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We appreciate the interest in our study from Takayasu and colleagues. They raise important issues which were largely addressed in our article but deserve further discussion. Takayasu and colleagues question whether variants detected in plasma in our study reflect tumor somatic mutations. We explain

Purpose: The role of plasma-based tumor mutation burden (pTMB) in predicting response to pembrolizumab-based first-line standard-of-care therapy for metastatic non–small cell lung cancer (mNSCLC) has not been explored. Experimental Design: A 500-gene next-generation sequencing panel was used to assess pTMB. Sixty-six patients with newly diagnosed mNSCLC starting first-line pembrolizumab-based therapy, either alone or in combination with chemotherapy, were enrolled (Clinicaltrial.gov identifier: NCT03047616). Response was assessed using RECIST 1.1. Associations were made for patient characteristics, 6-month durable clinical benefit (DCB), progression-free survival (PFS), and overall survival (OS). Results: Of 66 patients, 52 (78.8%) were pTMB-evaluable. Median pTMB was 16.8 mutations per megabase (mut/Mb; range, 1.9–52.5) and was significantly higher for patients achieving DCB compared with no durable benefit (21.3 mut/Mb vs. 12.4 mut/Mb, P = 0.003). For patients with pTMB ≥ 16 mut/Mb, median PFS was 14.1 versus 4.7 months for patients with pTMB < 16 mut/Mb [HR, 0.30 (0.16–0.60); P < 0.001]. Median OS for patients with pTMB ≥ 16 was not reached versus 8.8 months for patients with pTMB < 16 mut/Mb [HR, 0.48 (0.22–1.03); P = 0.061]. Mutations in ERBB2 exon 20, STK11, KEAP1, or PTEN were more common in patients with no DCB. A combination of pTMB ≥ 16 and absence of negative predictor mutations was associated with PFS [HR, 0.24 (0.11–0.49); P < 0.001] and OS [HR, 0.31 (0.13–0.74); P = 0.009]. Conclusions: pTMB ≥ 16 mut/Mb is associated with improved PFS after first-line standard-of-care pembrolizumab-based therapy in mNSCLC. STK11/KEAP1/PTEN and ERBB2 mutations may help identify pTMB-high patients unlikely to respond. These results should be validated in larger prospective studies.

Improved diagnostics for pancreatic ductal adenocarcinoma (PDAC) to detect the disease at earlier, curative stages and to guide treatments is crucial to progress against this disease. The development of a liquid biopsy for PDAC has proven challenging due to the sparsity and variable phenotypic expression of circulating biomarkers. Here we report methods we developed for isolating specific subsets of extracellular vesicles (EV) from plasma using a novel magnetic nanopore capture technique. In addition, we present a workflow for identifying EV miRNA biomarkers using RNA sequencing and machine-learning algorithms, which we used in combination to classify distinct cancer states. Applying this approach to a mouse model of PDAC, we identified a biomarker panel of 11 EV miRNAs that could distinguish mice with PDAC from either healthy mice or those with precancerous lesions in a training set of n = 27 mice and a user-blinded validation set of n = 57 mice (88% accuracy in a three-way classification). These results provide strong proof-of-concept support for the feasibility of using EV miRNA profiling and machine learning for liquid biopsy. Significance: These findings present a panel of extracellular vesicle miRNA blood-based biomarkers that can detect pancreatic cancer at a precancerous stage in a transgenic mouse model. Cancer Res; 78(13); 3688–97. ©2018 AACR.

Up to 30% of patients diagnosed with breast cancer will develop recurrent disease within their lifetime, and currently this form of the disease is incurable. There are unmet needs to better understand underlying metastatic biology, identify new therapeutic targets and develop better methods for monitoring changes in disease, both to monitor response and elucidate resistance mechanisms. To address these needs, the METAMORPH Study encompasses a comprehensive approach that combines serial molecular tissue profiling at the RNA and DNA level with circulating markers (DTCs, CTCs, plasma tumor DNA), and ongoing assessment of therapeutic response. METAMORPH is a prospective cohort study of women with suspected or confirmed recurrent breast cancer and accessible tumor by standard clinical biopsy, who are enrolled at the University of Pennsylvania prior to starting a new therapy for recurrent metastatic disease. The aims of this trial are to (1) evaluate the mechanisms through which recurrent breast cancer are genetically distinct from the primary tumor, (2) evaluate the circulating tumor biomarker trajectory of recurrent disease, (3) elucidate “escape pathways” of progressing tumors that emerge during the selective pressure of therapy, and (4) explore clinical utility of tumor and blood testing. The study protocol integrates research aims into clinical care, including a standardized approach to disease assessment and biopsy, pathologic confirmation of histology and receptor subtype, panel-based CLIA-approved genomic profiling, collection of research specimens, and standardized reporting of results, which are returned to patients and physicians. Patients are followed for treatment and outcome, and serial samples are collected at progression. A companion protocol, COMET, provides education about genomic testing and assesses patient understanding and impact of results. To date, 155 patients have enrolled, 142 (92%) have been biopsied, 120 (77%) have had sufficient DNA for molecular profiling and 109 (70%) have had genomic panel testing. Accrual is ongoing, with an initial target of 300 patients. Multiple sites within the UPHS Health System are enrolling. Contact information: angela.demichele@uphs.upenn.edu. Key words: Metastatic disease, tumor profiling. Citation Format: DeMichele A, Soucier-Ernst DJ, Clark C, Shih N, Stavropoulos W, Maxwell KN, Feldman M, Lierbamen D, Morrissette JJD, Paul MR, Pan T-C, Wang J, Belka GK, Chen Y, Yee S, Carpenter E, Fox K, Matro J, Clark A, Shah P, Domchek S, Bradbury A, Chodosh L. METAMORPH: METAstatic markers of recurrent tumor PHenotype for breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-06-03.

Simple Summary Discovery of predictive and prognostic radiomic features in cancer is currently of great interest to the radiologic and oncologic community. Tumor phenotypic and prognostic information can be obtained by extracting features on tumor segmentations, and it is typically imaging analysts, physician trainees, and attending physicians who provide these labeled datasets for analysis. The potential impact of level and type of specialty training on interobserver variability in manual segmentation of NSCLC was examined. Although there was some variability in segmentation between readers, the subsequently extracted radiomic features were overall well correlated. High fidelity radiomic feature extraction relies on accurate feature extraction from imaging that produce robust prognostic and predictive radiomic NSCLC biomarkers. This study concludes that this goal can be obtained using segmenters of different levels of training and clinical experience. Abstract This study tackles interobserver variability with respect to specialty training in manual segmentation of non-small cell lung cancer (NSCLC). Four readers included for segmentation are: a data scientist (BY), a medical student (LS), a radiology trainee (MH), and a specialty-trained radiologist (SK) for a total of 293 patients from two publicly available databases. Sørensen–Dice (SD) coefficients and low rank Pearson correlation coefficients (CC) of 429 radiomics were calculated to assess interobserver variability. Cox proportional hazard (CPH) models and Kaplan-Meier (KM) curves of overall survival (OS) prediction for each dataset were also generated. SD and CC for segmentations demonstrated high similarities, yielding, SD: 0.79 and CC: 0.92 (BY-SK), SD: 0.81 and CC: 0.83 (LS-SK), and SD: 0.84 and CC: 0.91 (MH-SK) in average for both databases, respectively. OS through the maximal CPH model for the two datasets yielded c-statistics of 0.7 (95% CI) and 0.69 (95% CI), while adding radiomic and clinical variables (sex, stage/morphological status, and histology) together. KM curves also showed significant discrimination between high- and low-risk patients (p-value < 0.005). This supports that readers’ level of training and clinical experience may not significantly influence the ability to extract accurate radiomic features for NSCLC on CT. This potentially allows flexibility in the training required to produce robust prognostic imaging biomarkers for potential clinical translation.

