Oncogenic BRAF mutations, including those in non-small cell lung cancer (NSCLC), are classified as Class I, II, or III. While approved therapies exist for BRAF Class I mutants, no approved therapies exist for Class II and III BRAF-mutated NSCLC. Analysis of a circulating tumor DNA database reveals Class II and III mutations comprise ~65% of BRAF-mutant NSCLC cases, with Class II patients showing worse outcomes than Class I. Exarafenib, a distinct pan-RAF inhibitor, demonstrates potent activity against BRAF Class II and III mutant preclinical models and initial clinical activity. Resistance studies reveal rewiring to an ARAF-mediated bypass pathway, characterized by RAS-mediated ARAF-KSR1 complexes maintaining MAPK signaling despite pan-RAF inhibitor treatment. RAS or MEK inhibition co-targeting is effective against this resistance mechanism. This study provides preclinical rationale for clinical testing of exarafenib in BRAF Class II/III cancers and unveils RAS-mediated ARAF-KSR1 complex formation as a resistance mechanism and rational co-therapy strategies.

The serine/threonine kinase BRAF is frequently mutated in several tumor types, including melanoma and non-small cell lung cancer (NSCLC). Oncogenic BRAF mutations can be classified as Class I, II, or III owing to differences in underlying oncogenic mechanisms. While there are approved targeted therapies for BRAF Class I mutants, there are no approved targeted therapy strategies for Class II and Class III BRAF mutated cancers. By leveraging analysis of a large, real world circulating tumor DNA (ctDNA) profiling database, we highlight a significant subset of NSCLC patients whose tumors harbored Class II and Class III BRAF mutations. These mutations comprise ~65% of BRAF-mutant NSCLC cases, with Class II patients showing significantly worse clinical outcomes compared to Class I patients. In several preclinical tumor systems with Class II and III BRAF mutations, exarafenib, a novel type 2 pan-RAF inhibitor that binds RAF proteins irrespective of isoform or dimerization state, demonstrates robust anti-tumor activity. Initial clinical evaluation also showed promising activity in Class II BRAF-mutant NSCLC patients. Mechanistic studies reveal that exarafenib resistance involves adaptive rewiring from conventional oncogenic BRAF-dependent signaling to an ARAF-mediated bypass pathway, characterized by drug-induced ARAF-KSR1 scaffolding complexes that maintain MAPK signaling despite pan-RAF inhibitor treatment. This resistance is driven by upstream RTK activation and RAS-GTP accumulation, which specifically promotes complex assembly under drug treatment. Based on these insights, we identified MEK inhibition as a rational combination strategy that overcomes resistance by targeting the convergence point of both signaling pathways. The exarafenib plus binimetinib combination demonstrated superior efficacy in diverse preclinical models. This study establishes ARAF-KSR1 complex formation as a novel resistance mechanism to pan-RAF inhibition and provides mechanistic rationale for combination strategies with potential to address the unmet clinical need for BRAF Class II and III-mutated NSCLC.

