Synergy of EGFR and AURKA Inhibitors in KRAS-mutated Non-small Cell Lung Cancers.

The most common oncogenic driver mutations for non-small cell lung cancer (NSCLC) activate EGFR or KRAS. Clinical trials exploring treatments for EGFR- or KRAS-mutated (EGFRmut or KRASmut) cancers have focused on small-molecule inhibitors targeting the driver mutations. Typically, these inhibitors perform more effectively based on combination with either chemotherapies, or other targeted therapies. For EGFRmut NSCLC, a combination of inhibitors of EGFR and Aurora-A kinase (AURKA), an oncogene commonly overexpressed in solid tumors, has shown promising activity in clinical trials. Interestingly, a number of recent studies have indicated that EGFR activity supports overall viability of tumors lacking EGFR mutations, and AURKA expression is abundant in KRASmut cell lines. In this study, we have evaluated dual inhibition of EGFR and AURKA in KRASmut NSCLC models. These data demonstrate synergy between the EGFR inhibitor erlotinib and the AURKA inhibitor alisertib in reducing cell viability and clonogenic capacity in vitro, associated with reduced activity of EGFR pathway effectors, accumulation of enhanced aneuploid cell populations, and elevated cell death. Importantly, the erlotinib-alisertib combination also synergistically reduces xenograft growth in vivo. Analysis of signaling pathways demonstrated that the combination of erlotinib and alisertib was more effective than single-agent treatments at reducing activity of EGFR and pathway effectors following either brief or extended administration of the drugs. In sum, this study indicates value of inhibiting EGFR in KRASmut NSCLC, and suggests the specific value of dual inhibition of AURKA and EGFR in these tumors. SIGNIFICANCE: The introduction of specific KRAS G12C inhibitors to the clinical practice in lung cancer has opened up opportunities that did not exist before. However, G12C alterations are only a subtype of all KRAS mutations observed. Given the high expression of AURKA in KRASmut NSCLC, our study could point to a potential therapeutic option for this subgroup of patients.

Source, co-occurrence, and prognostic value of PTEN mutations or loss in colorectal cancer.

Somatic PTEN mutations are common and have driver function in some cancer types. However, in colorectal cancers (CRCs), somatic PTEN-inactivating mutations occur at a low frequency (~8-9%), and whether these mutations are actively selected and promote tumor aggressiveness has been controversial. Analysis of genomic data from ~53,000 CRCs indicates that hotspot mutation patterns in PTEN partially reflect DNA-dependent selection pressures, but also suggests a strong selection pressure based on protein function. In microsatellite stable (MSS) tumors, PTEN alterations co-occur with mutations activating BRAF or PI3K, or with TP53 deletions, but not in CRC with microsatellite instability (MSI). Unexpectedly, PTEN deletions are associated with poor survival in MSS CRC, whereas PTEN mutations are associated with improved survival in MSI CRC. These and other data suggest use of PTEN as a prognostic marker is valid in CRC, but such use must consider driver mutation landscape, tumor subtype, and category of PTEN alteration.

Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing.

BACKGROUND: Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined. METHODS: Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes. RESULTS: Analysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of [Formula: see text]H2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased [Formula: see text]H2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities. CONCLUSIONS: Together, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC.

Acetyl-CoA Counteracts the Inhibitory Effect of Antiandrogens on Androgen Receptor Signaling in Prostate Cancer Cells.

The commonly used therapeutic management of PC involves androgen deprivation therapy (ADT) followed by treatment with AR signaling inhibitors (ARSI). However, nearly all patients develop drug-resistant disease, with a median progression-free survival of less than 2 years in chemotherapy-naïve men. Acetyl-coenzyme A (acetyl-CoA) is a central metabolic signaling molecule with key roles in biosynthetic processes and cancer signaling. In signaling, acetyl-CoA serves as the acetyl donor for acetylation, a critical post-translational modification. Acetylation affects the androgen receptor (AR) both directly and indirectly increasing expression of AR dependent genes. Our studies reveal that PC cells respond to the treatment with ARSI by increasing expression of ATP-citrate lyase (ACLY), a major enzyme responsible for cytosolic acetyl-CoA synthesis, and up-regulation of acetyl-CoA intracellular levels. Inhibition of ACLY results in a significant suppression of ligand-dependent and -independent routes of AR activation. Accordingly, the addition of exogenous acetyl-CoA, or its precursor acetate, augments AR transcriptional activity and diminishes the anti-AR activity of ARSI. Taken together, our findings suggest that PC cells respond to antiandrogens by increasing activity of the acetyl-coA pathway in order to reinstate AR signaling.

Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC. EXPERIMENTAL DESIGN: To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations. RESULTS: Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation. CONCLUSIONS: These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.

Comprehensive characterization of PTEN mutational profile in a series of 34,129 colorectal cancers.

Loss of expression or activity of the tumor suppressor PTEN acts similarly to an activating mutation in the oncogene PIK3CA in elevating intracellular levels of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), inducing signaling by AKT and other pro-tumorigenic signaling proteins. Here, we analyze sequence data for 34,129 colorectal cancer (CRC) patients, capturing 3,434 PTEN mutations. We identify specific patterns of PTEN mutation associated with microsatellite stability/instability (MSS/MSI), tumor mutational burden (TMB), patient age, and tumor location. Within groups separated by MSS/MSI status, this identifies distinct profiles of nucleotide hotspots, and suggests differing profiles of protein-damaging effects of mutations. Moreover, discrete categories of PTEN mutations display non-identical patterns of co-occurrence with mutations in other genes important in CRC pathogenesis, including KRAS, APC, TP53, and PIK3CA. These data provide context for clinical targeting of proteins upstream and downstream of PTEN in distinct CRC cohorts.

Efficient Hit-to-Lead Searching of Kinase Inhibitor Chemical Space via Computational Fragment Merging.

In early-stage drug discovery, the hit-to-lead optimization (or "hit expansion") stage entails starting from a newly identified active compound and improving its potency or other properties. Traditionally, this process relies on synthesizing and evaluating a series of analogues to build up structure-activity relationships. Here, we describe a computational strategy focused on kinase inhibitors, intended to expedite the process of identifying analogues with improved potency. Our protocol begins from an inhibitor of the target kinase and generalizes the synthetic route used to access it. By searching for commercially available replacements for the individual building blocks used to make the parent inhibitor, we compile an enumerated library of compounds that can be accessed using the same chemical transformations; these huge libraries can exceed many millions─or billions─of compounds. Because the resulting libraries are much too large for explicit virtual screening, we instead consider alternate approaches to identify the top-scoring compounds. We find that contributions from individual substituents are well described by a pairwise additivity approximation, provided that the corresponding fragments position their shared core in precisely the same way relative to the binding site. This key insight allows us to determine which fragments are suitable for merging into single new compounds and which are not. Further, the use of pairwise approximation allows interaction energies to be assigned to each compound in the library without the need for any further structure-based modeling: interaction energies instead can be reliably estimated from the energies of the component fragments, and the reduced computational requirements allow for flexible energy minimizations that allow the kinase to respond to each substitution. We demonstrate this protocol using libraries built from six representative kinase inhibitors drawn from the literature, which target five different kinases: CDK9, CHK1, CDK2, EGFRT790M, and ACK1. In each example, the enumerated library includes additional analogues reported by the original study to have activity, and these analogues are successfully prioritized within the library. We envision that the insights from this work can facilitate the rapid assembly and screening of increasingly large libraries for focused hit-to-lead optimization. To enable adoption of these methods and to encourage further analyses, we disseminate the computational tools needed to deploy this protocol.

Evaluating the impact of age on immune checkpoint therapy biomarkers.

