Method for Detecting Liver Diseases
20220033910 · 2022-02-03
Inventors
- David T. Ting (Dover, MA)
- Irun Bhan (Brookline, MA, US)
- Kelly Mosesso (Boston, MA, US)
- Martin Aryee (Cambridge, MA, US)
Cpc classification
G01N2800/56
PHYSICS
G01N2800/085
PHYSICS
C12Q2600/112
CHEMISTRY; METALLURGY
G01N2800/52
PHYSICS
International classification
G01N33/50
PHYSICS
Abstract
The present invention relates to methods for diagnosing, determining, or monitoring liver diseases and conditions based on the blood concentration of circulating epithelial cells in and their gene expression.
Claims
1. A method, comprising measuring expression levels of hepatocellular carcinoma (HCC) classifier genes in circulating epithelial cells (CECs) of a subject, wherein the HCC classifier genes comprise one or more of TESC, OSBP2, SLC6A8, SEPT5, F2RL3, E2F1, EZH2, CDC20, CCNA2, CCNB1, PLXNB3, CDC6, MYBL2, APOBEC3B, SPP1, AKR1B10, TOP2A, ASPM, SLC6A9, RECQL4, NUSAP1, PLVAP, FMO1, PDZK1IP1, and FBXO32.
2. The method of claim 1, wherein the HCC classifier genes consist of one or more of TESC, OSBP2, SLC6A8, SEPT5, F2RL3, E2F1, EZH2, CDC20, CCNA2, CCNB1, PLXNB3, CDC6, MYBL2, APOBEC3B, SPP1, AKR1B10, TOP2A, ASPM, SLC6A9, RECQL4, NUSAP1, PLVAP, FMO1, PDZK1IP1, and FBXO32.
3. The method of claim 1 or 2, wherein the HCC classifier genes consist of TESC, OSBP2, SLC6A8, SEPT5, F2RL3, E2F1, EZH2, CDC20, CCNA2, CCNB1, PLXNB3, CDC6, MYBL2, APOBEC3B, SPP1, AKR1B10, TOP2A, ASPM, SLC6A9, RECQL4, NUSAP1, PLVAP, FMO1, PDZK1IP1, and FBXO32.
4. The method of claim 1, wherein the HCC classifier genes further comprise one, two, three or more additional genes selected from the group consisting of ACTG2, ADM2, AFP, AGR2, ALDH3A1, ALPK3, AMIGO3, ANKRD65, ANLN, AP1M2, ARHGAP11A, ARHGEF39, ASF1B, ASPHD1, AURKA, AXIN2, BAIAP2L2, BEX2, C15orf48, C1orf106, C1QTNF3, C6orf223, CA12, CA9, CAMK2N2, CAP2, CBX2, CCDC170, CCDC28B, CCDC64, CCNE2, CCNF, CD109, CD34, CDC25A, CDC7, CDCA5, CDCA8, CDH13, CDK1, CDKN2A, CDKN2C, CDT1, CELF6, CENPF, CENPH, CENPL, CENPU, CENPW, CKB, CNNM1, COL15A1, COL4A5, COL7A1, COL9A2, CRIP3, CSPG4, CTNND2, CXorf36, CYP17A1, DLK1, DMKN, DSCC1, DTL, DUOX2, ECT2, EEF1A2, EFNA3, EPHB2, EPPK1, ETV4, FABP4, FAM111B, FAM3B, FAM83D, FANCD2, FANCI, FBXL18, FERMT1, FGF19, FLNC, FLVCR1, FOXD2-AS1, FOXM1, FXYD2, GABRE, GAL3ST1, GCNT3, GINS1, GJC1, GMNN, GNAZ, GOLGA2P7, GPC3, GPR64, GPSM1, HRCT1, IGF2BP2, IGSF1, IGSF3, IQGAP3, ITGA2, ITPKA, KIAA0101, KIF11, KIFC1, KIFC2, KNTC1, KRT23, LAMA3, LEF1, LGR5, LINC00152, LINGO1, LPL, LRRC1, LYPD1, MAD2L1, MAGED4, MAGED4B, MAPK12, MAPK8IP2, MAPT, MCM2, MDGA1, MDK, MFAP2, MISP, MKI67, MMP11, MNS1, MPZ, MSC, MSH5, MTMR11, MUC13, MUC5B, MYH4, NAALADL1, NAV3, NCAPG, NDUFA4L2, NEB, NKD1, NMB, NOTCH3, NOTUM, NPM2, NQO1, NRCAM, NT5DC2, NTS, OBSCN, OLFML2A, OLFML2B, PAQR4, PEG10, PI3, PLCE1, PLCH2, PLK1, PLXDC1, PODXL2, POLE2, PPAP2C, PRC1, PTGES, PTGFR, PTHLH, PTK7, PTP4A3, PTTG1, PYCR1, RACGAP1, RBM24, RHBG, RNF157, ROBO1, RP4-800G7.2, RPS6KL1, RRM2, S100A1, SCGN, 5-Sep, SERPINA12, SEZ6L2, SFN, SGOL2, SLC22A11, SLC51B, SLC6A2, SNCG, SOAT2, SP5, SPARCL1, SPINK1, STIL, STK39, SULT1C2, TCF19, TDGF1, THY1, TK1, TMC5, TMEM132A, TMEM150B, TNFRSF19, TNFRSF25, TONSL, TPX2, TRIM16, TRIM16L, TRIM31, TRIM45, TTC39A, UBD, UBE2C, UBE2T, UGT2B11, USH1C, VSIG10L, WDR62, WDR76, and ZWINT.
