Publications by authors named "Agnes Viale"

155 Publications

Tumor fraction-guided cell-free DNA profiling in metastatic solid tumor patients.

Genome Med 2021 May 31;13(1):96. Epub 2021 May 31.

Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA.

Background: Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth.

Methods: Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES).

Results: cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches.

Conclusions: cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.
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http://dx.doi.org/10.1186/s13073-021-00898-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165771PMC
May 2021

Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

Nucleic Acids Res 2021 Apr 20. Epub 2021 Apr 20.

Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.
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http://dx.doi.org/10.1093/nar/gkab262DOI Listing
April 2021

Targeting eIF4A-Dependent Translation of KRAS Signaling Molecules.

Cancer Res 2021 Apr 25;81(8):2002-2014. Epub 2021 Feb 25.

Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York.

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5' untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC. SIGNIFICANCE: These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137674PMC
April 2021

NRF2 Activation Confers Resistance to eIF4A Inhibitors in Cancer Therapy.

Cancers (Basel) 2021 Feb 5;13(4). Epub 2021 Feb 5.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Inhibition of the eIF4A RNA helicase with silvestrol and related compounds is emerging as a powerful anti-cancer strategy. We find that a synthetic silvestrol analogue (CR-1-31 B) has nanomolar activity across many cancer cell lines. It is especially active against aggressive MYC/BCL2 B cell lymphomas and this likely reflects the eIF4A-dependent translation of both MYC and BCL2. We performed a genome-wide CRISPR/Cas9 screen and identified mechanisms of resistance to this new class of therapeutics. We identify three negative NRF2 regulators (KEAP1, CUL3, CAND1) whose inactivation is sufficient to cause CR1-31-B resistance. NRF2 is known to alter the oxidation state of translation factors and cause a broad increase in protein production. We find that NRF2 activation particularly increases the translation of some eIF4A-dependent mRNAs and restores MYC and BCL2 production. We know that NRF2 functions depend on removal of sugar adducts by the frutosamine-3-kinase (FN3K). Accordingly, loss of FN3K results in NRF2 hyper-glycation and inactivation and resensitizes cancer cells to eIF4A inhibition. Together, our findings implicate NRF2 in the translation of eIF4A-dependent mRNAs and point to FN3K inhibition as a new strategy to block NRF2 functions in cancer.
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http://dx.doi.org/10.3390/cancers13040639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915661PMC
February 2021

Performance of Severe Acute Respiratory Syndrome Coronavirus 2 Real-Time RT-PCR Tests on Oral Rinses and Saliva Samples.

J Mol Diagn 2021 Jan 17;23(1):3-9. Epub 2020 Nov 17.

Department Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.

Access to rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is essential for controlling the current global pandemic of coronavirus disease 2019. In this study, the use of oral rinses (ORs) and posterior oropharyngeal saliva as an alternative to swab collection methods from symptomatic and asymptomatic health care workers for the detection of SARS-CoV-2 RNA by RT-PCR was evaluated. For saliva samples, the overall agreement with oropharyngeal swabs was 93% (Ƙ = 0.84), with a sensitivity of 96.7% (95% CI, 83.3%-99.8%). The agreement between saliva and nasopharyngeal swabs was 97.7% (Ƙ = 0.93), with a sensitivity of 94.1% (95% CI, 73.0%-99.7%). ORs were compared with nasopharyngeal swabs only, with an overall agreement of 85.7% (Ƙ = 0.65), and a sensitivity of 63% (95% CI, 46.6%-77.8%). The agreement between a laboratory-developed test based on the CDC RT-PCR and two commercial assays, the Xpert Xpress SARS-CoV-2 and the Cobas SARS-CoV-2, was also evaluated. The overall agreement was >90%. Finally, SARS-CoV-2 RNA in saliva samples was shown to be stable, with no changes in viral loads over 24 hours at both room temperature and 4°C. Although the dilution of SARS-CoV-2 in ORs precluded its acceptability as a sample type, posterior oropharyngeal saliva was an acceptable alternative sample type for SARS-CoV-2 RNA detection.
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http://dx.doi.org/10.1016/j.jmoldx.2020.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670901PMC
January 2021

Germ Cell Tumor Molecular Heterogeneity Revealed Through Analysis of Primary and Metastasis Pairs.

JCO Precis Oncol 2020 30;4. Epub 2020 Oct 30.

Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY.

Purpose: Although primary germ cell tumors (GCTs) have been extensively characterized, molecular analysis of metastatic sites has been limited. We performed whole-exome sequencing and targeted next-generation sequencing on paired primary and metastatic GCT samples in a patient cohort enriched for cisplatin-resistant disease.

Patients And Methods: Tissue sequencing was performed on 100 tumor specimens from 50 patients with metastatic GCT, and sequencing of plasma cell-free DNA was performed for a subset of patients.

Results: The mutational landscape of primary and metastatic pairs from GCT patients was highly discordant (68% of all somatic mutations were discordant). Whereas genome duplication was common and highly concordant between primary and metastatic samples, only 25% of primary-metastasis pairs had ≥ 50% concordance at the level of DNA copy number alterations (CNAs). Evolutionary-based analyses revealed that most mutations arose after CNAs at the respective loci in both primary and metastatic samples, with oncogenic mutations enriched in the set of early-occurring mutations versus variants of unknown significance (VUSs). pathway alterations were identified in nine cisplatin-resistant patients and had the highest degree of concordance in primary and metastatic specimens, consistent with their association with this treatment-resistant phenotype.

Conclusion: Analysis of paired primary and metastatic GCT specimens revealed significant molecular heterogeneity for both CNAs and somatic mutations. Among loci demonstrating serial genetic evolution, most somatic mutations arose after CNAs, but oncogenic mutations were enriched in the set of early-occurring mutations as compared with VUSs. Alterations in were clonal when present and shared among primary-metastasis pairs.
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http://dx.doi.org/10.1200/PO.20.00166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608584PMC
October 2020

Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes.

Nat Med 2020 10 3;26(10):1549-1556. Epub 2020 Aug 3.

Department of Hematology, Democritus University of Thrace Medical School, Alexandroupolis, Greece.

Tumor protein p53 (TP53) is the most frequently mutated gene in cancer. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease, rapid transformation to acute myeloid leukemia (AML), resistance to conventional therapies and dismal outcomes. Consistent with the tumor-suppressive role of TP53, patients harbor both mono- and biallelic mutations. However, the biological and clinical implications of TP53 allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS for TP53 mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third of TP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R). Surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcomes and response to therapy. This study shows that consideration of TP53 allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response.
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http://dx.doi.org/10.1038/s41591-020-1008-zDOI Listing
October 2020

Leveraging Systematic Functional Analysis to Benchmark an Framework Distinguishes Driver from Passenger MEK Mutants in Cancer.

Cancer Res 2020 10 8;80(19):4233-4243. Epub 2020 Jul 8.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York.

Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on and mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. prediction accurately distinguished functional from benign and mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response. SIGNIFICANCE: Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541597PMC
October 2020

Cell-free DNA profiling in retinoblastoma patients with advanced intraocular disease: An MSKCC experience.

Cancer Med 2020 09 7;9(17):6093-6101. Epub 2020 Jul 7.

Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Purpose: The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell-free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection.

Methods: Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra-ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA-cleared tumor sequencing assay, MSK-IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1.

Results: Tumor-guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence.

Conclusion: Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow-up schemes, and risk of metastasis.
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http://dx.doi.org/10.1002/cam4.3144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476838PMC
September 2020

Developing Quality Programs for Cell-Free DNA (cfDNA) Extraction from Peripheral Blood.

J Appl Lab Med 2020 07;5(4):788-797

Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY.

Background: Cell-free DNA (cfDNA) analysis using peripheral blood represents an exciting, minimally invasive technology for cancer diagnosis and monitoring. The reliability of testing is dependent on the accuracy and sensitivity of specific molecular analyses to detect tumor-associated genomic variants and on the quantity and quality of cfDNA available for testing. Specific guidelines for standardization and design of appropriate quality programs focused specifically on cfDNA isolation are lacking, as are standardized quality control reagents.

Content: This report describes and illustrates quality control and quality assurance processes, supported by generation of in-house quality control material, to ensure the reliability of the preanalytical phase of cfDNA analysis.

Summary: We have developed a robust quality program to support high-volume automated cfDNA extraction from peripheral blood by implementing processes and procedures designed to monitor the adequacy of specimen collection, specimen stability, efficiency of cfDNA extraction, and cfDNA quality.
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http://dx.doi.org/10.1093/jalm/jfaa050DOI Listing
July 2020

Coaltered and Is Associated with Extremes of Survivorship and Distinct Patterns of Metastasis in Patients with Metastatic Colorectal Cancer.