BACKGROUND Plasma circulating tumor DNA (ctDNA) is rarely detectable by traditional methods in patients with GBM. As a result, unlike in lung and other cancers, serial next generation sequencing of ctDNA for monitoring GBM tumor burden has been challenging. In light of the low tumor fraction (TF) of DNA fragments in GBM patient plasma and the urgent need to improve upon MRI for tracking GBM tumor burden, we conducted a pilot study in patients with newly diagnosed GBM using the C2 intelligence platform (C2i Genomics), which leverages genome-wide mutational integration for highly sensitive ctDNA detection. METHODS Plasma was collected pre- and post-operatively in patients with newly diagnosed GBM undergoing surgical resection/biopsy. cfDNA was extracted, quantified, and analyzed for fragment size. Genomic DNA (gDNA) was extracted from matched tumor tissue. Whole genome sequencing (WGS) was performed on both gDNA and cfDNA. A specific copy number alteration (CNA) compendium was created for each patient to generate a readout of TF (Zviran, Nat Medicine 2020). We assessed the association between TF at post-operative day 1 (a surrogate for residual disease) and OS, adjusting for other prognostic factors using Cox regression. RESULTS 37 patients were enrolled. For samples with high tumor fraction (n=5), a statistically significant (p< 1e-4) correlation between CNA profiles of tumor tissue and plasma samples was observed. Post-operative TF above the median value was associated with inferior OS (median 7.7 vs. 19.3 months, p=0.019). This association persisted after adjusting for age, O6-methylguanine-DNA methyltransferase methylation status, extent of resection, and performance status (adjusted HR 2.5, 95% CI 1.1-5.6, p=0.03). CONCLUSION Genome-wide mutational integration enables ultra-sensitive detection of ctDNA in GBM patient plasma. Post-operative TF measured by the C2i test is independently associated with OS in newly diagnosed GBM, providing the foundation to evaluate this technology for personalized prognostication and disease monitoring.

14 Background: Current NCCN guidelines recommend comprehensive molecular profiling for all newly diagnosed patients with metastatic non-squamous NSCLC to enable the delivery of personalized medicine. We have previously demonstrated that incorporation of plasma based next-generation gene sequencing (NGS) improves detection of clinically actionable mutations in patients with advanced NSCLC (Aggarwal et al, JAMA Oncology, 2018). To increase rates of comprehensive molecular testing at our institution, we adapted our clinical practice to include concurrent use of plasma (P) and tissue (T) based NGS upon initial diagnosis. P NGS testing was performed using a commercial 74 gene assay. We analyzed the impact of this practice change on guideline concordant molecular testing at our institution. Methods: A retrospective cohort study of patients with newly diagnosed metastatic non-squamous NSCLC following the implementation of this practice change in 12/2018 was performed. Tiers of NCCN guideline concordant testing were defined, Tier 1: complete EGFR, ALK, BRAF, ROS1, MET, RET, NTRK testing, Tier 2: included above, but with incomplete NTRK testing, Tier 3: > 2 genes tested, Tier 4: single gene testing, Tier 5: no testing. Proportion of patients with comprehensive molecular testing by modality (T NGS vs. T+P NGS) were compared using one-sided Fisher’s exact test. Results: Between 01/2019, and 12/2019, 170 patients with newly diagnosed metastatic non-Sq NSCLC were treated at our institution. Overall, 98.2% (167/170) patients underwent molecular testing, Tier 1: n = 100 (59%), Tier 2: n = 39 (23%), Tier 3/4: n = 28 (16.5%), Tier 5: n = 3 (2%). Amongst these patients, 43.1% (72/167) were tested with T NGS alone, 8% (15/167) with P NGS alone, and 47.9% (80/167) with T+P NGS. A higher proportion of patients underwent comprehensive molecular testing (Tiers 1+2) using T+P NGS: 95.7% (79/80) compared to T alone: 62.5% (45/72), p < 0.0005. Prior to the initiation of first line treatment, 72.4% (123/170) patients underwent molecular testing, Tier 1: n = 73 (59%), Tier 2: n = 27 (22%) and Tier 3/4: n = 23 (18%). Amongst these, 39% (48/123) were tested with T NGS alone, 7% (9/123) with P NGS alone and 53.6% (66/123) with T+P NGS. A higher proportion of patients underwent comprehensive molecular testing (Tiers 1+2) using T+P NGS, 100% (66/66) compared to 52% (25/48) with T NGS alone (p < 0.0005). Conclusions: Incorporation of concurrent T+P NGS testing in treatment naïve metastatic non-Sq NSCLC significantly increased the proportion of patients undergoing guideline concordant molecular testing, including prior to initiation of first-line therapy at our institution. Concurrent T+P NGS should be adopted into institutional pathways and routine clinical practice.

To determine the feasibility of liquid biopsy for monitoring of patients with advanced melanoma, cell-free DNA was extracted from plasma for 25 Stage III/IV patients, most (84.0%) having received previous therapy. DNA concentrations ranged from 0.6 to 390.0 ng/ml (median = 7.8 ng/ml) and were positively correlated with tumor burden as measured by imaging (Spearman rho = 0.5435, p = .0363). Using ultra-deep sequencing for a 61-gene panel, one or more mutations were detected in 12 of 25 samples (48.0%), and this proportion did not vary significantly for patients on or off therapy at the time of blood draw (52.9% and 37.5% respectively; p = .673). Sixteen mutations were detected in eight different genes, with the most frequent mutations detected in BRAF, NRAS, and KIT. Allele fractions ranged from 1.1% to 63.2% (median = 29.1%). Among patients with tissue next-generation sequencing, nine of 11 plasma mutations were also detected in matched tissue, for a concordance of 81.8%.

PURPOSE We have previously demonstrated the potential role of liquid biopsy, specifically plasma cell-free DNA (cfDNA), as a non-invasive biomarker for prognostication in patients with glioblastoma. In separate prior studies, we have also developed MRI-based radiomic signatures to predict survival outcomes in glioblastoma. In this study, for the first time, we evaluated the potential of combining radiomic signatures, epidemiological and clinical variables, and plasma cfDNA quantification for upfront prediction of overall survival (OS) in patients with newly diagnosed glioblastoma. METHODS Quantitative radiomic features were extracted from multiparametric MRI (T1, T1Gd, T2, T2-FLAIR) scans of a discovery cohort of 505 and an independent replication cohort of 50 IDH-wildtype glioblastoma patients. For the independent replication cohort, pre-surgical plasma cfDNA was extracted and quantified. In the first stage, a radiomic signature was created for stratification of patients into categories of short (OS ≤ 6 months) and long (OS ≥ 18 months) survivors using a cross-validated XGBoost method based on the discovery cohort, which was tested independently on the replication cohort. In the second stage, the radiomic signature and clinical variables were integrated to build a second-stage signature using a cross-validated support vector machine (SVM) classifier to stratify the patients into short and long survivor categories. In the third stage, the value of the second-stage signature integrated with cfDNA concentration was assessed through a cross-validated SVM regression method. RESULTS The combination of radiomic, clinical, and cfDNA variables resulted in the best overall predictive accuracy, with Pearson’s correlation coefficient of 0.59 (p< 0.0001) between actual and predicted OS. CONCLUSION In this study, we evaluated the value of combining plasma cfDNA, radiomic, and clinical variables for predicting OS, and showed that it could act as an effective non-invasive prognostic and patient stratification tool in patients with newly diagnosed glioblastoma.

5544 Background: All cells release volatile organic compounds (VOCs) which emanate from body fluids. Our previous preliminary proof of concept study demonstrated that VOCs released from tissue and plasma from ovarian cancer patients are distinct from those released from samples of patients with benign tumors and controls. We seek to create a sensitive and specific, high-throughput screening test for cancer based on analysis of VOCs using novel nanosensors, first targeting cancers with limited clinical screening modalities. In this study we use these sensors to distinguish vapor characteristics in plasma samples from patients with ovarian and pancreatic cancer from benign specimens and controls. Methods: VOCs emanating from.5 mL of thawed, previously banked plasma samples from 93 total individuals were analyzed using a 10-channel nanoelectronic olfaction (“e-nose”) system based on single-stranded DNA-decorated single-walled carbon nanotube (DNA-NT) vapor sensors. Analysis was performed on samples from 20 patients with ovarian cancer, 20 with benign ovarian tumors and 20 age-matched women as well as 13 patients with pancreatic cancer, 10 patients with benign pancreatic disease, and 10 age- and sex-matched controls. All ovarian cancer patients and comparators were non-smokers, while 1 pancreatic patient and 1 corresponding control were current smokers. The sample set included cancer patients with both early- and late-stage disease. All cancer specimens were obtained proximal to initial diagnosis and prior to initiation of therapy. With a test time of approximately 20 minutes per sample, the array output for each individual sample creates a vector in a 10-dimensional sensor space. The ability of the nanosensor array to discriminate between malignant, benign, and healthy groups was investigated using linear discriminant analysis (LDA), support vector machine (SVM), k-nearest neighbors (KNN), and random forest classification algorithms. Each algorithm was trained and tested according to leave-one-out and repeated stratified k-fold cross-validation methods. Results: Compared to their corresponding benign and control specimens, the DNA-NT sensor array was able to discriminate the VOCs from ovarian cancer with 95% accuracy and pancreatic cancer with 90% accuracy. Plasma samples from patients with early-stage ovarian and pancreatic cancers were correctly identified by the algorithms. Conclusions: Nano-enabled DNA coated vapor sensors were able to distinguish the VOC pattern between cancer, benign and control samples in both ovarian and pancreatic cancer. We provide strong evidence that ovarian and pancreatic cancer alters the VOC pattern emanating from plasma. Our results provide optimism that a diagnostic approach based on vapor detection of ovarian and pancreatic cancer is achievable.

