BackgroundNeomorphic isocitrate dehydrogenase (IDH) mutations lead to the accumulation of 2-hydroxyglutarate (2-HG), an oncometabolite implicated in tumor progression via inhibitory effects on alpha-ketoglutarate. Moreover, mutant IDH-dependent accumulation of 2-HG results in homologous recombination deficiency (HRD), which preclinically renders tumors sensitive to poly(adenosine diphosphate ribose) polymerase inhibitors. Here, the results of the cholangiocarcinoma (CCA) arm of the National Cancer Institute (NCI) 10129 olaparib in IDH-mutant solid tumors basket trial are reported.MethodsOlaparib 300 mg twice daily was evaluated in an open-label, phase 2 clinical trial for treatment-refractory IDH-mutant solid tumors. Patients in the IDH-mutant CCA arm enrolled in two cohorts: (1) IDH inhibitor (IDHi) pretreated and (2) IDHi untreated, with a primary end point of overall response rate.ResultsNCI 10129 enrolled 30 patients with IDH-mutant CCA with no objective responses seen, and recruitment was closed early. Median progression-free survival (PFS) was 2.4 months (95% CI, 1.9 to 6.5 months) and median overall survival was 12.9 months (95% CI, 6.3 months to not reached). Eight patients (27%) had clinical benefit (CB), with a PFS of >= 6 months. Patients with CB had lower baseline 2-HG levels compared to those without CB (1.4 vs. 5.9 mu mol/L; p = .01).ConclusionsOlaparib does not have sufficient single-agent activity to warrant further development in IDH-mutant CCA. However, a subgroup of patients demonstrated CB, and exploratory analysis revealed this subgroup to be enriched for lower baseline 2-HG levels. Future clinical trials leveraging the HRD properties of IDH mutations are warranted with enhanced patient selection and novel combination therapies.

Detecting somatic mutations in normal tissues is challenging due to sequencing errors and the low allele fractions of post-zygotic variants. Duplex sequencing greatly reduces errors and can detect mutations at any allele fraction, but systematic, cross-platform comparisons are lacking. We present a comprehensive benchmarking of six duplex sequencing technologies used by the SMaHT Network: CODEC, CompDuplex-seq, HiDEF-seq, NanoSeq, ppmSeq, and VISTA-seq. We evaluated their performance using cord blood DNA, a tumor-normal cell line mixture, and homogenates from six human tissues. Each method shows distinct profiles in genomic footprint, sensitivity, and cost. Despite differences in library construction and sequencing platforms, estimates of mutation rates and mutational signatures are highly concordant. Integration with ultra-deep whole-genome sequencing shows that duplex approaches sensitively capture mutations and signatures beyond embryonic or clonally expanded variants. These results provide a foundation for selecting duplex methods and interpreting their data, enabling scalable single-molecule analyses of somatic mutation landscapes.

PURPOSECell-free circulating tumor DNA (ctDNA) has shown its potential as a quantitative biomarker for longitudinal monitoring of response to anticancer therapies. However, ctDNA dynamics have not been studied in patients with heavily pretreated, advanced solid tumors, for whom therapeutic responses can be weak. We investigated whether changes in ctDNA could predict clinical outcomes in such a cohort treated with combined poly(ADP-ribose) polymerase/vascular endothelial growth factor receptor inhibitor therapy.MATERIALS AND METHODSPatients with metastatic pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer (TNBC), small-cell lung cancer (SCLC), or non-small-cell lung cancer (NSCLC) received up to 7 days of cediranib 30 mg orally once daily monotherapy lead-in followed by addition of olaparib 200 mg orally twice daily. Patients had progressed on a median of three previous lines of therapy. Plasma samples were collected before and after cediranib monotherapy lead-in and on combination therapy at 7 days, 28 days, and every 28 days thereafter. ctDNA was quantified from plasma samples using a multigene mutation-based assay. Radiographic assessment was performed every 8 weeks.RESULTSctDNA measurements were evaluable in 63 patients. The median baseline ctDNA variant allele fractions (VAFs) were 20%, 28%, 27%, and 34% for PDAC, TNBC, SCLC, and NSCLC, respectively. No association was observed between baseline VAF and radiographic response, progression-free survival, or overall survival (OS). Similarly, no association was found between ctDNA decline and radiographic response or survival. However, an increase in ctDNA at 56 days of combination therapy was associated with disease progression and inferior OS in a landmark analysis.CONCLUSIONctDNA levels or dynamics did not correlate with radiographic response or survival outcomes in patients with advanced metastatic malignancies treated with olaparib and cediranib.

