tolerability and secondary endpoints include: pharmacokinetics (PK), reduction of circulating C5 and complement activity, as measured by CAP/CCP Wieslab ELISA assays and sheep erythrocyte hemolysis assay, as well as reduction in LDH. Results: Part C included 6 patients with PNH (treatment naïve n=3; patients receiving eculizumab n=3), including 1 patient who was experiencing breakthrough hemolysis despite receiving 1200mg eculizumab q2wk. ALN-CC5 was generally well tolerated in patients with PNH after multiple doses with the majority of adverse events (AEs) being mild or moderate in severity. There were no serious AEs or discontinuations due to AEs. One patient, who was also taking eculizumab, cyclosporine and anabolic steroids as concomitant medications, experienced a transient asymptomatic grade 3 elevation of liver transaminases that was deemed possibly related to study drug. In eculizumab naïve patients (n=3), ALN-CC5 monotherapy achieved a mean maximum C5 knockdown of 98.2% ± 0.3%, residual C5 levels of 0.9 mcg/mL and a mean maximum CCP inhibition of 94.2 ± 1.7%. During treatment with ALN-CC5, maximum reduction in LDH was 37% and 50% in 2 patients who received 17 doses of ALN-CC5 but remained above the goal of less than 1.5 times the ULN. In the remaining eculizumab naïve patient, LDH lowering was not observed following 8 doses of ALN-CC5. After completion of ALN-CC5 dosing and in the setting of ongoing >95% ALN-CC5-mediated KD of serum C5, treatment naïve patients received a single 600 mg dose of eculizumab (labeled induction dose is 600 mg weekly x 4) for the treatment of residual hemolysis followed by close clinical monitoring. An exploratory analysis was conducted to understand the potential for ALN-CC5 to reduce eculizumab dose and frequency. All 3 patients achieved sustained lowering of LDH <1.5 ULN for out to 4 weeks. In patients entering the study on a stable dose of eculizumab (n=3), ALN-CC5 was also able to achieve a robust C5 KD (mean max 86.7% ± 5.6%) with residual complement activity of <2% as measured by CCP assay from day 21 onward. The addition of ALN-CC5 resulted in LDH lowering to within reference range by day 35 post treatment (maintained out to Day 112) in an inadequate response patient who entered the study on higher than labeled dose of eculizumab. As a result, this patient's subsequent dosing of eculizumab was lowered from 1200mg q2wk to 900mg q2wk. Updated safety, pharmacodynamics (PD) and clinical activity for all 6 patients with PNH in Part C of this ongoing Phase 1/2 study will be presented. Conclusion: ALN-CC5 was shown to be generally well tolerated in patients with PNH. The PD effects of ALN-CC5 were found to be durable, with clamped C5 knockdown and complement activity inhibition. Collectively, the data suggest that clamped inhibition of hepatic C5 synthesis may provide the foundation to potentially reduce the dose and frequency of eculizumab administration for patients with PNH, and to improve disease control in patients with inadequate response to eculizumab. Disclosures Hill: Alnylam Pharmaceuticals: Consultancy, Honoraria. Griffin:Alexion Pharmaceuticals: Honoraria, Other: Conference support. Munir:Alexion pharmaceuticals: Honoraria. Borodovsky:Alnylam Pharmaceuticals: Employment, Equity Ownership. Kawahata:Alnylam Pharmaceuticals: Employment, Equity Ownership. Mclean:Alnylam Pharmaceuticals: Employment, Equity Ownership. Shi:Alnylam Pharmaceuticals: Employment, Equity Ownership. Partisano:Alnylam: Employment, Equity Ownership. Kim:Alnylam Pharmaceuticals: Employment, Equity Ownership. Najafian:Alnylam Pharmaceuticals: Employment, Equity Ownership.

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Progression By Lymphocytosis Correlates with Favourable Long-Term Clinical Outcomes in Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v128.22.4352.4352 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4352-4352

Author(s):

Alexandra Bazeos ◽

Craig Gower ◽

Francine Swann ◽

Peter C Trask ◽

Mark Dixon ◽

...