Background: MAPK activating mutations are common in melanoma, with 40% of cases attributed to oncogenic BRAF mutations and 20-25% NRAS mutations. Secondary MAPK activation is a known resistance mechanism to approved BRAF inhibitors in BRAFV600 melanoma. While BRAF inhibitors are approved for Class I BRAFV600 melanomas, patients with dimer-driven BRAF Class II/III and RAF1-dependent NRAS activated melanomas lack approved targeted therapy. Development of next-gen pan-RAF inhibitors targeting all RAF proteins and mutant dimers remains a priority. Emerging clinical data from pan-RAF inhibitors combined with MEK inhibitors suggests increased benefit for MAPK-altered melanoma patients. Exarafenib (KIN-2787) is a clinical stage, novel, highly selective pan-RAF inhibitor designed to be effective in RAF-dependent cancers.Methods: KIN-2787 was evaluated using enzyme assays across the human kinome and activity against oncogenic RAF alterations were validated in BaF3 cells. MAPK pathway suppression and cell growth inhibition were assessed across a panel of human tumor cell lines. Combination dose matrices were performed with KIN-2787 and binimetinib (bini) to evaluate synergistic cell growth inhibition. Extended cell growth studies were performed by Incucyte. In vivo KIN-2787 efficacy was evaluated in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of human BRAF and NRAS mutant cancer.Results: Exarafenib demonstrated exceptional kinome selectivity with minimal off-target kinases significantly inhibited relative to BRAF. Exarafenib potently inhibited a broad panel of oncogenic BRAF mutations in biochemical and BaF3 cell assays. Functional MAPK signaling and viability studies in human tumor cell lines highlighted exarafenib activity in BRAF and NRAS mutant melanoma with minimal activity in normal (BRAF WT) cells. Synergy with the MEK inhibitor bini was determined and the exarafenib + bini combination durably inhibited growth in NRAS mutant melanoma cell lines. In line with cellular studies, treatment with exarafenib demonstrated significant tumor growth inhibition at 30 mg/kg BID in CDX and PDX models of human NRAS mutant melanoma. 10 mg/kg BID exarafenib combined with a clinically relevant dose of bini resulted in combination benefit and durable suppression of the MAPK pathway, relative to either agent alone.Conclusions: The superior kinome selectivity of exarafenib and its activity across multiple RAF-dependent melanoma models position it as a potentially class-leading pan-RAF inhibitor. In addition to efficacy in BRAF mutant tumors, these data support use of exarafenib in combination therapy with MEK inhibitors in NRAS mutant melanoma. A Ph I dose escalation clinical trial evaluating the safety and efficacy of exarafenib in monotherapy and in combination with binimetinib is ongoing (NCT04913285). Citation Format: Tim S. Wang, Catherine Lee, Paul Severson, Robert J. Pelham, Richard Williams, Nichol L. G. Miller. Exarafenib (KIN-2787) is a potent, selective pan-RAF inhibitor with activity in preclinical models of BRAF class II/III mutant and NRAS mutant melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4927.

Abstract Background: MAPK activating mutations are common in melanoma, with 40% of cases attributed to oncogenic BRAF mutations and 20-25% NRAS mutations. Secondary MAPK activation is a known resistance mechanism to approved BRAF inhibitors in BRAFV600 melanoma. While BRAF inhibitors are approved for Class I BRAFV600 melanomas, patients with dimer-driven BRAF Class II/III and RAF1-dependent NRAS activated melanomas lack approved targeted therapy. Development of next-gen pan-RAF inhibitors targeting all RAF proteins and mutant dimers remains a priority. Emerging clinical data from pan-RAF inhibitors combined with MEK inhibitors suggests increased benefit for MAPK-altered melanoma patients. Exarafenib (KIN-2787) is a clinical stage, novel, highly selective pan-RAF inhibitor designed to be effective in RAF-dependent cancers.Methods: KIN-2787 was evaluated using enzyme assays across the human kinome and activity against oncogenic RAF alterations were validated in BaF3 cells. MAPK pathway suppression and cell growth inhibition were assessed across a panel of human tumor cell lines. Combination dose matrices were performed with KIN-2787 and binimetinib (bini) to evaluate synergistic cell growth inhibition. Extended cell growth studies were performed by Incucyte. In vivo KIN-2787 efficacy was evaluated in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of human BRAF and NRAS mutant cancer.Results: Exarafenib demonstrated exceptional kinome selectivity with minimal off-target kinases significantly inhibited relative to BRAF. Exarafenib potently inhibited a broad panel of oncogenic BRAF mutations in biochemical and BaF3 cell assays. Functional MAPK signaling and viability studies in human tumor cell lines highlighted exarafenib activity in BRAF and NRAS mutant melanoma with minimal activity in normal (BRAF WT) cells. Synergy with the MEK inhibitor bini was determined and the exarafenib + bini combination durably inhibited growth in NRAS mutant melanoma cell lines. In line with cellular studies, treatment with exarafenib demonstrated significant tumor growth inhibition at 30 mg/kg BID in CDX and PDX models of human NRAS mutant melanoma. 10 mg/kg BID exarafenib combined with a clinically relevant dose of bini resulted in combination benefit and durable suppression of the MAPK pathway, relative to either agent alone.Conclusions: The superior kinome selectivity of exarafenib and its activity across multiple RAF-dependent melanoma models position it as a potentially class-leading pan-RAF inhibitor. In addition to efficacy in BRAF mutant tumors, these data support use of exarafenib in combination therapy with MEK inhibitors in NRAS mutant melanoma. A Ph I dose escalation clinical trial evaluating the safety and efficacy of exarafenib in monotherapy and in combination with binimetinib is ongoing (NCT04913285). Citation Format: Tim S. Wang, Catherine Lee, Paul Severson, Robert J. Pelham, Richard Williams, Nichol L. G. Miller. Exarafenib (KIN-2787) is a potent, selective pan-RAF inhibitor with activity in preclinical models of BRAF class II/III mutant and NRAS mutant melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4927.

