Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance.

Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug-resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient-matched tumor cells obtained at diagnosis. Electron microscopy identified reduced ER-mitochondria-associated membranes (MAMs; ER-mitochondria contacts, ERMCs) in therapy-resistant cells, and genetically or biochemically reducing MAMs in therapy-sensitive tumors phenocopied resistance. MAMs serve as platforms to transfer Ca2+ and bioactive lipids to mitochondria. Reduced Ca2+ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ER-mitochondria-associated membranes as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.

Hepatocytes direct the formation of a pro-metastatic niche in the liver.

The liver is the most common site of metastatic disease1. Although this metastatic tropism may reflect the mechanical trapping of circulating tumour cells, liver metastasis is also dependent, at least in part, on the formation of a 'pro-metastatic' niche that supports the spread of tumour cells to the liver2,3. The mechanisms that direct the formation of this niche are poorly understood. Here we show that hepatocytes coordinate myeloid cell accumulation and fibrosis within the liver and, in doing so, increase the susceptibility of the liver to metastatic seeding and outgrowth. During early pancreatic tumorigenesis in mice, hepatocytes show activation of signal transducer and activator of transcription 3 (STAT3) signalling and increased production of serum amyloid A1 and A2 (referred to collectively as SAA). Overexpression of SAA by hepatocytes also occurs in patients with pancreatic and colorectal cancers that have metastasized to the liver, and many patients with locally advanced and metastatic disease show increases in circulating SAA. Activation of STAT3 in hepatocytes and the subsequent production of SAA depend on the release of interleukin 6 (IL-6) into the circulation by non-malignant cells. Genetic ablation or blockade of components of IL-6-STAT3-SAA signalling prevents the establishment of a pro-metastatic niche and inhibits liver metastasis. Our data identify an intercellular network underpinned by hepatocytes that forms the basis of a pro-metastatic niche in the liver, and identify new therapeutic targets.

Protein biomarkers and alternatively methylated cell-free DNA detect early stage pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed. DESIGN: To assess circulating marker discrimination in training, testing and validation patient cohorts (total n=426 patients), plasma markers were measured among PDAC cases and patients with chronic pancreatitis, colorectal cancer (CRC), and healthy controls. Using CA19-9 as an anchor marker, measurements were made of two protein markers (TIMP1, LRG1) and cell-free DNA (cfDNA) pancreas-specific methylation at 9 loci encompassing 61 CpG sites. RESULTS: Comparative methylome analysis identified nine loci that were differentially methylated in exocrine pancreas DNA. In the training set (n=124 patients), cfDNA methylation markers distinguished PDAC from healthy and CRC controls. In the testing set of 86 early stage PDAC and 86 matched healthy controls, CA19-9 had an area under the receiver operating characteristic curve (AUC) of 0.88 (95% CI 0.83 to 0.94), which was increased by adding TIMP1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.06), LRG1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02) or exocrine pancreas-specific cfDNA methylation markers at nine loci (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02). In the validation set of 40 early stage PDAC and 40 matched healthy controls, a combined panel including CA19-9, TIMP1 and a 9-loci cfDNA methylation panel had greater discrimination (AUC 0.86, 95% CI 0.77 to 0.95) than CA19-9 alone (AUC 0.82; 95% CI 0.72 to 0.92). CONCLUSION: A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC.

Molecular response assessment using circulating tumor DNA (ctDNA) in advanced solid tumors.

The therapeutic landscape for patients with advanced malignancies has changed dramatically over the last twenty years. The growing number of targeted therapies and immunotherapeutic options available have improved response rates and survival for a subset of patients, however determining which patients will experience clinical benefit from these therapies in order to avoid potential toxicities and reduce healthcare costs remains a clinical challenge. Cell-free circulating tumor DNA (ctDNA) is shed by tumor cells into systemic circulation and is already an integral part of routine clinical practice for the non-invasive tumor genotyping in advanced non-small cell lung cancer as well as other malignancies. The short half-life of ctDNA offers a unique opportunity to utilize early on-treatment changes in ctDNA for real-time assessment of therapeutic response and outcome, termed molecular response. Here, we provide a summary and review of the use of molecular response for the prediction of outcomes in patients with advanced cancer, including the current state of science, its application in clinic, and next steps for the development of this predictive tool.

Outcomes and clinicopathologic characteristics associated with disseminated tumor cells in bone marrow after neoadjuvant chemotherapy in high-risk early stage breast cancer: the I-SPY SURMOUNT study.