Keyword(s):

Board Of Directors ◽

Physical Symptoms ◽

Response To Therapy ◽

Phase Iii ◽

Rank Test ◽

Outcome Analysis ◽

Employment Equity ◽

Advisory Committees ◽

Histological Transformation ◽

Equity Ownership

Abstract BACKGROUND The evaluation of response to therapy in CLL is widely assessed according to the iwCLL guidelines which define progressive disease (PD) as advancing lymphocytosis, lymphadenopathy, organomegaly, cytopenias or histological transformation (Hallek, Blood, 2008). At the time of PD, considerable heterogeneity exists; patients (pts) with asymptomatic lymphocytosis seem to have a more indolent clinical course than those who progress by other means. On this basis, we hypothesized that the type of PD might impact on subsequent post-progression pt outcomes including the time to next treatment (TTNT) and overall survival (OS). METHODS All analyses were performed on data collected from pts enrolled in the CLL11 trial (NCT01010061), an open-label, randomized, pivotal phase III study comparing the efficacy and safety of obinutuzumab (GA101; GAZYVA) plus chlorambucil (Clb) with rituximab and Clb or Clb alone in treatment-naïve pts with CLL and pre-existing comorbidities (Goede, NEJM, 2014). Pts with defined PD, excluding death, were identified and assigned to 1 of 2 groups according to whether PD was by absolute lymphocyte count (ALC), or other, (non-ALC) causes, as per iwCLL criteria. Individuals could be allocated to only 1 group. To test whether subgroups were balanced at baseline (BL), characteristics between groups were compared for proportional differences using a Pearson chi-square test. Post-progression Kaplan-Meier survival curves for TTNT and OS were plotted by PD type. The log-rank test was used to detect significant differences in the treatment timings and survival distributions between groups, respectively. Group differences in the proportion of pts with cytopenia due to bone marrow (BM) infiltration (excluding autoimmune causes) were tested at BL and the time of PD. Similarly, B symptoms were calculated as the proportion of pts reporting disease-attributable fevers, night sweats or weight loss. Health-related quality of life (HRQoL) data were extracted from the EORTC QLQ-C30 and -CLL16 questionnaires. The mean and mean change scores by progression type were derived from BL and PD assessments. RESULTSOf the 781 pts enrolled in CLL11, progression data were available for 507 (64.9%) subjects. Of these, a total of 329 (64.9%) pts progressed by ALC, while the remaining 178 (35.1%) had PD from an alternative, non-ALC cause. At study BL, there were no differences in the demographics or disease characteristics between groups. The median post-progression TTNT for the ALC group was 373 days (95% CI [320, 449]) versus 120 days (95% CI [101, 209]) for the non-ALC group, (p < 0.0001) (Fig 1). Type of PD was also associated with a better OS in the ALC group (p = 0.0014) but the median time could not be accurately estimated due to insufficient events (Fig 2). A higher proportion of non-ALC pts demonstrated evidence of disease-related BM infiltration at PD (0.38 vs 0.24, p = 0.0013) with anemia (0.24 vs 0.10, p < 0.001) and neutropenia (0.16 vs 0.06, p < 0.001) representing the greatest differences between groups (Fig 3). Despite a trend towards a higher proportion of B symptoms in the non-ALC group at PD, significance was not achieved (p = 0.0624). Mean absolute HRQoL scores at BL, highlighted trends towards a higher level of functioning (higher scores) and milder physical symptoms (lower scores) in the ALC group. At the time of PD, those progressing by ALC reported clinically meaningful (6 point) higher role and physical functioning and measurably less fatigue, insomnia, dyspnea and pain. Within-group, mean score changes between BL and PD highlighted an overall trend towards improved functioning (QLQ-C30) over the course of treatment in the ALC group and a decline in function in the non-ALC group although differences were small. In general, both groups reported milder disease-related symptoms (QLQ-C30 and -CLL16) at PD with pts in the ALC group showing clinically meaningful improvements in fatigue and appetite. CONCLUSION These data provide the first comprehensive outcome analysis in CLL based on the mode of first progression for pts receiving upfront chemoimmunotherapy. We have shown that progression by ALC is consistently associated with a favourable clinical profile but whether our findings apply to pts in the relapsed setting or to those receiving other novel therapies is yet to be determined. An accurate estimate of survival by PD type might guide physician choice/timing of next treatments. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Bazeos: Roche: Employment. Gower:Roche Products Ltd: Employment. Swann:Roche: Employment. Trask:Genentech, Inc.: Employment, Equity Ownership. Dixon:Roche Products Limited: Employment, Equity Ownership. Crompton:Roche: Employment. Kinnersley:Roche-Genentech: Employment, Equity Ownership. Al-Sawaf:Gilead: Other: Travel grants. Goede:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria; Roche: Consultancy, Honoraria, Other: Travel grant, Research Funding. Fingerle-Rowson:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Humphrey:Genentech, Inc.: Employment.