DNA damage caused by the alkylator TMZ can sensitize tumors to PARP inhibitors. Pamiparib, an investigational oral PARP1/2 inhibitor, has shown PARP-DNA complex trapping activity, brain penetration, and synergistic cytotoxicity with LD TMZ in nonclinical studies and preliminary antitumor activity in pts with solid tumors. This ongoing phase Ib study consists of a dose-escalation (3+3 design) and dose-expansion phase. In dose escalation, pts received pamiparib 60 mg PO BID on Days 1-28 and LD TMZ at escalating doses PO QD on Days 1-7, 1-14, or 1-28 of each 28-day cycle. Dose-expansion pts, including pts with gastric cancer and SCLC with 1-2 prior lines of chemotherapy, were treated at the recommended phase II dose of pamiparib 60 mg PO BID on Days 1-28 and LD TMZ 60 mg PO QD on Days 1-7. Tumor assessments occurred every 8 weeks. Endpoints were safety/tolerability (CTCAE v4.03) and antitumor activity (RECIST v1.1). Biomarker assessments included determination of DDR mutational status (SNV/CNV homozygous loss) of 16 core DDR genes in circulating tumor DNA and genomic instability score (GIS) by the Myriad myChoice® HRD test. Herein, we present data from the biomarker analysis. As of 10 April 2020, 114 pts were enrolled (n=66, dose escalation; n=48, dose expansion). Median follow-up was 8.5 mo (range: 0.3, 26.5). Of 36 pts analyzed for GIS, 11 (31%) were GIS positive (GIS+ ≥33), with an ORR of 82% and disease control rate (DCR) of 91% across multiple tumor types. Antitumor activity was observed in BRCAm/GIS+ (n=5; ORR and DCR, 100%) and BRCAwt/GIS+ pts (n=6; ORR, 67%; DCR, 83%). Responses were observed in 3 GIS– pts with pancreatic cancer, pheochromocytoma, and nonsquamous NSCLC (ORR=12%; DCR, 52%). Of 104 pts analyzed for DDR mutational status, 27 (26%) were DDR+, with an ORR of 26% and DCR of 52%. In DDR– pts, ORR was 14% and DCR was 67%. Five pts were both GIS+ and DDR+. In this limited subset of pts analyzed for GIS status, GIS+ pts derived superior benefit from pamiparib + LD TMZ, irrespective of BRCA status. GIS status appears to be the most robust biomarker to predict response to pamiparib + LD TMZ.