PURPOSE: Disseminated tumor cells (DTCs) expressing epithelial markers in the bone marrow are associated with recurrence and death, but little is known about risk factors predicting their occurrence. We detected EPCAM+/CD45- cells in bone marrow from early stage breast cancer patients after neoadjuvant chemotherapy (NAC) in the I-SPY 2 Trial and examined clinicopathologic factors and outcomes. METHODS: Patients who signed consent for SURMOUNT, a sub-study of the I-SPY 2 Trial (NCT01042379), had bone marrow collected after NAC at the time of surgery. EPCAM+CD45- cells in 4 mLs of bone marrow aspirate were enumerated using immunomagnetic enrichment/flow cytometry (IE/FC). Patients with > 4.16 EPCAM+CD45- cells per mL of bone marrow were classified as DTC-positive. Tumor response was assessed using the residual cancer burden (RCB), a standardized approach to quantitate the extent of residual invasive cancer present in the breast and the axillary lymph nodes after NAC. Association of DTC-positivity with clinicopathologic variables and survival was examined. RESULTS: A total of 73 patients were enrolled, 51 of whom had successful EPCAM+CD45- cell enumeration. Twenty-four of 51 (47.1%) were DTC-positive. The DTC-positivity rate was similar across receptor subtypes, but DTC-positive patients were significantly younger (p = 0.0239) and had larger pretreatment tumors compared to DTC-negative patients (p = 0.0319). Twenty of 51 (39.2%) achieved a pathologic complete response (pCR). While DTC-positivity was not associated with achieving pCR, it was significantly associated with higher RCB class (RCB-II/III, 62.5% vs. RCB-0/I; 33.3%; Chi-squared p = 0.0373). No significant correlation was observed between DTC-positivity and distant recurrence-free survival (p = 0.38, median follow-up = 3.2 years). CONCLUSION: DTC-positivity at surgery after NAC was higher in younger patients, those with larger tumors, and those with residual disease at surgery.

Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus argonaute with applications in cancer diagnostics.

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 (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); 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.

CD40 agonistic monoclonal antibody APX005M (sotigalimab) and chemotherapy, with or without nivolumab, for the treatment of metastatic pancreatic adenocarcinoma: an open-label, multicentre, phase 1b study.

BACKGROUND: Standard chemotherapy remains inadequate in metastatic pancreatic adenocarcinoma. Combining an agonistic CD40 monoclonal antibody with chemotherapy induces T-cell-dependent tumour regression in mice and improves survival. In this study, we aimed to evaluate the safety of combining APX005M (sotigalimab) with gemcitabine plus nab-paclitaxel, with and without nivolumab, in patients with pancreatic adenocarcinoma to establish the recommended phase 2 dose. METHODS: This non-randomised, open-label, multicentre, four-cohort, phase 1b study was done at seven academic hospitals in the USA. Eligible patients were adults aged 18 years and older with untreated metastatic pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status score of 0-1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. All patients were treated with 1000 mg/m2 intravenous gemcitabine and 125 mg/m2 intravenous nab-paclitaxel. Patients received 0·1 mg/kg intravenous APX005M in cohorts B1 and C1 and 0·3 mg/kg in cohorts B2 and C2. In cohorts C1 and C2, patients also received 240 mg intravenous nivolumab. Primary endpoints comprised incidence of adverse events in all patients who received at least one dose of any study drug, incidence of dose-limiting toxicities (DLTs) in all patients who had a DLT or received at least two doses of gemcitabine plus nab-paclitaxel and one dose of APX005M during cycle 1, and establishing the recommended phase 2 dose of intravenous APX005M. Objective response rate in the DLT-evaluable population was a key secondary endpoint. This trial (PRINCE, PICI0002) is registered with ClinicalTrials.gov, NCT03214250 and is ongoing. FINDINGS: Between Aug 22, 2017, and July 10, 2018, of 42 patients screened, 30 patients were enrolled and received at least one dose of any study drug; 24 were DLT-evaluable with median follow-up 17·8 months (IQR 16·0-19·4; cohort B1 22·0 months [21·4-22·7], cohort B2 18·2 months [17·0-18·9], cohort C1 17·9 months [14·3-19·7], cohort C2 15·9 months [12·7-16·1]). Two DLTs, both febrile neutropenia, were observed, occurring in one patient each for cohorts B2 (grade 3) and C1 (grade 4). The most common grade 3-4 treatment-related adverse events were lymphocyte count decreased (20 [67%]; five in B1, seven in B2, four in C1, four in C2), anaemia (11 [37%]; two in B1, four in B2, four in C1, one in C2), and neutrophil count decreased (nine [30%]; three in B1, three in B2, one in C1, two in C2). 14 (47%) of 30 patients (four each in B1, B2, C1; two in C2) had a treatment-related serious adverse event. The most common serious adverse event was pyrexia (six [20%] of 30; one in B2, three in C1, two in C2). There were two chemotherapy-related deaths due to adverse events: one sepsis in B1 and one septic shock in C1. The recommended phase 2 dose of APX005M was 0·3 mg/kg. Responses were observed in 14 (58%) of 24 DLT-evaluable patients (four each in B1, C1, C2; two in B2). INTERPRETATION: APX005M and gemcitabine plus nab-paclitaxel, with or without nivolumab, is tolerable in metastatic pancreatic adenocarcinoma and shows clinical activity. If confirmed in later phase trials, this treatment regimen could replace chemotherapy-only standard of care in this population. FUNDING: Parker Institute for Cancer Immunotherapy, Cancer Research Institute, and Bristol Myers Squibb.