5. A method for detecting the presence of HCC in a subject having chronic liver disease (CLD), the method comprising: (a) measuring expression levels of the HCC classifier genes of any one of claims 1-4 in CECs of the subject; and (b) comparing the expression levels of the HCC classifier genes in the CECs of the subject with reference expression levels of HCC classifier genes thereby determining the presence of HCC.
6. The method of claim 5, wherein the expression levels of HCC classifier genes are used to calculate a HCC score, and the calculated HCC score is compared with a reference score, wherein the presence of HCC is determined based on the presence of a HCC score above the reference score.
7. The method of claim 6, wherein the HCC score is calculated using a random forest analysis.
8. The method of claim 5, wherein the expression levels of HCC classifier genes are compared with the reference expression levels of HCC classifier genes using a multivariate logistic regression modeling approach.
9. The method of any one of claims 1-8, wherein the expression levels of HCC classifier genes in circulating epithelial cells (CECs) are measured by: (a) obtaining a sample comprising blood from the subject; (b) removing red blood cells, platelets, and plasma from the sample by size-based exclusion; (c) removing white blood cells (WBCs) from the sample by magnetophoresis; and (d) measuring the expression of a set of genes in the CECs using RNA-sequencing, qRT-PCT, RNA in situ hybridization, protein microarray, or mass spectrometry and protein profiling.
10. The method of any one of claims 5-9, wherein the HCC being detected is an early stage HCC.
11. The method of any one of claims 5-9, wherein the HCC being detected is a late stage HCC.
12. The method of any one of claims 5-11 further comprising: (a) confirming or having confirmed the presence of HCC in the patient by ultrasound imaging, dynamic CT, MRI imaging, needle biopsy, and/or biopsy; and (b) if the presence of HCC in the patient is confirmed, treating or having the subject treated for HCC by surgical removal of the HCC tissue, radiofrequency ablation of the HCC tissue, embolization of the HCC tissue; embolization of HCC tissue, chemotherapy, and/or cryotherapy.
13. A method of monitoring a subject having CLD for development of HCC, the method comprising: (a) performing the method of claim 6 or 7 at an initial time point, and if the HCC score is below the reference score, then (b) performing the method of claim 6 or 7 at one or more subsequent time points.
14. The method of claim 13, wherein step (b) is performed at one or more subsequent time points until the presence of HCC is determined.
15. The method of claim 13 or 14, wherein the initial and each subsequent time point is about three months, six months, or a year apart.
16. A method of distinguishing between the presence of early stage liver fibrosis and late stage liver fibrosis in a subject having CLD, the method comprising: (a) detecting a concentration of CECs in a blood sample of the subject; (b) comparing the concentration of CECs in the blood sample of the subject with a reference value; (c) diagnosing the subject with early stage fibrosis if the subject has concentration of CECs in the blood sample that is below the reference value; and (d) diagnosing the subject with late stage fibrosis if the subject has concentration of CECs in the blood sample that is above the reference value.
17. The method of claim 16, wherein the subject has hepatitis B.
18. The method of claim 16 or 17, wherein the concentration of CECs is measured by immunofluorescence.
19. The method of any one of claims 16-18, wherein the concentration of CECs is measured by detecting glypican-3 (GPC3) and/or cytokeratins (CKs).
20. A method of monitoring a subject having CLD for development of advanced fibrosis, the method comprising: (a) performing the method of any one of claims 16-19; and if the concentration of CECs in the blood sample of the subject is lower than the reference value, then (b) performing the method of any one of claims 16-19 at one or more subsequent time points.
21. The method of claim 20, wherein step (b) is performed at one or more subsequent time points until the subject is diagnosed with late stage fibrosis.
22. The method of any one of claims 16-20, wherein the initial and each subsequent time point is about three months, six months, or a year apart.
23. A method of monitoring a subject having CLD being treated to prevent the progression of fibrosis or HCC, the method comprising: (a) performing the method of any one of claims 16-19; and if the concentration of CECs in the blood sample of the subject is lower than the reference value, then performing the method of any one of claims 16-19 at one or more subsequent time point; and (b) performing the method of claim 6 or 7 at an initial time point, and if the expression levels of the HCC score is below the reference score, then performing the method of claim 6 or 7 at one or more subsequent time points.
24. The method of claim 23, wherein step (a) is performed at one or more subsequent time points until the subject is diagnosed with late stage fibrosis, and/or wherein step (b) is performed at one or more subsequent time points until the presence of HCC is determined.
25. The method of claim 24, wherein the first initial and each subsequent time point for performing step (a) or step (b) of claim 23 is about three months, six months, or a year apart, and the second initial and each subsequent time point is about three months, six months, or a year apart.
26. The method of any one of claims 1-25, wherein the CECs in the blood are purified or enriched using a microfluidic device.