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Characterization of the Mutational Landscape of Multiple Myeloma Using Comprehensive Genomic Profiling

Blood ◽

10.1182/blood.v124.21.3418.3418 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3418-3418 ◽

Cited By ~ 3

Author(s):

Christoph Heuck ◽

Donald Johann ◽

Brian A Walker ◽

Caleb K Stein ◽

Yogesh Jethava ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Mutant Allele ◽

Genomic Profiling ◽

Employment Equity ◽

Advisory Committees ◽

Comprehensive Genomic Profiling ◽

Equity Ownership

Abstract Introduction: Multiple myeloma (MM) is a neoplastic disease of the bone marrow characterized by a malignant transformation of plasma cells. Many patients relapse after initial treatment and require additional therapies. Impaired cell cycle regulation and DNA repair mechanisms as well as exposure to genotoxic drugs leads to accumulation of genomic alterations with progressive disease. Pressure from antineoplastic agents, including novel agents, eventually leads to the selection of resistant clones. Assessing acquired somatic mutations in MM patients can identify key genomic drivers and guide the development of a rational, individualized therapy plan for each patient with advanced disease. Here we report on the mutational landscape of cancer-associated genes in 214 patients who underwent comprehensive genomic profiling. Methods: Review of this data was approved by the UAMS institutional review board. DNA and RNA were extracted from CD138+ selected cells from bone marrow aspirates. Adaptor ligated sequencing libraries from extracted nucleic acids were captured by solution hybridization using bait sets targeting 405 cancer-related and 265 frequently rearranged genes (FoundationOne Heme®; Foundation Medicine ). For samples with low cell yield only the DNA portion was performed. All samples were sequenced in a CLIA-certified, CAP-accredited laboratory to an average depth >500x. Results We identified 147 clinically relevant alterations with an average of 3 alterations per patient ranging from 1 to 8. The most frequently altered genes were KRAS (29% of cases), NRAS (23%), TP53 (19%), RB1 (10%), BRAF (8%), TRAF3(8%), CDKN2C (7%), DNMT3A (5%), NF1, FAF1 and TET2 (4% each). While RAS, RAF, RB1 and TP53 mutations are also found in previously untreated patients, albeit in lower frequencies, mutations of DNTM3A and TET2 are rarely reported in the early phase of the disease, arguing for the accumulation of genomic alterations over time. We found concomitant alterations in KRAS and BRAF in 5, KRAS and NRAS in 3, and NRAS and BRAF in 2 patients. The vast majority of RAS alterations occurred at hotspots resulting in activating alterations at codons 12, 13 or 61 with mutant allele frequencies ranging from 0.01 to 0.92 with an average of 0.30. In the 17 patients with BRAF alterations the hotspot mutation V600E was found in 7 with mutant allele frequencies ranging from 0.01 to 0.48 with an average of 0.32. Overall the MAPK pathway was affected in 128 of 214 patients. 61 patients had alterations of genes associated with DNA damage repair. Among the 10 patients with DNMT3A alterations 2 also had alterations of TET2 suggesting significant epigenetic deregulation in a subset of patients. Data on subclonal structure and correlation of mutation status with paired gene expression profiles will be presented as well, as will be selected responses of patients treated on the basis of these results. Conclusion Subjecting CD138 selected bone marrow cells to comprehensive genomic profiling allows for the identification of clinically relevant alterations, which deregulate critical pathways in multiple myeloma. Small molecule inhibitors that target key genes in these affected pathways (MEK, BRAF) have recently been approved for therapy in other cancers or are being actively developed (PI3K, AKT, PARP). This comprehensive genomic characterization allows rational development of individualized clinical strategies using molecular targets for MM patients who are refractory to standard of care therapies. Disclosures Walker: Onyx Pharmaceuticals: Consultancy, Honoraria. van Rhee:Senesco: PI Other. Zangari:Norvartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Morgan:Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees. Barlogie:Celgene: Consultancy, Patents & Royalties, Research Funding; Millenium: Consultancy, Patents & Royalties, Research Funding.