Plasma Cell-Free DNA Genotyping: From an Emerging Concept to a Standard-of-Care Tool in Metastatic Non-Small Cell Lung Cancer.

Plasma cell-free DNA (cfDNA) genotyping is an alternative to tissue genotyping, particularly when tissue specimens are insufficient or unavailable, and provides critical information that can be used to guide treatment decisions in managing patients with non-small cell lung cancer (NSCLC). In this article, we review the evolution of plasma cfDNA genotyping from an emerging concept, through development of analytical methods, to its clinical applications as a standard-of-care tool in NSCLC. The number of driver or resistance mutations recommended for testing in NSCLC continues to increase. Because of the expanding list of therapeutically relevant variants, comprehensive testing to investigate larger regions of multiple genes in a single run is often preferable and saves on time and cost, compared with performing serial single-gene assays. Recent advances in nucleic acid next-generation sequencing have led to a rapid expansion in cfDNA genotyping technologies. Analytic assays that have received regulatory approval are now routinely used as diagnostic companions in the setting of metastatic NSCLC. As the demand for plasma-based technologies increases, more regulatory approvals of cfDNA genotyping assays are expected in the future. Plasma cfDNA genotyping is currently aiding oncologists in the delivery of personalized care by facilitating matching of patients with targeted therapy and monitoring emergence of resistance to therapy in NSCLC. Further advances currently underway to increase assay sensitivity and specificity will potentially expand the use of plasma cfDNA genotyping in early cancer detection, monitoring response to therapy, detection of minimal residual disease, and measurement of tumor mutational burden in NSCLC. IMPLICATIONS FOR PRACTICE: Plasma cell-free DNA (cfDNA) genotyping offers an alternative to tissue genotyping, particularly when tissue specimens are insufficient or unavailable. Advances in cfDNA genotyping technologies have led to analytic assays that are now routinely used to aid oncologists in the delivery of personalized care by facilitating matching of patients with targeted therapy and monitoring emergence of resistance to therapy. Further advances underway to increase assay sensitivity and specificity will potentially expand the use of plasma cfDNA genotyping in early cancer detection, monitoring response to therapy, detection of minimal residual disease, and evaluation of tumor mutational burden in non-small cell lung cancer.

An integrated enrichment system to facilitate isolation and molecular characterization of single cancer cells from whole blood.

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.

Flow Cytometric Methods for Circulating Tumor Cell Isolation and Molecular Analysis.

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, DNA, and mRNA: Potential for Clinical Utility in Patients With Melanoma.

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.

Inflammatory and tissue injury marker dynamics in pediatric acute respiratory distress syndrome.