27. The method of claim 26, wherein the microfluidic device is an iChip device.
Description
DESCRIPTION OF DRAWINGS
[0032] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION
[0048] The present invention is based, at least in part, on the discovery that hCECs are not exclusive to carcinogenesis, but also can be present in subjects having non-cancer diseases or conditions such as chronic liver disease (CLD). Furthermore, the present invention is based, at least in part, on the discovery that the hCECs in subjects with CLD can be analyzed quantitatively or qualitatively to accurately detect the presence of cancer such as hepatocellular carcinoma (HCC) and/or to accurately characterize the stage (e.g., early or late stage) of a liver disease or liver condition such as liver fibrosis.
[0049] As demonstrated herein, cells from diseased livers circulating in the bloodstream (i.e., hCECs) are detected both quantitatively (e.g., by immunofluorescence) and qualitatively (e.g., gene expression profile or expression levels of HCC classifier genes) for use in diagnosis of HCC and CLD. Important applications of this liquid biopsy include detection or diagnosis of a liver disease or condition such as HCC, CLD etiology determination, liver fibrosis staging, and HCC surveillance or monitoring. The present invention can be applied to both diagnosis and monitoring of patients with liver conditions such as CLDs.
[0050] As used herein, the phrases “accurately diagnose” and “accurately detect” with respect to a disease or a condition refer to predicting the presence of the disease or the condition with a high degree of sensitivity (i.e., true positive rate or detecting a disease or a condition when the disease or the condition is present) or a high degree of specificity (i.e., true negative rate or not detecting a disease or a condition when the disease or the condition is not present). In some embodiments, the phrases “accurately diagnose” and “accurately detect” can also mean being able to detect the presence of a disease or a condition with a true positive rate of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and at least about 99.9%. In some embodiments, the phrases “accurately diagnose” and “accurately detect” can mean being able to detect the presence of a disease or a condition with a true negative rate of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and at least about 99.9%.
[0051] As used herein, the phrase “accurately distinguish” with respect to two diseases or conditions, can refer to detecting the presence of a first disease or a first condition with a high degree of sensitivity (i.e., detecting a first disease or condition when the first disease or condition is present, i.e., true positive rate) or a high degree of specificity (i.e., not detecting a first disease or condition when the first disease or condition is not present, i.e., true negative rate), regardless of whether the second disease or condition is also present or absent. In some embodiments, the phrase “accurately distinguish” can mean being able to detect the presence of a disease or a condition with a true positive rate of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and at least about 99.9%. In some embodiments, the phrase “accurately distinguish” can mean being able to detect the presence of a disease or a condition with a true negative rate of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and at least about 99.9%.
[0052] As used herein, the phrase “accurately distinguish” with respect to different stages of a disease or a condition can refer to detecting the presence of a particular stage of the disease (e.g., advanced fibrosis in liver) with a high degree of sensitivity (i.e., detecting the stage of a disease or condition when the disease or condition is present at that stage, i.e., true positive rate) or a high degree of specificity (i.e., not detecting a stage of a disease or condition when the disease or condition is not present at that stage, i.e., true negative rate) so that the particular stage of the condition or disease can be predicted. In some embodiments, the phrase “accurately distinguish” can mean being able to detect the presence of a stage of a disease or a condition with a true positive rate of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and at least about 99.9%. In some embodiments, the phrase “accurately diagnose” can mean being able to detect the presence of a disease or a condition with a true negative rate of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and at least about 99.9%.
[0053] As used herein, the term “circulating epithelial cells (CECs)” can refer to cells of epithelial origin that are shed from a tissue (e.g., diseased tissue, tumor tissue, or non-tumor tissue) and present in the blood, i.e. in circulation. Cell markers (e.g. marker genes) that can be used to identify and/or isolate CECs from other components of the blood are described below herein. In some embodiments, the CECs from a subject with a liver disease (e.g., HCC and/or CLD) are predominantly hepatic CECs (hCECs), for example, as determined by immunofluorescence staining of the CECs with genes expressed in hepatocytes (e.g., GPC3 and CKs).
[0054] As used herein, the term “chronic liver disease (CLD)” refers to a disease process of the liver involving progressive destruction and regeneration of the liver parenchyma. In some embodiments, CLD can lead to fibrosis cirrhosis. In some other embodiments, CLD can result in complications such as portal hypertension (e.g., ascites, hyperplenism, and lower esophageal varices and rectal varices) hepatopulmonary syndrome, hepatorenal syndrome, encephalopathy, or HCC. CLD can also refer to disease of the liver which lasts over a period of six months, one year, two years, three years, four years, five years, or more than five years. CLD can be caused by hepatitis B, hepatitis C, cytomegalovirus, Epstein Bar virus, yellow fever viruses, alcoholic liver disease, and/or drug induced liver disease from methotrexate, amiodarone, nitrofurantoin, or acetaminophen. In other embodiments, CLD can be caused by non-alcoholic fatty liver disease, haemochromatosis, Wilson's disease, or autoimmune responses such as primary biliary cholangitis or primary sclerosing cholangitis.