4122 Background: Traditional imaging-guided therapeutic decision-making for patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) may lag and, on occasion, be misleading. The concept of liquid biopsy-based molecular response holds promise for proximate and accurate therapy monitoring and assessment of emerging resistance to therapy. Here we investigate the association between baseline (pre-treatment) level and early, on-treatment changes in plasma circulating cell-free DNA (ccfDNA) mutant KRAS (ctKRAS) with progression-free survival (PFS) and overall survival (OS) in mPDAC. Methods: 189 plasma samples were analyzed from 123 total patients with mPDAC. An initial cohort included 54 patients treated at the University of Pennsylvania who received first-line standard of care (SOC) regimens and had a baseline plasma sample. Of these, 21 also had an on-therapy sample collected at ̃8 weeks. We also analyzed an independent cohort of 69 patients enrolled in the PRINCE trial (NCT03214250) who had a baseline sample, of which 45 also had an on-treatment sample at ̃8 weeks. PRINCE trial patients received gemcitabine/nab-paclitaxel with immunotherapy (I/O) agents (APX005M and/or nivolumab). ctKRAS variant allele fraction (VAF) was quantified by droplet digital PCR on pre-amplified ccfDNA. Baseline ctKRAS was dichotomized at 5% VAF. ctKRAS clearance was defined as detectable ctKRAS at baseline followed by ctKRAS becoming undetectable in the on-treatment sample. Results: Baseline ctKRAS (above/below 5% VAF) and ctKRAS clearance were associated with PFS and OS in both cohorts (Table). Further, in a multivariate cox regression model, ctKRAS clearance associated with improved PFS (HR 3.8, 1.4-10.9 or 3.6, 1.8-7.2) in both the SOC and I/O cohorts, respectively, and OS in the SOC cohort (HR 5.5, 1.5-20.8) after adjusting for baseline VAF. Conclusions: Baseline ctKRAS is significantly associated with OS and PFS in mPDAC in both independent cohorts. Further, early on-treatment ctKRAS clearance is strongly associated with improved PFS and OS, independent of baseline ctKRAS VAF. These data strongly support further investigation of ccfDNA as a biomarker of response and resistance to therapy.[Table: see text]

Purpose: The expanding number of targeted therapeutics for non–small cell lung cancer (NSCLC) necessitates real-time tumor genotyping, yet tissue biopsies are difficult to perform serially and often yield inadequate DNA for next-generation sequencing (NGS). We evaluated the feasibility of using cell-free circulating tumor DNA (ctDNA) NGS as a complement or alternative to tissue NGS. Experimental Design: A total of 112 plasma samples obtained from a consecutive study of 102 prospectively enrolled patients with advanced NSCLC were subjected to ultra-deep sequencing of up to 70 genes and matched with tissue samples, when possible. Results: We detected 275 alterations in 45 genes, and at least one alteration in the ctDNA for 86 of 102 patients (84%), with EGFR variants being most common. ctDNA NGS detected 50 driver and 12 resistance mutations, and mutations in 22 additional genes for which experimental therapies, including clinical trials, are available. Although ctDNA NGS was completed for 102 consecutive patients, tissue sequencing was only successful for 50 patients (49%). Actionable EGFR mutations were detected in 24 tissue and 19 ctDNA samples, yielding concordance of 79%, with a shorter time interval between tissue and blood collection associated with increased concordance (P = 0.038). ctDNA sequencing identified eight patients harboring a resistance mutation who developed progressive disease while on targeted therapy, and for whom tissue sequencing was not possible. Conclusions: Therapeutically targetable driver and resistance mutations can be detected by ctDNA NGS, even when tissue is unavailable, thus allowing more accurate diagnosis, improved patient management, and serial sampling to monitor disease progression and clonal evolution. Clin Cancer Res; 22(23); 5772–82. ©2016 AACR.

Noninvasive isolation of circulating tumor cells (CTCs) from patient blood samples allows for interrogation of valuable molecular and phenotypic information useful for disease diagnosis and monitoring response to therapy. However, CTCs are extremely rare relative to red and white blood cells (R/WBC), thus making CTC isolation from unmanipulated whole blood very time-consuming. Moreover, single CTC analysis often requires hand-picking, a step that can result in more CTC loss and compromised cell integrity. Here we describe an automated flow cytometry-based approach for isolation and analysis of single, viable CTCs that combines gentle RBC lysis and magnetic, no-wash negative-depletion of WBCs, followed by a highly adaptable sorting protocol for rare cells of interest. Multiparametric flow-cytometric panels allow probing of numerous extracellular markers for immunophenotyping, while whole transcriptome analysis contributes to molecular characterization of individual CTCs. Index sorting links single CTC proteogenomics information.

Purpose: The clinical utility of plasma cell-free DNA (cfDNA) has not been assessed prospectively in patients with glioblastoma (GBM). We aimed to determine the prognostic impact of plasma cfDNA in GBM, as well as its role as a surrogate of tumor burden and substrate for next-generation sequencing (NGS). Experimental Design: We conducted a prospective cohort study of 42 patients with newly diagnosed GBM. Plasma cfDNA was quantified at baseline prior to initial tumor resection and longitudinally during chemoradiotherapy. Plasma cfDNA was assessed for its association with progression-free survival (PFS) and overall survival (OS), correlated with radiographic tumor burden, and subjected to a targeted NGS panel. Results: Prior to initial surgery, GBM patients had higher plasma cfDNA concentration than age-matched healthy controls (mean 13.4 vs. 6.7 ng/mL, P < 0.001). Plasma cfDNA concentration was correlated with radiographic tumor burden on patients' first post-radiation magnetic resonance imaging scan (ρ = 0.77, P = 0.003) and tended to rise prior to or concurrently with radiographic tumor progression. Preoperative plasma cfDNA concentration above the mean (>13.4 ng/mL) was associated with inferior PFS (median 4.9 vs. 9.5 months, P = 0.038). Detection of ≥1 somatic mutation in plasma cfDNA occurred in 55% of patients and was associated with nonstatistically significant decreases in PFS (median 6.0 vs. 8.7 months, P = 0.093) and OS (median 5.5 vs. 9.2 months, P = 0.053). Conclusions: Plasma cfDNA may be an effective prognostic tool and surrogate of tumor burden in newly diagnosed GBM. Detection of somatic alterations in plasma is feasible when samples are obtained prior to initial surgical resection.

Background: Lung cancer is one of the most common cancers in the United States and the most fatal, with 142,670 deaths in 2019. Accurately determining tumor response is critical to clinical treatment decisions, ultimately impacting patient survival. To better differentiate between non-small cell lung cancer (NSCLC) responders and non-responders to therapy, radiomic analysis is emerging as a promising approach to identify associated imaging features undetectable by the human eye. However, the plethora of variables extracted from an image may actually undermine the performance of computer-aided prognostic assessment, known as the curse of dimensionality. In the present study, we show that correlative-driven hierarchical clustering improves high-dimensional radiomics-based feature selection and dimensionality reduction, ultimately predicting overall survival in NSCLC patients. Methods: To select features for high-dimensional radiomics data, a correlation-incorporated hierarchical clustering algorithm automatically categorizes features into several groups. The truncation distance in the resulting dendrogram graph is used to control the categorization of the features, initiating low-rank dimensionality reduction in each cluster, and providing descriptive features for Cox proportional hazards (CPH)-based survival analysis. Using a publicly available non- NSCLC radiogenomic dataset of 204 patients’ CT images, 429 established radiomics features were extracted. Low-rank dimensionality reduction via principal component analysis (PCA) was employed (𝒌 = 𝟏, 𝒏 < 𝟏) to find the representative components of each cluster of features and calculate cluster robustness using the relative weighted consistency metric. Results: Hierarchical clustering categorized radiomic features into several groups without primary initialization of cluster numbers using the correlation distance metric (as a function) to truncate the resulting dendrogram into different distances. The dimensionality was reduced from 429 to 67 features (for truncation distance of 0.1). The robustness within the features in clusters was varied from -1.12 to -30.02 for truncation distances of 0.1 to 1.8, respectively, which indicated that the robustness decreases with increasing truncation distance when smaller number of feature classes (i.e., clusters) are selected. The best multivariate CPH survival model had a C-statistic of 0.71 for truncation distance of 0.1, outperforming conventional PCA approaches by 0.04, even when the same number of principal components was considered for feature dimensionality. Conclusions: Correlative hierarchical clustering algorithm truncation distance is directly associated with robustness of the clusters of features selected and can effectively reduce feature dimensionality while improving outcome prediction.