Clin Cancer Res 2020 03 12;26(5):1077-1085. Epub 2019 Nov 12.

Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: We aimed to investigate genomic correlates underlying extremes of survivorship in metastatic colorectal cancer and their applicability in informing survival in distinct subsets of patients with metastatic colorectal cancer.

Experimental Design: We examined differences in oncogenic somatic alterations between metastatic colorectal cancer cohorts demonstrating extremes of survivorship following complete metastasectomy: ≤2-year ( = 17) and ≥10-year ( = 18) survivors. Relevant genomic findings, and their association with overall survival (OS), were validated in two independent datasets of 935 stage IV and 443 resected stage I-IV patients.

Results: In the extremes-of-survivorship cohort, significant co-occurrence of hotspot mutations and alterations was observed in ≤2-year survivors ( < 0.001). When validating these findings in the independent cohort of 935 stage IV patients, incorporation of the cumulative effect of any oncogenic (i.e., either , or ) and alteration generated three prognostic clusters: (i) -altered alone (median OS, 132 months); (ii) -altered alone (65 months) or - and pan-wild-type (60 months); and (iii) coaltered - (40 months; < 0.0001). Coaltered - was independently associated with mortality (HR, 2.47; 95% confidence interval, 1.91-3.21; < 0.001). This molecular profile predicted survival in the second independent cohort of 443 resected stage I-IV patients. Coaltered - was associated with worse OS in patients with liver ( = 490) and lung ( = 172) but not peritoneal surface ( = 149) metastases. Moreover, coaltered - tumors were significantly more likely to involve extrahepatic metastatic sites with limited salvage options.

Conclusions: Genomic analysis of extremes of survivorship following colorectal cancer metastasectomy identifies a prognostic role for coaltered - and its association with distinct patterns of colorectal cancer metastasis.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056517PMC
March 2020

Detection of Early Human Papillomavirus-Associated Cancers by Liquid Biopsy.

JCO Precis Oncol 2019 3;3. Epub 2019 Apr 3.

Memorial Sloan-Kettering Cancer Center, New York, NY.

Purpose: A circulating tumor DNA (ctDNA) test to detect plasma Epstein-Barr viral DNA can be used to screen for early nasopharyngeal cancers; however, the reported sensitivity of viral ctDNA tests to detect human papillomavirus (HPV)-associated cancers is modest. We assessed the utility of droplet digital polymerase chain reaction (ddPCR) to detect early-stage HPV-associated cancers using sequential HPV16 and HPV33 assays that account for HPV subtype distribution and subtype sequence variants.

Patients And Methods: We collected plasma specimens from 97 HPV-positive patients with oropharyngeal squamous cell carcinoma and eight patients with HPV-positive anal squamous cell carcinoma, each with locoregionally confined disease. Negative controls included samples from seven patients with HPV-negative head and neck cancers and 20 individuals without cancer.

Results: Of 97 patients with nonmetastatic, locoregionally confined oropharyngeal squamous cell carcinoma, 90 patients had detectable HPV16 ctDNA and three patients had HPV33 ctDNA, indicating an overall sensitivity of 95.6%. Seven of eight patients with early anal cancer were HPV16 ctDNA positive. No HPV ctDNA was detected in 27 negative controls, indicating 100% specificity. HPV16 ctDNA was detected in 19 of 19 patients with low-volume disease, defined as patients with a single, asymptomatic positive lymph node (N1) or an isolated T1-2 asymptomatic primary tumor. HPV16 ctDNA levels directly corresponded to tumor responses to chemoradiation and surgery.

Conclusion: With an updated understanding of HPV subtypes and sequence variation, HPV ctDNA by ddPCR is highly sensitive and specific, identifying HPV16 and HPV33 subtypes in a similar distribution as reported in major genomic profiling studies. The detection of small tumors indicates that HPV16 and HPV33 ctDNA ddPCR could be readily used in early detection screening trials and in disease response monitoring, analogous to Epstein-Barr virus DNA.
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http://dx.doi.org/10.1200/PO.18.00276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726127PMC
April 2019

The Oncogenic Action of NRF2 Depends on De-glycation by Fructosamine-3-Kinase.