TPS5086 Background: Men with mCRPC who have a BRCA1/2 mutation ( BRCA1/2mut) or mutations in other genes resulting in HRD have a poor prognosis. A novel liquid biopsy test (EPIC Sciences) identifies CTCs with an HRD phenotype. Preliminary studies showed that these men may respond to treatment with a PARP inhibitor. Pamiparib, an investigational PARP1/2 inhibitor, has shown brain penetration and potent PARP–DNA complex trapping in nonclinical studies. In early phase clinical studies (NCT02361723; NCT03333915), pamiparib was generally well tolerated and showed preliminary antitumor activity; 60 mg orally twice daily (BID) was established as the recommended investigational dose. Methods: This open-label, global, phase 2 study (NCT03712930) evaluates the antitumor activity and safety/tolerability of pamiparib in mCRPC patients (pts) with CTC-HRD, assessed by the CTC-HRD assay, or deleterious germline/somatic mutations in BRCA1/2. Patients must have progressed on/after ≥1 androgen receptor-targeted therapy, received ≥1 taxane-based therapy, and have prostate-specific antigen (PSA) progression per PCWG3 criteria. Four cohorts of pts will receive pamiparib 60 mg BID in 28-day cycles. Cohort 1 will include ~50 pts with CTC-HRD+ +/- BRCA1/2mut mCRPC with measurable metastatic disease; Cohort 2 will include ~30 pts with CTC-HRD+ +/- BRCA1/2mut mCRPC with bone-only disease; Cohorts 3 & 4 will include ~20 pts with CTC-HRD-/unk + BRCA1/2mut mCRPC with measurable metastatic disease (Cohort 3), or bone-only disease (Cohort 4). Disease status will be assessed every 8 wks for 24 wks, then every 12 wks; PSA levels will be tested every 4 wks. Co-primary endpoints are radiographic ORR assessed by IRC (pts with measurable disease) and confirmed PSA response rate per PCWG3 criteria (pts +/- measurable disease). Secondary endpoints include ORR, time to PSA response/progression, duration of PSA response, time to symptomatic skeletal event, radiographic progression-free survival, overall survival, and safety. As of 05 December 2018, this study is actively enrolling. Clinical trial information: NCT03712930.

Introduction: Multiple myeloma (MM) is a highly prevalent hematological malignancy, with a worldwide incidence of over 62,000 patients in 2012. Despite significant progress in the development of MM treatments in the last decade, there is a great unmet medical need for MM patients who relapse on existing therapies. Given the greater time needed for relapses to manifest when minimal residual disease (MRD) is reduced, new therapies should be aimed at eliminating MRD. Bispecific T cell engager (BiTE®) molecules recruit T cells to cancer cells and trigger T cell-dependent cellular cytotoxicity (TDCC). BiTEs are clinically validated for the treatment of hematological malignancies and have the potential to greatly reduce MRD. CD38 is a membrane protein normally expressed by plasma cells, lymphocytes, and other immune cells and is a clinically validated tumor-associated antigen expressed at high levels on MM cells. Herein, we describe for the first time AMG 424, a novel, humanized T cell-recruiting bispecific anti-CD3/CD38 antibody containing an XmAb® Fc domain that cross-reacts with nonhuman primate (NHP) CD3 and CD38. AMG 424 was selected for its affinity for CD3 and CD38 in an effort to overcome the challenges associated with CD38 and CD3 binding.