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Predictive and Prognostic Significance of Comprehensive Genomic Profiling in Patients with Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.2651.2651 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2651-2651

Author(s):

Jie He ◽

Shan Zhong ◽

Michelle Nahas ◽

Mark Bailey ◽

Jon Chung ◽

...

Keyword(s):

Copy Number ◽

Cell Lymphoma ◽

Point Mutations ◽

B Cell Lymphoma ◽

Genomic Profiling ◽

First Line ◽

Employment Equity ◽

Large B Cell Lymphoma ◽

Comprehensive Genomic Profiling ◽

Equity Ownership

Abstract Introductions: We assessed the benefit of adding comprehensive genomic profiling (CGP) to improve clinical outcomes in both first-line and relapse/refractory settings in adult patients with diffuse large B-cell lymphoma (DLBCL). Current standard first-line therapy affords long term remission in only 55-60% of patients, and less than half of these cases can be treated successfully following relapse. Thus, there is a clear unmet clinical need to improve the standard of care for all patients with DLBCL. We interrogated specimens from 125 patients with DLBCL using a novel next generation sequencing (NGS)-based CGP assay (FoundationOne® Heme) to identify potential clinically relevant therapeutic targets and improve risk stratification. Methods: Genomic profiles from 125 DLBCL specimens received in a CLIA-certified, CAP-accredited, NYS-approved laboratory were successfully characterized by FoundationOne® Heme. Sequencing libraries targeting 405 cancer-related genes by DNA-seq and 265 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 498x for DNA and ~7M on-target unique pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, copy number alterations and gene rearrangements. Additionally, relative mRNA expression of MYC, BCL2 and BCL6 was quantified by normalizing with the median ppm of each gene in the cohort. The statistical significance of expression between the rearrangement positive cohort and rearrangement negative cohort was determined by one-sided Wilcoxon rank-sum test. Results: Somatic driver alterations were identified in all 125 cases (including 28 relapse/refractory patients, 28 transformed from low grade lymphoma and remainder are being assessed) with clinically relevant genomic alterations observed in 97 (78%) patients. The cohort includes 79 male and 46 female patients with a median age of 63. The mean mutation burden averaged 6.5 alterations per patient. Genomic alterations previously described in DLBCL were confirmed in this study, including changes in MLLT3 (39.2%), TP53 (31.2%), CDKN2A (30.4%), BCL2 (28.8%), BCL6 (20%), CREBBP (20%) and MYC (12%). Importantly, alterations in several genes with known therapeutic associations were also detected, including EZH2 (11.2%), TET2 (8.8%), CD79 (7.2%) and PTEN (4.8%) (Fig 1). Therapeutically targetable kinase mutations classically associated with solid tumors (e.g. RET, ALK, BRAF, HRAS, NRAS, FGFR2 and ERBB2 amp) were also identified, though at lower frequency (<3%). Strong correlation was observed between genomic rearrangements and over-expression in MYC (p=4.64e-06) and BCL2 (p=1.39e-05) but not in BCL6 (p=6.64e-02) from the integrated NGS-based platform. In 10 samples, focal CNA or point mutation leads to overexpression of BCL,2 and point mutations occur in 18/125 (14%) of patients and co-occur with BCL2 rearrangement significantly (p < 1e-04) (Fig 2). Conclusions: Comprehensive genomic profiling identifies a complex genomic landscape in DLBCL that may impact diagnosis and prognosis, deepen our understanding of underlying biologic pathways, and guide targeted combination therapy in relapsed patients with limited treatment options. Additionally, our data suggests that focal copy number amplification, point mutations and rearrangements can serve as distinct/interactive mechanism to potentially activate MYC and BCL2 and may explain the challenges currently encountered in subclassification and prognostication (e.g. double-hit) of DLBCL. Further functional validation of these MYC, BCL2 and BCL6 alterations through comparing orthogonal clinical test results and clinical outcomes could help further refine the subclassification in DLBCL. Disclosures He: Foundation Medicine, Inc.: Employment, Equity Ownership. Zhong:Foundation Medicine Inc: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc.: Employment, Equity Ownership. Bailey:Foundation Medicine Inc: Employment, Equity Ownership. Chung:Foundation Medicine Inc: Employment, Equity Ownership. Otto:Foundation Medicine Inc: Employment, Equity Ownership. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc: Employment, Equity Ownership. Mughal:Foundation Medicine, Inc.: Employment, Equity Ownership. Vergilio:Foundation Medicine, Inc.: Employment, Equity Ownership.