[0055] As used herein, the term “monitoring” or “surveillance” refers to periodically assessing a subject or a patient (e.g., a subject who is at risk of developing a condition) for the presence of a disease or a condition. In some embodiments, the periodic assessment can occur about every day, about every other day, about once a week, about once every other week, about every month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every year, about every 18 months, about every 2 years, about every 3 years, about every 4 years, about every 5 years, about every 6 years, about every 7 years, about every 8 years, about every 9 years, or about every 10 years. This recurring assessment of a subject or a patient for the presence of a disease or a condition can continue until (1) the disease or the condition is detected in the subject or the patient; (2) the patient is no longer at risk of developing the disease or the condition; (3) at the discretion of the subject receiving the monitoring or the person administering the monitoring; or (4) discontinuation of the recurring assessment is necessary due to other reasons. The interval with which a subject is assessed for the presence of a disease or a condition can be adjusted during the course of the monitoring.
[0056] As used herein the term “ensemble learning method” refers to a supervised learning algorithm such as random forest that can be trained and then used to make predictions.
[0057] As used herein the term “hepatocellular carcinoma (HCC)” refers to a type of primary liver cancer prevalent in subjects with CLD. HCC can develop in patients with underlying cirrhotic liver disease of various etiologies, including patients with negative markers for HBV infection and who have HBV DNA integrated in the hepatocyte genome. Epidemiology, etiology, and carcinogenesis of HCC has been described in Ghouri Y A, et al., J Carcinog 2017; 16:1, which is incorporated by reference herein.
[0058] As used herein, the phrase “early stage HCC” can refer to HCC being within the Milan criteria. As used herein, the phrase “late stage HCC” can refer to HCC being outside of the Milan criteria. Milan criteria requires the subject with HCC meet the following criteria: HCC being one lesion smaller than 5 cm or up to 3 lesions, each smaller than 3 cm; no extrahepatic manifestations; and no evidence of gross vascular invasion. In other words, “early stage HCC” meets all Milan criteria and “late stage HCC” does not meet all Milan criteria.
[0059] As used herein, the term “early stage liver fibrosis” and “late stage liver fibrosis” refer to F1 or F2 stages, and F3 or F4 stages, respectively, as defined by the METAVIR classification.
[0060] The methods described herein can be used to accurately diagnose or predict the presence of cancer, e.g., HCC, in a patient with a non-cancerous disease condition, e.g., CLD, by detecting and analyzing expression of a set of genes in the CECs of the patient using a classifier that is based on an ensemble learning method such as random forest classifier.
[0061] In some embodiments, hCECs from subjects with CLD (e.g., subjects with Hepatitis B or subjects who are infected with Hepatitis B Virus) can be analyzed (e.g., qualitatively) to accurately distinguish between subjects with and without HCC. In other embodiments, hCECs from subjects with CLD can be quantitatively measured to accurately distinguish between subjects with early stage liver fibrosis and subjects with late stages liver fibrosis.
[0062] As demonstrated herein, the presence of cancer, e.g., HCC, and the presence of non-cancer diseases or conditions, e.g., CLD, are associated with the increased presence of CECs. The increased presence of CECs is also associated with the previous presence of cancer (e.g., HCC) which was treated to result in no clinical evidence of disease (e.g., in HCC patients who underwent curative treatment and had no clinical evidence of the disease).
[0063] Thus the methods can include the detection and analysis of a set of genes (e.g., HCC classifier genes) using a variety of statistical and computational prediction method (e.g., an ensemble learning method such as random forest classifier or a statistical method such as multivariable logistic regression), to detect the presence of a cancer, e.g., HCC.
[0064] The method can, in some embodiments, detect the presence of cancer at an early stage, which may otherwise be difficult to detect using a currently known method such as ultrasound imaging, dynamic CT, MRI imaging, needle biopsy, or biopsy.
[0065] In some embodiments, microfluidic (e.g., “lab-on-a-chip” or the iChip device) can be used in the present methods to separate, purify, enrich, or prepare CECs. Such devices have been successfully used for microfluidic flow cytometry, continuous size-based separation, chromatographic, or magnetophoretic separation. For Example, the iChip device and various other embodiments of such devices are described in U.S. Patent Application US2016/0312298 A1 (which is incorporated herein by reference) can be used for separating hCECs from a mixture of cells, or preparing an enriched population of hCECs. In particular, such devices can be used for the isolation of hCECs from complex mixtures such as whole blood.
[0066] In some embodiments, the devices retain at least 75%, e.g., 80%, 90%, 95%, 98%, or 99% of the desired cells compared to the initial sample mixture, while enriching the population of desired cells by a factor of at least 100, e.g., by 1000, 10,000, 100,000, or even 1,000,000 relative to one or more non-desired cell types. In one example, a detection module can be in fluid communication with a separation or enrichment device. The detection module can operate using any method of detection disclosed herein, or other methods known in the art. For example, the detection module includes a microscope, a cell counter, a magnet, a biocavity laser (see, e.g., Gourley et al., J. Phys. D: Appl. Phys., 36: R228-R239 (2003)), a mass spectrometer, a PCR device, an RT-PCR device, a microarray, a device for performing RNA in situ hybridization, or a hyperspectral imaging system (see, e.g., Vo-Dinh et al., IEEE Eng. Med. Biol. Mag., 23:40-49 (2004)). In some embodiments, a computer terminal can be connected to the detection module. For instance, the detection module can detect a label that selectively binds to cells, proteins, or nucleic acids of interest, e.g., transcripts of HCC classifier genes or encoded proteins.
