Publications by authors named "AmirAli Talasaz"

37 Publications

Minimal Residual Disease Detection using a Plasma-only Circulating Tumor DNA Assay in Patients with Colorectal Cancer.

Clin Cancer Res 2021 Oct 29;27(20):5586-5594. Epub 2021 Apr 29.

Department of Medicine, Division of Hematology and Oncology, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts.

Purpose: Detection of persistent circulating tumor DNA (ctDNA) after curative-intent surgery can identify patients with minimal residual disease (MRD) who will ultimately recur. Most ctDNA MRD assays require tumor sequencing to identify tumor-derived mutations to facilitate ctDNA detection, requiring tumor and blood. We evaluated a plasma-only ctDNA assay integrating genomic and epigenomic cancer signatures to enable tumor-uninformed MRD detection.

Experimental Design: A total of 252 prospective serial plasma specimens from 103 patients with colorectal cancer undergoing curative-intent surgery were analyzed and correlated with recurrence.

Results: Of 103 patients, 84 [stage I (9.5%), II (23.8%), III (47.6%), IV (19%)] had evaluable plasma drawn after completion of definitive therapy, defined as surgery only ( = 39) or completion of adjuvant therapy ( = 45). In "landmark" plasma drawn 1-month (median, 31.5 days) after definitive therapy and >1 year follow-up, 15 patients had detectable ctDNA, and all 15 recurred [positive predictive value (PPV), 100%; HR, 11.28 ( < 0.0001)]. Of 49 patients without detectable ctDNA at the landmark timepoint, 12 (24.5%) recurred. Landmark recurrence sensitivity and specificity were 55.6% and 100%. Incorporating serial longitudinal and surveillance (drawn within 4 months of recurrence) samples, sensitivity improved to 69% and 91%. Integrating epigenomic signatures increased sensitivity by 25%-36% versus genomic alterations alone. Notably, standard serum carcinoembryonic antigen levels did not predict recurrence [HR, 1.84 ( = 0.18); PPV = 53.9%].

Conclusions: Plasma-only MRD detection demonstrated favorable sensitivity and specificity for recurrence, comparable with tumor-informed approaches. Integrating analysis of epigenomic and genomic alterations enhanced sensitivity. These findings support the potential clinical utility of plasma-only ctDNA MRD detection..
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http://dx.doi.org/10.1158/1078-0432.CCR-21-0410DOI Listing
October 2021

Spectrum of driver mutations and clinical impact of circulating tumor DNA analysis in non-small cell lung cancer: Analysis of over 8000 cases.

Cancer 2020 07 4;126(14):3219-3228. Epub 2020 May 4.

Division of Hematology-Oncology, Department of Internal Medicine, University of California Davis Comprehensive Cancer Center, Sacramento, California.

Background: Circulating cell-free tumor DNA (ctDNA)-based mutation profiling, if sufficiently sensitive and comprehensive, can efficiently identify genomic targets in advanced lung adenocarcinoma. Therefore, the authors investigated the accuracy and clinical utility of a commercially available digital next-generation sequencing platform in a large series of patients with non-small cell lung cancer (NSCLC).

Methods: Plasma-based comprehensive genomic profiling results from 8388 consecutively tested patients with advanced NSCLC were analyzed. Driver and resistance mutations were examined with regard to their distribution, frequency, co-occurrence, and mutual exclusivity.

Results: Somatic alterations were detected in 86% of samples. The median variant allele fraction was 0.43% (range, 0.03%-97.62%). Activating alterations in actionable oncogenes were identified in 48% of patients, including EGFR (26.4%), MET (6.1%), and BRAF (2.8%) alterations and fusions (ALK, RET, and ROS1) in 2.3%. Treatment-induced resistance mutations were common in this cohort, including driver-dependent and driver-independent alterations. In the subset of patients who had progressive disease during EGFR therapy, 64% had known or putative resistance alterations detected in plasma. Subset analysis revealed that ctDNA increased the identification of driver mutations by 65% over standard-of-care, tissue-based testing at diagnosis. A pooled data analysis on this plasma-based assay demonstrated that targeted therapy response rates were equivalent to those reported from tissue analysis.

Conclusions: Comprehensive ctDNA analysis detected the presence of therapeutically targetable driver and resistance mutations at the frequencies and distributions predicted for the study population. These findings add support for comprehensive ctDNA testing in patients who are incompletely tested at the time of diagnosis and as a primary option at the time of progression on targeted therapies.
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http://dx.doi.org/10.1002/cncr.32876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383626PMC
July 2020

Lung cancer and a bold new vision.

Authors:
AmirAli Talasaz

Future Oncol 2020 Apr 20;16(12):701-703. Epub 2019 Sep 20.

Guardant Health, Inc., 505 Penobscot Dr., Redwood City, CA 94063, USA.

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http://dx.doi.org/10.2217/fon-2019-0323DOI Listing
April 2020

Validation of Microsatellite Instability Detection Using a Comprehensive Plasma-Based Genotyping Panel.

Clin Cancer Res 2019 12 4;25(23):7035-7045. Epub 2019 Aug 4.

The University of Texas MD Anderson Cancer Center, Houston, Texas.

Purpose: To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing.