BACKGROUNDThe molecular signature of pediatric acute respiratory distress syndrome (ARDS) is poorly described, and the degree to which hyperinflammation or specific tissue injury contributes to outcomes is unknown. Therefore, we profiled inflammation and tissue injury dynamics over the first 7 days of ARDS, and associated specific biomarkers with mortality, persistent ARDS, and persistent multiple organ dysfunction syndrome (MODS).METHODSIn a single-center prospective cohort of intubated pediatric patients with ARDS, we collected plasma on days 0, 3, and 7. Nineteen biomarkers reflecting inflammation, tissue injury, and damage-associated molecular patterns (DAMPs) were measured. We assessed the relationship between biomarkers and trajectories with mortality, persistent ARDS, or persistent MODS using multivariable mixed effect models.RESULTSIn 279 patients (64 [23%] nonsurvivors), hyperinflammatory cytokines, tissue injury markers, and DAMPs were higher in nonsurvivors. Survivors and nonsurvivors showed different biomarker trajectories. IL-1α, soluble tumor necrosis factor receptor 1, angiopoietin 2 (ANG2), and surfactant protein D increased in nonsurvivors, while DAMPs remained persistently elevated. ANG2 and procollagen type III N-terminal peptide were associated with persistent ARDS, whereas multiple cytokines, tissue injury markers, and DAMPs were associated with persistent MODS. Corticosteroid use did not impact the association of biomarker levels or trajectory with mortality.CONCLUSIONSPediatric ARDS survivors and nonsurvivors had distinct biomarker trajectories, with cytokines, endothelial and alveolar epithelial injury, and DAMPs elevated in nonsurvivors. Mortality markers overlapped with markers associated with persistent MODS, rather than persistent ARDS.FUNDINGNIH (K23HL-136688, R01-HL148054).

Electronic Medical Record-Based Nudge Intervention to Increase Comprehensive Molecular Genotyping in Patients With Metastatic Non-Small Cell Lung Cancer: Results From a Prospective Clinical Trial.

PURPOSE: Less than half of the patients with newly diagnosed metastatic non-small cell lung cancer (NSCLC) undergo comprehensive molecular testing. We designed an electronic medical record (EMR)-based "nudge intervention" to prompt plasma-based molecular testing at the time of initial medical oncology consultation. METHODS: A nonrandomized prospective trial was conducted at the University of Pennsylvania's academic practice and two affiliated community practices. Molecular genotyping was performed by tissue- and/or plasma-based next generation sequencing methods. Comprehensive testing was defined as testing for EGFR, ALK, BRAF, ROS1, MET, RET, KRAS, and NTRK. Guideline-concordant treatment was defined as the use of the appropriate first-line (1L) therapy as per the National Comprehensive Cancer Network (NCCN) guidelines. Proportion of patients with comprehensive molecular genotyping results available at any time, molecular results available before 1L therapy, and guideline-concordant 1L treatment were compared between the preintervention and postintervention cohorts using Fisher's exact test or Pearson's chi-squared test. RESULTS: Five hundred and thirty-three patients were included, 376 in the preintervention cohort and 157 in the postintervention cohort. After implementation of the EMR-based nudge, a higher proportion of patients underwent comprehensive molecular testing in the postintervention versus the preintervention cohort (100% v 88%, P = <.001), had results of comprehensive molecular testing available before initiating 1L treatment (97.3% v 91.6%, P = .026), and received NCCN guideline-concordant care (89.8% v 78.2%, P = .035). CONCLUSION: Across three practice sites in a large health system, implementation of a provider team-focused EMR-based nudge intervention was feasible, and led to a higher number of patients with NSCLC undergoing comprehensive molecular genotyping. These findings demonstrate that behavioral nudges can promote molecular testing and should be studied further as a tool to improve guideline-concordant care in both community and academic sites.

Combined JAK inhibition and PD-1 immunotherapy for non-small cell lung cancer patients.

Persistent inflammation driven by cytokines such as type-one interferon (IFN-I) can cause immunosuppression. We show that administration of the Janus kinase 1 (JAK1) inhibitor itacitinib after anti-PD-1 (programmed cell death protein 1) immunotherapy improves immune function and antitumor responses in mice and results in high response rates (67%) in a phase 2 clinical trial for metastatic non-small cell lung cancer. Patients who failed to respond to initial anti-PD-1 immunotherapy but responded after addition of itacitinib had multiple features of poor immune function to anti-PD-1 alone that improved after JAK inhibition. Itacitinib promoted CD8 T cell plasticity and therapeutic responses of exhausted and effector memory-like T cell clonotypes. Patients with persistent inflammation refractory to itacitinib showed progressive CD8 T cell terminal differentiation and progressive disease. Thus, JAK inhibition may improve the efficacy of anti-PD-1 immunotherapy by pivoting T cell differentiation dynamics.

Circulating KRAS G12D but not G12V is associated with survival in metastatic pancreatic ductal adenocarcinoma.