Cell 2019 08;178(4):807-819.e21

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.
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http://dx.doi.org/10.1016/j.cell.2019.07.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693658PMC
August 2019

Tumour lineage shapes BRCA-mediated phenotypes.

Nature 2019 07 10;571(7766):576-579. Epub 2019 Jul 10.

Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Mutations in BRCA1 and BRCA2 predispose individuals to certain cancers, and disease-specific screening and preventative strategies have reduced cancer mortality in affected patients. These classical tumour-suppressor genes have tumorigenic effects associated with somatic biallelic inactivation, although haploinsufficiency may also promote the formation and progression of tumours. Moreover, BRCA1/2-mutant tumours are often deficient in the repair of double-stranded DNA breaks by homologous recombination, and consequently exhibit increased therapeutic sensitivity to platinum-containing therapy and inhibitors of poly-(ADP-ribose)-polymerase (PARP). However, the phenotypic and therapeutic relevance of mutations in BRCA1 or BRCA2 remains poorly defined in most cancer types. Here we show that in the 2.7% and 1.8% of patients with advanced-stage cancer and germline pathogenic or somatic loss-of-function alterations in BRCA1/2, respectively, selective pressure for biallelic inactivation, zygosity-dependent phenotype penetrance, and sensitivity to PARP inhibition were observed only in tumour types associated with increased heritable cancer risk in BRCA1/2 carriers (BRCA-associated cancer types). Conversely, among patients with non-BRCA-associated cancer types, most carriers of these BRCA1/2 mutation types had evidence for tumour pathogenesis that was independent of mutant BRCA1/2. Overall, mutant BRCA is an indispensable founding event for some tumours, but in a considerable proportion of other cancers, it appears to be biologically neutral-a difference predominantly conditioned by tumour lineage-with implications for disease pathogenesis, screening, design of clinical trials and therapeutic decision-making.
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http://dx.doi.org/10.1038/s41586-019-1382-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048239PMC
July 2019

Genomic and Transcriptomic Characterization of Papillary Microcarcinomas With Lateral Neck Lymph Node Metastases.

J Clin Endocrinol Metab 2019 10;104(10):4889-4899

Memorial Sloan Kettering Cancer Center, New York, New York.

Context: Most papillary microcarcinomas (PMCs) are indolent and subclinical. However, as many as 10% can present with clinically significant nodal metastases.

Objective And Design: Characterization of the genomic and transcriptomic landscape of PMCs presenting with or without clinically important lymph node metastases.

Subjects And Samples: Formalin-fixed paraffin-embedded PMC samples from 40 patients with lateral neck nodal metastases (pN1b) and 71 patients with PMC with documented absence of nodal disease (pN0).

Outcome Measures: To interrogate DNA alterations in 410 genes commonly mutated in cancer and test for differential gene expression using a custom NanoString panel of 248 genes selected primarily based on their association with tumor size and nodal disease in the papillary thyroid cancer TCGA project.

Results: The genomic landscapes of PMC with or without pN1b were similar. Mutations in TERT promoter (3%) and TP53 (1%) were exclusive to N1b cases. Transcriptomic analysis revealed differential expression of 43 genes in PMCs with pN1b compared with pN0. A random forest machine learning-based molecular classifier developed to predict regional lymph node metastasis demonstrated a negative predictive value of 0.98 and a positive predictive value of 0.72 at a prevalence of 10% pN1b disease.

Conclusions: The genomic landscape of tumors with pN1b and pN0 disease was similar, whereas 43 genes selected primarily by mining the TCGA RNAseq data were differentially expressed. This bioinformatics-driven approach to the development of a custom transcriptomic assay provides a basis for a molecular classifier for pN1b risk stratification in PMC.
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http://dx.doi.org/10.1210/jc.2019-00431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733494PMC
October 2019

SET: a robust 18-gene predictor for sensitivity to endocrine therapy for metastatic breast cancer.

NPJ Breast Cancer 2019 30;5:16. Epub 2019 May 30.

1Department of Pathology and Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX USA.

