To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility.
Analytic validation was ...conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples.
Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity prevalence-adjusted positive predictive values (PPV) >98%. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker.
High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery.
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(HER2) amplification is an emerging biomarker in colon cancer, conferring sensitivity to combination anti-HER2 therapy. Measurement of HER2 copy number is typically performed using surgical ...specimens, but cell-free circulating tumor DNA (ctDNA) analysis may be a noninvasive alternative. We determined the sensitivity of plasma copy number (pCN) for detecting
amplifications and whether pCN correlated with tissue-detected copy number. We also assessed response to HER2-targeted therapy based on pCN and suggest a pCN threshold predictive of response.
Forty-eight pretreatment and progression plasma samples from 29 HER2-positive patients in the HERACLES A clinical trial were tested using the Guardant360 cfDNA assay. We correlated
pCN with progression-free survival (PFS) and best objective response (BOR) and applied an adjustment method based on tumor DNA shedding using the maximum mutant allele fraction as a surrogate for tumor content to accurately determine the pCN threshold predictive of response.
Forty-seven of 48 samples had detectable ctDNA, and 46 of 47 samples were
-amplified on the basis of cfDNA 2.55-122 copies; 97.9% sensitivity (95% confidence interval, 87.2%-99.8%). An adjusted
pCN of ≥25.82 copies correlated with BOR and PFS (
= 0.0347).
cfDNA is a viable alternative to tissue-based genotyping in the metastatic setting. The cfDNA platform utilized correctly identified 28 of 29 (96.6%) of pretreatment samples as
-amplified and predicted benefit from HER2-targeted therapy. In this study, an observed pCN of 2.4 and an adjusted pCN of 25.82 copies of
are proposed to select patients who will benefit from HER2-targeted therapy.
Addition of trastuzumab to first-line chemotherapy improves overall survival in patients with HER2-positive metastatic gastric cancer. We assessed the safety and activity of pembrolizumab in ...combination with trastuzumab and chemotherapy in first-line HER2-positive metastatic oesophagogastric (gastric, oesophageal, or gastroesophageal junction) cancer.
This study was an investigator-initiated, open-label, non-randomised, single-arm, single centre, phase 2 trial in patients aged 18 years or older with HER2-positive metastatic oesophagogastric cancer. Eligible patients had measurable or evaluable non-measurable disease, Eastern Cooperative Oncology Group performance status of 0, 1, or 2, and left ventricular ejection fraction of at least 53%. Patients were eligible to receive an initial induction cycle of 200 mg flat dose of intravenous pembrolizumab and 8 mg/kg loading dose of intravenous trastuzumab. For subsequent cycles, patients received 130 mg/m2 of intravenous oxaliplatin or 80 mg/m2 of cisplatin on day 1, 850 mg/m2 of oral capecitabine twice a day for 2 weeks followed by 1 week off (or intravenous 5-fluorouracil, 800 mg/m2 per day on days 1–5), and a 200 mg flat dose of intravenous pembrolizumab, and 6 mg/kg of trastuzumab, administered on day 1 of each 3-week cycle. The primary endpoint was 6-month progression-free survival, defined as the proportion of patients alive and free of progression at 6 months, assessed in patients who received at least one dose of trastuzumab and pembrolizumab. The regimen would be considered worthy of further investigation if 26 or more of 37 patients were progression-free at 6 months. This trial is registered with ClinicalTrials.gov, NCT02954536, and is ongoing, but closed to enrolment.
Between Nov 11, 2016, and Jan 23, 2019, 37 patients were enrolled. At the time of data cutoff on Aug 6, 2019, median follow-up among survivors was 13·0 months (IQR 11·7–23·5). The primary endpoint was achieved; 26 (70%; 95% CI 54–83) of 37 patients were progression-free at 6 months. The most common treatment-related adverse event of any grade was neuropathy, which was reported in 36 (97%) of 37 patients. The most common grade 3 or 4 adverse events were lymphocytopenia (seven 19% patients with grade 3 and two 5% with grade 4), grade 3 decreased electrolytes (six 16% patients), and grade 3 anaemia (four 11% patients). Serious adverse events occurred in two patients patients (both grade 3 nephritis leading to treatment discontinuation). Four patients discontinued pembrolizumab because of immune-related adverse events. There were no treatment-related deaths.
Pembrolizumab can be safely combined with trastuzumab and chemotherapy and has promising activity in HER2-positive metastatic oesophagogastric cancer. A randomised phase 3 clinical trial assessing the efficacy and safety of pembrolizumab versus placebo in combination with trastuzumab and chemotherapy in first-line HER2-positive metastatic oesophagogastric cancer is underway.
Merck & Co.
Cell-free DNA (cfDNA) sequencing provides a noninvasive method for obtaining actionable genomic information to guide personalized cancer treatment, but the presence of multiple alterations in ...circulation related to treatment and tumor heterogeneity complicate the interpretation of the observed variants.
We describe the somatic mutation landscape of 70 cancer genes from cfDNA deep-sequencing analysis of 21,807 patients with treated, late-stage cancers across >50 cancer types. To facilitate interpretation of the genomic complexity of circulating tumor DNA in advanced, treated cancer patients, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality.