Experimental Design: Pan-cancer MSI detection using Guardant360 was analytically validated according to established guidelines and clinically validated using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated.

Results: cfDNA MSI evaluation was shown to have high specificity, precision, and sensitivity, with a limit of detection of 0.1% tumor content. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit.

Conclusions: cfDNA-based MSI detection using Guardant360 is highly concordant with tissue-based testing, enabling highly accurate detection of MSI status concurrent with comprehensive genomic profiling and expanding access to immunotherapy for patients with advanced cancer for whom current testing practices are inadequate..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1324DOI Listing
December 2019

Molecular Profiling of Hepatocellular Carcinoma Using Circulating Cell-Free DNA.

Clin Cancer Res 2019 10 30;25(20):6107-6118. Epub 2019 Jul 30.

Center for Personalized Cancer Therapy and Division of Hematology and Oncology, University of California San Diego, Moores Cancer Center, La Jolla, California.

Purpose: Molecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies.

Experimental Design: We analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA).

Results: A total of 153/206 (74.3%) were men; median age, 62 years (range, 18-91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1-13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%-55.03%). was the common altered gene [>120 alterations (non-unique)] followed by [20-38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in . In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have alterations, 35.7% (5/14) versus 8.8% HBV-negative ( = 0.04).

Conclusions: This study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3341DOI Listing
October 2019

Tumor Genomic Profiling Guides Patients with Metastatic Gastric Cancer to Targeted Treatment: The VIKTORY Umbrella Trial.

Cancer Discov 2019 10 17;9(10):1388-1405. Epub 2019 Jul 17.

Clinical, Research and Early Development, Oncology R&D, AstraZeneca, Cambridge, United Kingdom.

The VIKTORY (targeted agent eValuation In gastric cancer basket KORea) trial was designed to classify patients with metastatic gastric cancer based on clinical sequencing and focused on eight different biomarker groups ( aberration, mutation, mutation/amplification, amplification, MET overexpression, all negative, deficient, or amplification) to assign patients to one of the 10 associated clinical trials in second-line (2L) treatment. Capivasertib (AKT inhibitor), savolitinib (MET inhibitor), selumetinib (MEK inhibitor), adavosertib (WEE1 inhibitor), and vistusertib (TORC inhibitor) were tested with or without chemotherapy. Seven hundred seventy-two patients with gastric cancer were enrolled, and sequencing was successfully achieved in 715 patients (92.6%). When molecular screening was linked to seamless immediate access to parallel matched trials, 14.7% of patients received biomarker-assigned drug treatment. The biomarker-assigned treatment cohort had encouraging response rates and survival when compared with conventional 2L chemotherapy. Circulating tumor (ctDNA) analysis demonstrated good correlation between high copy number by ctDNA and response to savolitinib. SIGNIFICANCE: Prospective clinical sequencing revealed that baseline heterogeneity between tumor samples from different patients affected response to biomarker-selected therapies. VIKTORY is the first and largest platform study in gastric cancer and supports both the feasibility of tumor profiling and its clinical utility..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0442DOI Listing
October 2019

Genomic Assessment of Blood-Derived Circulating Tumor DNA in Patients With Colorectal Cancers: Correlation With Tissue Sequencing, Therapeutic Response, and Survival.

JCO Precis Oncol 2019 25;3. Epub 2019 Jan 25.

Shumei Kato, Maria C. Schwaederlé, Paul T. Fanta, Ryosuke Okamura, Lawrence Leichman, and Scott M. Lippman, Razelle Kurzrock, University of California San Diego Moores Cancer Center, La Jolla; and Richard B. Lanman, Victoria M. Raymond, and AmirAli Talasaz, Guardant Health, Redwood City, CA.

Purpose: Genomic alterations in blood-derived circulating tumor DNA (ctDNA) from patients with colorectal cancers were correlated with clinical outcomes.

Patients And Methods: Next-generation sequencing of ctDNA (54- to 73-gene panel) was performed in 94 patients with colorectal cancer.

Results: Most patients (96%) had metastatic or recurrent disease at the time of blood draw. The median number of nonsynonymous alterations per patient was three (range, zero to 30). The most frequently aberrant genes were (52.1% of patients), (34%), and (28.7%). Concordance between tissue and blood next-generation sequencing ranged from 63.2% () to 85.5% (). Altogether, 74 patients (79%) had one or more nonsynonymous alterations, 69 (73%) had one or more potentially actionable alterations, and 61 (65%) had an alteration actionable by a drug approved by the US Food and Drug Administration (on or off label). Lung metastases correlated with improved survival from diagnosis in univariable analysis. ctDNA of 5% or more from blood tests as well as and (HER2) nonsynonymous alterations correlated with worse survival (but only remained significant in multivariable analysis). No two patients had identical molecular portfolios. Overall, 65% versus 31% of patients treated with matched (n = 17) versus unmatched therapy (n = 18) after ctDNA testing achieved stable disease for 6 months or more, partial response, or complete response ( = .045); progression-free survival, 6.1 versus 2.3 months ( = .08); and survival not reached versus 9.4 months ( = .146; all by multivariable analysis).

