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PDF file - 395KB, Figure S1. Pre-surgery plasma ddPCR analysis for PIK3CA mutations. Figure S2. Positive and negative controls for droplet thresholds and counts. Figure S3. Lower Limit of Detection for PIK3CA Exon 9. Supplementary Table S1: Primers used to amplify and sequence FFPE specimens. Supplementary Table S2: Primers and probes used for ddPCR on FFPE samples. Supplementary Table S3: Primers and probes for ddPCR of plasma DNA. Supplementary Table S4: Fractional abundance and 95% confidence intervals of positive ptDNA samples.

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection.Experimental Design: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR.Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery.Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients. Clin Cancer Res; 20(10); 2643–50. ©2014 AACR.

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer.Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR.Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%).Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

Supplementary Methods Figure S1 ESR1 Y537SY537N and D538G probes are mutation specific. Figure S2 Patient 1 and Patient 14's plasma DNA. Table S1 Additional patient characteristics. Table S2 Primers used in this study.

Patients hospitalized with COVID-19 are at significant risk for superimposed bacterial pneumonia. However, diagnosing superinfection is challenging due to its clinical resemblance to severe COVID-19. We therefore evaluated whether the immune biomarker, procalcitonin, could facilitate the diagnosis of bacterial superinfection. To do so, we identified 185 patients with severe COVID-19 who underwent lower respiratory culture; 85 had superinfection. Receiver operating characteristic curve analysis showed that procalcitonin at the time of culture was incapable of distinguishing patients with bacterial infection (AUC, 0.52). We conclude that static measurement of procalcitonin does not aid in the diagnosis of superinfection in severe COVID-19.

IntroductionProcalcitonin expression is thought to be stimulated by bacteria and suppressed by viruses via interferon signalling. Consequently, during respiratory viral illness, clinicians often interpret elevated procalcitonin as evidence of bacterial coinfection, prompting antibiotic administration. We sought to evaluate the validity of this practice and the underlying assumption that viral infection inhibits procalcitonin synthesis.MethodsWe conducted a retrospective cohort study of patients hospitalised with pure viral infection (n=2075) versus bacterial coinfection (n=179). The ability of procalcitonin to distinguish these groups was assessed. In addition, procalcitonin and interferon gene expression were evaluated in murine and cellular models of influenza infection.ResultsPatients with bacterial coinfection had higher procalcitonin than those with pure viral infection, but also more severe disease and higher mortality (pDiscussionThese studies reveal that procalcitonin rises during pure viral infection in proportion to disease severity and is not suppressed by interferon signalling, in contrast to prior models of procalcitonin regulation. Applied clinically, our data suggest that procalcitonin represents a better indicator of disease severity than bacterial coinfection during viral respiratory infection.

BackgroundPatients with active acute myelogenous leukemia (AML) are at risk for leukemic infiltration (LI) into the lung and acute tumor lysis pneumopathy (ATLP) following chemotherapy. Fulminant presentations of these leukemic lung diseases are well-described, but indolent forms have not yet been studied. Therefore, we sought to elucidate the clinical features of mild-to-moderate LI and ATLP.MethodsA retrospective cohort analysis was performed on 51 hospitalized patients with AML, circulating blast count ≥3%, non-critical illness, and receipt of bronchoscopy between 2015-2019. Diagnoses of LI and ATLP were made via retrospective chart review by a multidisciplinary team of physicians.Results19 cases of leukemic lung disease were identified: 14 with LI and 5 with ATLP. The clinical presentations closely resembled pneumonia, with the majority demonstrating respiratory symptoms (63%), hypoxemia (63%), fever (84%), and pulmonary opacities (100%). All patients were presumptively diagnosed with infection, leading to an average of 18 days of broad-spectrum antibiotic therapy and multiple instances of delayed chemotherapy in treatment candidates. Although most patients were near the end-of-life (90% died within 1 year), transitions to comfort care were infrequent (25%) and hospitalizations were protracted (median 25 days).ConclusionsLI and ATLP are common yet under-recognized pulmonary complications in patients with active AML. When presenting indolently, these conditions are difficult to distinguish from lung infection, leading to missed diagnosis, inappropriate antibiosis, chemotherapy deferrals, and prolonged hospitalizations. Greater awareness and consensus definitions of LI and ATLP are therefore needed to improve care of this population.

