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Variant Ciz1 Is a Circulating Biomarker for Early-Stage Lung Cancer

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Variant Ciz1 Is a Circulating Biomarker for Early-Stage Lung Cancer Variant Ciz1 is a circulating biomarker for early-stage lung cancer Gillian Higginsa,b, Katherine M. Ropera,b, Irene J. Watsona,b, Fiona H. Blackhallc, William N. Romd, Harvey I. Passe, Justin F. X. Ainscoughf, and Dawn Coverleya,b,1 aCizzle Biotech, University of York, Yorkshire YO10 5DD, United Kingdom; bDepartment of Biology, University of York, Yorkshire YO10 5YW, United Kingdom; cPaterson Institute for Cancer Research, University of Manchester, Lancashire M20 4BX, United Kingdom; dDivision of Pulmonary, Critical Care and Sleep Medicine, New York University School of Medicine, New York, NY 10016; eNew York University Langone Medical Center, New York, NY 10016; and fSchool of Medicine, Leeds University, Yorkshire LS2 9JT, United Kingdom Edited by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved September 20, 2012 (received for review June 15, 2012) There is an unmet need for circulating biomarkers that can detect CDK2, and with the cyclin-dependent kinase inhibitor p21 (7) early-stage lung cancer. Here we show that a variant form of the and also plays an indirect role in DNA replication by modulating nuclear matrix-associated DNA replication factor Ciz1 is present in the expression of genes, including cyclin D, that influence cell 34/35 lung tumors but not in adjacent tissue, giving rise to stable proliferation (8). Normally, Ciz1 is attached to the salt- and protein quantifiable by Western blot in less than a microliter of nuclease-resistant protein component of the nucleus referred plasma from lung cancer patients. In two independent sets, with to as the “nuclear matrix” and resides within foci that partially 170 and 160 samples, respectively, variant Ciz1 correctly identified colocalize with sites of DNA replication (9), implicating Ciz1 in patients who had stage 1 lung cancer with clinically useful accuracy. the spatial organization of DNA replication. Here we describe a For set 1, mean variant Ciz1 level in individuals without diagnosed Ciz1 variant that lacks part of a C-terminal domain involved in tumors established a threshold that correctly classified 98% of nuclear matrix attachment (Fig. 1A). Expression of this stable small cell lung cancers (SCLC) and non-SCLC patients [receiver variant is apparently restricted to tumor cells, making clinical operator characteristic area under the curve (AUC) 0.958]. Within exploitation as a cancer biomarker highly tractable. set2,comparisonofpatientswithstage1non-SCLCwithasymp- tomatic age-matched smokers or individuals with benign lung Results nodules correctly classified 95% of patients (AUCs 0.913 and 0.905), As part of a gene-focused analysis of function, we cloned human with overall specificity of 76% and 71%, respectively. Moreover, Ciz1 from a SCLC cell line and recovered multiple variants, in- using the mean of controls in set 1, we achieved 95% sensitivity cluding a prevalent transcript in which 24 nucleotides from the 3′ among patients with stage 1 non-SCLC patients in set 2 with 74% end of exon 14 (2475_2498del) is excluded, leading to in-frame specificity, demonstrating the robustness of the classification. RNAi- deletion of eight amino acids (VEEELCKQ). Analysis of the mediated selective depletion of variant Ciz1 is sufficient to restrain sequence surrounding exons 14 and 15 revealed a second splice the growth of tumor cells that express it, identifying variant Ciz1 donor site within exon 14 (2475/6) that could support alternative as a functionally relevant driver of cell proliferation in vitro and in splicing. Location identifiers refer to Ciz1 reference sequence vivo. The data show that variant Ciz1 is a strong candidate for a NM_012127.2. We refer to the whole of predicted exon 14 as cancer-specific single marker capable of identifying early-stage lung “14a,” the shorter alternative as “14b,” and Ciz1 transcripts cancer within at-risk groups without resort to invasive procedures. harboring 14b as “b-variant.” Transcript frequencies among ESTs that map to the Ciz1 Unigene cluster Hs. 212395 suggested that ung cancer is the leading cause of cancer death worldwide. b-variant is prevalent in neuroendocrine lung tumors, and this fi LApproximately 80% of lung tumors are classified as nonsmall prevalence was con rmed by analysis of SCLC cell lines using cell lung cancer (NSCLC), including squamous cell carcinoma independent detection methods (Fig. S1 and below). and adenocarcinoma, and the remainder as small cell type As a nuclear matrix protein characterized by resistance to (SCLC). SCLCs are primarily neuroendocrine in origin, ranging harsh extraction conditions, b-variant could offer a robust bio- from low-grade typical carcinoid to high-grade neuroendocrine marker with potential to remain stable and detectable in body fluids. Consistent with this idea, an affinity-purified polyclonal tumors (HGNTs), although some HGNTs are classified as large antibody directed against the unique peptide encoded at the cell type (1, 2). The risk of lung cancer is increased dramatically junction of exon 14b/exon15 (Fig. S2) detected b-variant protein by smoking, and genetic factors appear to play a role in our by Western blot in 1 μL of plasma from patients with SCLC and ability to deal with smoking-related damage. However, clearly NSCLC but not from healthy individuals (Fig. 1B). A diffuse but heritable forms of increased lung cancer risk are not common. specific band of 50–60 kDa (Fig. S2) is reproducibly seen and Lung cancer diagnosis and staging relies heavily on imaging, remained stable even after extended periods of storage at 4 °C. suggesting that imaging may offer a route to early detection in high-risk groups. Although the impact of early detection on sur- vival has been questioned, several studies have looked at the fi Author contributions: J.F.X.A. and D.C. designed research; G.H., K.M.R., I.J.W., and D.C. potential bene t (3), and it recently became clear that screening performed research; F.H.B., W.N.R., and H.I.P. contributed new patient samples; G.H. and with low-dose spiral CT can achieve a significant reduction in D.C. analyzed data; and J.F.X.A. and D.C. wrote the paper. mortality among heavy smokers (4). However, because around a Conflict of interest statement: The findings reported in this paper arise directly out of quarter of individuals require follow-up procedures to investigate basic research at the University of York (York, Yorkshire, United Kingdom) on the Ciz1 suspicious imaging results, the cost of this approach is extremely protein and its role in the spatial and temporal organization of DNA replication. The findings are the result of an academic and commercial collaboration, funded in part by high, highlighting the need for a second-line noninvasive test that Cizzle Biotech, which is a spin-out company of the University of York. G.H. and D.C. are can confirm malignancy. Here we present evidence that protein- partially funded by Cizzle Biotech. D.C. and J.F.X.A hold shares in Cizzle Biotech. level detection of a variant form of the nuclear matrix protein This article is a PNAS Direct Submission. Ciz1 has the potential to meet this need. Ciz1 promotes initiation 1To whom correspondence should be addressed. E-mail: [email protected]. of mammalian DNA replication, where it helps coordinate the See Author Summary on page 18263 (volume 109, number 45). sequential functions of cyclin E- and A-dependent protein This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. kinases (5). It interacts directly with cyclins E and A (6), with 1073/pnas.1210107109/-/DCSupplemental. E3128–E3135 | PNAS | Published online October 16, 2012 www.pnas.org/cgi/doi/10.1073/pnas.1210107109 Downloaded by guest on September 26, 2021 PNAS PLUS Fig. 1. Variant Ciz1 protein in 170 samples in plasma set 1. (A) Ciz1 gene showing translated exons (numbered) and the alternative-splicing event at the exon 14/15 junction which gives rise to b-variant Ciz1. Exons that encode DNA replication domain (5) and nuclear matrix anchor domain (9) are indicated by dotted lines above. Exons that are commonly excluded from known Ciz1 variants (23) are shaded in dark gray. A complete representation of Ciz1 alternative splice variants was assembled previously (24), and transcript diversity was discussed recently (25, 26). (B) Western blot showing b-variant protein detected with antibody 2B in 1 μL of plasma from patients with SCLC or NSCLC plus five samples from individuals with no diagnosed disease. Endogenous Ig is used to normalize for loading (control). (C) Box-and-whisker plots showing the median, upper, and lower quartile, range, and outliers for data derived from Western blots by densitometry. Results for a total of 119 pretreatment lung cancer patients (Left) with the indicated type and stage of disease (Right), plus 51 samples from individuals with no disease or from patients with COPD, asthma, or anemia are shown. Individual sample values are given in Dataset S1. Using a threshold set at the mean of the noncancer samples, the test correctly classified 98% of all 119 lung cancer patients, with specificity of 85%. (D) ROC curve, with 95% confidence interval, generated for all 170 samples (AUC is 0.958). Student’s two-tailed t test with unequal variance returned a P value of <0.0001 for the noncancer samples compared with all sample sets from individuals with lung cancer. Quantification of b-variant protein in 119 SCLC and NSCLC patients with early-stage lung cancer and those without lung samples (summary information in Table 1 with sample-specific cancer. However, for maximum clinical utility a blood test must information in Dataset S1) gave a mean signal intensity of 95.8 be able to identify individuals with early-stage lung cancer within densitometry units with an SD of 51.8, indicating considerable populations that are at high risk of developing the disease.
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