Abstract Background: In the phase 3 ENDEAVOR trial, treatment with carfilzomib administered at 56 mg/m2 twice weekly in combination with dexamethasone (Kd56) significantly improved progression-free survival (PFS) compared to treatment with bortezomib and dexamethasone (Vd) in patients with relapsed or refractory multiple myeloma (RRMM) (Dimopoulos MA, et al. Lancet Oncol . 2016;17:27-38). In this substudy of ENDEAVOR, we used whole transcriptome RNA sequencing (RNA-seq) to identify genes whose baseline expression levels in CD138+ cells were predictive of PFS in patients treated with Kd56 or Vd. The objective of this study was to develop a genomic classifier that could be used to stratify patients for benefit with Kd56 or Vd therapy. Methods: Patients were randomized to receive Kd56 or Vd at a 1:1 ratio. Patients who consented for this biomarker study and provided samples (Kd56, n = 155; Vd, n = 148) were included. CD138+ cells were isolated from bone marrow aspirate collected at baseline. Sequencing libraries for isolated RNA samples were prepared using an Illumina TruSeq RNA library construction kit and sequenced on an Illumina HiSeq 2500 platform. Sequencing reads were aligned against the human reference genome GRCh38 using STAR RNA-seq aligner and annotated with GENCODE v24 at the gene level. Expression counts were estimated using RSEM software and converted to counts per million for subsequent analyses using the edgeR package. Cox proportional hazard regression analysis with LASSO was used to model the relationship between patients' baseline gene expression and PFS. A classifier was established and its predictive performance was assessed using the cross-validation scheme outlined by Simon et al (Brief Bioinform . 2011;12:203-214). The statistical significance of the cross-validated Kaplan-Meier curves and corresponding log-rank statistic was estimated by generating an approximate null distribution of the cross-validated log-rank statistic through 500 random permutations. For each permutation, the patients' baseline gene expression profiles and treatment assignments were randomly re-shuffled against patients' survival times and event indicators, and the same cross-validation procedures used in the model performance assessment were repeated to compute the cross-validated log-rank statistic for the permuted data. Results: Among the 303 Kd56 or Vd patients included in this biomarker study, patients in the Kd56 arm had a 58% reduced risk of progression or death compared with patients in the Vd arm (hazard ratio [HR]: 0.42; 95% confidence interval [CI]: 0.30-0.59; P= 4.5 x 10-7). We developed a linearized classifier using patients' baseline gene expression (n = 303) to stratify patients for PFS benefit from Kd56 or Vd therapy. The cross-validated Kaplan-Meier curves and log-rank statistic for the classifier were statistically significant at P < 0.001. A 13-gene classifier derived from the whole data set could separate patients from the Kd56 arm (n = 155) into two distinct subgroups, in which one with 113 (73%) patients had a PFS benefit over the other with 42 (27%) patients (HR: 0.13; 95% CI: 0.06-0.26; P= 3.3 x 10-13). When these 42 patients were excluded from the Kd56 arm, the PFS benefit for the Kd56 arm (n = 113) over the Vd arm (n = 148) was improved by 52% (HR: 0.20; 95% CI: 0.12-0.31; P= 2.0 x 10-14). The classifier was unable to stratify patients in the Vd arm for high or low PFS benefit. The 13 genes included in the classifier were ACOXL, CLEC2B, CLIP4, COCH, FRK, IGHD, ITPRIPL2, NAP1L5, RNASE6, SH3RF3, SHROOM3, TCF7, and UGT3A2 . Several genes in this classifier, including CLIP4, IGHD, and SH3RF3, have been previously implicated in myeloma biology and in vitroresistance to proteasome inhibitors. Individually, each gene showed similar ability to stratify patients from the Kd56 arm, but the cross-validated Kaplan-Meier curves for the individual genes were not significant at P < 0.05. Conclusions: We identified a classifier with a set of genes whose baseline expression could potentially be used to stratify RRMM patients for greater treatment benefit with Kd56. As only one patient cohort was used for this study, the classifier identified here should be validated in prospective studies and with independent sets of patient cohorts. Further study of this group of genes may provide additional insights into the biology of multiple myeloma and how mechanism of action differs between carfilzomib and bortezomib. Disclosures Pelham: Amgen: Employment, Equity Ownership. Hu: Amgen: Employment, Equity Ownership. Moreau: Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Janssen: Consultancy, Honoraria; Celgene, Janssen, Takeda, Novartis, Amgen, Roche: Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceutical: Consultancy, Honoraria. Oriol: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia, Speakers Bureau; Celgene: Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored symposia, Speakers Bureau. Quach: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Takeda: Honoraria. Kovacsovics: Seattle Genetics: Research Funding; Celgene: Consultancy; Flexus: Research Funding. Keats: Amgen: Research Funding. Feng: Amgen: Employment, Equity Ownership. Kimball: Amgen: Employment, Equity Ownership. Dimopoulos: Novartis: Consultancy, Honoraria; Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology: Consultancy, Honoraria, Other: Advisory Committee: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology; Genesis Pharma: Research Funding.

Pathogenic gene fusions have been identified in several histologic types of salivary gland neoplasia, but not previously in acinic cell carcinoma (AcCC). To discover novel gene fusions, we performed whole-transcriptome sequencing surveys of three AcCC archival cases. In one specimen we identified a novel HTN3-MSANTD3 gene fusion, and in another a novel PRB3-ZNF217 gene fusion. The structure of both fusions was consistent with the promoter of the 5’ partner (HTN3 or PRB3), both highly expressed salivary gland genes, driving overexpression of full-length MSANTD3 or ZNF217. By fluorescence in situ hybridization of an expanded AcCC case series, we observed MSANTD3 rearrangements altogether in 3 of 20 evaluable cases (15%), but found no additional ZNF217 rearrangements. MSANTD3 encodes a previously uncharacterized Myb/SANT domain-containing protein. Immunohistochemical staining demonstrated diffuse nuclear MSANTD3 expression in 8 of 27 AcCC cases (30%), including the three cases with MSANTD3 rearrangement. MSANTD3 displayed heterogeneous expression in normal salivary ductal epithelium, as well as among other histologic types of salivary gland cancer though without evidence of translocation. In a broader survey, MSANTD3 showed variable expression across a wide range of normal and neoplastic human tissue specimens. In preliminary functional studies, engineered MSANTD3 overexpression in rodent salivary gland epithelial cells did not enhance cell proliferation, but led to significant upregulation of gene sets involved in protein synthesis. Our findings newly identify MSANTD3 rearrangement as a recurrent event in salivary gland AcCC, providing new insight into disease pathogenesis, and identifying a putative novel human oncogene.