[0067] In some embodiments, the microfluidic system includes (i) a device for separation or enrichment of CECs (e.g., hCECs); (ii) a device for lysis of the enriched CECs; and (iii) a device for detection of gene transcripts (e.g., transcripts of HCC classifier genes) or encoded proteins.
[0068] In some embodiments, a population of CECs prepared using a microfluidic device as described herein is used for analysis of expression of gene transcripts or proteins using known molecular biological techniques, e.g., as described above and in Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press; 3rd edition (Jan. 15, 2001)); and Short Protocols in Molecular Biology, Ausubel et al., eds. (Current Protocols; 52 edition (Nov. 5, 2002)).
[0069] In general, devices for detection and/or quantification of expression of classifier genes useful for cancer diagnosis or encoded proteins in an enriched population of CECs (e.g., CTCs) are described herein and can be used for the early detection of cancer, e.g., tumors of epithelial origin, e.g., early detection of liver, pancreatic, lung, breast, prostate, renal, ovarian or colon cancer.
[0070] As described herein, the phrase “differential expression analysis” can refer to performing computational or statistical analysis on expression level of individual genes (e.g., individual HCC classifier genes) and/or expression patterns of multiple genes (e.g., multiple HCC classifier genes) in a sample (e.g., cell, e.g., CEC, e.g., hCEC). The term “differential expression” can mean over-expression (expressing a gene at a higher level than the reference value) or under-expression (expressing a gene at a lower level than the reference value). In some embodiments, a differential expression analysis can compare the expression levels or patterns in a sample with a reference value (e.g., expression levels or patterns of one or more genes in a sample from a non-diseased counterpart cell or tissue). In other embodiments, the expression levels or patterns can be normalized to expression levels of one or more control genes, or may be quantified in a non-relative manner (e.g., transcript copies per volume or absolute copy number). The gene expression levels can be measured by any of the known methods, such RNA-sequencing, qRT-PCT, RNA in situ hybridization, protein microarray, and/or mass spectrometry and protein profiling. Other known biochemical, or molecular biology techniques can be used to detect the expression of genes. In some embodiments, RNA-sequencing and qRT-PCT is the preferred method for measuring gene expression levels.
[0071] The differential expression analysis can be performed by any one of the known statistical or computational methods, for example, an ensemble learning method such as random forest classifier or a statistical method such as multivariable logistic regression.
[0072] In one aspect, the present invention provides methods including measuring expression levels of hepatocellular carcinoma (HCC) classifier genes in circulating epithelial cells (CECs) of a subject. The overexpression of HHC classifier genes by the CECs of subjects was determined as being highly predictive of the presence of HCC in the subjects (see e.g., Example 1-4). In some embodiment, the HCC classifier genes include one, two, three, or more of (e.g., all of) TESC, OSBP2, SLC6A8, SEPT5, F2RL3, E2F1, EZH2, CDC20, CCNA2, CCNB1, PLXNB3, CDC6, MYBL2, APOBEC3B, SPP1, AKR1B10, TOP2A, ASPM, SLC6A9, RECQL4, NUSAP1, PLVAP, FMO1, PDZK1IP1, and FBXO32. In some embodiments, the HCC classifier genes can be include all of TESC, OSBP2, SLC6A8, SEPT5, F2RL3, E2F1, EZH2, CDC20, CCNA2, CCNB1, PLXNB3, CDC6, MYBL2, APOBEC3B, SPP1, AKR1B10, TOP2A, ASPM, SLC6A9, RECQL4, NUSAP1, PLVAP, FMO1, PDZK1IP1. In other embodiments, the HCC classifier genes can also include one or more other genes that are overexpressed in HCC, e.g., one or more of ACTG2, ADM2, AFP, AGR2, ALDH3A1, ALPK3, AMIGO3, ANKRD65, ANLN, AP1M2, ARHGAP11A, ARHGEF39, ASF1B, ASPHD1, AURKA, AXIN2, BAIAP2L2, BEX2, C15orf48, C1orf106, C1QTNF3, C6orf223, CA12, CA9, CAMK2N2, CAP2, CBX2, CCDC170, CCDC28B, CCDC64, CCNE2, CCNF, CD109, CD34, CDC25A, CDC7, CDCA5, CDCA8, CDH13, CDK1, CDKN2A, CDKN2C, CDT1, CELF6, CENPF, CENPH, CENPL, CENPU, CENPW, CKB, CNNM1, COL15A1, COL4A5, COL7A1, COL9A2, CRIP3, CSPG4, CTNND2, CXorf36, CYP17A1, DLK1, DMKN, DSCC1, DTL, DUOX2, ECT2, EEF1A2, EFNA3, EPHB2, EPPK1, ETV4, FABP4, FAM111B, FAM3B, FAM83D, FANCD2, FANCI, FBXL18, FERMT1, FGF19, FLNC, FLVCR1, FOXD2-AS1, FOXM1, FXYD2, GABRE, GAL3ST1, GCNT3, GINS1, GJC1, GMNN, GNAZ, GOLGA2P7, GPC3, GPR64, GPSM1, HRCT1, IGF2BP2, IGSF1, IGSF3, IQGAP3, ITGA2, ITPKA, KIAA0101, KIF11, KIFC1, KIFC2, KNTC1, KRT23, LAMA3, LEF1, LGR5, LINC00152, LINGO1, LPL, LRRC1, LYPD1, MAD2L1, MAGED4, MAGED4B, MAPK12, MAPK8IP2, MAPT, MCM2, MDGA1, MDK, MFAP2, MISP, MKI67, MMP11, MNS1, MPZ, MSC, MSH5, MTMR11, MUC13, MUC5B, MYH4, NAALADL1, NAV3, NCAPG, NDUFA4L2, NEB, NKD1, NMB, NOTCH3, NOTUM, NPM2, NQO1, NRCAM, NT5DC2, NTS, OBSCN, OLFML2A, OLFML2B, PAQR4, PEG10, PI3, PLCE1, PLCH2, PLK1, PLXDC1, PODXL2, POLE2, PPAP2C, PRC1, PTGES, PTGFR, PTHLH, PTK7, PTP4A3, PTTG1, PYCR1, RACGAP1, RBM24, RHBG, RNF157, ROBO1, RP4-800G7.2, RPS6KL1, RRM2, S100A1, SCGN, 