There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SET index to measure gene expression microarray probe sets that were correlated with hormone receptors ( and ) and robust to preanalytical and analytical influences. We tested SET index in biopsies of metastastic HR+/HER2- breast cancer against the treatment outcomes in 140 patients. Then we customized the SET assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SET index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955,  = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631,  = 0.001). Mutated LBD was detected in 8/53 (15%) of metastases, involving 1-98% of transcripts (all had high SET index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SET was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2- breast cancer, especially in the absence of mutated transcript.
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http://dx.doi.org/10.1038/s41523-019-0111-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542807PMC
May 2019

c-MYC regulates mRNA translation efficiency and start-site selection in lymphoma.

J Exp Med 2019 07 29;216(7):1509-1524. Epub 2019 May 29.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY

The oncogenic c-MYC (MYC) transcription factor has broad effects on gene expression and cell behavior. We show that MYC alters the efficiency and quality of mRNA translation into functional proteins. Specifically, MYC drives the translation of most protein components of the electron transport chain in lymphoma cells, and many of these effects are independent from proliferation. Specific interactions of MYC-sensitive RNA-binding proteins (e.g., SRSF1/RBM42) with 5'UTR sequence motifs mediate many of these changes. Moreover, we observe a striking shift in translation initiation site usage. For example, in low-MYC conditions, lymphoma cells initiate translation of the CD19 mRNA from a site in exon 5. This results in the truncation of all extracellular CD19 domains and facilitates escape from CD19-directed CAR-T cell therapy. Together, our findings reveal MYC effects on the translation of key metabolic enzymes and immune receptors in lymphoma cells.
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http://dx.doi.org/10.1084/jem.20181726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605752PMC
July 2019

Author Correction: The Rho GTPase Rnd1 suppresses mammary tumorigenesis and EMT by restraining Ras-MAPK signalling.

Nat Cell Biol 2019 04;21(4):534

Cell Biology Program, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York, 10065, USA.

In the version of this Article originally published the same blot was inadvertently presented as both p-Rb and Cyclin A in Fig. 2a. This blot corresponds to the p-Rb panel, as can be seen in the unprocessed version of these blots in Supplementary Fig. 9. The corrected version of the panel is shown below, together with a completely uncropped image of both blots. In addition, in the 'Viral transduction' section of the Methods, the pLKO.1 plasmids encoding short hairpin RNAs targeting human Rnd1 were incorrectly listed as clones TRCN0000018338 and TRCN0000039977. The correct clone numbers are TRCN0000047434 and TRCN0000047435.
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http://dx.doi.org/10.1038/s41556-019-0288-3DOI Listing
April 2019

Peripheral Circulating Tumor DNA Detection Predicts Poor Outcomes After Liver Resection for Metastatic Colorectal Cancer.

Ann Surg Oncol 2019 Jun 31;26(6):1824-1832. Epub 2019 Jan 31.

Department of Surgery, Hepatopancreatobiliary Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Background: Liver resection can be curative for well-selected metastatic colorectal cancer (CRC) patients. Circulating tumor DNA (ctDNA) has shown promise as a biomarker for tumor dynamics and recurrence following CRC resection. This prospective pilot study investigated the use of ctDNA to predict disease outcome in resected CRC patients.

Methods: Between November 2014 and November 2015, 60 patients with CRC were identified and prospectively enrolled. During liver resection, blood was drawn from peripheral (PERIPH), portal (PV), and hepatic (HV) veins, and 3-4 weeks postoperatively from a peripheral vein (POSTOP). Kappa statistics were used to compare mutated (mt) genes in tissue and ctDNA. Disease-specific and disease-free survival (DSS and DFS) were assessed from surgery with Kaplan-Meier and Cox methods.

Results: For the 59 eligible patients, the most commonly mutated genes were TP53 (mtTP53: 47.5%) and APC (mtAPC: 50.8%). Substantial to almost-perfect agreement was seen between ctDNA from PERIPH and PV (mtTP53: 89.8%, κ = 0.73, 95% confidence interval [CI] 0.53-0.93; mtAPC: 94.9%, κ = 0.83, 95% CI 0.64-1.00), as well as HV (mtTP53: 91.5%, κ = 0.78, 95% CI 0.60-0.96; mtAPC: 91.5%, κ = 0.73, 95% CI 0.51-0.95). Tumor mutations and PERIPH ctDNA had fair-to-moderate agreement (mtTP53: 72.9%, κ = 0.44, 95% CI 0.23-0.66; mtAPC: 61.0%, κ = 0.23, 95% CI 0.04-0.42). Detection of PERIPH mtTP53 was associated with worse 2-year DSS (mt+ 79% vs. mt- 90%, P = 0.024).