Introduction: In the randomized phase 3 ENDEAVOR study, treatment with carfilzomib (56 mg/m2) and dexamethasone (Kd56) resulted in a superior progression-free survival (PFS; median 18.7 vs 9.4 months, respectively; hazard ratio (HR), 0.53; 95% confidence interval (CI), 0.44-0.65; one-sided P Methods: Patients in the Kd56 arm received intravenous (IV) carfilzomib as a 30-min infusion on days (D) 1, 2, 8, 9, 15, and 16 of 28-day cycles (20 mg/m2 on D1 and 2 of cycle 1 [C1]; 56 mg/m2 thereafter) and dexamethasone (20 mg) was given on D1, 2, 8, 9, 15, 16, 22, and 23. In the Vd arm, bortezomib (1.3 mg/m2; IV or subcutaneous administration) was given on D1, 4, 8, and 11 and dexamethasone (20 mg) was given on D1, 2, 4, 5, 8, 9, 11, and 12 of a 21-day cycle. Patients in both arms received treatment until withdrawal of consent, progression of disease, or unacceptable toxicity. Population PK and PDn were evaluated as exploratory endpoints. Blood for plasma PK assessments was collected from a subset of 133 Kd56 patients and analyzed by population PK analysis. PDn in the form of proteasome inhibition was measured in whole blood and isolated peripheral blood mononuclear cells (PBMCs) collected from 8 Kd56- and 9 Vd-treated patients at select sites during C1, 2, 3, 5, and 7, using LLVY-AMC as a substrate to measure chymotrypsin-like (CT-L) proteasome activity (Lightcap ES, et al. Clin Chem . 2000;46:673-83). Results: At the 56 mg/m2 dose, carfilzomib was eliminated with a half-life of approximately 1 hr; clearance at 56 mg/m2 was similar to that observed at 27 mg/m2 and 70 mg/m2 and was not impacted by infusion duration (10 min vs 30 min). Treatment with Kd56 resulted in reduction of CT-L activity in whole blood to undetectable levels by 1 hr post-dose on C1D8 (Figure). The extent of proteasome inhibition was greater at all time points for Kd56 vs Vd. Maximal inhibition of the activity of the β5 subunit in whole blood was higher in Kd56-treated pts (100% of C1D1 pre-dose activity) vs Vd-treated pts (74%). Although CT-L activity returned to pre-treatment levels prior to Kd56 dosing on C2D1 and C3D1, activity was reduced to undetectable levels post-dose on these days. Substantial recovery of β5 subunit activity in whole blood was observed in Vd-treated patients at the start of cycles 3, 5, and 7. In contrast, sustained inhibition of >50% was observed in the whole blood of Kd56-treated patients. A limitation of these data is that whole blood is composed primarily of anucleated cells. Prolonged and deeper suppression of proteasome activity by Kd56 compared with Vd demonstrated using whole blood LLVY-AMC methodology was also supported by PBMC assays. Conclusion: Carfilzomib induced a deep and prolonged proteasome inhibition compared with bortezomib. The proteasome inhibition profile of carfilzomib may be consistent with superiority in PFS and OS demonstrated by Kd56 relative to Vd. Figure. Comparison of CT-L proteasome activity between Kd56- and Vd- treated patients in C1, 3, 5, and 7 Disclosures Ludwig: Bristol-Meyers: Speakers Bureau; Celgene: Speakers Bureau; Janssen-Cilag: Consultancy, Speakers Bureau; AMGEN: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau. Spencer: Amgen: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kovacsovics: Seattle Genetics: Research Funding; Celgene: Consultancy; Flexus: Research Funding. Minuk: Celgene, Janssen, Amgen, BMS: Other: Personal fees, Research Funding. Kimball: Amgen: Employment, Equity Ownership. Pelham: Amgen Inc.: Employment, Equity Ownership. Ou: Amgen Inc.: Employment, Equity Ownership. Orlowski: BioTheryX: Consultancy, Membership on an entity9s Board of Directors or advisory committees.