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Clinical Relevant Alterations Identified By Comprehensive Genomic Profiling Can Potentially Improve Therapeutic Option and Change Prognosis in Hematologic Malignancies

Blood ◽

10.1182/blood.v128.22.5109.5109 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5109-5109

Author(s):

Jie He ◽

Minal Patel ◽

Irene Phillip ◽

Mark Bailey ◽

Ellin Berman ◽

...

Keyword(s):

Targeted Therapies ◽

Copy Number ◽

Research Funding ◽

Standard Of Care ◽

Genomic Profiling ◽

Employment Equity ◽

Comprehensive Genomic Profiling ◽

High Concordance ◽

Equity Ownership ◽

Copy Number Amplification

Abstract Background: Hybrid capture based comprehensive genomic profiling (CGP) in a spectrum of human leukemias has led to a deepened understanding of biological mechanism of leukemogenesis and improved risk stratification and has provided additional options for targeted therapy beyond standard chemotherapy or transplantation. However, most adults with leukemia still relapse after initial therapy, and incorporating comprehensive genomic profiling results into clinical practice still requires clear guidelines and evidence on how this compares with the conventional diagnosis approach. Here we analyzed a consecutive series of 116 acute leukemia patients profiled by FoundationOne Heme to learn how the results compared with current standard of care diagnosis and how any additional insights into the genomic alterations may lead to a changed or improved diagnosis, therapy and prognosis. Methods: A total of 116 consecutive newly diagnosed or relapse/refractory leukemia patients from Memorial Sloan Kettering Cancer Center were profiled by FoundationOne Heme. DNA and RNA integrated next-generation sequencing was performed in a CLIA-certified, CAP-accredited, NYS-approved laboratory for comprehensive genomic profiling. All captured libraries were sequenced to high depth averaging 569X for DNA (405 genes) and >3M unique pairs for RNA (265 genes). Somatic variants identified included short variants, copy number amplification and loss and rearrangements. Ninety-nine patients had Karyotyping and/or FISH performed at the same time to allow comparing results between platforms. Results: The age of this clinical sample cohort range from 19 to 85, with median age of 54. The diagnosis included AML (49), ALL (29), LGL (12), CML (7), MDS (4), and 16 other subtypes. CGP was performed at the time of diagnosis (47 patients, 41%), relapsed/refractory (58 patients, 50%), stable disease (7 patients, 6%) and complete remission (3 patients, 3%). CGP successfully reported 308 alterations in 103 patients with an average of 3.0 alterations per patient, including 158 base substitutions, 75 indels, 21 copy number amplification or loss, and 54 rearrangements. High concordance of known translocations, including BCR-ABL1, RUNX1T1-RUNX1, PML-RARA, MLLT10-MICALM, MLL-rearrangement, ETV-6 rearrangement and EVI1 rearrangement, were observed between FoundationOne Heme and Karyotpye/FISH in 76 out of 78 cases (97%). In addition to genomic abnormalities identified by Karyotype/FISH, CGP identified additional clinically relevant alterations in 61 cases (59%). These alterations included genes associated with new targeted therapies, such as FLT3, IDH1/2, KRAS/NRAS/BRAF and KIT and known/novel prognostic biomarkers, including TP53, NF1, CDKN2A/B, and RB1 (Figure 1). Novel fusions involved in NUP98, ABL1, PDGFRB, JAK2 were found in 12 patients, and 4 known/novel Ph-like ALL signature fusions were identified which can inform the use of clinically available targeted therapies for these patients with high-risk ALL. Conclusion: CGP has high concordance with standard of care testing (Karyotyping/FISH) with respect to the detection of known translocations/fusion genes. More importantly, CGP allowed for the identification of additional clinically relevant genomic alterations in a substantial fraction of leukemia patients seen in routine clinical care. These data demonstrate CGP can inform novel therapeutic interventions, improve accuracy of clinical diagnosis, and provide added value to improve prognosis prediction in adult leukemia. Figure 1 Figure 1. Disclosures He: Foundation Medicine, Inc: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Park:Amgen: Consultancy; Genentech/Roche: Research Funding; Juno Therapeutics: Consultancy, Research Funding. Douer:Shire: Consultancy, Speakers Bureau; Pfizer: Consultancy; Jazz Pharmaceuticals: Honoraria; Gilled Sciences, Inc: Consultancy; Spectrum: Consultancy. Nahas:Foundation medicine: Employment. Otto:Foundation Medicine, Inc: Employment. Vergilio:Foundation Medicine: Employment. Mughal:Foundation Medicine: Employment, Equity Ownership. Ross:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership. Lipson:Foundation Medicine, Inc: Employment.

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Genetic and Transcript Changesin Cereblon in IMiD-Treated Myeloma Patients

Blood ◽

10.1182/blood-2019-126828 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1793-1793

Author(s):

Sarah Gooding ◽

Naser Ansari-Pour ◽

Fadi Towfic ◽

Maria Ortiz ◽

Dan Rozelle ◽

...

Keyword(s):