Conclusion: Patients with colorectal cancer have heterogeneous ctDNA profiles, and most harbor potentially actionable ctDNA alterations. Matched therapy yielded higher rates of stable disease for 6 months or more, partial response, or complete response. ctDNA assessment may have clinical utility and merits further investigation.
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http://dx.doi.org/10.1200/PO.18.00158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484865PMC
January 2019

Circulating tumor DNA alterations in patients with metastatic castration-resistant prostate cancer.

Cancer 2019 05 8;125(9):1459-1469. Epub 2019 Jan 8.

Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina.

Background: Because cell-free DNA (cfDNA) analysis facilitates the noninvasive genomic profiling of metastatic castration-resistant prostate cancer (mCRPC), the authors evaluated the association between cfDNA alterations and outcomes and evolution with therapy.

Methods: Patients with mCRPC underwent cfDNA genomic profiling using Guardant360, which examines major cancer-associated genes. Clinical factors, therapy information, failure-free survival, and overall survival (OS) were obtained for select patients. The association between genomic alterations and outcomes was investigated.

Results: Of 514 men with mCRPC, 482 (94%) had ≥1 circulating tumor DNA (ctDNA) alteration. The most common recurrent somatic mutations were in TP53 (36%), androgen receptor (AR) (22%), adenomatous polyposis coli (APC) (10%), neurofibromin 1 (NF1) (9%), epidermal growth factor receptor (EGFR), catenin beta-1 (CTNNB1), and AT-rich interactive domain-containing protein 1A (ARID1A) (6% each); and BRCA1, BRCA2, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (5% each) The most common genes with increased copy numbers were AR (30%), MYC (20%), and BRAF (18%). Clinical outcomes were available for 163 patients, 46 of whom (28.8%) were untreated for mCRPC. A higher number of ctDNA alterations, AR alterations, and amplifications of MYC and BRAF were associated with worse failure-free survival and/or OS. On multivariable analysis, MYC amplification remained significantly associated with OS. Prior therapy and serial profiling demonstrated the evolution of alterations in AR and other genes.

Conclusions: ctDNA frequently was detected in this large cohort of "real-world" patients with mCRPC, and the alterations appeared to be similar to previously reported tumor tissue alterations. A higher number of alterations, and AR and MYC alterations, appear to compromise clinical outcomes, suggesting a role for immune checkpoint inhibitors and novel AR and BET inhibitors in selected patients.
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http://dx.doi.org/10.1002/cncr.31959DOI Listing
May 2019

Targeted Tissue and Cell-Free Tumor DNA Sequencing of Advanced Lung Squamous-Cell Carcinoma Reveals Clinically Significant Prevalence of Actionable Alterations.

Clin Lung Cancer 2019 01 5;20(1):30-36.e3. Epub 2018 Sep 5.

The University of Texas MD Anderson Cancer Center, Houston, TX. Electronic address:

Background: Major guidelines do not recommend routine molecular profiling of lung squamous-cell carcinoma (LUSC) because the prevalence of actionable alterations is thought to be low. Increased utilization of next-generation sequencing (NGS), particularly with cell-free circulating tumor DNA, facilitates reevaluation of this premise. PATIENTS AND METHODS: We retrospectively evaluated the prevalence of actionable alterations in 2 distinct LUSC cohorts totaling 492 patients. A total of 410 consecutive patients with stage 3B or 4 LUSC were tested with a targeted cell-free circulating DNA NGS assay, and 82 patients with LUSC of any stage were tested with a tissue NGS cancer panel.

Results: In the overall cohort, 467 patients (94.9%) had a diagnosis of LUSC, and 25 patients (5.1%) had mixed histology with a squamous component. A total of 10.5% of the LUSC subgroup had somatic alterations with therapeutic relevance, including in EGFR (2.8%), ALK/ROS1 (1.3%), BRAF (1.5%), and MET amplification or exon 14 skipping (5.1%). Sixteen percent of patients with mixed histology had an actionable alteration. In the LUSC subgroup, 3 evaluable patients were treated with targeted therapy for an actionable alteration; all of them experienced partial response.

Conclusion: In this large, real-world LUSC cohort, we observed a clinically significant prevalence of actionable alterations. Accurate local histopathologic assessment in advanced-stage LUSC can be challenging. Further evaluation of the genomic landscape in this setting is warranted to potentially identify underappreciated treatment options.
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http://dx.doi.org/10.1016/j.cllc.2018.08.020DOI Listing
January 2019

Comprehensive molecular characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer.

Nat Med 2018 09 16;24(9):1449-1458. Epub 2018 Jul 16.

Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

Clinical studies support the efficacy of programmed cell death 1 (PD-1) targeted therapy in a subset of patients with metastatic gastric cancer (mGC). With the goal of identifying determinants of response, we performed molecular characterization of tissues and circulating tumor DNA (ctDNA) from 61 patients with mGC who were treated with pembrolizumab as salvage treatment in a prospective phase 2 clinical trial. In patients with microsatellite instability-high and Epstein-Barr virus-positive tumors, which are mutually exclusive, dramatic responses to pembrolizumab were observed (overall response rate (ORR) 85.7% in microsatellite instability-high mGC and ORR 100% in Epstein-Barr virus-positive mGC). For the 55 patients for whom programmed death-ligand 1 (PD-L1) combined positive score positivity was available (combined positive score cut-off value ≥1%), ORR was significantly higher in PD-L1(+) gastric cancer when compared to PD-L1(-) tumors (50.0% versus 0.0%, P value <0.001). Changes in ctDNA levels at six weeks post-treatment predicted response and progression-free survival, and decreased ctDNA was associated with improved outcomes. Our findings provide insight into the molecular features associated with response to pembrolizumab in patients with mGC and provide biomarkers potentially relevant for the selection of patients who may derive greater benefit from PD-1 inhibition.
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http://dx.doi.org/10.1038/s41591-018-0101-zDOI Listing
September 2018

The Landscape of Actionable Genomic Alterations in Cell-Free Circulating Tumor DNA from 21,807 Advanced Cancer Patients.