Both tumors and aging alter the immune landscape of tissues. These interactions may play an important role in tumor progression among elderly patients and may suggest considerations for patient care. We leverage large-scale genomic and clinical databases to perform comprehensive comparative analysis of molecular and cellular markers of immune checkpoint blockade (ICB) response with patient age. These analyses demonstrate that aging is associated with increased tumor mutational burden, increased expression and decreased promoter methylation of immune checkpoint genes, and increased interferon gamma signaling in older patients in many cancer types studied, all of which are expected to promote ICB efficacy. Concurrently, we observe age-related alterations that might be expected to reduce ICB efficacy, such as decreases in T cell receptor diversity. Altogether, these changes suggest the capacity for robust ICB response in many older patients, which may warrant large-scale prospective study on ICB therapies among patients of advanced age.

Design, synthesis and biological evaluation of 2-quinolyl-1,3-tropolone derivatives as new anti-cancer agents.

Tropolones are promising organic compounds that can have important biologic effects. We developed a series of new 2-quinolyl-1,3-tropolones derivatives that were prepared by the acid-catalyzed reaction of 4,7-dichloro-2-methylquinolines with 1,2-benzoquinones. 2-Quinolyl-1,3-tropolones have been synthesized and tested for their anti-proliferative activity against several human cancer cell lines. Two compounds (3d and mixture B of 3i-k) showed excellent activity against six cancer cell lines of different tissue of origin. The promising compounds 3d and mixture B of 3i-k also demonstrated induction of apoptotic cell death of ovarian cancer (OVCAR-3, OVCAR-8) and colon cancer (HCT 116) cell lines and affected ERK signaling. In summary, 2-quinolyl-1,3-tropolones are promising compounds for development of effective anticancer agents.

Nedd9 Restrains Autophagy to Limit Growth of Early Stage Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. With overall 5-year survival estimated at <17%, it is critical to identify factors that regulate NSCLC disease prognosis. NSCLC is commonly driven by mutations in KRAS and TP53, with activation of additional kinases such as SRC promoting tumor invasion. In this study, we investigated the role of NEDD9, a SRC activator and scaffolding protein, in NSCLC tumorigenesis. In an inducible model of NSCLC dependent on Kras mutation and Trp53 loss (KP mice), deletion of Nedd9 (KPN mice) led to the emergence of larger tumors characterized by accelerated rates of tumor growth and elevated proliferation. Orthotopic injection of KP and KPN tumors into the lungs of Nedd9-wild-type and -null mice indicated the effect of Nedd9 loss was cell-autonomous. Tumors in KPN mice displayed reduced activation of SRC and AKT, indicating that activation of these pathways did not mediate enhanced growth of KPN tumors. NSCLC tumor growth has been shown to require active autophagy, a process dependent on activation of the kinases LKB1 and AMPK. KPN tumors contained high levels of active LKB1 and AMPK and increased autophagy compared with KP tumors. Treatment with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors. These data for the first time identify NEDD9 as a negative regulator of LKB1/AMPK-dependent autophagy during early NSCLC tumor growth. SIGNIFICANCE: This study demonstrates a novel role for the scaffolding protein NEDD9 in regulating LKB1-AMPK signaling in early stage non-small cell lung cancer, suppressing autophagy and tumor growth.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

CRISPR/Cas9 genome-wide loss-of-function screening identifies druggable cellular factors involved in sunitinib resistance in renal cell carcinoma.

BACKGROUND: Multi-targeted tyrosine kinase inhibitors (TKIs) are the standard of care for patients with advanced clear cell renal cell carcinoma (ccRCC). However, a significant number of ccRCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilisation nor clinical benefits. METHODS: We used CRISPR/Cas9-based high-throughput loss of function (LOF) screening to identify cellular factors involved in the resistance to sunitinib. Next, we validated druggable molecular factors that are synthetically lethal with sunitinib treatment using cell and animal models of ccRCC. RESULTS: Our screening identified farnesyltransferase among the top hits contributing to sunitinib resistance in ccRCC. Combined treatment with farnesyltransferase inhibitor lonafarnib potently augmented the anti-tumour efficacy of sunitinib both in vitro and in vivo. CONCLUSION: CRISPR/Cas9 LOF screening presents a promising approach to identify and target cellular factors involved in the resistance to anti-cancer therapeutics.

Identification of the KRIT1 Protein by LexA-Based Yeast Two-Hybrid System.