We performed an analysis of patients with stage IVWe identified 131 consecutive patients withThe presence of

Noninvasive isolation of circulating tumor cells (CTCs) from patient blood samples allows for interrogation of valuable molecular and phenotypic information useful for disease diagnosis and monitoring response to therapy. However, CTCs are extremely rare relative to red and white blood cells (R/WBC), thus making CTC isolation from unmanipulated whole blood very time-consuming. Moreover, single CTC analysis often requires hand-picking, a step that can result in more CTC loss and compromised cell integrity. Here we describe an automated flow cytometry-based approach for isolation and analysis of single, viable CTCs that combines gentle RBC lysis and magnetic, no-wash negative-depletion of WBCs, followed by a highly adaptable sorting protocol for rare cells of interest. Multiparametric flow-cytometric panels allow probing of numerous extracellular markers for immunophenotyping, while whole transcriptome analysis contributes to molecular characterization of individual CTCs. Index sorting links single CTC proteogenomics information.

Detection of disease-associated, cell-free nucleic acids enables early diagnostics, genotyping, and personalized therapy, but is challenged by their low concentration and sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for Nucleic Acid enrichment Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo) that allows for specific cleavage of guide-complementary DNA and RNA with single nucleotide precision. NAVIGATER greatly increases the fractions of rare alleles, enhancing the sensitivity of downstream detection such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of KRAS G12D and nearly 100-fold increased sensitivity of clamped-PCR (PNA and XNA-PCR), enabling detection of low-frequency (0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-DASH, identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require the target to contain any specific (PAM-like) motifs; is a multi-turnover enzyme; cleaves ssDNA, dsDNA, and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases. The here-described NAVIGATER approach has important advantages over other enrichment methods.

Circulating tumor cells (CTCs) carry valuable biological information. While enumeration of CTCs in peripheral blood is an FDA-approved prognostic indicator of survival in metastatic prostate and other cancers, analysis of CTC phenotypic and genomic markers is needed to identify cancer origin and elucidate pathways that can guide therapeutic selection for personalized medicine. Given the emergence of single-cell mRNA sequencing technologies, a method is needed to isolate CTCs with high sensitivity and specificity as well as compatibility with downstream genomic analysis. Flow cytometry is a powerful tool to analyze and sort single cells, but pre-enrichment is required prior to flow sorting for efficient isolation of CTCs due to the extreme low frequency of CTCs in blood (one in billions of blood cells). While current enrichment technologies often require many steps and result in poor recovery, we demonstrate a magnetic separator and acoustic microfluidic focusing chip integrated system that enriches rare cells in-line with FACS™ (fluorescent activated cell sorting) and single-cell sequencing. This system analyzes, isolates, and index sorts single cells directly into 96-well plates containing reagents for Molecular Indexing (MI) and transcriptional profiling of single cells. With an optimized workflow using the integrated enrichment-FACS system, we performed a proof-of-concept experiment with spiked prostate cancer cells in peripheral blood and achieved: (i) a rapid one-step process to isolate rare cancer cells from lysed whole blood; (ii) an average of 92% post-enrichment cancer cell recovery (R2 = 0.9998) as compared with 55% recovery for a traditional benchtop workflow; and (iii) detection of differentially expressed genes at a single cell level that are consistent with reported cell-type dependent expression signatures for prostate cancer cells. These model system results lay the groundwork for applying our approach to human blood samples from prostate and other cancer patients, and support the enrichment-FACS system as a flexible solution for isolation and characterization of CTCs for cancer diagnosis. © 2018 International Society for Advancement of Cytometry.

Importance The clinical implications of adding plasma-based circulating tumor DNA next-generation sequencing (NGS) to tissue NGS for targetable mutation detection in non–small cell lung cancer (NSCLC) have not been formally assessed. Objective To determine whether plasma NGS testing was associated with improved mutation detection and enhanced delivery of personalized therapy in a real-world clinical setting. Design, Setting, and Participants This prospective cohort study enrolled 323 patients with metastatic NSCLC who had plasma testing ordered as part of routine clinical management. Plasma NGS was performed using a 73-gene commercial platform. Patients were enrolled at the Hospital of the University of Pennsylvania from April 1, 2016, through January 2, 2018. The database was locked for follow-up and analyses on January 2, 2018, with a median follow-up of 7 months (range, 1-21 months). Main Outcomes and Measures The number of patients with targetable alterations detected with plasma and tissue NGS; the association between the allele fractions (AFs) of mutations detected in tissue and plasma; and the association of response rate with the plasma AF of the targeted mutations. Results Among the 323 patients with NSCLC (60.1% female; median age, 65 years [range, 33-93 years]), therapeutically targetable mutations were detected inEGFR,ALK,MET,BRCA1,ROS1, RET, ERBB2,orBRAFfor 113 (35.0%) overall. Ninety-four patients (29.1%) had plasma testing only at the discretion of the treating physician or patient preference. Among the 94 patients with plasma testing alone, 31 (33.0%) had a therapeutically targetable mutation detected, thus obviating the need for an invasive biopsy. Among the remaining 229 patients who had concurrent plasma and tissue NGS or were unable to have tissue NGS, a therapeutically targetable mutation was detected in tissue alone for 47 patients (20.5%), whereas the addition of plasma testing increased this number to 82 (35.8%). Thirty-six of 42 patients (85.7%) who received a targeted therapy based on the plasma result achieved a complete or a partial response or stable disease. The plasma-based targeted mutation AF had no correlation with depth of Response Evaluation Criteria in Solid Tumors response (r = −0.121;P = .45). Conclusions and Relevance Integration of plasma NGS testing into the routine management of stage IV NSCLC demonstrates a marked increase of the detection of therapeutically targetable mutations and improved delivery of molecularly guided therapy.

125 Background: Prostate specific membrane antigen (PSMA) is a highly expressed tumor-associated antigen potentially amenable to chimeric antigen receptor-modified T (CAR-T) cell therapy for castration-resistant prostate cancer (CRPC). However, a primary challenge to the success of CAR-T therapy in CRPC is the immunosuppressive microenvironment, characterized by high levels of TGFβ. The immunosuppressive functions of TGFβ can be inhibited in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. Methods: We conducted a first-in-human phase 1 clinical trial to evaluate the feasibility, safety and preliminary efficacy of PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in patients with metastatic CRPC (NCT03089203). In a 3+3 dose-escalation design, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting (LD) chemotherapy. In Cohort 3, one patient received 1-3 x 108/m2 CART-PSMA-TGFβRdn cells following a LD chemotherapy regimen of cyclophosphamide and fludarabine (Cy/Flu). In Cohort -3, three patients received 1-3 x 107/m2 CART-PSMA-TGFβRdn cells following Cy/Flu. Patients underwent metastatic tumor biopsies at baseline and on day 10 following treatment. Quantitative PCR of CART-PSMA-TGFβRdn DNA was performed at serial timepoints to evaluate for CAR-T expansion and persistence in peripheral blood and trafficking to target tissues. Multiplex cytokine analysis assessed CART-PSMA-TGFβRdn bioactivity. Results: Ten patients received CART-PSMA-TGFβRdn therapy across dose-level cohorts. All CART-PSMA-TGFβRdn infusion products met target transduction efficiency. Evaluation of CAR-T cellular kinetics demonstrated dose-dependent peripheral blood T cell expansion, as well as tumor tissue trafficking in post-treatment tumor biopsies. At Cohort 2 and above, 5 of 7 treated patients developed grade ≥2 cytokine release syndrome (CRS). Marked increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) correlated with high-grade CRS events. One grade 5 adverse event (sepsis) occurred in Cohort 3. PSA decline was observed in 6 of 10 patients (median decline -33.2%, range -11.6% to -98.3%), and PSA30 response occurred in 4 of 10 patients (including one patient achieving PSA < 0.1 ng/mL). Conclusions: Adoptive cellular therapy with CART-PSMA-TGFβRdn is safe and feasible in patients with metastatic CRPC. A dose-dependent and lymphodepletion chemotherapy-dependent relationship was observed with CART-PSMA-TGFβRdn cell expansion, cytokine expression, CRS, and anti-tumor effect. Correlative cell trafficking and paired tumor Nanostring analyses will be presented. Future clinical investigations seek to enhance anti-tumor efficacy, while optimizing the therapeutic window. Clinical trial information: NCT03089203.