Characterization of the Osimertinib resistant H1975 cells.

PDF file - 76K, Scheme for appending modular barcodes to gene-specific primers

Table of Mutant Allele Fractions

Detailed Individual Characteristics of 49 Enrolled Patients

PDF file - 63K, ctDNA levels in patients with fewer than 3 time-points

Apoptosis susceptibility of KRAS mutant A549 cell line following treatment with NGI-1, Erlotinib or both.

Legends for supplementary figures 1 and 2

Deep sequencing of lung cancer cell lines to determine the proportion of EGFR mutations.

Results of the Lung Cancer Cell Line Screen for OST Inhibitor Sensitivity

PURPOSE Discordant responses between brain metastases and extracranial tumors can arise from branched tumor evolution, underscoring the importance of profiling mutations to optimize therapy. However, the morbidity of brain biopsies limits their use. We investigated whether cell-free DNA (cfDNA) in CSF could serve as an effective surrogate marker for genomic profiling of intraparenchymal (IP) brain metastases. METHODS CSF and blood were collected simultaneously from patients with progressive brain metastases undergoing a craniotomy or lumbar puncture. Mutations in both biofluids were measured using an error-suppressed deep sequencing method previously published by our group. Forty-three regions of 24 cancer-associated genes were assayed. RESULTS This study enrolled 14 patients with either IP brain metastases (n = 12) or cytology-positive leptomeningeal disease (LMD, n = 2) and two controls with normal pressure hydrocephalus. Primary cancer types were lung, melanoma, renal cell, and colorectal. cfDNA was measurable in all sixteen samples of CSF. Cancer-associated mutations were found in the CSF of ten patients (eight with IP [67%] and two with LMD [100%]) and plasma of five patients (five with IP [42%] and none with LMD). All patients with plasma cfDNA had extracranial tumors. Among the five patients in the cohort who also had mutation data from time-matched brain metastasis tissue, four patients (80%) had matching mutations detected in CSF and brain, whereas only one patient (20%) had matching mutations detected in plasma and brain. CONCLUSION The detection of mutational DNA in CSF is not restricted to LMD and was found in two thirds of patients with IP brain metastases in our cohort. Analysis of CSF can be a viable alternative to biopsy for detection of somatic mutations in brain metastases.