BACKGROUND:The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS:Three stage I and one stage II primary NSCLC tumors were subjected to multiregion whole-exome sequencing (WES) and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants (SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing (mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained before surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers. RESULTS:Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R(2) = 0.8722, P < 0.0001). Sixteen (43%) out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) range of 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R(2) = 0.5144; P = 0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6 copies). CONCLUSIONS:The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was able to detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required to define its potential use in clinical practice.

We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.

Nat. Genet. 46, 722–725 (2014); published online 25 May 2014; corrected after print 12 November 2014 In the version of this article initially published, the introductory paragraph mistakenly stated that ameloblasts were “cells in the tooth roots of the upper (maxilla) and lower (mandible) jaw responsible for depositing enamel during tooth development (odontogenesis).

Abstract Genomic instability, the hallmark of cancer, presents with a variety of mutation types, most commonly single nucleotide variations (SNVs) and copy number variations (CNVs), which traditionally have required different methods for identification. It has proven challenging to simultaneously achieve sufficient breadth to detect CNVs and depth to detect SNVs on samples of limited input amount. The objective of this study was to validate a new methodology for detection of SNVs and CNVs in a single assay. We used a massively multiplex PCR/NGS approach combining an SNV panel covering 585 point mutation hotspots in breast cancer (Cosmic) and a CNV panel targeting 28,000 SNPs designed to detect copy number at chromosomes 1, 2, 13, 18, 21, and X, and focal regions 4p16, 5p15, 7q11, 15q, 17p, 22q11, and 22q13. We applied these panels to breast cancer cell lines and fresh frozen (FF) breast tumor samples; the presence of CNVs in circulating cell-free tumor DNA (ctDNA) in the plasma of breast cancer patients was also investigated. The CNV assay methodology was validated using genomic DNA isolated from 96 human samples with known karyotype; sensitivity to single region deletions or duplications was 100% (71/71) and specificity was 100% for normal regions in the same samples. Single-molecule sensitivity for the detection of CNVs was established by analyzing isolated single cells. Performance of the mutation assay was demonstrated with the analysis of 5 matched tumor and normal cell lines, with 24 out of 27 SNVs known to be present in these cell lines detected. The 3 undetected SNVs were determined to be a result of assay design failure. Also, multiple somatic CNVs (median: 13) were detected in all 5 tumor cell lines. Analysis of the normal cell lines found no cancer related SNVs or CNVs. In 32 FF tumor samples, 78.1% (25/32) had SNVs detected; of samples with SNVs, 88% (22/25) had SNVs in TP53 or PIK3CA. Of the same 32 FF breast tumor samples, 96.9% (31/32) showed full or partial CNVs in at least 1 and up to 15 regions; of the 31 samples with detected CNVs, 93.5% had a CNV of either 1q or 17p, two of the three most prevalent breast cancer CNVs (the 16q region was not represented in this panel). Overall, a combination of SNV and CNV testing allowed identification of genetic changes in 100% of the breast tumor samples, a significant improvement in diagnostic yield than using SNV detection alone. Of the 12 breast cancer patients with matched tumor tissue and plasma samples, 83.3% (10/12) had CNVs detected in tissue. The CNVs present in each primary tumor sample were identified in corresponding plasma ctDNA samples (1 stage IIa, 7 stage IIb, and 2 stage III). The ctDNA fractions in these samples ranged from 0.58 to 4.33%; detection required as few as 86 heterozygous SNPs per CNV. Analysis of ctDNA for cancer-associated mutations may allow earlier, safer and more accurate profiling and monitoring of breast cancer. Thus, this targeted PCR approach offers the promise of an assay able to detect both cancer-associated SNVs and CNVs in the same sample with good sensitivity and specificity, and improved detection rates compared to assays that only detect SNVs. Citation Format: Robert J Pelham, Bernhard G Zimmermann, Eser Kirkizlar, Ryan K Swenerton, Bin Hoang, Onur Sakarya, Joshua E Babiarz, Nicholas Wayham, Tudor Constantin, Styrmir Sigurjonsson, Matthew Rabinowitz, Matthew Hill. Detection of single nucleotide variations and copy number variations in breast cancer tissue and ctDNA samples using single-nucleotide polymorphism-targeted massively multiplexed PCR [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-03.