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system that is associated with leaky capillaries, and a predisposition to serious clinical conditions including intracerebral hemorrhage and seizures. Germline or sporadic mutations in the CCM1/KRIT1 gene are responsible for the majority of cases of CCM. In this article, we describe the original characterization of the CCM1/KRIT1 gene. This cloning was done through the use of a variant of the yeast two-hybrid screen known as the interaction trap, using the RAS-family GTPase KREV1/RAP1A as a bait. The partial clone of KRIT1 (Krev1 Interaction Trapped) initially identified was extended through 5'RACE and computational analysis to obtain a full-length cDNA, then used in a sequential screen to define the integrin-associated ICAP1 protein as a KRIT1 partner protein. We discuss how these interactions are relevant to the current understanding of KRIT1/CCM1 biology, and provide a protocol for library screening with the Interaction Trap.

RNA interference screening methods to identify proliferation determinants and mechanisms of resistance to immune attack.

We have used RNA interference (RNAi) screening technology to reveal unknown components of biological signaling pathways including survival mechanisms of estrogen-independent breast cancer cell growth and cancer cell resistance to immune attack. In this chapter, a detailed protocol describing the use of RNAi screening to identify factors important for the proliferation of estrogen-independent MCF7 breast cancer cells will be described. Resistance to therapies that target the estrogen pathway remains a challenge in the treatment of estrogen receptor-positive breast cancer. To address this challenge, small interfering-RNA (siRNA)-based libraries targeting an estrogen receptor (ER)- and aromatase-centered network, including 631 genes relevant to estrogen signaling, was designed and constructed for RNAi screening. This protocol will include the following parts: (1) selection of RNAi transfection reagent for specific cells; (2) optimization of RNAi screening conditions using Z'-factor; (3) procedure of ER-network gene siRNA library screening using automated machines under optimized experimental conditions; and (4) method of analysis for RNAi screening data to identify specific determinants important for cell proliferation. 46 genes were found to be selectively required for the survival of estrogen-independent MCF7-derived cells.

Comprehensive characterization of RAS mutations in colon and rectal cancers in old and young patients.

Colorectal cancer (CRC) is increasingly appreciated as a heterogeneous disease, with factors such as microsatellite instability (MSI), cancer subsite within the colon versus rectum, and age of diagnosis associated with specific disease course and therapeutic response. Activating oncogenic mutations in KRAS and NRAS are common in CRC, driving tumor progression and influencing efficacy of both cytotoxic and targeted therapies. The RAS mutational spectrum differs substantially between tumors arising from distinct tissues. Structure-function analysis of relatively common somatic RAS mutations in G12, Q61, and other codons is characterized by differing potency and modes of action. Here we show the mutational profile of KRAS, NRAS, and the less common HRAS in 13,336 CRC tumors, comparing the frequency of specific mutations based on age of diagnosis, MSI status, and colon versus rectum subsite. We identify mutation hotspots, and unexpected differences in mutation spectrum, based on these clinical parameters.

Comprehensive Genomic Landscapes in Early and Later Onset Colorectal Cancer.

PURPOSE: The incidence rates of colorectal cancers are increasing in young adults. The objective of this study was to investigate genomic differences between tumor samples collected from younger and older patients with colorectal cancer. EXPERIMENTAL DESIGN: DNA was extracted from 18,218 clinical specimens, followed by hybridization capture of 3,769 exons from 403 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer. Genomic alterations (GA) were determined, and association with patient age and microsatellite stable/microsatellite instability high (MSS/MSI-H) status established. RESULTS: Overall genomic alteration rates in the younger (<40) and older (≥50) cohorts were similar in the majority of the genes analyzed. Gene alteration rates in the microsatellite stable (MSS) younger and older cohorts were largely similar, with several notable differences. In particular, TP53 (FDR < 0.01) and CTNNB1 (FDR = 0.01) alterations were more common in younger patients with colorectal cancer, and APC (FDR < 0.01), KRAS (FDR < 0.01), BRAF (FDR < 0.01), and FAM123B (FDR < 0.01) were more commonly altered in older patients with colorectal cancer. In the MSI-H cohort, the majority of genes showed similar rate of alterations in all age groups, but with significant differences seen in APC (FDR < 0.01), BRAF (FDR < 0.01), and KRAS (FDR < 0.01). CONCLUSIONS: Tumors from younger and older patients with colorectal cancer demonstrated similar overall rates of genomic alteration. However, differences were noted in several genes relevant to biology and response to therapy. Further study will need to be conducted to determine whether the differences in gene alteration rates can be leveraged to provide personalized therapies for young patients with early-onset sporadic colorectal cancer.