Clin Cancer Res 2018 08 18;24(15):3528-3538. Epub 2018 May 18.

Guardant Health, Inc., Redwood City, California.

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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http://dx.doi.org/10.1158/1078-0432.CCR-17-3837DOI Listing
August 2018

Multiplex Gene Profiling of Cell-Free DNA in Patients With Metastatic Melanoma for Monitoring Disease.

JCO Precis Oncol 2018 17;2. Epub 2018 May 17.

, , , , , and , John Wayne Cancer Institute at Providence Saint John's Health Center, Santa Monica; and , Guardant Health, Redwood City, CA; and , Medical Data Research Center at Providence Saint Joseph's Health, Portland, OR.

Purpose: Hotspot blood cell-free DNA (cfDNA) biomarker assays have limited utility in profiling tumor heterogeneity and burden and in capturing regional metastasis with low disease burden in patients with melanoma. We investigated the utility of a sensitive 54-cancer gene digital next-generation sequencing approach targeting blood cfDNA single nucleotide variants (SNVs) and copy number amplification for monitoring disease in patients with melanoma with regional or distant organ metastasis (DOM).

Patients And Methods: A total of 142 blood samples were evaluated by digital next-generation sequencing across two patient cohorts. Cohort 1 contained 44 patients with stage II, III, or IV disease with matched tumor DNA at the time of surgery or DOM. Cohort 2 consisted of 12 overlapping patients who were longitudinally monitored after complete lymph node dissection to DOM.

Results: In cohort 1, cfDNA SNVs were detected in 75% of patients. Tumor-cfDNA somatic SNV concordance was 85% at a variant allele fraction of ≥ 0.5%. An SNV load (number of unique SNVs detected) of greater than two SNVs and an SNV burden (total cumulative SNV VAF) of > 0.5% were significantly associated with worse overall survival ( < .05) in stage IV patients. In cohort 2, 98 longitudinal blood samples along with matched regional and distant metastases from 12 stage III patients were analyzed before complete lymph node dissection and throughout disease progression. cfDNA SNV levels correlated with tumor burden ( = .019), enabled earlier detection of recurrence compared with radiologic imaging ( < .01), captured tumor heterogeneity, and identified increasing SNVs levels before recurrence.

Conclusion: This study demonstrates significant utility for cfDNA profiling in patients with melanoma with regional and/or distant metastasis for earlier detection of recurrence and progression and in capturing tumor evolution and heterogeneity, thus impacting how patients with melanoma are monitored.
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http://dx.doi.org/10.1200/PO.17.00225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446321PMC
May 2018

Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies.

Clin Cancer Res 2018 08 24;24(15):3539-3549. Epub 2018 Apr 24.

Guardant Health, Redwood City, California.

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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http://dx.doi.org/10.1158/1078-0432.CCR-17-3831DOI Listing
August 2018

Next-Generation Sequencing of Circulating Tumor DNA Reveals Frequent Alterations in Advanced Hepatocellular Carcinoma.

Oncologist 2018 05 27;23(5):586-593. Epub 2018 Feb 27.

Center for Personalized Cancer Therapy, Division of Hematology/Oncology, Department of Medicine, University of California San Diego Moores Cancer Center, La Jolla, California, USA

Background: Because imaging has a high sensitivity to diagnose hepatocellular carcinoma (HCC) and tissue biopsies carry risks such as bleeding, the latter are often not performed in HCC. Blood-derived circulating tumor DNA (ctDNA) analysis can identify somatic alterations, but its utility has not been characterized in HCC.

Materials And Methods: We evaluated 14 patients with advanced HCC (digital ctDNA sequencing [68 genes]). Mutant relative to wild-type allele fraction was calculated.

Results: All patients (100%) had somatic alterations (median = 3 alterations/patient [range, 1-8]); median mutant allele fraction, 0.29% (range, 0.1%-37.77%). Mutations were identified in several genes: (57% of patients), (29%), (7%), (7%), (7%), and (7%); amplifications, in (14%), (14%), (14%), (7%), (7%), (7%), (7%), (7%), and (7%). Eleven patients (79%) had ≥1 theoretically actionable alteration. No two patients had identical genomic portfolios, suggesting the need for customized treatment. A patient with a -inactivating and a -activating mutation received matched treatment: palbociclib (CDK4/6 inhibitor) and celecoxib (COX-2/Wnt inhibitor); des-gamma-carboxy prothrombin level decreased by 84% at 2 months (1,410 to 242 ng/mL [normal: ≤7.4 ng/mL]; alpha fetoprotein [AFP] low at baseline). A patient with a -inactivating and a -activating mutation (an effect suggested by in silico molecular dynamic simulations) received sirolimus (mechanistic target of rapamycin inhibitor) and cabozantinib (MET inhibitor); AFP declined by 63% (8,320 to 3,045 ng/mL [normal: 0-15 ng/mL]).