Research Funding ◽

E3 Ligase ◽

Resistance Mechanisms ◽

Response To Therapy ◽

Employment Equity ◽

Transcript Variants ◽

Equity Ownership ◽

The Right ◽

The Impact ◽

Exon 10

Myeloma survival has been significantly improved by novel therapies over the past two decades. Immunomodulatory agents (IMiDs, Lenalidomide (LEN) and Pomalidomide (POM)) are a backbone of current treatment strategies. But myeloma (MM) remains incurable, because patients ultimately relapse. IMiD drug resistance mechanisms are multifactorial, and can be either dependent or independent of IMiDs binding to Cereblon (CRBN) (a component of an E3 ligase) in tumor and immune cells. A small series of relapsed patients demonstrated sub-clonal mutation rates of 12% in CRBN and 10% in other CRBN pathway genes (Kortum et al, Blood 2016), but the clinical implication of this observation is unknown. In contrast, baseline gene expression of CRBN was not associated with clinical outcome to POM-DEX therapy (Qian et al, Leukemia & Lymphoma 2018). An integrated assessment of the burden of different mechanisms of CRBN function loss, the selective pressure for their survival, and their effects on response to CRBN-modulating agents, is lacking. For patients that progress on IMiD-based therapies, there is a need to develop drugs that will overcome their resistance. To appropriately target the right novel agents to the right patients, resistance mechanisms must be understood. A deeper understanding of the mechanistic basis for IMiD-specific resistance mechanisms is critical for differentiating new Cereblon modulating agents (eg CELMoDs) from the IMiDs. Here, we present the largest comprehensive analysis of the burden of CRBN mutation or transcript variants in relapsed refractory myeloma (RRMM) patients. We analysed WGS and RNASeq data from 298 MM samples from 268 patients across 4 clinical trials (CC-4047-MM-010 (N=226), CC-4047-MM-013 (N=17), CC-220-MM-001 (N=45) and CC-122-ST-001MM2 (N=10), for whom outcome data were available. All patients had been exposed to LEN-based therapy and a subset (69/268) were also exposed to POM-based therapy. The overall incidence of single nucleotide variants in CRBN was 17/298 (5.7%). The incidence of at least monoalleleic deletion at the CRBN gene locus was 21/298 (5.5%). CRBN has different transcript isoforms (Gandhi et al, BJHaem 2014). As high levels of isoform ENST00000424814.5, with deletion of exon 10, was previously correlated with poorer survival (Neri et al, Blood 2016), we assessed incidence of a high ratio (>2) of exon 10-deleted CRBN transcript to full length CRBN transcript. 