PURPOSE Androgen receptor ( AR) gene alterations, including ligand-binding domain mutations and copy number (CN) gain, have yet to be fully established as predictive markers of resistance to enzalutamide and abiraterone in men with metastatic castration-resistant prostate cancer (mCRPC). The goal of this study was to validate AR gene alterations detected in cell-free DNA (cfDNA) as markers of enzalutamide and abiraterone resistance in patients with mCRPC. METHODS Patients with mCRPC (N = 62) were prospectively enrolled between 2014 and 2018. Blood was collected before therapies—enzalutamide (n = 25), abiraterone (n = 35), or enzalutamide and abiraterone (n = 2) —and at disease progression. We used deep next-generation sequencing to analyze cfDNA for sequence variants and CN status in AR and 45 additional cancer-associated genes. Primary end points were prostate-specific antigen response, progression-free survival (PFS), and overall survival (OS). RESULTS Elevated tumor-specific cfDNA (circulating tumor DNA) was associated with a worse prostate-specific antigen response (hazard ratio [HR], 3.17; 95% CI, 1.11 to 9.05; P = .031), PFS (HR, 1.76; 95% CI, 1.03 to 3.01; P = .039), and OS (HR, 2.92; 95% CI, 1.40 to 6.11; P = .004). AR ligand-binding domain missense mutations (HR, 2.51; 95% CI, 1.15 to 5.72; P = .020) were associated with a shorter PFS in multivariable models. AR CN gain was associated with a shorter PFS; however, significance was lost in multivariable modeling. Genetic alterations in tumor protein p53 (HR, 2.70; 95% CI, 1.27 to 5.72; P = .009) and phosphoinositide 3-kinase pathway defects (HR, 2.62; 95% CI, 1.12 to 6.10; P = .026) were associated with a worse OS in multivariable models. CONCLUSION These findings support the conclusion that high circulating tumor DNA burden is associated with worse outcomes to enzalutamide and abiraterone in men with mCRPC. Tumor protein p53 loss and phosphoinositide 3-kinase pathway defects were associated with worse OS in men with mCRPC. AR status associations with outcomes were not robust, and additional validation is needed.

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer. Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR. Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%). Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection. Experimental Design: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR. Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery. Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients. Clin Cancer Res; 20(10); 2643–50. ©2014 AACR.

// Alexa Goldstein 1, 2, 3 , Patricia Valda Toro 1, 2, 3 , Justin Lee 2, 3 , John L. Silberstein 1, 3 , Mary Nakazawa 1, 3 , Ian Waters 2, 3 , Karen Cravero 2, 3 , David Chu 2, 3 , Rory L. Cochran 2, 3 , Minsoo Kim 2, 3 , Daniel Shinn 2, 3 , Samantha Torquato 1, 2, 3 , Robert M. Hughes 1, 2, 3 , Aparna Pallavajjala 4 , Michael A. Carducci 2, 3 , Channing J. Paller 2, 3 , Samuel R. Denmeade 2, 3 , Bruce Kressel 2, 3 , Bruce J. Trock 1 , Mario A. Eisenberger 1, 2, 3 , Emmanuel S. Antonarakis 1, 2, 3 , Ben H. Park 2, 3, 5 , Paula J. Hurley 1, 2, 3 1 The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA 2 The Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA 3 The Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA 4 The Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA 5 The Whiting School of Engineering, Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA Correspondence to: Paula J. Hurley, email: phurley2@jhmi.edu Keywords: next generation sequencing, droplet digital PCR, circulating tumor DNA, androgen receptor, DNA polymerase Received: September 22, 2016 Accepted: December 25, 2016 Published: January 31, 2017 ABSTRACT Somatic genetic alterations including copy number and point mutations in the androgen receptor ( AR) are associated with resistance to therapies targeting the androgen/AR axis in patients with metastatic castration resistant prostate cancer (mCRPC). Due to limitations associated with biopsying metastatic lesions, plasma derived cell-free DNA (cfDNA) is increasingly being used as substrate for genetic testing. AR mutations detected by deep next generation sequencing (NGS) of cfDNA from patients with mCRPC have been reported at allelic fractions ranging from over 25% to below 1%. The lower bound threshold for accurate mutation detection by deep sequencing of cfDNA has not been comprehensively determined and may have locus specific variability. Herein, we used NGS for AR mutation discovery in plasma-derived cfDNA from patients with mCRPC and then used droplet digital polymerase chain reaction (ddPCR) for validation. Our findings show the AR (tTC>cTC) F877L hotspot was prone to false positive mutations during NGS. The rate of error at AR (tTC>cTC) F877L during amplification prior to ddPCR was variable among high fidelity polymerases. These results highlight the importance of validating low-abundant mutations detected by NGS and optimizing and controlling for amplification conditions prior to ddPCR.

Objectives Circulating plasma DNA is being increasingly used for biomedical and clinical research as a substrate for genetic testing. However, cell lysis can occur hours after venipuncture when using standard tubes for blood collection, leading to an increase in contaminating cellular DNA that may hinder analysis of circulating plasma DNA. Cell stabilization agents can prevent cellular lysis for several days, reducing the need for immediate plasma preparation after venipuncture, thereby facilitating the ease of blood collection and sample preparation for clinical research. However, the majority of cell stabilizing reagents have not been formally tested for their ability to preserve circulating plasma tumor DNA. Design & methods In this study, we compared the properties of two cell stabilizing reagents, the cell-free DNA BCT tube and the PAXgene tube, by collecting blood samples from metastatic breast cancer patients and measuring genome equivalents of plasma DNA by droplet digital PCR. We compared wild type PIK3CA genome equivalents and also assayed for two PIK3CA hotspot mutations, E545K and H1047R. Results Our results demonstrate that blood stored for 7 days in BCT tubes did not show evidence of cell lysis, whereas PAXgene tubes showed an order of magnitude increase in genome equivalents, indicative of considerable cellular lysis. Conclusions We conclude that BCT tubes can prevent lysis and cellular release of genomic DNA of blood samples from cancer patients when stored at room temperature, and could therefore be of benefit for blood specimen collections in clinical trials.