5-Sep, SERPINA12, SEZ6L2, SFN, SGOL2, SLC22A11, SLC51B, SLC6A2, SNCG, SOAT2, SP5, SPARCL1, SPINK1, STIL, STK39, SULT1C2, TCF19, TDGF1, THY1, TK1, TMC5, TMEM132A, TMEM150B, TNFRSF19, TNFRSF25, TONSL, TPX2, TRIM16, TRIM16L, TRIM31, TRIM45, TTC39A, UBD, UBE2C, UBE2T, UGT2B11, USH1C, VSIG10L, WDR62, WDR76, and ZWINT.
[0073] In another aspect, the present invention provides methods for detecting the presence of HCC in subjects having a chronic liver disease (CLD). The methods can include: (a) measuring expression levels of HCC classifier genes in CECs of a subject; and (b) comparing the expression levels of HCC classifier genes in the CECs of the subject with reference expression levels of HCC classifier genes thereby determining the presence of HCC.
[0074] In another aspect, the present invention provides methods of monitoring subjects having CLD for development of HCC. The methods can include: (a) measuring expression levels of HCC classifier genes in CECs of a subject and comparing the expression levels of HCC classifier genes in the CECs of the subject with reference expression levels of HCC classifier genes at an initial time point; and if the expression levels of the HCC classifier genes are below the reference level, then (b) performing the step again at a subsequent time point, and optionally at additional time points, e.g., until the expression levels of HCC classifier genes are above the reference level. This assessment can be formed by first calculating a HCC score (e.g., the vote fraction from the RF classifier) or other metrics that indicate the degree of differential expression of HCC classifier genes in the subject's CECs, as compared to a reference score, or other reference metrics values.
[0075] In another aspect, the present invention provides methods of distinguishing between the presence of early stage liver fibrosis and late stage liver fibrosis in a subject having CLD. The methods can include: (a) detecting a concentration of CECs in a blood sample of a subject; (b) comparing the concentration of CECs in the blood sample of the subject with a reference value; (c) diagnosing the subject with early stage fibrosis if the subject's blood concentration of CECs is below the reference value; and (d) diagnosing the subject with late stage fibrosis if the subject's blood concentration of CECs is above the reference value.
[0076] In another aspect, the present invention provides methods of monitoring a subject having CLD for development of advanced fibrosis. The methods can include: (a) detecting a concentration of CECs in a blood sample of a subject and comparing the blood CEC concentration to a reference value; and if the concentration of CECs in the blood sample of the subject is lower than the reference value, then (b) performing the same detection and comparison step at one or more subsequent time points, e.g., until the concentration of CECs in the blood sample of the subject is higher than the reference value.
[0077] In some embodiments, the expression levels of HCC classifier genes are used to calculate a HCC score, preferably using a random forest analysis, and the method includes comparing the HCC score with a reference score, wherein the presence of HCC is determined based on the presence of a HCC score above the reference score.
[0078] In some embodiments, the expression levels of HCC classifier genes are compared with the reference expression levels of HCC classifier genes using a multivariate logistic regression modeling approach.
[0079] In some embodiments, the expression levels of HCC classifier genes in circulating epithelial cells (CECs) are measured by: (a) obtaining a sample comprising blood from the subject; (b) removing red blood cells, platelets, and plasma from the sample by size-based exclusion; (c) removing white blood cells (WBCs) from the sample by magnetophoresis; and (d) measuring the expression of a set of genes in the CECs using RNA-sequencing, qRT-PCT, RNA in situ hybridization, protein microarray, or mass spectrometry and protein profiling.
[0080] In some embodiments, the HCC being detected is an early stage HCC or a late stage HCC.