Tumor heterogeneity - resulting from genetic and epigenetic alterations acquired during tumor progression - is a critical driver of phenotypic diversity in most cancers. A lethal consequence of tumor heterogeneity is the acquisition of metastatic traits by tumor cells, leading to poor clinical outcomes. This remains a major problem in pancreatic ductal adenocarcinoma (PDAC), which continues to have the worst prognosis of any major cancer type. While most cases of PDAC present with metastatic disease at the time of diagnosis, the patterns and burden of metastasis can vary widely, with some patients exhibiting a limited metastatic burden while others have more extensive spread, which impacts clinical outcomes. However, the biological and functional differences that drive metastatic heterogeneity are poorly understood. One barrier to understanding metastatic heterogeneity has been a paucity of model systems that capture this natural variation and allow for direct assessment of paired primary tumors and metastases. We previously developed an autochthonous model of PDAC – the KPCX model – that employs multiplexed fluorescence-based labeling to track the contribution of multiple distinct tumor populations to metastasis. Importantly, this technique allows for ascertainment of primary-metastatic lineage relationships in vivo, so that primary tumor clones with substantial metastatic potential can be distinguished with those having poor metastatic potential. To understand the factors underlying differences in metastatic potential, we analyzed paired primary tumors and metastases in the KPCX model and from a cohort of 398 PDAC patients. Genomic and transcriptomic analysis of murine and human metastatic PDAC revealed an association between the highly metastatic state and gene amplification or transcriptional upregulation of MYC and its transcriptional targets. Functional assessments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was also associated of MYC signaling pathways and enrichment for MYC amplification in metastasis. Collectively, these results implicate MYC activity as a major determinant of metastatic burden and heterogeneity in advanced PDA. Citation Format: Ravikanth Maddipati, Robert J. Norgard, Timour Baslan, Komal S. Rathi, Amy Zhang, Pichai Raman, Max D. Wengyn, Taiji Yamazoe, Jinyang Li, David Balli, Michael J. LaRiviere, Ian W. Folkert, Ian D. Millstein, Jonathan Bermeo, Erica L. Carpenter, Scott Lowe, Christine Iacobuzio-Donahue, Faiyaz Notta, Ben Z. Stanger. MYC Influences metastatic heterogeneity in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-053.

Tumor heterogeneity - resulting from genetic and epigenetic alterations acquired during tumor progression - is a critical driver of phenotypic diversity in most cancers. A lethal consequence of tumor heterogeneity is the acquisition of metastatic traits by tumor cells, leading to poor clinical outcomes. This remains a major problem in pancreatic ductal adenocarcinoma (PDAC), which continues to have the worst prognosis of any major cancer type. While most cases of PDAC present with metastatic disease at the time of diagnosis, the patterns and burden of metastasis can vary widely, with some patients exhibiting a limited metastatic burden while others have more extensive spread, which impacts clinical outcomes. However, the biological and functional differences that drive metastatic heterogeneity are poorly understood. Citation Format: Ravikanth Maddipati, Robert J. Norgard, Timour Baslan, Komal S. Rathi, Amy Zhang, Pichai Raman, Max D. Wengyn, Taiji Yamazoe, Jinyang Li, David Balli, Michael LaRiviere, Ian W. Folkert, Ian D. Millstein, Jonathan Bermeo, Erica L. Carpenter, Scott Lowe, Christine Iacobuzio-Donahue, Faiyaz Notta, Ben Z. Stanger. MYC influences metastatic heterogeneity in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-071.

UNLABELLED : Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and messenger RNA (mRNA), collectively termed circulating tumor products (CTPs), represent areas of immense interest from scientists' and clinicians' perspectives. In melanoma, CTP analysis may have clinical utility in many areas, from screening and diagnosis to clinical decision-making aids, as surveillance biomarkers or sources of real-time genetic or molecular characterization. In addition, CTP analysis can be useful in the discovery of new biomarkers, patterns of treatment resistance, and mechanisms of metastasis development. Here, we compare and contrast CTCs, ctDNA, and mRNA, review the extent of translational evidence to date, and discuss how future studies involving both scientists and clinicians can help to further develop this tool for the benefit of melanoma patients. IMPLICATIONS FOR PRACTICE Scientific advancement has enabled the rapid development of tools to analyze circulating tumor cells, tumor DNA, and messenger RNA, collectively termed circulating tumor products (CTPs). A variety of techniques have emerged to detect and characterize melanoma CTPs; however, only a fraction has been applied to human subjects. This review summarizes the available human data that investigate clinical utility of CTP in cancer screening, melanoma diagnosis, prognosis, prediction, and genetic or molecular characterization. It provides a rationale for how CTPs may be useful for future research and discusses how clinicians can be involved in developing this exciting new technology.

2508 Background: Due to significant interpatient heterogeneity, survival outcomes vary widely in patients with GBM. Novel prognostic biomarkers are needed. We aimed to determine the prognostic impact of baseline plasma cfDNA concentration in patients with GBM. Methods: We analyzed 84 patients with newly diagnosed GBM and at least 7 months of follow-up time. The first 41 patients comprised a previously published derivation cohort (Bagley, Clin Cancer Res 2020). The subsequent 43 patients served as an independent validation cohort. cfDNA was extracted from plasma collected prior to initial surgical resection and quantified by qPCR for a 115 bp amplicon of the human ALU repeat element. Receiver operating characteristic (ROC) curve analysis was used in the derivation cohort to (1) assess the accuracy of plasma cfDNA concentration for predicting progression-free survival status at 7 months (PFS-7), a landmark based on the median PFS for newly diagnosed GBM (Stupp, N Engl J Med 2005), and (2) derive the optimal cutoff for dichotomizing patients into high- and low-cfDNA groups. In the validation cohort, logistic regression was used to measure the association of plasma cfDNA concentration (high vs. low) with PFS-7, adjusted for age, isocitrate dehydrogenase ( IDH) 1/2 mutational status, 0-6-methylguanine-methyltransferase ( MGMT) methylation, extent of resection, and performance status. Multivariate Cox regression was used for overall survival (OS) analysis. Results: In the derivation cohort, the optimal cutoff for plasma cfDNA was 25.0 ng/mL (area under the curve [AUC] = 0.663), with inferior PFS and OS in patients with cfDNA above this cutoff (PFS, median 4.9 vs. 9.5 months, log-rank p = 0.001; OS, median 8.5 vs. 15.5 months, log-rank p = 0.03). In the validation cohort, baseline plasma cfDNA concentration over the cutoff was independently associated with a lower likelihood of being alive and progression-free at 7 months (adjusted OR 0.13, 95% CI 0.02 – 0.75, p = 0.02). OS was also worse in in the validation cohort in patients with high plasma cfDNA (adjusted HR 3.0, 95% CI 1.1 – 8.0, p = 0.03). Conclusions: In patients with newly diagnosed GBM, high baseline plasma cfDNA concentration is associated with worse survival outcomes independent of other prognostic factors. Further validation in a larger, multicenter study is warranted.

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (

OBJECTIVES: A malignant pleural effusion (MPE) is a common complication in non-small cell lung cancer (NSCLC) with important staging and prognostic information. Patients with MPEs are often candidates for advanced therapies, however, the current gold standard, cytological analysis of pleural fluid samples, has limited sensitivity. We aimed to demonstrate the feasibility of non-invasive enumeration and immunophenotyping of EpCAM-positive cells in pleural fluid samples for the diagnosis of a MPE in NSCLC patients. MATERIALS AND METHODS: Pleural fluid specimens were prospectively collected from patients with NSCLC and the CellSearch® technology was utilized for the enumeration of pleural EpCAM-positive cells (PECs) and determination of PD-L1 expression on PECs from pleural fluid samples. The diagnostic performance of the enumeration of single PECs and PEC clusters was assessed using receiver operating characteristic (ROC) curves. The Kaplan-Meier method and Cox proportional hazards model was used to assess the impact of PECs and PEC clusters on overall survival (OS). RESULTS: 101 NSCLC patients were enrolled. The median number of PECs was significantly greater in the malignant (n=84) versus non-malignant group (n=17) (730 PECs/mL vs 1.0 PEC/mL, p

BACKGROUND Liquid biopsy has been not been widely utilized in patients with glioblastoma (GBM) compared to other solid tumors. However, the clinical utility of plasma cell-free DNA (cfDNA) in GBM has not been assessed prospectively or at the time of initial diagnosis. METHODS We conducted a prospective cohort study of patients with newly diagnosed GBM. Whole blood was collected in Streck® tubes at baseline prior to initial surgical resection and longitudinally during the course of adjuvant chemoradiotherapy. Plasma cfDNA concentration (ng/mL) was quantified by qPCR for a 115 bp amplicon of the human ALU repeat element, correlated with radiographic tumor burden by volumetry at multiple time points using Spearman rank correlation, and assessed for its impact on progression-free (PFS) and overall survival (OS) by Cox regression. RESULTS Prior to initial resection, GBM patients (N=42) had higher plasma cfDNA concentration compared to age-matched healthy controls (N=42) (mean 13.43 vs. 6.70 ng/mL, p< 0.001). Plasma cfDNA concentration was correlated with radiographic tumor burden on subjects’ first post-radiation MRI scan (r=0.77, p=0.003) and tended to rise prior to or concurrently with radiographic tumor progression. Pre-operative plasma cfDNA concentration above the mean (>13.4 ng/mL) was associated with inferior PFS (median 4.9 vs. 9.5 months, p=0.038) and OS (median 8.9 vs. 14.8 months, p=0.078). The impact on PFS persisted after adjusting for age, extent of resection, performance status, MGMT promoter methylation, and IDH1/2 mutational status (HR 2.48, 95% CI 1.1–6.1, p=0.046). CONCLUSIONS Plasma cfDNA may be an effective prognostic tool and noninvasive surrogate of tumor burden in newly diagnosed GBM. Tumor tissue samples from our cohort have been subjected to targeted next generation sequencing (NGS), and baseline plasma samples have been sent to Guardant Health for NGS. Plasma NGS results and concordance with matched tissue NGS will be included at time of presentation.