92 samples had sufficient purity (>90% tumor cells) for this analysis. 13/92 samples (14.1%) had a high exon10-deleted transcript ratio. Overall, 43/268 (16.0%) patients had genetic or transcript variants in CRBN. In contrast, 27/514 (5.2%) newly diagnosed myeloma (NDMM) patients from the Myeloma Genome Project had genetic or transcript variants in CBRN; 2/514 (0.4%) had CRBN mutations, 11/514 (2.1%) had CRBN gene deletion and 14/514 (2.7%) had a high exon10-deleted transcript ratio. Thus, there was an increase in CRBN variants from NDMM to RRMM. In patients exposed to LEN but not POM (219/268), there were 5 CRBN mutations, 14 monoallelic CRBN deletions and 13/92 patients with high exon10-deleted transcript ratio. In 69/268 patients exposed to POM (baseline from CC-220-MM-001 (N=35), CC-122-ST-001MM2 (N=10), or follow up samples from CC-4047-MM-010 (N=24)), there were 12 CRBN mutations in 8 patients (11.6%) and 7 CRBN deletions (10.1%), approximately double the incidence seen in the whole cohort. 3 CRBN deletions were homozygous, which was not observed in non-POM-exposed individuals. Sample purity was insufficient to measure transcript ratios. In summary, 16.0% of RRMM patients that received LEN or POM have genetic or transcript variants in CRBN, a higher proportion than in NDMM. The impact of these aberrations on CRBN function, especially related to binding of CRBN-modulating drugs, remains to be ascertained. Analysis of the correlation between CRBN variation and response to therapy, clinical outcomes, and the incidence and effect of mutation or copy loss of CRBN interactors (E3 Ligase members and regulators, CRBN substrates) is underway and will be presented. Disclosures Gooding: Celgene: Research Funding. Ansari-Pour:Celgene Corporation: Consultancy. Towfic:Celgene Corporation: Employment, Equity Ownership. Ortiz:Celgene Corporation: Employment, Equity Ownership. Rozelle:Celgene Corporation: Other: Contractor for Celgene. Zadorozhny:Celgene Corporation: Other: Contractor for Celgene. Amatangelo:Celgene Corporation: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Tsai:Celgene Corporation: Employment, Equity Ownership. Neri:Celgene, Janssen: Consultancy, Honoraria, Research Funding. Bahlis:AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Vyas:Novartis: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding. Thakurta:Celgene: Employment, Equity Ownership.

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