While high circulating tumor DNA (ctDNA) levels are associated with poor survival for multiple cancers, variant-specific differences in the association of ctDNA levels and survival have not been examined. Here we investigate KRAS ctDNA (ctKRAS) variant-specific associations with overall and progression-free survival (OS/PFS) in first-line metastatic pancreatic ductal adenocarcinoma (mPDAC) for patients receiving chemoimmunotherapy ("PRINCE", NCT03214250), and an independent cohort receiving standard of care (SOC) chemotherapy. For PRINCE, higher baseline plasma levels are associated with worse OS for ctKRAS G12D (log-rank p = 0.0010) but not G12V (p = 0.7101), even with adjustment for clinical covariates. Early, on-therapy clearance of G12D (p = 0.0002), but not G12V (p = 0.4058), strongly associates with OS for PRINCE. Similar results are obtained for the SOC cohort, and for PFS in both cohorts. These results suggest ctKRAS G12D but not G12V as a promising prognostic biomarker for mPDAC and that G12D clearance could also serve as an early biomarker of response.

Modeling and optimization of parallelized immunomagnetic nanopore sorting for surface marker specific isolation of extracellular vesicles from complex media.

The isolation of specific subpopulations of extracellular vesicles (EVs) based on their expression of surface markers poses a significant challenge due to their nanoscale size (< 800 nm), their heterogeneous surface marker expression, and the vast number of background EVs present in clinical specimens (1010-1012 EVs/mL in blood). Highly parallelized nanomagnetic sorting using track etched magnetic nanopore (TENPO) chips has achieved precise immunospecific sorting with high throughput and resilience to clogging. However, there has not yet been a systematic study of the design parameters that control the trade-offs in throughput, target EV recovery, and ability to discard background EVs in this approach. We combine finite-element simulation and experimental characterization of TENPO chips to elucidate design rules to isolate EV subpopulations from blood. We demonstrate the utility of this approach by reducing device background > 10× relative to prior published designs without sacrificing recovery of the target EVs by selecting pore diameter, number of membranes placed in series, and flow rate. We compare TENPO-isolated EVs to those of gold-standard methods of EV isolation and demonstrate its utility for wide application and modularity by targeting subpopulations of EVs from multiple models of disease including lung cancer, pancreatic cancer, and liver cancer.

Parallelized immunomagnetic nanopore sorting: modeling, scaling, and optimization of surface marker specific isolation of extracellular vesicles from complex media.

The isolation of specific subpopulations of extracellular vesicles (EVs) based on their expression of surface markers poses a significant challenge due to their nanoscale size (< 800 nm), their heterogeneous surface marker expression, and the vast number of background EVs present in clinical specimens (10 10 -10 12 EVs/mL in blood). Highly parallelized nanomagnetic sorting using track etched magnetic nanopore (TENPO) chips has achieved precise immunospecific sorting with high throughput and resilience to clogging. However, there has not yet been a systematic study of the design parameters that control the trade-offs in throughput, target EV recovery, and specificity in this approach. We combine finite-element simulation and experimental characterization of TENPO chips to elucidate design rules to isolate EV subpopulations from blood. We demonstrate the utility of this approach by increasing specificity > 10x relative to prior published designs without sacrificing recovery of the target EVs by selecting pore diameter, number of membranes placed in series, and flow rate. We compare TENPO-isolated EVs to those of gold-standard methods of EV isolation and demonstrate its utility for wide application and modularity by targeting subpopulations of EVs from multiple models of disease including lung cancer, pancreatic cancer, and liver cancer.

The Clinical Implications of Reversions in Patients with Advanced Pancreatic Cancer and Pathogenic Variants in BRCA1, BRCA2, or PALB2 after Progression on Rucaparib.

PURPOSE: PARP inhibitors (PARPi) provide an effective maintenance option for patients with BRCA- or PALB2-mutated pancreatic cancer. However, mechanisms of PARPi resistance and optimal post-PARPi therapeutic strategies are poorly characterized. EXPERIMENTAL DESIGN: We collected paired cell-free DNA samples and post-PARPi clinical data on 42 patients with advanced, platinum-sensitive pancreatic cancer who were treated with maintenance rucaparib on NCT03140670, of whom 32 developed progressive disease. RESULTS: Peripherally detected, acquired BRCA or PALB2 reversion variants were uncommon (5/30; 16.6%) in patients who progressed on rucaparib. Reversions were significantly associated with rapid resistance to PARPi treatment (median PFS, 3.7 vs. 12.5 months; P = 0.001) and poor overall survival (median OS, 6.2 vs. 23.0 months; P < 0.0001). All patients with reversions received rechallenge with platinum-based chemotherapy following PARPi progression and experienced faster progression on this therapy than those without reversion variants (real-world time-to-treatment discontinuation, 2.4 vs. 5.8 months; P = 0.004). Of the patients who progressed on PARPi and received further chemotherapy, the OS from initiation of second-line therapy was significantly lower in those with reversion variants than in those without (5.5 vs. 12.0 months, P = 0.002). Finally, high levels of tumor shedding were independently associated with poor outcomes in patients who received rucaparib. CONCLUSIONS: Acquired reversion variants were uncommon but detrimental in a population of patients with advanced BRCA- or PALB2-related pancreatic ductal adenocarcinoma who received maintenance rucaparib. Reversion variants led to rapid progression on PARPi, rapid failure of subsequent platinum-based treatment, and poor OS of patients. The identification of such variants in the blood may have both predictive and prognostic value. See related commentary by Tsang and Gallinger, p. 5005.