Conclusion: ctDNA derived from noninvasive blood tests can provide exploitable genomic profiles in patients with HCC.

Implications For Practice: This study reports that blood-derived circulating tumor DNA can provide therapeutically exploitable genomic profiles in hepatocellular cancer, a malignancy that is known to be difficult to biopsy.
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http://dx.doi.org/10.1634/theoncologist.2017-0479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947459PMC
May 2018

Genomic Landscape of Cell-Free DNA in Patients with Colorectal Cancer.

Cancer Discov 2018 02 1;8(2):164-173. Epub 2017 Dec 1.

The University of Texas MD Anderson Cancer Center, Houston, Texas.

"Liquid biopsy" approaches analyzing cell-free DNA (cfDNA) from the blood of patients with cancer are increasingly utilized in clinical practice. However, it is not yet known whether cfDNA sequencing from large cohorts of patients with cancer can detect genomic alterations at frequencies similar to those observed by direct tumor sequencing, and whether this approach can generate novel insights. Here, we report next-generation sequencing data from cfDNA of 1,397 patients with colorectal cancer. Overall, frequencies of genomic alterations detected in cfDNA were comparable to those observed in three independent tissue-based colorectal cancer sequencing compendia. Our analysis also identified a novel cluster of extracellular domain (ECD) mutations in , mediating resistance by blocking binding of anti-EGFR antibodies. Patients with ECD mutations displayed striking tumor heterogeneity, with 91% harboring multiple distinct resistance alterations (range, 1-13; median, 4). These results suggest that cfDNA profiling can effectively define the genomic landscape of cancer and yield important biological insights. This study provides one of the first examples of how large-scale genomic profiling of cfDNA from patients with colorectal cancer can detect genomic alterations at frequencies comparable to those observed by direct tumor sequencing. Sequencing of cfDNA also generated insights into tumor heterogeneity and therapeutic resistance and identified novel ectodomain mutations. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-1009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809260PMC
February 2018

Clinical utility of circulating cell-free DNA in advanced colorectal cancer.

PLoS One 2017 29;12(8):e0183949. Epub 2017 Aug 29.

Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

Background: Circulating cell-free DNA (cfDNA) isolated from the plasma of cancer patients (pts) has been shown to reflect the genomic mutation profile of the tumor. However, physician and patient assessment of clinical utility of these assays in patients with metastatic colorectal cancer (mCRC) has not been previously described.

Methods: Patients were prospectively consented to a prospective genomic matching protocol (Assessment of Targeted Therapies Against Colorectal Cancer [ATTACC]), with collection of blood for cfDNA extraction and sequencing of a 54-gene panel in a CLIA-certified lab. Formalin-fixed, paraffin-embedded (FFPE) tissue from prior resections or biopsies underwent 50-gene sequencing. Results from both assays were returned to the treating physicians for patient care and clinical trial selection. Follow-up surveys of treating physicians and chart reviews assessed clinical utility.

Results: 128 mCRC pts were enrolled between 6/2014 and 1/2015. Results were returned in median of 13 and 26 days for cfDNA and FFPE sequencing, respectively. With cfDNA sequencing, 78% (100/128) of samples had a detectable somatic genomic alteration. 50% of cfDNA cases had potentially actionable alterations, and 60% of these could be genomically matched to at least one clinical trial in our institution. 50% (15/30) of these pts enrolled onto an identified matched trial. Physicians reported that the cfDNA testing improved the quality of care they could provide in 73% of the cases, and that 89% of pts reported greater satisfaction with the efforts to personalize experimental therapeutic agents.

Conclusions: cfDNA sequencing can provide timely information on potentially actionable mutations and amplifications, thereby facilitating clinical trial enrollment and improving the perceived quality of care.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183949PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574560PMC
October 2017

Genomic Alterations in Circulating Tumor DNA from Diverse Cancer Patients Identified by Next-Generation Sequencing.

Cancer Res 2017 10 14;77(19):5419-5427. Epub 2017 Aug 14.

Center for Personalized Cancer Therapy and Division of Hematology and Oncology, UCSD Moores Cancer Center, La Jolla, California.

Noninvasive genomic profiling of tumors may be possible with next-generation sequencing (NGS) of blood-derived circulating tumor DNA (ctDNA), but proof of concept in a large cohort of patients with diverse cancers has yet to be reported. Here we report the results of an analysis of plasma-derived ctDNA from 670 patients with diverse cancers. The tumors represented in the patient cohort were mainly gastrointestinal (31.8%), brain (22.7%), or lung (20.7%). ctDNA obtained from most patients [ = 423 (63%)] displayed at least one alteration. The most frequent alterations seen, as characterized mutations or variants of unknown significance, occurred in (32.5% of patients), (13%), (12.5%), and (9.1%); for characterized alterations, 30.7% (), 7.6% (), 12.2% (), and 7.7% (). We found that 32% of brain tumors had at least one ctDNA alteration. Head and neck tumors were independently associated with a higher number of alterations in a multivariable analysis ( = 0.019). Notably, 320/670 (48%) of patients displayed potentially actionable alterations, with 241 patients possible candidates for on-label or off-label treatment with an FDA-approved drug. Several illustrations of the clinical utility of the information obtained for improving treatment of specific patients is provided. Our findings demonstrate the feasibility and impact of genomic profiling of tumors by ctDNA NGS, greatly encouraging broader investigations of the application of this technology for precision medicine in cancer management. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-0885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626633PMC
October 2017

Prospective Feasibility Study for Using Cell-Free Circulating Tumor DNA-Guided Therapy in Refractory Metastatic Solid Cancers: An Interim Analysis.