3035 Background: Circulating tumor DNA (ctDNA) has emerged as a potential biomarker to monitor treatment response in solid tumors. Our group previously showed that changes in ctDNA levels were predictive of radiographic response and survival in NSCLC patients receiving immunotherapy. Here we evaluated whether ctDNA dynamics could similarly be used to assess response in a PARP inhibitor-based therapy. Methods: A total of 122 patients with NSCLC, TNBC, PDAC or SCLC received cediranib (C) 30mg daily and Olaparib (O) 200mg twice daily in a phase II study NCI9881. Using a multigene NGS assay, ctDNA was measured longitudinally at baseline (T 0 ), after 3 to 7 days of C monotherapy (T 1 ), after 1 week of C+O combination (T 2 ), after 4 weeks of C+O (T 3 ), and every 4 weeks (T 4+ ) thereafter. The first radiographic assessment was done after 8 weeks of C+O and every 8-12 weeks thereafter. CtDNA was quantified by determining the allele fraction of cancer-associated somatic mutations in plasma. We defined an early ctDNA response (e-ctDNA-R) as a >10% decrease in mutant allele fraction from T 0 to T 2 , and an early ctDNA progression (e-ctDNA-P) as a > 10% increase; otherwise, it was stable ctDNA (e-ctDNA-S). Results: In total, 493 samples were analyzed from 94 patients, and 40 unique patients had both T 0 and T 2 ctDNA measurements, as well as corresponding radiographic assessments. These included 10 NSCLC, 17 TNBC, 3 SCLC and 10 PDAC. Of these patients, 4, 21, and 15 patients had PR, SD, and PD as best overall radiographic response respectively. Twenty-three (57.5%) patients had either e-ctDNA-R (17, 42.5%) or e-ctDNA-S (6, 15.0%), 18 (78.3%) of whom subsequently had either radiographic partial response (PR) or stable disease (SD). Seventeen (42.5%) patients had e-ctDNA-P, 10 (58.8%) of whom then had PD. A fair agreement was observed between e-ctDNA-R/S or e-ctDNA-P and radiographic PR/SD or PD with Cohen’s k 0.38 (70% agreement). The correlation between early ctDNA changes and PFS/OS are summarized in the table. All 25 patients with PR/SD eventually progressed. Of these, 17 (68%) had >10% increase in ctDNA from the nadir prior to disease progression (median 94.6%, 95%CI 38.2%-389.1%). The time between ctDNA progression and ctDNA nadir was significantly shorter (median 21 days, 95%CI 21-28) than the time between radiographic/clinical progression and initial PR/SD (median 107 days, 95%CI 56-204, P=0.0015). Conclusions: Longitudinal ctDNA measurements could enable early assessment of treatment response, resistance, and disease progression in patients treated with PARP inhibitor-based therapy. However, in this study, tumor responses and ctDNA changes were generally not as robust as have been observed with other classes of therapy.[Table: see text]

Abstract INTRODUCTION Discordant response between brain and systemic metastases occur in patients receiving targeted therapies and repeat tumor profiling of the progressing site could guide further therapy. We propose that circulating tumor DNA (ctDNA) might be detectable in the cerebrospinal fluid (CSF) and reflective of the genetic profile of intraparenchymal brain metastases. METHODS Patients with brain metastases undergoing a craniotomy or lumbar puncture were enrolled between July 2018 to April 2019 under an IRB-approved protocol. CSF and blood were collected simultaneously. Cell-free DNA (cfDNA) were extracted and ctDNA were identified and quantified using an Error-Suppressed Deep Sequencing method previously published by our group. Forty-three mutation-prone regions of 24 cancer-associated genes were assayed, and the allelic fractions were calculated against wild-type sequence counts. RESULTS Sixteen patients were enrolled in this study - 12 patients with intraparenchymal brain metastases, two patients with CSF cytology-positive leptomeningeal disease (LMD) and 2 patients with normal pressure hydrocephalus (NPH) as controls. Primary cancer types were lung (n=10), melanoma (n=2), renal cell (n=1) and colorectal (n=1) cancers. cfDNA was found in all sixteen samples of CSF. CSF ctDNA were found in eight patients (67%) and plasma ctDNA were only found in five patients (42%) with intraparenchymal tumors. In six patients with additional time-matched brain metastasis tissue, four were found to have congruent mutations in the CSF, while only one harbored such mutation in the plasma. DISCUSSION Analysis of CSF can be a viable alternative to obtaining brain metastasis tissue for DNA profiling in the detection of novel and resistance mutations. The presence CSF ctDNA is not restricted to LMD and were isolated from two-thirds of patients with intraparenchymal disease in our cohort. Furthermore, CSF remains a better source than plasma for the detection of ctDNA across multiple brain metastases tumor subtypes.