Conclusions: Peripheral blood reflects the perihepatic ctDNA signature. Disagreement between tissue and ctDNA mutations may reflect the true natural history of tumor genes or an assay limitation. Peripheral ctDNA detection before liver resection is associated with worse DSS.
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http://dx.doi.org/10.1245/s10434-019-07201-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511310PMC
June 2019

Tracking tumour evolution in glioma through liquid biopsies of cerebrospinal fluid.

Nature 2019 01 23;565(7741):654-658. Epub 2019 Jan 23.

Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Diffuse gliomas are the most common malignant brain tumours in adults and include glioblastomas and World Health Organization (WHO) grade II and grade III tumours (sometimes referred to as lower-grade gliomas). Genetic tumour profiling is used to classify disease and guide therapy, but involves brain surgery for tissue collection; repeated tumour biopsies may be necessary for accurate genotyping over the course of the disease. While the detection of circulating tumour DNA (ctDNA) in the blood of patients with primary brain tumours remains challenging, sequencing of ctDNA from the cerebrospinal fluid (CSF) may provide an alternative way to genotype gliomas with lower morbidity and cost. We therefore evaluated the representation of the glioma genome in CSF from 85 patients with gliomas who underwent a lumbar puncture because they showed neurological signs or symptoms. Here we show that tumour-derived DNA was detected in CSF from 42 out of 85 patients (49.4%) and was associated with disease burden and adverse outcome. The genomic landscape of glioma in the CSF included a broad spectrum of genetic alterations and closely resembled the genomes of tumour biopsies. Alterations that occur early during tumorigenesis, such as co-deletion of chromosome arms 1p and 19q (1p/19q codeletion) and mutations in the metabolic genes isocitrate dehydrogenase 1 (IDH1) or IDH2, were shared in all matched ctDNA-positive CSF-tumour pairs, whereas growth factor receptor signalling pathways showed considerable evolution. The ability to monitor the evolution of the glioma genome through a minimally invasive technique could advance the clinical development and use of genotype-directed therapies for glioma, one of the most aggressive human cancers.
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http://dx.doi.org/10.1038/s41586-019-0882-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457907PMC
January 2019

Phase 1b trial of an ibrutinib-based combination therapy in recurrent/refractory CNS lymphoma.

Blood 2019 01 19;133(5):436-445. Epub 2018 Dec 19.

Department of Neurology and.

Ibrutinib is a first-in-class inhibitor of Bruton tyrosine kinase (BTK) and has shown single-agent activity in recurrent/refractory central nervous system (CNS) lymphoma. Clinical responses are often transient or incomplete, suggesting a need for a combination therapy approach. We conducted a phase 1b clinical trial to explore the sequential combination of ibrutinib (560 or 840 mg daily dosing) with high-dose methotrexate (HD-MTX) and rituximab in patients with CNS lymphoma (CNSL). HD-MTX was given at 3.5 g/m every 2 weeks for a total of 8 doses (4 cycles; 1 cycle = 28 days). Ibrutinib was held on days of HD-MTX infusion and resumed 5 days after HD-MTX infusion or after HD-MTX clearance. Single-agent daily ibrutinib was administered continuously after completion of induction therapy until disease progression, intolerable toxicity, or death. We also explored next-generation sequencing of circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF) before and during treatment. The combination of ibrutinib, HD-MTX, and rituximab was tolerated with an acceptable safety profile (no grade 5 events, 3 grade 4 events). No dose-limiting toxicity was observed. Eleven of 15 patients proceeded to maintenance ibrutinib after completing 4 cycles of the ibrutinib/HD-MTX/rituximab combination. Clinical responses occurred in 12 of 15 patients (80%). Sustained tumor responses were associated with clearance of ctDNA from the CSF. This trial was registered at www.clinicaltrials.gov as #NCT02315326.
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http://dx.doi.org/10.1182/blood-2018-09-875732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356986PMC
January 2019

Isoform Switching as a Mechanism of Acquired Resistance to Mutant Isocitrate Dehydrogenase Inhibition.

Cancer Discov 2018 12 24;8(12):1540-1547. Epub 2018 Oct 24.