Combined inhibition of Aurora A and p21-activated kinase 1 as a new treatment strategy in breast cancer.

PURPOSE: The serine-threonine kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast tumors, with overexpression promoting aggressive breast cancer phenotypes and poor clinical outcomes. Besides the well-defined roles of these proteins in control of cell division, proliferation, and invasion, both kinases support MAPK kinase pathway activation and can contribute to endocrine resistance by phosphorylating estrogen receptor alpha (ERα). PAK1 directly phosphorylates AURKA and its functional partners, suggesting potential value of inhibiting both kinases activity in tumors overexpressing PAK1 and/or AURKA. Here, for the first time, we evaluated the effect of combining the AURKA inhibitor alisertib and the PAK inhibitor FRAX1036 in preclinical models of breast cancer. METHODS: Combination of alisertib and FRAX1036 was evaluated in a panel of 13 human breast tumor cell lines and BT474 xenograft model, with assessment of the cell cycle by FACS, and signaling changes by immunohistochemistry and Western blot. Additionally, we performed in silico analysis to identify markers of response to alisertib and FRAX1036. RESULTS: Pharmacological inhibition of AURKA and PAK1 synergistically decreased survival of multiple tumor cell lines, showing particular effectiveness in luminal and HER2-enriched models, and inhibited growth and ERα-driven signaling in a BT474 xenograft model. In silico analysis suggested cell lines with dependence on AURKA are most likely to be sensitive to PAK1 inhibition. CONCLUSION: Dual targeting of AURKA and PAK1 may be a promising therapeutic strategy for treatment of breast cancer, with a particular effectiveness in luminal and HER2-enriched tumor subtypes.

Circulating tumor cell and cell-free RNA capture and expression analysis identify platelet-associated genes in metastatic lung cancer.

BACKGROUND: Circulating tumor cells (CTC) and plasma cell-free RNA (cfRNA) can serve as biomarkers for prognosis and treatment response in lung cancer. One barrier to the selected or routine use of CTCs and plasma cfRNA in precision oncology is the limited quantity of both, and CTCs are only seen in metastatic disease. As capture of CTCs and plasma cfRNA presents an opportunity to monitor and assess malignancies without invasive procedures, we compared two methods for CTC capture and identification, and profiled mRNA from CTCs and plasma cfRNA to identify potential tumor-associated biomarkers. METHODS: Peripheral blood was collected from ten patients with small cell lung cancer (SCLC), ten patients with non-small cell lung cancer (NSCLC) and four healthy volunteers. Two methods were used for CTC capture: the standard epithelial cell adhesion molecule (EpCam) CellSearch kit (unicapture) and EpCAM plus HER2, EGFR and MUC-1 specific combined ferrofluid capture (quadcapture). For the quadcapture, anti-cytokeratin 7 (CK7) was additionally used to assist in CTC identification. NanoString analysis was performed on plasma cfRNA and on mRNA from combined ferrofluid isolated CTCs. Expression data was analyzed using STRING and Reactome. RESULTS: Unicapture detected CTCs in 40% of NSCLC and 60% of SCLC; whereas, quadcapture/CK7 identified CTCs in 20% of NSCLC and 80% of SCLC. Bioinformatic analysis of NanoString data identified high expression of a platelet factor 4 (PF4)-related group of transcripts. CONCLUSIONS: Quadcapture ferrofluid reagent did not significantly improve CTC capture efficacy. NanoString analysis based on CTC and plasma cfRNA data highlighted an intriguing PF-4-centric network in patients with metastatic lung cancer.