Patterns and prevalence of cfDNA alterations in major driver genes for non-small cell lung, breast, and colorectal cancer largely recapitulated those from tumor tissue sequencing compendia (The Cancer Genome Atlas and COSMIC;
= 0.90-0.99), with the principal differences in alteration prevalence being due to patient treatment. This highly sensitive cfDNA sequencing assay revealed numerous subclonal tumor-derived alterations, expected as a result of clonal evolution, but leading to an apparent departure from mutual exclusivity in treatment-naïve tumors. Upon applying novel cfDNA clonality and copy-number driver identification methods, robust mutual exclusivity was observed among predicted truncal driver cfDNA alterations (FDR = 5 × 10
for
and
), in effect distinguishing tumor-initiating alterations from secondary alterations. Treatment-associated resistance, including both novel alterations and parallel evolution, was common in the cfDNA cohort and was enriched in patients with targetable driver alterations (>18.6% patients).
Together, these retrospective analyses of a large cfDNA sequencing data set reveal subclonal structures and emerging resistance in advanced solid tumors.
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Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells ...and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.
The present study investigated the role of adaptability in helping high school students navigate their online learning during a period of COVID-19 that entailed fully or partially remote online ...learning. Drawing on Job Demands-Resources theory and data from a sample of 1,548 Australian high school students in nine schools, we examined the role of adaptability in predicting students’ online learning self-efficacy in mathematics and their end of year mathematics achievement. It was found that beyond the effects of online learning demands, online and parental learning support, and background attributes, adaptability was significantly associated with higher levels of online learning self-efficacy and with gains in later achievement; online learning self-efficacy was also significantly associated with gains in achievement—and significantly mediated the relationship between adaptability and achievement. These findings confirm the role of adaptability as an important personal resource that can help students in their online learning, including through periods of remote instruction, such as during COVID-19.
Conflation over motivation and engagement has historically impeded research and practice. One reason for this is because definition and measurement have often been too general or diffuse-especially ...in the case of engagement. Recently conceptual advances aimed at disentangling facets of engagement and motivation have highlighted a need for better psychometric precision-particularly in the case of engagement. To the extent that engagement is inadequately assessed, motivation research involving engagement continues to be hampered. The present study investigates multidimensional effort (a specific facet of engagement) and how it relates to motivation. In particular, we examine the associations between specific positive and negative motivation factors and dimensions of effort, thereby shedding further insight into how different types of motivation interplay with different types of engagement. Drawing on data from a sample of 946 Australian high school students in 59 mathematics classrooms at five schools, this study hypothesized a tripartite model of academic effort in terms of operative, cognitive, and social-emotional dimensions. A novel nine-item self-report Effort Scale measuring each of the three factors was developed and tested for internal and external validity-including its relationship with multidimensional motivation. Multilevel confirmatory factor analyses were conducted to test the factor structure and validity of multidimensional effort. Additionally, doubly-latent multilevel structural equation models were conducted to explore the hypothesized motivation → engagement (effort) process, and the role of student- and classroom-level background attributes as predictors of both motivation and effort. Results supported the hypothesized model of tripartite effort and its distinctiveness from motivation, and showed that key dimensions of motivation predicted effort at student- and classroom-levels. This study provides implications and suggestions for future motivation research and theorizing by (1) establishing evidence for the validity of a novel engagement framework (multidimensional effort), and (2) supporting future measurement and practice in academic engagement juxtaposed with multidimensional motivation-critical for better understanding engagement, and motivation itself.
Liquid biopsy provides a real-time assessment of metastatic breast cancer (MBC). We evaluated the utility of combining circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) to predict ...prognosis in MBC.
We conducted a retrospective study of 91 patients with locally advanced breast cancer and MBC. CTCs were enumerated by CellSearch; the plasma-based assay was performed utilizing Guardant360 and the survival analysis using Kaplan-Meier curves.
Eighty-four patients had stage IV cancer, and 7 patients had no metastases. Eighty patients had CTC analysis: median number 2 (0-5,612). Blood samples 232 of 277 (84%) had mutations. The average ctDNA fraction was 4.5% (0-88.2%) and number of alterations 3 (0-27); the most commonly mutated genes were
(52%),
(40%), and
(20%). At the time of analysis, 36 patients (39.6%) were dead. The median follow-up for CTCs was 9 months; for ctDNA, it was 9.9 months. For CTCs and ctDNA, respectively, progression-free survival (PFS) was 4.2 and 5.2 months and overall survival (OS) was 18.7 and 21.5 months. There was a statistically significant difference in PFS and OS for baseline CTCs < 5 versus CTCs ≥ 5 (
= 0.021 and
= 0.0004, respectively); %ctDNA < 0.5 versus ≥ 0.5 (
= 0.003 and
= 0.012); number of alterations < 2 versus ≥ 2 (
= 0.059 borderline and
= 0.0015). A significant association by Fisher exact test was found between the number of alterations and the %ctDNA in the baseline sample (
< 0.0001).
The study demonstrated that liquid biopsy is an effective prognostic tool.
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Most
-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors.
amplification has been described in patients progressing on ALK inhibitors, ...but frequency of this event has not been comprehensively assessed.
We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (
= 101) or plasma (
= 106) specimens from patients with ALK-positive lung cancer to detect
genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with
alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance.
amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop
amplification than those who had received next-generation ALK inhibitors after crizotinib (
= 0.019). Two tumor specimens harbored an identical
rearrangement, one of which had concurrent
amplification. Expressing
in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both
and
amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired
alterations achieved rapid responses to ALK/MET combination therapy.
Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired
alterations may derive clinical benefit from therapies that target both ALK and MET.
Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform ...time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame
deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC.
Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease.
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