The PIK3CA gene encodes for the p110 alpha isoform of PI3 kinase and is one of the most frequently mutated oncogenes in human cancers. However, the mechanisms by which PIK3CA mutations activate cell signaling are not fully understood. Here we used a phosphoproteomic approach to compare differential phosphorylation patterns between human breast epithelial cells and two isogenic somatic cell knock in derivatives, each harboring a distinct PIK3CA mutation. We demonstrated differential phosphorylation patterns between isogenic cell lines containing a PIK3CA helical domain mutation (E545K) compared to cells with a PIK3CA kinase domain mutation (H1047R). In particular, the receptor tyrosine kinase, HER3, showed increased phosphorylation at tyrosine 1328 in H1047R cells versus E545K cells. Genetic studies using shRNA demonstrated that H1047R cells have a profound decrease in growth factor independent proliferation upon HER3 knock down, but this effect was attenuated in E545K cells. In addition, HER3 knock down led to reductions in both PI3 kinase and MAP kinase pathway activation in H1047R cells, but in E545K cells only PI3 kinase pathway diminution was observed. These studies demonstrate the power of using paired isogenic cell lines for proteomic analysis to gain new insights into oncogenic signal transduction pathways.

Loss-of-heterozygosity (LOH) analysis of archival tumor tissue can aid in determining the clinical significance of BRCA variants. Here we describe an approach for assessing LOH in formalin-fixed, paraffin-embedded (FFPE) tissues using variant-specific probes and droplet digital polymerase chain reaction (ddPCR). We evaluated LOH in 2 related breast cancer patients harboring a rare missense BRCA2 variant of unknown clinical significance (c.6966G>T; M2322I). Conventional PCR followed by Sanger sequencing suggested a change in allelic abundance in the FFPE specimens. However, we found no evidence of LOH as determined by the allelic ratio (wild type-variant) for BRCA2 in both patients' archival tumor specimens and adjacent normal control tissues using ddPCR. In summary, these experiments demonstrate the utility of ddPCR to quickly and accurately assess LOH in archival FFPE tumor tissue.

Digital polymerase chain reaction is a new technology that enables detection and quantification of cancer DNA molecules from peripheral blood. Using this technique, we identified mutant PIK3CA DNA in circulating ptDNA (plasma tumor DNA) from a patient with concurrent early stage breast cancer and non-small cell lung cancer. The patient underwent successful resection of both her breast and lung cancers, and using standard Sanger sequencing the breast cancer was shown to harbor the identical PIK3CA mutation identified in peripheral blood. This case report highlights potential applications and concerns that can arise with the use of ptDNA in clinical oncology practice.

11019 Background: PIK3CA is mutated in up to 30% of breast cancers. Classically somatic mutations are identified by Sanger sequencing of the primary tumor specimen. However third generation droplet digital PCR technologies offer a novel platform for quantitative mutation detection with improved sensitivity. Methods: Thirty stage I-III breast cancer patients were consented on an IRB-approved prospective repository study at Johns Hopkins for collection of their primary breast tumor specimen. Formalin-fixed paraffin embedded (FFPE) samples were analyzed by standard sequencing for three PIK3CA hotspot mutations. The DNA from these samples was then analyzed using the RainDrop digital PCR platform with TaqMan probes in a triplex format to simultaneously detect and quantitate hotspot mutations and genome equivalents. Results are expressed as a percentage of mutant to wild-type PIK3CA molecules for each sample. Results: Standard sequencing of all tumors (n=30) identified seven PIK3CA Exon 20 mutations (H1047R) and three Exon 9 mutations (E545K). Samples were scored as PIK3CA mutation positive by digital PCR if the tumor DNA contained at least 5% mutant molecules. All ten mutations identified by sequencing were verified by digital PCR with quantities of mutant molecules ranging from 20.3-55.6% in a given sample. Digital PCR identified additional PIK3CA mutations that were wild type by standard sequencing including three mutant Exon 20 samples, two mutant Exon 9 samples and one sample with an Exon 20 and Exon 9 mutation. Quantities of mutant molecules in these additional samples ranged from 5-28.9%. Conclusions: RainDrop digital PCR offers improved sensitivity and quantification for detecting PIK3CA mutations in FFPE samples using nanograms of DNA. Additional mutations identified by digital PCR may reflect genetic heterogeneity or possibly tissue contamination. The clinical utility of identifying a small proportion of mutations is unknown but may impact eligibility for targeted therapies and clinical trials. Ongoing studies will also address whether the identification of solid tumor mutations in circulating cell-free plasma DNA by digital PCR can improve diagnostics and aid in therapeutic decisions.