Rare cancer types are not only understudied, but are typically represented by formalin‐fixed paraffin‐embedded (FFPE) (rather than freshly‐frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non‐surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole‐transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 22 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog pathway mutation confirmed increased basal pathway activity, and defined the response profile to various pharmacologic Hedgehog inhibitors. Together, our results define new ameloblastoma drivers and nominate new molecularly‐directed therapies for this rare but disfiguring disease. More generally, our findings validate a robust approach for discovering driver mutations in rare cancers.Grant Funding Source: Supported by Stanford University Department of Pathology

Background: Prostate tumor heterogeneity and biopsy undersampling pose challenges to accurate, individualized risk assessment for men with localized disease.Objective: To identify and validate a biopsy-based gene expression signature that predicts clinical recurrence, prostate cancer (PCa) death, and adverse pathology.Design, setting, and participants: Gene expression was quantified by reverse transcription- polymerase chain reaction for three studies-a discovery prostatectomy study (n = 441), a biopsy study (n = 167), and a prospectively designed, independent clinical validation study (n = 395)-testing retrospectively collected needle biopsies from contemporary (1997-2011) patients with low to intermediate clinical risk who were candidates for active surveillance (AS).Outcome measures and statistical analysis: The main outcome measures defining aggressive PCa were clinical recurrence, PCa death, and adverse pathology at prostatectomy. Cox proportional hazards regression models were used to evaluate the association between gene expression and time to event end points. Results from the prostatectomy and biopsy studies were used to develop and lock a multigene-expression-based signature, called the Genomic Prostate Score (GPS); in the validation study, logistic regression was used to test the association between the GPS and pathologic stage and grade at prostatectomy. Decision-curve analysis and risk profiles were used together with clinical and pathologic characteristics to evaluate clinical utility.Results and limitations: Of the 732 candidate genes analyzed, 288 (39%) were found to predict clinical recurrence despite heterogeneity and multifocality, and 198 (27%) were predictive of aggressive disease after adjustment for prostate-specific antigen, Gleason score, and clinical stage. Further analysis identified 17 genes representing multiple biological pathways that were combined into the GPS algorithm. In the validation study, GPS predicted high-grade (odds ratio [OR] per 20 GPS units: 2.3; 95% confidence interval [CI], 1.5-3.7; p < 0.001) and high-stage (OR per 20 GPS units: 1.9; 95% CI, 1.3-3.0; p = 0.003) at surgical pathology. GPS predicted high-grade and/ or high-stage disease after controlling for established clinical factors (p < 0.005) such as anOR of 2.1 (95% CI, 1.4-3.2) when adjusting for Cancer of the Prostate Risk Assessment score. A limitation of the validation study was the inclusion of men with low-volume intermediate-risk PCa (Gleason score 3 + 4), for whom some providers would not consider AS.Conclusions: Genes representing multiple biological pathways discriminate PCa aggressiveness in biopsy tissue despite tumor heterogeneity, multifocality, and limited sampling at time of biopsy. The biopsy-based 17-gene GPS improves prediction of the presence or absence of adverse pathology and may help men with PCa make more informed decisions between AS and immediate treatment.Patient summary: Prostate cancer (PCa) is often present in multiple locations within the prostate and has variable characteristics. We identified genes with expression associated with aggressive PCa to develop a biopsy-based, multigene signature, the Genomic Prostate Score (GPS). GPS was validated for its ability to predict men who have high-grade or highstage PCa at diagnosis and may help men diagnosed with PCa decide between active surveillance and immediate definitive treatment. (C) 2014 European Association of Urology. Published by Elsevier B. V. All rights reserved.