Comprehensive molecular analysis of rare circulating tumor cells (CTCs) and cell clusters is often hampered by low throughput and purity, as well as cell loss. To address this, we developed a fully integrated platform for flow cytometry-based isolation of CTCs and clusters from blood that can be combined with whole transcriptome analysis or targeted RNA transcript quantification. Downstream molecular signature can be linked to cell phenotype through index sorting. This newly developed platform utilizes in-line magnetic particle-based leukocyte depletion, and acoustic cell focusing and washing to achieve >98% reduction of blood cells and non-cellular debris, along with >1.5 log-fold enrichment of spiked tumor cells. We could also detect 1 spiked-in tumor cell in 1 million WBCs in 4/7 replicates. Importantly, the use of a large 200μm nozzle and low sheath pressure (3.5 psi) minimized shear forces, thereby maintaining cell viability and integrity while allowing for simultaneous recovery of single cells and clusters from blood. As proof of principle, we isolated and transcriptionally characterized 63 single CTCs from a genetically engineered pancreatic cancer mouse model (n = 12 mice) and, using index sorting, were able to identify distinct epithelial and mesenchymal sub-populations based on linked single cell protein and gene expression.

BACKGROUND: Distinguishing between radiographic pseudoprogression and true tumor progression is challenging in patients with GBM. Recent data suggests that plasma cfDNA concentration may serve as a viable surrogate for tumor burden in other malignancies. We performed a pilot study to determine the feasibility of detecting cfDNA in patients with newly diagnosed GBM and explored its correlation with radiographic tumor burden and other relevant clinical variables. METHODS: We collected blood in Streck cfDNA tubes from patients with radiographically suspected high-grade glioma prior to planned initial surgical resection. Plasma was isolated using a 3-step centrifugation protocol. cfDNA was extracted from using a QIAamp Circulating Nucleic Acid Kit. cfDNA concentration (ng DNA/mL) was determined by quantitative real-time PCR (qPCR) for the ALU repeat element. Tumor burden was defined as the sum of products of diameters (SPD) of target enhancing lesions plus the SPD of the T2 FLAIR signal abnormality on preoperative MRI. RESULTS: 22 preoperative patients were enrolled and diagnosed histopathologically with GBM. The median cfDNA concentration was 10.9 ng/mL (IQR 7.2–23.6, range 0.48–37.6). There was a significant correlation between radiographic tumor burden and cfDNA concentration (Spearmans rho = 0.46, p = 0.03). In a multiple linear regression model, cfDNA concentration remained significantly associated with radiographic tumor burden (beta coefficient 1.87, p=0.03) after adjusting for age, sex, tumor Ki-67 proliferation rate, weight, and glomerular filtration rate (GFR). GFR was also independently (negatively) associated with cfDNA concentration. CONCLUSIONS: In this small pilot study, we demonstrated that plasma cfDNA is easily detected and quantified in patients with newly diagnosed GBM prior to initial surgical resection. In addition, plasma cfDNA concentration was independently associated with radiographic tumor burden. A prospective, longitudinal study monitoring cfDNA dynamics during standard treatment for GBM, including gold standard tissue correlates for tumor progression vs. pseudoprogression, is ongoing at our institution.

The identification of therapeutically targetable mutations in circulating tumor cells (CTCs) from cancer patient blood is increasingly used to personalize patient care. Here, we describe a novel approach for the enumeration, capture, and molecular analysis of CTCs from blood using an FDA-approved CTC enrichment and enumeration platform followed by dielectrophoretic capture and next-generation sequencing.

The use of microtechnology for the highly selective isolation and sensitive detection of circulating tumor cells has shown enormous promise. One challenge for this technology is that the small feature sizes – which are the key to this technology's performance – can result in low sample throughput and susceptibility to clogging. Additionally, conventional molecular analysis of CTCs often requires cells to be taken off-chip for sample preparation and purification before analysis, leading to the loss of rare cells. To address these challenges, we have developed a microchip platform that combines fast, magnetic micropore based negative immunomagnetic selection (>10 mL h−1) with rapid on-chip in situ RNA profiling (>100× faster than conventional RNA labeling). This integrated chip can isolate both rare circulating cells and cell clusters directly from whole blood and allow individual cells to be profiled for multiple RNA cancer biomarkers, achieving sample-to-answer in less than 1 hour for 10 mL of whole blood. To demonstrate the power of this approach, we applied our device to the circulating tumor cell based diagnosis of pancreatic cancer. We used a genetically engineered lineage-labeled mouse model of pancreatic cancer (KPCY) to validate the performance of our chip. We show that in a cohort of patient samples (N = 25) that this device can detect and perform in situ RNA analysis on circulating tumor cells in patients with pancreatic cancer, even in those with extremely sparse CTCs (

Circulating tumor cells provide a non-invasive source of tumor material that can be valuable at all stages of disease management, including screening and early diagnosis, monitoring response to therapy, identifying therapeutic targets, and assessing development of drug resistance. Cells isolated from the blood of cancer patients can be used for phenotypic analysis, tumor genotyping, transcriptional profiling, as well as for ex vivo culture of isolated cells. There are a variety of novel technologies currently being developed for the detection and analysis of rare cells in circulation of cancer patients. Flow cytometry is a powerful cell analysis platform that is increasingly being used in this field of study due to its relatively high throughput and versatility with respect to the large number of commercially available antibodies and fluorescent probes available to translational and clinical researchers. More importantly, it offers the ability to easily recover viable cells with high purity that are suitable for downstream molecular analysis, thus making it an attractive technology for cancer research and as a diagnostic tool.

Circulating tumor cells provide a non-invasive source of tumor material that can be valuable at all stages of disease management, including screening and early diagnosis, monitoring response to therapy, identifying therapeutic targets, and assessing development of drug resistance. Cells isolated from the blood of cancer patients can be used for phenotypic analysis, tumor genotyping, transcriptional profiling, as well as for ex vivo culture of isolated cells. There are a variety of novel technologies currently being developed for the detection and analysis of rare cells in circulation of cancer patients. Flow cytometry is a powerful cell analysis platform that is increasingly being used in this field of study due to its relatively high throughput and versatility with respect to the large number of commercially available antibodies and fluorescent probes available to translational and clinical researchers. More importantly, it offers the ability to easily recover viable cells with high purity that are suitable for downstream molecular analysis, thus making it an attractive technology for cancer research and as a diagnostic tool.

The identification of therapeutically targetable mutations in circulating tumor cells (CTCs) from cancer patient blood is increasingly used to personalize patient care. Here, we describe a novel approach for the enumeration, capture, and molecular analysis of CTCs from blood using an FDA-approved CTC enrichment and enumeration platform followed by dielectrophoretic capture and next-generation sequencing.