JCO Precis Oncol 2017 26;1. Epub 2017 Jun 26.

, , , , , , , , , and , Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; and , , and , Guardant Health, Redwood City, CA.

Purpose: Retrospective studies have demonstrated that cell-free circulating tumor DNA (ctDNA) hotspot testing predicts matched therapy response to first- and second-line therapies in patients with advanced non-small-cell lung cancer (NSCLC). However, no prospective outcomes studies have evaluated ctDNA-guided matched therapy decision making on the basis of comprehensive plasma genomic testing including all four major classes of alterations. Here, we report the clinical utility of this approach in advanced solid tumor cancers.

Patients And Methods: We conducted a multiple parallel cohort, open-label, clinical trial using ctDNA-guided matched therapy when tissue was insufficient or unobtainable for next-generation sequencing. Plasma-based digital sequencing identified point mutations in 70 genes and indels, fusions, and copy number amplifications in selected genes. Patients with prespecified targetable alterations in metastatic NSCLC, gastric cancer (GC), and other cancers were matched to several independent targeted agent trials at a tertiary academic center.

Results: Somatic alterations were detected in 59 patients with GC (78%), and 25 patients (33%) had targetable alterations (, n = 11; , n = 5; , n = 3; , n = 6). In NSCLC, 62 patients (85%) had somatic alterations, and 34 (47%) had targetable alterations (, n = 29; , n = 2; , n = 1; , n = 2). After confirmation of ctDNA findings on tissue (to meet trial eligibility criteria), 10 patients with GC and 17 patients with NSCLC received molecularly matched therapy. Response rate and disease control rate were 67% and 100%, respectively, in GC and 87% and 100%, respectively, in NSCLC. Response was independent of targeted alteration variant allele fraction in NSCLC ( = .63).

Conclusion: To our knowledge, this is the first prospective feasibility study of comprehensive ctDNA-guided treatment in advanced GC and lung cancers. Response rates in this interim analysis are similar to those in tissue-based targeted therapy studies.
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http://dx.doi.org/10.1200/PO.16.00059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446388PMC
June 2017

Utility of Genomic Assessment of Blood-Derived Circulating Tumor DNA (ctDNA) in Patients with Advanced Lung Adenocarcinoma.

Clin Cancer Res 2017 Sep 24;23(17):5101-5111. Epub 2017 May 24.

Center for Personalized Cancer Therapy and Division of Hematology and Oncology, UCSD Moores Cancer Center, La Jolla, California.

Genomic alterations in blood-derived circulating tumor DNA (ctDNA) from patients with non-small cell lung adenocarcinoma (NSCLC) were ascertained and correlated with clinical characteristics and therapeutic outcomes. Comprehensive plasma ctDNA testing was performed in 88 consecutive patients; 34 also had tissue next-generation sequencing; 29, other forms of genotyping; and 25 (28.4%) had no tissue molecular tests because of inadequate tissue or biopsy contraindications. Seventy-two patients (82%) had ≥1 ctDNA alteration(s); among these, 75% carried alteration(s) potentially actionable by FDA-approved (61.1%) or experimental drug(s) in clinical trials (additional 13.9%). The most frequent alterations were in the (44.3% of patients), (27.3%), (14.8%), (13.6%), and (6.8%) genes. The concordance rate for alterations was 80.8% (100% vs. 61.5%; ≤1 vs. >1 month between ctDNA and tissue tests; = 0.04) for patients with any detectable ctDNA alterations. Twenty-five patients (28.4%) received therapy matching ≥1 ctDNA alteration(s); 72.3% ( = 16/22) of the evaluable matched patients achieved stable disease ≥6 months (SD) or partial response (PR). Five patients with ctDNA-detected T790M were subsequently treated with a third generation EGFR inhibitor; all five achieved SD ≥ 6 months/PR. Patients with ≥1 alteration with ≥5% variant allele fraction (vs. < 5%) had a significantly shorter median survival ( = 0.012). ctDNA analysis detected alterations in the majority of patients, with potentially targetable aberrations found at expected frequencies. Therapy matched to ctDNA alterations demonstrated appreciable therapeutic efficacy, suggesting clinical utility that warrants future prospective studies. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-2497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581668PMC
September 2017

Biopsy-free circulating tumor DNA assay identifies actionable mutations in lung cancer.

Oncotarget 2016 Oct;7(41):66880-66891

City of Hope, Duarte, CA 91010, USA.

Introduction: The potential of oncogene-driven targeted therapy is perhaps most fully realized in non-small cell lung cancer (NSCLC), given the number of genomic targets and approved matched therapies. However, invasive tissue biopsy at the time of each disease progression may not be possible and is associated with high morbidity and cost. Use of newly available "liquid biopsies" can circumvent these issues.