[0081] In some embodiments, the method also includes (a) confirming or having confirmed the presence of HCC in the patient by ultrasound imaging, dynamic CT, MIR imaging, needle biopsy, and/or biopsy; and (b) if the presence of HCC in the patient is confirmed, treating or having the subject treated for HCC by surgical removal of the HCC tissue, radiofrequency ablation of the HCC tissue, embolization of the HCC tissue; embolization of HCC tissue, chemotherapy, and/or cryotherapy.
[0082] In some embodiments, the initial and each subsequent time point for measuring and comparing the blood CEC concentration or for measuring and comparing HCC classifier gene is about three months, six months, or a year apart. In some embodiments, the subject has hepatitis B or not have hepatitis B. In some embodiments, the concentration of CECs is measured by immunofluorescence. In some embodiments, the concentration of CECs is measured by detecting glypican-3 (GPC3) and/or cytokeratins (CKs).
Diagnosis and Treatment of Liver Diseases
[0083] Once a liver disease such as CLD or HCC are detected in a subject, the presence of the disease such as CLD or HCC may be confirmed using other methods.
[0084] Diagnosis or Detection of HCC
[0085] HCC can be further confirmed or diagnosed by analyzing blood sample using traditional methods, including a complete blood count (CBC), electrolytes, liver function tests (LFTs), coagulation studies (e.g., international normalized ratio (INR) and partial thromboplastin time (PTT)), and alpha-fetoprotein (AFP) determination.
[0086] Various imaging techniques can be used to diagnose HCC. For example, ultrasonography offers a relatively inexpensive method of screening without the cost of magnetic resonance imaging (MRI) or the exposure to radiation and potentially nephrotoxic contrast agents required for computed tomography (CT). Ultrasonography as a screening method is reported to have 60% sensitivity and 97% specificity in the cirrhotic population, and it has been demonstrated to be cost-effective. Due to this low-sensitivity, findings on ultrasound examination should be confirmed with further imaging studies and potentially biopsy.
[0087] HCC can be detected using CT imaging, preferably with early enhancement on the arterial phase with rapid washout of contrast on the portal venous phase of a three-phase contrast scan. HCC can also be detected using MRI.
[0088] HCC can be detected by biopsy, especially for subjects with HCCs that are larger than 2 cm with low levels of alpha-fetoprotein or in whom ablative treatment or transplant is contraindicated.
[0089] In patients with elevated AFP and consistent imaging characteristics, patients can be treated presumptively for HCC without a biopsy. Patients preferably can also undergo evaluation for extrahepatic disease (primarily pulmonary metastasis) with cross-sectional imaging; this would preclude curative locoregional therapy
Treatment of HCC
[0090] HCC can be treated using a number of methods known in the art, including by liver transplantation-however a limited supply of donor organs limit the availability of transplantation as an option for many subjects. HCC can also be treated using resection, radiofrequency ablation (RFA). Systemic therapy with sorafenib (or, if sorafenib fails, with regorafenib, nivolumab, or lenvatinib), can be used to bridge patients to transplant or to delay recurrence of HCC. In patients who experience a recurrence following resection or transplantation, aggressive surgical treatment appears to be associated with the best possible outcome.
[0091] HCC can be treated by transcatheter arterial chemoembolization, which selectively cannulates the feeding artery to the tumor and delivers high local doses of chemotherapy, including doxorubicin, cisplatin, or mitomycin C. To prevent systemic toxicity, the feeding artery is occluded with gel foam or coils to prevent flow.
[0092] HCC can be treated by chemotherapy-however, HCC is minimally responsive to systemic chemotherapy. For example, doxorubicin-based regimens, which appears to have the greatest efficacy, has a response rates of 20-30% and a minimal impact on survival.
[0093] For patients with Child class C cirrhosis and contraindications for transplantation, HCC can be managed by focusing on pain control, ascites, edema, and portosystemic encephalopathy management.
[0094] HCC can be treated surgically. Presently, in view of the absence of effective chemotherapy and the insensitivity of HCC to radiotherapy, complete tumor extirpation is the only option for a long-term cure. Resection of the tumor by partial hepatectomy can be accomplished in a limited number of patients (generally <15-30%) due to the degree of underlying cirrhosis.
Diagnosis and Treatment of Liver Cirrhosis
[0095] Chronic liver disease can include liver cirrhosis, which is characterized by fibrosis and the conversion of normal liver architecture into structurally abnormal nodules. The progression of liver injury to cirrhosis may occur over weeks to years. In addition to fibrosis, the complications of cirrhosis include, but are not limited to, portal hypertension, ascites, hepatorenal syndrome, and hepatic encephalopathy.
[0096] Liver cirrhosis can occur in Hepatitis C alcoholic liver disease, NASH; and Hepatitis B. Hepatic fibrosis can occur due to alteration in the normally balanced processes of extracellular matrix production and degradation in liver. In liver cirrhosis, stellate cells can become activated into collagen-forming cells by a variety of paracrine factors. Such factors can be released by hepatocytes, Kupffer cells, and sinusoidal endothelium following liver injury. For example, increased levels of the cytokine transforming growth factor beta1 (TGF-beta1) are observed in patients with chronic hepatitis C and those with cirrhosis. TGF-beta1, in turn, stimulates activated stellate cells to produce type I collagen.