Background: Checkpoint inhibitors such as anti-PD-1 are ineffective as single agents for patients (pts) with PDAC. Preclinical data suggest that chemotherapy with agonistic CD40 antibodies can be combined with anti-PD-1 to trigger effective T cell immunity. We conducted a multi-center, open label clinical trial to evaluate the combination of APX005M with nivo and standard chemotherapy (gem and NP). Herein, we report safety and efficacy from the ongoing study. Methods: Pts with previously untreated PDAC were enrolled in 4 cohorts: Gem/NP/APX005M 0.1 mg/kg (B1), Gem/NP/APX005M 0.3 mg/kg (B2), Gem/NP/Nivo/APX005M 0.1 mg/kg (C1) and Gem/NP/Nivo/APX005M 0.3 mg/kg (C2). Primary objectives were to evaluate safety and determine the recommended Phase II dose (RP2D) of APX005M. Secondary objectives included tumor response and immune pharmacodynamics. Analyses were performed on dose-limiting toxicity (DLT)-evaluable subjects, defined as receiving 1 dose of APX005M and ≥ 2 doses of gem/NP during Cycle 1 and remaining on study through Cycle 2 Day 1. Results: N=30 pts dosed; 24 DLT-evaluable (6 per cohort). Median follow up is 32.2 weeks. N=13 (54%) pts experienced an AE leading to discontinuation, and 10 (42%) pts experienced a treatment-related serious AE. Two DLTs were observed, 1 each in cohorts B2 and C1 (grade 3 and 4 febrile neutropenia, respectively). AE rates were similar across cohorts. Four (17%) subjects died (2 each in Cohorts B1 and C1) due to disease progression (n=2) and AE (n=2, sepsis and septic shock in the setting of neutropenia). Within the DLT-evaluable population, best overall responses included 14 (58%) PR (11 confirmed, 3 unconfirmed), 8 (33%) SD, 1 (4%) PD and 1 (4%) NE. Post-baseline tumor assessments were available for 2 of the 6 DLT-inevaluable pts (best overall responses of SD and confirmed PR). Multiplexed IHC analysis of baseline tumors revealed a low CD8 T cell and high macrophage infiltrate. Analysis of circulating mutant KRAS DNA showed marked and rapid decrease with therapy in some pts. Immune profiling of PBMCs at baseline and on-treatment by CyTOF revealed remodeling of the myeloid compartment in response to treatment, with rapid activation of dendritic cells in most pts. Conclusions: Gem/NP/APX005M ± nivo demonstrated manageable safety profiles and promising antitumor activity in untreated metastatic PDAC pts. APX005M 0.3 mg/kg was selected as the dose for a randomized Phase II study in which the primary endpoint is 1-year overall survival. Clinical trial information: NCT02482168. Citation Format: Mark H. O'Hara, Eileen M. O'Reilly, Mick Rosemarie, Gauri Varadhachary, Zev A. Wainberg, Andrew Ko, George A. Fisher, Osama Rahma, Jaclyn P. Lyman, Christopher R. Cabanski, Erica L. Carpenter, Travis Hollmann, Pier Federico Gherardini, Lacey Kitch, Cheryl Selinsky, Theresa LaVallee, Ovid C. Trifan, Ute Dugan, Vanessa M. Hubbard-Lucey, Robert H. Vonderheide. A Phase Ib study of CD40 agonistic monoclonal antibody APX005M together with gemcitabine (Gem) and nab-paclitaxel (NP) with or without nivolumab (Nivo) in untreated metastatic ductal pancreatic adenocarcinoma (PDAC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT004.

Background: PARP inhibitors have activity in multiple BRCA-related malignancies and have recently demonstrated dramatic efficacy as a maintenance strategy for platinum-sensitive ovarian cancer. Between 5-8% of patients with pancreatic cancer (PC) have a pathogenic mutation in BRCA1, BRCA2 or PALB2. Therefore, we initiated a single arm phase II clinical trial of maintenance monotherapy rucaparib in patients with advanced PC and a pathogenic germline or somatic BRCA or PALB2 mutation, whose cancer had not progressed following at least four months of platinum-based chemotherapy (NCT 03140670). Methods: Patients were enrolled and treated with rucaparib 600mg PO BID until disease progression or unacceptable toxicity. The primary endpoint is progression free survival (PFS). Patients have previously received >4 months of platinum-based chemotherapy without evidence of disease progression. However, patients with a medical contraindication to receiving the full four months of platinum have been permitted to enroll at the discretion of the primary investigator. Responses were determined using RECIST v1.1. Results: As of December 31st, 2018, we have enrolled 24 of the planned 42 patients, of which 19 are evaluable for PFS at the time of this interim analysis. For these patients, the mutational distribution includes: 13 germline BRCA2, 3 germline BRCA1, 2 germline PALB2, 1 somatic BRCA2. Patients were predominantly female (84.2%) with a median age of 61 years (range: 35-81). Patients had received a median of four months (range 0.5-32 months) of prior platinum therapy for advanced disease. All patients were evaluable for toxicity. Overall, treatment with rucaparib was well tolerated without dose limiting toxicities. The most common adverse events that were at least possibly related to treatment included nausea (grade 1, 41.6%; grade 2, 4.2%), dysgeusia (grade 1, 33.3%) and fatigue (grade 1, 25%). One patient required dose reduction for nausea. The median PFS was 9.1 months from the start of rucaparib therapy with an ORR of 36.8% (six PRs; one CR). Disease control rate (CR + PR + SD) was 89.5% for at least eight weeks. Two patients (10.5%) had progressive disease at first follow-up scan two months after beginning treatment. Eight patients have been on rucaparib for >6 months and two patients remain on treatment for >1 year (13 months and 15 months). The seven responding patients include those with germline BRCA2 mutations (4 patients), germline PALB2 mutations (2 patients) and somatic BRCA2 mutation (1 patient). Conclusions: Based on these early data, maintenance rucaparib following induction with platinum-based chemotherapy shows encouraging disease control with minimal toxicity in patients with platinum-sensitive advanced PC and a pathogenic mutation in BRCA1, BRCA2 or PALB2. Citation Format: Kim A. Reiss Binder, Rosemarie Mick, Mark O'Hara, Ursina Teitelbaum, Thomas Karasic, Charles Schneider, Peter J. O'Dwyer, Erica Carpenter, Austin Pantel, Mehran Makvandi, David Mankoff, Katherine Nathanson, Kara Maxwell, Stacy Cowden, Mary Jane Fuhrer, Janae Romeo, Gregory L. Beatty, Susan Domchek. A Phase II, single arm study of maintenance rucaparib in patients with platinum-sensitive advanced pancreatic cancer and a pathogenic germline or somatic mutation in BRCA1, BRCA2 or PALB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT234.

3040 Background: Circulating tumor DNA next generation sequencing (ctDNA NGS) is increasingly being used to detect mutations (MT) in patients (pts) with metastatic (m) NSCLC. Limited data exist on the correlation of baseline ctDNA NGS profile and serial ctDNA NGS monitoring to response to immunotherapy. Methods: We conducted a prospective study in pts with mNSCLC receiving pembrolizumab monotherapy. Plasma was collected at weeks 0 (T0), 9 (T1), and 18 (T2). ctDNA NGS was performed using a 73 gene panel. Number of MTs and variant allelic fraction (VAF) were determined at baseline, and serially; change in mean VAF was calculated between T1-T0, and T2-T0. Response rate (RR) was assessed using RECIST 1.1. Correlations were made for pt characteristics, RR, progression free survival (PFS), and overall survival (OS). Results: We analyzed 95 samples from 33 pts, 21 female, median age 69 (range 51-89 years), smokers (n = 29), adenocarcinoma (n = 23), 25 pts enrolled at initial diagnosis, majority had high PD-L1 ≥50% (n = 29, 88%). At T0, 32 pts had detectable MT, median number of MTs was 4 (range 0-21), (non-synonymous MT = 3), most common MT was TP53 (n = 21). Confirmed PR was 27% (n = 9), clinical benefit rate (SD+PR) was 64% (n = 21), and 2 pts were not evaluable for response. Smokers were more likely to have higher number of MT at T0 (4 vs. 1 p = 0.003); there was no correlation with smoking and overall RR (p = 0.17). RR was not related to number of MT at T0, p = 0.37. A decrease in ctDNA VAF was seen in 6/9 pts with PR (mean VAF change range -0.11 to -0.001); 2/5 pts with PD showed an increase in mean VAF while 3 showed decrease. At median follow up of 9.26 months (mos), median PFS and OS were 7.4 and 10.5 mos, respectively. Median PFS was longer for pts with a decrease in ctDNA VAF at both T1-T0 (8.9 vs. 5 mos, p = 0.02) and T2-T0 (9.1 vs. 5.5 mos, p = 0.006). OS and additional biomarker analyses including correlation of response to a 2mb ctDNA plasma-based NGS panel will be reported at the meeting. Conclusions: Our results demonstrate that it is feasible to serially monitor plasma NGS, decline in mean ctDNA VAF correlates with radiographic response and PFS on immunotherapy with pembrolizumab.