INTRODUCTION: Cell-free mutational deoxyribonucleic acid (DNA) has been shown to be detectable in the cerebrospinal fluid (CSF) of patients with intra-axial gliomas. The same might be possible in brain metastasis (BrM). Identification of tumor-specific DNA in the CSF and correlation of these findings with CSF cytokine profiles might enable minimally invasive monitoring of treatment response and failure, which is particularly important in the era of targeted and immunotherapies where radiographic changes are becoming increasingly difficult to interpret. METHODS: BrM patients were consented under an Institutional Review Board-approved protocol. Circulating tumor DNA (ctDNA) and secreted cytokines were profiled in the CSF and plasma from 31 patients. To isolate ctDNA, a novel method was developed, which applies molecular tagging and computational error-suppression techniques to next-generation DNA sequencing data. Concentrations of 42 cytokines and chemokines in the CSF and plasma were quantified by Luminex (Eve Technologies). Nineteen patients had concurrent tissue pathology. RESULTS: Of the 31 patients with BrM, most had lung cancer, melanoma, and breast cancer. Preliminary analysis detected secreted cytokines in CSF and plasma in all three cancer types. As little as 5 mL and 75 μL of CSF are required for genomic and proteomic analysis, respectively. To date, cell-free DNA was found in 78% of CSF samples analyzed (N = 18), with 45% harboring mutational DNA. Notably, all patients with cytology-positive leptomeningeal disease (N = 4) also had driver mutations in their CSF. Of the 42 cytokines profiled, 31 were reliably detected in CSF and plasma across multiple primary subtypes, tumor volumes, and extent of disease. CONCLUSION: ctDNA and cytokines can be detected in CSF of patients with both intraparenchymal and leptomeningeal disease. Further analysis of the biomarker differential between CSF and plasma may assist in diagnosis and understanding of the immune response to BrM.

META RNA profiling is a simple and inexpensive method to measure the expression of multiple targeted RNAs across many samples. By assigning sample-specific tags up-front during reverse-transcription, cDNAs from multiple samples can be pooled prior to amplification and deep sequencing. Such early parallelization of samples simplifies the workflow, minimizes cross-sample experimental variability, and reduces reagent and sequencing costs. Herein we describe the theoretical framework of the method and provide a detailed protocol to facilitate its implementation.

Abstract Asparagine (N)-linked glycosylation is a posttranslational modification essential for the function of complex transmembrane proteins. However, targeting glycosylation for cancer therapy has not been feasible due to generalized effects on all glycoproteins. Here, we perform sensitivity screening of 94 lung cancer cell lines using NGI-1, a small-molecule inhibitor of the oligosaccharyltransferase (OST) that partially disrupts N-linked glycosylation, and demonstrate a selective loss of tumor cell viability. This screen revealed NGI-1 sensitivity in just 11 of 94 (12%) cell lines, with a significant correlation between OST and EGFR inhibitors. In EGFR-mutant non-small cell lung cancer with EGFR tyrosine kinase inhibitor (TKI) resistance (PC9-GR, HCC827-GR, and H1975-OR), OST inhibition maintained its ability to induce cell-cycle arrest and a proliferative block. Addition of NGI-1 to EGFR TKI treatment was synthetic lethal in cells resistant to gefitinib, erlotinib, or osimertinib. OST inhibition invariably disrupted EGFR N-linked glycosylation and reduced activation of receptors either with or without the T790M TKI resistance mutation. OST inhibition also dissociated EGFR signaling from other coexpressed receptors like MET via altered receptor compartmentalization. Translation of this approach to preclinical models was accomplished through synthesis and delivery of NGI-1 nanoparticles, confirmation of in vivo activity through molecular imaging, and demonstration of significant tumor growth delay in TKI-resistant HCC827 and H1975 xenografts. This therapeutic strategy breaks from kinase-targeted approaches and validates N-linked glycosylation as an effective target in tumors driven by glycoprotein signaling. Significance: EGFR-mutant NSCLC is incurable despite the marked sensitivity of these tumors to EGFR TKIs. These findings identify N-linked glycosylation, a posttranslational modification common to EGFR and other oncogenic signaling proteins, as an effective therapeutic target that enhances tumor responses for EGFR-mutant NSCLC. Cancer Res; 78(17); 5094–106. ©2018 AACR.