Association Between Availability of Molecular Genotyping Results and Overall Survival in Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancer.

PURPOSE: Current guidelines recommend molecular genotyping for patients newly diagnosed with metastatic nonsquamous (mNSq) non-small-cell lung cancer (NSCLC). The association between availability of molecular genotyping before first line (1L) therapy and overall survival (OS) is not known. METHODS: We conducted a real-world cohort study using electronic health records in patients newly diagnosed with mNSq NSCLC. Cox proportional-hazards multivariable regression models were constructed to examine the association between OS and test result availability before 1L therapy, adjusting for covariates. Additional analyses were conducted to assess the consistency and strength of the relationship. Multivariable logistic regression models were used to examine the association between concurrent tissue and plasma testing (v tissue alone) and result availability. RESULTS: Three hundred twenty-six patients were included, 80% (261/326) with results available before 1L (available testing group), and 20% (65/326) without results available (unavailable testing group). With 14.2-month median follow-up, patients in the available testing group had significantly longer OS relative to the unavailable testing group (adjusted hazard ratio, 0.43; 95% CI, 0.30 to 0.62; P < .0001). The adjusted odds of availability of results before 1L therapy was higher with concurrent tissue and plasma testing (v tissue testing alone; adjusted odds ratio, 2.06; 95% CI, 1.09 to 3.90; P = .026). CONCLUSION: Among patients with mNSq NSCLC in a real-world cohort, availability of molecular genotyping results before 1L therapy was associated with significantly better OS. Concurrent tissue and plasma testing was associated with a higher odds of availability of results before 1L therapy. These findings warrant renewed attention to the completion of molecular genotyping before 1L therapy.

Distinguishing Progression from Pseudoprogression in Glioblastoma Using 18F-Fluciclovine PET.

Accurate differentiation between tumor progression (TP) and pseudoprogression remains a critical unmet need in neurooncology. 18F-fluciclovine is a widely available synthetic amino acid PET radiotracer. In this study, we aimed to assess the value of 18F-fluciclovine PET for differentiating pseudoprogression from TP in a prospective cohort of patients with suspected radiographic recurrence of glioblastoma. Methods: We enrolled 30 glioblastoma patients with radiographic progression after first-line chemoradiotherapy for whom surgical resection was planned. The patients underwent preoperative 18F-fluciclovine PET and MRI. The relative percentages of viable tumor and therapy-related changes observed in histopathology were quantified and categorized as TP (≥50% viable tumor), mixed TP (<50% and >10% viable tumor), or pseudoprogression (≤10% viable tumor). Results: Eighteen patients had TP, 4 had mixed TP, and 8 had pseudoprogression. Patients with TP/mixed TP had a significantly higher 40- to 50-min SUVmax (6.64 + 1.88 vs. 4.11 ± 1.52, P = 0.009) than patients with pseudoprogression. A 40- to 50-min SUVmax cutoff of 4.66 provided 90% sensitivity and 83% specificity for differentiation of TP/mixed TP from pseudoprogression (area under the curve [AUC], 0.86). A maximum relative cerebral blood volume cutoff of 3.672 provided 90% sensitivity and 71% specificity for differentiation of TP/mixed TP from pseudoprogression (AUC, 0.779). Combining a 40- to 50-min SUVmax cutoff of 4.66 and a maximum relative cerebral blood volume of 3.67 on MRI provided 100% sensitivity and 80% specificity for differentiating TP/mixed TP from pseudoprogression (AUC, 0.95). Conclusion: 18F-fluciclovine PET uptake can accurately differentiate pseudoprogression from TP in glioblastoma, with even greater accuracy when combined with multiparametric MRI. Given the wide availability of 18F-fluciclovine, larger, multicenter studies are warranted to determine whether amino acid PET with 18F-fluciclovine should be used in the routine posttreatment assessment of glioblastoma.