Gastrointestinal Oncology Service, Memorial Sloan Kettering Cancer Center, New York, New York.

Somatic mutations in cytosolic or mitochondrial isoforms of isocitrate dehydrogenase ( or , respectively) contribute to oncogenesis via production of the metabolite 2-hydroxyglutarate (2HG). Isoform-selective IDH inhibitors suppress 2HG production and induce clinical responses in patients with - and -mutant malignancies. Despite the promising activity of IDH inhibitors, the mechanisms that mediate resistance to IDH inhibition are poorly understood. Here, we describe four clinical cases that identify mutant IDH isoform switching, either from mutant IDH1 to mutant IDH2 or vice versa, as a mechanism of acquired clinical resistance to IDH inhibition in solid and liquid tumors. SIGNIFICANCE: IDH-mutant cancers can develop resistance to isoform-selective IDH inhibition by "isoform switching" from mutant IDH1 to mutant IDH2 or vice versa, thereby restoring 2HG production by the tumor. These findings underscore a role for continued 2HG production in tumor progression and suggest therapeutic strategies to prevent or overcome resistance..
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http://dx.doi.org/10.1158/2159-8290.CD-18-0877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699636PMC
December 2018

The Genomic Landscape of Endocrine-Resistant Advanced Breast Cancers.

Cancer Cell 2018 09;34(3):427-438.e6

Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

We integrated the genomic sequencing of 1,918 breast cancers, including 1,501 hormone receptor-positive tumors, with detailed clinical information and treatment outcomes. In 692 tumors previously exposed to hormonal therapy, we identified an increased number of alterations in genes involved in the mitogen-activated protein kinase (MAPK) pathway and in the estrogen receptor transcriptional machinery. Activating ERBB2 mutations and NF1 loss-of-function mutations were more than twice as common in endocrine resistant tumors. Alterations in other MAPK pathway genes (EGFR, KRAS, among others) and estrogen receptor transcriptional regulators (MYC, CTCF, FOXA1, and TBX3) were also enriched. Altogether, these alterations were present in 22% of tumors, mutually exclusive with ESR1 mutations, and associated with a shorter duration of response to subsequent hormonal therapies.
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http://dx.doi.org/10.1016/j.ccell.2018.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327853PMC
September 2018

Lobular Carcinomas Display Intralesion Genetic Heterogeneity and Clonal Evolution in the Progression to Invasive Lobular Carcinoma.

Clin Cancer Res 2019 01 5;25(2):674-686. Epub 2018 Sep 5.

Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: Lobular carcinoma (LCIS) is a preinvasive lesion of the breast. We sought to define its genomic landscape, whether intralesion genetic heterogeneity is present in LCIS, and the clonal relatedness between LCIS and invasive breast cancers. We reanalyzed whole-exome sequencing (WES) data and performed a targeted amplicon sequencing validation of mutations identified in 43 LCIS and 27 synchronous more clinically advanced lesions from 24 patients [9 ductal carcinomas (DCIS), 13 invasive lobular carcinomas (ILC), and 5 invasive ductal carcinomas (IDC)]. Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic trees were defined using validated computational methods.

Results: WES of 43 LCIS lesions revealed a genomic profile similar to that previously reported for ILCs, with mutations present in 81% of the lesions. Forty-two percent (18/43) of LCIS were found to be clonally related to synchronous DCIS and/or ILCs, with clonal evolutionary patterns indicative of clonal selection and/or parallel/branched progression. Intralesion genetic heterogeneity was higher among LCIS clonally related to DCIS/ILC than in those nonclonally related to DCIS/ILC. A shift from aging to APOBEC-related mutational processes was observed in the progression from LCIS to DCIS and/or ILC in a subset of cases.

Conclusions: Our findings support the contention that LCIS has a repertoire of somatic genetic alterations similar to that of ILCs, and likely constitutes a nonobligate precursor of breast cancer. Intralesion genetic heterogeneity is observed in LCIS and should be considered in studies aiming to develop biomarkers of progression from LCIS to more advanced lesions.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726436PMC
January 2019

Germline Lysine-Specific Demethylase 1 () Mutations Confer Susceptibility to Multiple Myeloma.

Cancer Res 2018 05 20;78(10):2747-2759. Epub 2018 Mar 20.

University of Chicago, Chicago, Illinois.