Results: 83% of subjects had at least one genomic alteration identified in plasma. Most commonly mutated genes were TP53, KRAS and EGFR. Subjects with no detectable ctDNA were more likely to have small volume disease, lepidic growth pattern, mucinous tumors or isolated leptomeningeal disease.

Methods: Subjects were individuals with NSCLC undergoing analysis of cell-free circulating tumor DNA using a validated, commercially-available next-generation sequencing assay at a single institution. Demographic, clinicopathologic information and results from tissue and plasma-based genomic testing were reviewed for each subject.

Conclusions: This is the first clinic-based series of NSCLC patients assessing outcomes of targeted therapies using a commercially available ctDNA assay. Over 80% of patients had detectable ctDNA, concordance between paired tissue and blood for truncal oncogenic drivers was high and patients with biomarkers identified in plasma had PFS in the expected range. These data suggest that biopsy-free ctDNA analysis is a viable first choice when the diagnostic tissue biopsy is insufficient for genotyping or at the time of progression when a repeated invasive tissue biopsy is not possible/preferred.
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http://dx.doi.org/10.18632/oncotarget.11801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341844PMC
October 2016

Detection rate of actionable mutations in diverse cancers using a biopsy-free (blood) circulating tumor cell DNA assay.

Oncotarget 2016 Mar;7(9):9707-17

Center for Personalized Cancer Therapy and Division of Hematology and Oncology, UCSD Moores Cancer Center, La Jolla, CA, USA.

Analysis of cell-free DNA using next-generation sequencing (NGS) is a powerful tool for the detection/monitoring of alterations present in circulating tumor DNA (ctDNA). Plasma extracted from 171 patients with a variety of cancers was analyzed for ctDNA (54 genes and copy number variants (CNVs) in three genes (EGFR, ERBB2 and MET)). The most represented cancers were lung (23%), breast (23%), and glioblastoma (19%). Ninety-nine patients (58%) had at least one detectable alteration. The most frequent alterations were TP53 (29.8%), followed by EGFR (17.5%), MET (10.5%), PIK3CA (7%), and NOTCH1 (5.8%). In contrast, of 222 healthy volunteers, only one had an aberration (TP53). Ninety patients with non-brain tumors had a discernible aberration (65% of 138 patients; in 70% of non-brain tumor patients with an alteration, the anomaly was potentially actionable). Interestingly, nine of 33 patients (27%) with glioblastoma had an alteration (6/33 (18%) potentially actionable). Overall, sixty-nine patients had potentially actionable alterations (40% of total; 69.7% of patients (69/99) with alterations); 68 patients (40% of total; 69% of patients with alterations), by a Food and Drug Administration (FDA) approved drug. In summary, 65% of diverse cancers (as well as 27% of glioblastomas) had detectable ctDNA aberration(s), with the majority theoretically actionable by an approved agent.
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http://dx.doi.org/10.18632/oncotarget.7110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891078PMC
March 2016

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

PLoS One 2015 16;10(10):e0140712. Epub 2015 Oct 16.

Department of Research and Bioinformatics, Guardant Health, Inc., Redwood City, California, United States of America; Administration, Guardant Health, Inc., Redwood City, California, United States of America.

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140712PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608804PMC
June 2016

Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients.

Oncotarget 2015 Nov;6(37):40360-9

Guardant Health Inc., Redwood City, CA, USA.

Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.
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http://dx.doi.org/10.18632/oncotarget.5465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741900PMC
November 2015

A Multicenter, Open-Label Phase II Clinical Trial of Combined MEK plus EGFR Inhibition for Chemotherapy-Refractory Advanced Pancreatic Adenocarcinoma.

Clin Cancer Res 2016 Jan 6;22(1):61-8. Epub 2015 Aug 6.

Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California.

Purpose: On the basis of preclinical evidence of synergistic activity between MEK and EGFR inhibitors in pancreatic ductal adenocarcinoma (PDAC), we evaluated the safety and efficacy of selumetinib, a MEK1/2 inhibitor, plus erlotinib in patients with previously treated advanced PDAC.

Experimental Design: In this single-arm phase II trial, eligible patients received the combination of erlotinib 100 mg plus selumetinib 100 mg daily in 3-week cycles. Study assessments included measurement of clinical outcomes, with a primary endpoint of overall survival, and exploration of potential molecular predictors of treatment benefit.

Results: Forty-six patients were enrolled and received a median of two cycles (range, 1-7). Although no objective responses were observed, 19 patients (41%) showed evidence of stable disease for ≥6 weeks, and 13 of 34 patients (38%) had a CA19-9 decline ≥50%. Median progression-free survival was 1.9 months [95% confidence interval (CI), 1.4-3.3 months], with a median overall survival of 7.3 months (95% CI, 5.2-8.0 months). Common adverse events included rash, diarrhea, and nausea/vomiting. Patients with tumors exhibiting an epithelial phenotype (demonstrated by a high level of E-cadherin expression) were more likely to be sensitive to study treatment. Tumor-derived DNA was detectable in plasma from the majority of patients using next-generation digital DNA sequencing, and its relative abundance correlated with tumor burden.