TPS4161 Background: The treatment paradigm for advanced pancreatic ductal adenocarcinoma (PDAC) typically involves ongoing chemotherapy until either disease progression or clinical deterioration. A subset of patients with advanced PDAC have exceptional responses to platinum-based chemotherapy. We hypothesized that durable platinum sensitivity in patients with advanced PDAC might be indicative of a DNA repair deficiency, and that these patients may respond to a combination of niraparib, a PARP inhibitor, plus immune checkpoint blockade. Methods: We have enrolled 25 of 84 planned patients on study NCT 03404960. Eligibility criteria include inoperable PDAC and stability on platinum-based chemotherapy for ≥16 weeks without evidence of progressive disease. Patients who have progressed on platinum-based treatment or who have received prior therapy with PARP inhibitors are excluded. Patients are randomized to receive oral niraparib 200mg PO daily plus nivolumab 240mg IV every two weeks in continuous 28 day cycles or oral niraparib 200mg PO daily plus ipilimumab 3mg/kg IV every three weeks for four doses in continuous 21 day cycles. The primary endpoint is progression-free survival at 6 months. Secondary endpoints include response rate, duration of response and overall survival. Paired biopsies are obtained, as well as serial blood collections for circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and peripheral blood mononuclear cells (PBMCs). Correlative assays will include germline whole exome sequencing and analyses of serially collected PBMCs, CTCs and ctDNA to identify genomic and immunologic innate and adaptive resistance mechanisms. Clinical trial information: NCT 03404960.

e16529 Background: Prostate cancer is the most common cancer in men in the U.S., with 30% 5-year overall survival (OS) for patients (pts) with metastases. To take a precision medicine approach to the management of metastatic castrate-resistant prostate cancer (mCRPC), we developed a blood circulating tumor cell (CTC)-based test to identify mCRPC pts most likely to benefit from abiraterone (abi) or enzalutamide (enza). Methods: In this multi-institution prospective study, men with mCRPC were enrolled prior to starting abi (1,000 mg/d plus prednisone 10 mg/d) or enza (160 mg/d). At baseline (BL), 12 w, and progression, blood samples were collected for CellSearch-based CTC enumeration and qPCR-based gene expression analysis. Results: 69 pts (median age 68 y [50-82]) received abi (n = 25) or enza (n = 44) and had evaluable blood samples. Consistent with prior publications, among 43 pts with BL CTC > 0, clearance of detectable CTCs (BL CTCs > 0 and 12 w CTCs = 0), was achieved in 24 patients (55.8%), and was associated with greater median OS (31 mo vs. 18 mo, log-rank p = 0.03). The 43 pts with BL CTC > 0 were then randomly divided into training (n = 31) and validation (n = 12) sets. Baseline gene expression data for the training set was used to develop a model to predict CTC clearance, starting with a panel of 141 expressed genes/isoforms including those associated with prostate cancer. Of the models tested, random forest yielded the best performance, with respective training and validation set sensitivity of 0.7 and 1, specificity 0.75 and 0.71, AUC 0.88 and 0.91. Top genes identified include those previously associated with disease – HOXB13, ESRP2, KLK3, GRHL2, and KRT19, among others. Conclusions: A gene expression signature from a baseline blood sample with CellSearch-enriched CTCs can predict clearance of detectable CTCs in response to abi/enza with high AUC and may give insight into molecular mechanisms of response. A prospective study with a larger number of patients will be required to further validate our findings. Ultimately, this blood test has the potential to select the patients most likely to benefit from second-generation antiandrogen vs. non-hormonal systemic treatment.

TPS347 Background: Adoptive immunotherapy with Chimeric Antigen Receptor (CAR)-T cells has transformative potential for the treatment of cancer. However, a primary challenge to the success of these therapies in prostate cancer is the immunosuppressive microenvironment, including high levels of Transforming Growth Factor-beta (TGFβ), encountered by re-directed T cells upon tumor infiltration. Importantly, these immunosuppressive functions of TGFβ can be abrogated in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. In in vivo disseminated prostate cancer models, co-expression of TGFβRdn on PSMA-redirected CAR-T cells led to increased T cell proliferation, enhanced cytokine secretion, resistance to exhaustion, long-term persistence, and greater tumor eradication. Methods: We initiated a first-in-human phase 1 clinical trial to evaluate the safety and preliminary efficacy of lentivirally-transduced PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in men with metastatic CRPC. In preliminary dose-escalation cohorts, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting chemotherapy in a 3+3 design. In Cohort 3, patients will receive the MTD of CART-PSMA-TGFβRdn following a lymphodepleting regimen of cyclophosphamide and fludarabine. All patients provide newly obtained metastatic tumor biopsies at baseline, as well as on day +10 following the CAR-T cell infusion and at disease progression. CAR-T expansion and persistence in peripheral blood and trafficking to target tissues is evaluated via quantitative PCR of CART-PSMA-TGFβRdn DNA. Bioactivity of CART-PSMA-TGFβRdn cells is evaluated via multiplex immunoassays. Additional correlative studies include enumeration and phenotyping of circulating tumor cells and DNA. Cohorts 1 and 2 have been completed without observed DLT. Interestingly, a reversible cytokine release syndrome has been observed that is responsive to tocilizumab. Enrollment in Cohort 3 began in September 2018. Cohort expansions will examine serial CART-PSMA-TGFβRdn re-treatment strategies. Clinical trial information: NCT03089203.

Purpose: Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell-cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes. Experimental Procedures: We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011 (Novartis Oncology), a highly specific CDK4/6 inhibitor. Results: Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nmol/L in sensitive lines). LEE011 caused cell-cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. Although our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (P = 0.01), the identification of additional clinically accessible biomarkers is of high importance. Conclusions: Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. Clin Cancer Res; 19(22); 6173–82. ©2013 AACR.

Background Next-generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low-input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching. Methods To address this, we have developed a novel approach combining tumor cell isolation from preserved blood with Repli-G WGA and Illumina TruSeq Amplicon Cancer Panel-based NGS. We purified cell pools ranging from 10 to 1000 cells from three different cell lines, and quantitatively demonstrate comparable quality of DNA extracted from preserved versus unpreserved samples. Results Preservation and WGA were compatible with the generation of high-quality libraries. Known point mutations and gene amplification were detected for libraries that had been prepared from amplified DNA from preserved blood. Conclusion These spiking experiments provide proof of concept of a clinically applicable workflow for real-time monitoring of patient tumor using noninvasive liquid biopsies.

Despite improvements in cancer therapies in the past 50 years, neuroblastoma remains a devastating clinical problem and a leading cause of childhood cancer deaths. Advances in treatments for children with high-risk neuroblastoma have, until recently, involved addition of cytotoxic therapy to dose-intensive regimens. In this era of targeted therapies, substantial efforts have been made to identify optimal targets for different types of cancer. The discovery of hereditary and somatic activating mutations in the oncogene ALK has now placed neuroblastoma among other cancers, such as melanoma and non-small-cell lung cancer (NSCLC), which benefit from therapies with oncogene-specific small-molecule tyrosine kinase inhibitors. Crizotinib, a small-molecule inhibitor of ALK, has transformed the landscape for the treatment of NSCLC harbouring ALK translocations and has demonstrated activity in preclinical models of ALK-driven neuroblastomas. However, inhibition of mutated ALK is complex when compared with translocated ALK and remains a therapeutic challenge. This Review discusses the biology of ALK in the development of neuroblastoma, preclinical and clinical progress with the use of ALK inhibitors and immunotherapy, challenges associated with resistance to such therapies and the steps being taken to overcome some of these hurdles.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies-as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis and hepatocellular carcinoma (HCC). Both advanced solid tumors and HCV have previously been associated with memory B-cell dysfunction. In this study, we sought to dissect the effect of viral infection, cirrhosis, and liver cancer on memory B-cell frequency and function in the spectrum of HCV disease. Peripheral blood from healthy donors, HCV-infected patients with F1-F2 liver fibrosis, HCV-infected patients with cirrhosis, patients with HCV-related HCC, and non-HCV-infected cirrhotics were assessed for B-cell phenotype by flow cytometry. Isolated B cells were stimulated with anti–cluster of differentiation (CD)40 antibodies and Toll-like receptor (TLR)9 agonist for assessment of costimulation marker expression, cytokine production, immunoglobulin (Ig) production, and CD4+ T-cell allostimulatory capacity. CD27+ memory B cells and, more specifically, CD27+IgM+ B cells were markedly less frequent in cirrhotic patients independent of HCV infection. Circulating B cells in cirrhotics were hyporesponsive to CD40/TLR9 activation, as characterized by CD70 up-regulation, tumor necrosis factor beta secretion, IgG production, and T-cell allostimulation. Last, blockade of TLR4 and TLR9 signaling abrogated the activation of healthy donor B cells by cirrhotic plasma, suggesting a role for bacterial translocation in driving B-cell changes in cirrhosis. Conclusion: Profound abnormalities in B-cell phenotype and function occur in cirrhosis independent of HCV infection. These B-cell defects may explain, in part, the vaccine hyporesponsiveness and susceptibility to bacterial infection in this population. (HEPATOLOGY 2012)