Given the frequent and largely incurable occurrence of multiple myeloma, identification of germline genetic mutations that predispose cells to multiple myeloma may provide insight into disease etiology and the developmental mechanisms of its cell of origin, the plasma cell (PC). Here, we identified familial and early-onset multiple myeloma kindreds with truncating mutations in lysine-specific demethylase 1 (LSD1/KDM1A), an epigenetic transcriptional repressor that primarily demethylates histone H3 on lysine 4 and regulates hematopoietic stem cell self-renewal. In addition, we found higher rates of germline truncating and predicted deleterious missense KDM1A mutations in patients with multiple myeloma unselected for family history compared with controls. Both monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma cells have significantly lower KDM1A transcript levels compared with normal PCs. Transcriptome analysis of multiple myeloma cells from KDM1A mutation carriers shows enrichment of pathways and MYC target genes previously associated with myeloma pathogenesis. In mice, antigen challenge followed by pharmacologic inhibition of KDM1A promoted PC expansion, enhanced secondary immune response, elicited appearance of serum paraprotein, and mediated upregulation of MYC transcriptional targets. These changes are consistent with the development of MGUS. Collectively, our findings show that KDM1A is the first autosomal-dominant multiple myeloma germline predisposition gene providing new insights into its mechanistic roles as a tumor suppressor during post-germinal center B-cell differentiation. KDM1A is the first germline autosomal dominant predisposition gene identified in multiple myeloma and provides new insights into multiple myeloma etiology and the mechanistic role of KDM1A as a tumor suppressor during post-germinal center B-cell differentiation. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-1900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955848PMC
May 2018

Clinical Sequencing Defines the Genomic Landscape of Metastatic Colorectal Cancer.

Cancer Cell 2018 01;33(1):125-136.e3

Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Departments of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Metastatic colorectal cancers (mCRCs) are clinically heterogeneous, but the genomic basis of this variability remains poorly understood. We performed prospective targeted sequencing of 1,134 CRCs. We identified splice alterations in intronic regions of APC and large in-frame deletions in CTNNB1, increasing oncogenic WNT pathway alterations to 96% of CRCs. Right-sided primary site in microsatellite stable mCRC was associated with shorter survival, older age at diagnosis, increased mutations, and enrichment of oncogenic alterations in KRAS, BRAF, PIK3CA, AKT1, RNF43, and SMAD4 compared with left-sided primaries. Left-sided tumors frequently had no identifiable genetic alteration in mitogenic signaling, but exhibited higher mitogenic ligand expression. Our results suggest different pathways to tumorigenesis in right- and left-sided microsatellite stable CRC that may underlie clinical differences.
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http://dx.doi.org/10.1016/j.ccell.2017.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765991PMC
January 2018

Four-month course of adjuvant dabrafenib in patients with surgically resected stage IIIC melanoma characterized by a BRAFV600E/K mutation.

Oncotarget 2017 Dec 16;8(62):105000-105010. Epub 2017 Sep 16.

Memorial Sloan Kettering Cancer Center, New York, New York, USA.

Background: We tested the hypothesis that a 4-month course of adjuvant dabrafenib in stage IIIC BRAF-mutated melanoma would improve 2 year RFS from 24% to 51%, and that tumor-derived cell free DNA (cfDNA) in plasma would correlate with and predict recurrence.

Methods: Patients with stage IIIC BRAF V600E/K mutated melanoma who were free of disease after surgical resection received 4 months of adjuvant dabrafenib. Patients were evaluated with imaging at baseline, at the end of cycles 2, 4, 6, then every 3 months until disease relapse or 2 years, whichever came first. Serial blood samples were collected for evaluation of cfDNA at the same time.

Results: 21/23 patients enrolled were evaluable; 2 patients withdrew consent during the first week of treatment. The 2 year RFS was 28.6% (95% CI 12-48%). The estimated overall survival at 2 years was 78% (95% CI 51-91%). cfDNA detection had a 53% sensitivity in relapsing patients but cfDNA detection did not provide lead-time advantage over CT scanning.

Conclusion: A 4-month course of adjuvant dabrafenib did not result in a detectable improvement in 2-year RFS. cfDNA was less sensitive than standard CT imaging and did not provide a lead-time advantage in detecting relapse.
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http://dx.doi.org/10.18632/oncotarget.21072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739615PMC
December 2017