Conclusions: A therapeutic strategy of dual targeted inhibition of the MEK and EGFR pathways shows modest antitumor activity in pancreatic cancer. Specific molecular subtypes may derive greatest benefit from this combination. Further exploration, both with more potent MEK inhibitors and in molecularly enriched patient subsets, is warranted.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-0979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703532PMC
January 2016

Cell-Free DNA Next-Generation Sequencing in Pancreatobiliary Carcinomas.

Cancer Discov 2015 Oct 24;5(10):1040-8. Epub 2015 Jun 24.

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California. Department of Medicine, University of California, San Francisco, California.

Unlabelled: Patients with pancreatic and biliary carcinomas lack personalized treatment options, in part because biopsies are often inadequate for molecular characterization. Cell-free DNA (cfDNA) sequencing may enable a precision oncology approach in this setting. We attempted to prospectively analyze 54 genes in tumor and cfDNA for 26 patients. Tumor sequencing failed in 9 patients (35%). In the remaining 17, 90.3% (95% confidence interval, 73.1%-97.5%) of mutations detected in tumor biopsies were also detected in cfDNA. The diagnostic accuracy of cfDNA sequencing was 97.7%, with 92.3% average sensitivity and 100% specificity across five informative genes. Changes in cfDNA correlated well with tumor marker dynamics in serial sampling (r = 0.93). We demonstrate that cfDNA sequencing is feasible, accurate, and sensitive in identifying tumor-derived mutations without prior knowledge of tumor genotype or the abundance of circulating tumor DNA. cfDNA sequencing should be considered in pancreatobiliary cancer trials where tissue sampling is unsafe, infeasible, or otherwise unsuccessful.

Significance: Precision medicine efforts in biliary and pancreatic cancers have been frustrated by difficulties in obtaining adequate tumor tissue for next-generation sequencing. cfDNA sequencing reliably and accurately detects tumor-derived mutations, paving the way for precision oncology approaches in these deadly diseases.
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http://dx.doi.org/10.1158/2159-8290.CD-15-0274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592417PMC
October 2015

Transcriptome analysis of CD133-positive stem cells and prognostic value of survivin in colorectal cancer.

Cancer Genomics Proteomics 2014 Sep-Oct;11(5):259-66

Division of Colorectal Surgery, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Colorectal Cancer Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.

Background/aim: CD133 is an important, but not exclusive, biomarker of colorectal cancer (CRC) stem cells.

Materials And Methods: In order to identify other CRC stem cell-specific genes, we performed a comparative expression profiling of CD133(+) and CD133(-) cell populations in primary and metastatic tumors from four patients with CRC. CD133(+) and CD133(-) CRC cells were isolated using MagSweeper and used for whole-transcriptome analysis with RNA-Seq.

Results: We found that in CD133(+) cells, 17 genes (RNASE2, PRB2, IL4, MGC27382, CLEC4C, SALL3, GIMAP1, ISG15, LOC728875, ZIK1, ICAM2, CCDC7, CDYL2, LRRC2, ZEB1, OSTF1 and CCDC144B) were significantly up-regulated compared to CD133(-) CRC cells. Among them, IL4 has been known as an inducer of survivin implicated in the survival and proliferation of cancer cells. However, the prognostic value of survivin in CRC is controversial. We evaluated survivin expression in formalin-fixed paraffin-embedded tumor samples of 188 patients with CRC by immunohistochemistry. Survivin over-expression was detected in 85 patients (45.2%) and was significantly associated with primary tumor sites (p=0.028), lymph node metastasis (p=0.029) and advanced III/IV CRC stages (AJCC 7; p=0.001). Furthermore, survivin up-regulation correlated with reduced disease-free survival (DFS; p=0.021) and overall survival (OS; p<0.000) and was proved to be an independent prognostic factor for both DFS and OS in multivariate analysis.

Conclusion: Our data suggest that CD133(+) CRC stem cells have a distinct expression pattern and that survivin, up-regulated by differentially expressed IL-4, is a candidate biomarker for the prediction of recurrence and survival in CRC.
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May 2015

mRNA-Seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer.

PLoS One 2012 7;7(11):e49144. Epub 2012 Nov 7.

Department of Diagnostic Research, Illumina, Inc., Hayward, California, United States of America.

Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049144PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492322PMC
May 2013

Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines.

PLoS One 2012 7;7(5):e33788. Epub 2012 May 7.

Department of Surgery, Stanford University School of Medicine, Stanford, California, USA.

Background: To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.

Methodology/principal Findings: We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.

Conclusions/significance: For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033788PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346739PMC
September 2012

A Microfluidic Platform for Characterization of Protein-Protein Interactions.

IEEE Sens J 2009 Aug;9(8):883-891

M. Javanmard and F. Pease are with the Department of Electrical Engineering, Stanford University, Stanford, CA 94305 USA ( ; ). A. H. Talasaz, M. Nemat-Gorgani, D. E. Huber, and R. W. Davis are with the Stanford Genome Technology Center, Palo Alto, CA 94304 USA ( ; ; ; ). M. Ronaghi is with Illumina Inc., San Diego, CA 92121 USA ( ).

Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein-protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein-glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules.
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http://dx.doi.org/10.1109/JSEN.2009.2022558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2868195PMC
August 2009
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