[0097] Diagnosis of Liver Cirrhosis
[0098] Severity of liver cirrhosis is commonly assessed using the Child-Turcotte-Pugh (CTP) system, a scoring system for assessing the severity of cirrhosis by considering the clinical variables encephalopathy, presence and/or severity of ascites, levels of bilirubin and albumin levels in blood, and prothrombin time.
[0099] Severity of liver cirrhosis can also be assessed using the Model for End-Stage Liver Disease (MELD) scoring system, by considering the clinical variables of number of times dialysis was needed, blood levels of creatinine, bilirubin levels, sodium, and prothrombin time.
[0100] Treatment of Liver Cirrhosis
[0101] Subjects with severe CLD (e.g., decompensated cirrhosis) can be treated using liver transplantation. Liver transplantation has a 1-year survival rate of 85-90% and a 5-year survival rate of higher than 70%. Quality of life after liver transplant is good or excellent in most cases. However, a limited supply of donor organs limit the availability of transplantation as an option for many subjects.
[0102] A number of therapies are available to prevent or delay the development of cirrhosis in subjects with CLD: prednisone and azathioprine for treating autoimmune hepatitis, interferon and other antiviral agents for treating hepatitis B and C, phlebotomy for hemochromatosis, ursodeoxycholic acid for primary biliary cirrhosis, and trientine and zinc for Wilson disease. NASH is an advanced form of nonalcoholic fatty liver disease (NAFLD), which are being evaluated for treatment using allosteric Acetyl-CoA Carboxylase (ACC) inhibitors (e.g., NDI-010976/GS-0976), obeticholic acid, thiazolidinediones (e.g., pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone, balaglitazone), elafibranor (GFT505), obeticholic acid (OCA), apoptosis signal-regulating kinase 1 (ASK1) inhibitor (selonsertib), dual CCR2/CCR5 inhibitor cenicriviroc (CVC, also TBR-652 or TAK-652), and vitamin E.
[0103] These therapies are less effective if chronic liver disease evolves into cirrhosis. Once cirrhosis develops, treatment is aimed at the management of complications arising from cirrhosis. For example, cirrhosis-related zinc deficiency can be treated with zinc sulfate at 220 mg orally twice daily to improve dysgeusia and to stimulate appetite. Furthermore, zinc is effective in the treatment of muscle cramps and is adjunctive therapy for hepatic encephalopathy. Pruritus in subjects with CLD (e.g., cholestatic liver diseases or Hepatitis C) can be treated with Cholestyramine, antihistamines (eg, diphenhydramine, hydroxyzine) and ammonium lactate 12% skin cream (Lac-Hydrin), include ursodeoxycholic acid, doxepin, and rifampin. Naltrexone may be effective but is often poorly tolerated. Gabapentin is an unreliable therapy. Patients with severe pruritus may require institution of ultraviolet light therapy or plasmapheresis. Hypogonadism in male subjects with CLD can be treated with topical testosterone preparations. Osteoporosis in subjects with CLD (especially chronic cholestasis or primary biliary cirrhosis) can be treated with calcium and vitamin D supplements. In addition, patients with CLD can be vaccinated against hepatitis A.
Examples
[0104] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Methods
[0105] The following materials and methods were used in the Examples set forth below.
Clinical Protocol
[0106] Patient medical data were collected from patient electronic medical record with patient permission and a maximum of 20 ml of blood was obtained from patients at any given blood draw in two 10-mL EDTA tubes, and approximately 8-15 ml of blood was processed per patient.
Microfluidic Purification of CECs from Whole Blood Using the iChip Device
[0107] Biotinylated primary antibodies against anti-human CD45 antibody (clone 2D1, R&D Systems, BAM1430) and anti-human CD66b antibody (Abd Serotec, 80H3) were spiked into whole blood (5-10 mL total volume) at 100 fg/WBC and 37.5 fg/WBC, respectively, and incubated rocking at room temperature for 20 min. Dynabeads MyOne Strepavidin T1 (Life Technologies, 65602) magnetic beads were then added and incubated rocking at room temperature for an additional 20 min. The total blood volume (5-10 mL) was then run on the iChip device as previously described..sup.8
Immunofluorescent Staining of CECs
[0108] Cells in an aliquot of the iChip device-processed blood samples were fixed with 2% paraformaldehyde for 10 min and then applied to glass slides via cytospin using a Shandon EZ Megafunnel (ThermoFisher A78710001) at 2000 rpm for 5 min. Slides were washed with PBS and blocked with 5% donkey serum+0.3% Triton-X in PBS for 1 hr at room temperature (RT). Primary antibodies (each at 1:50 dilution in PBS, 0.1% BSA, 0.3% Triton-X) against wide spectrum cytokeratin (WS CK, Abcam ab9377), glypican-3 (Abcam ab81263), and CD45 (Becton Dickenson 555480) were then added and incubated for 1 hr at RT. Secondary antibodies (each at 1:200 dilution in PBS, 0.1% BSA, 0.3% Triton-X) directed against each of the primary antibodies were then used for fluorescent labelling, incubated for 1 hr at RT protected from light: 1) cytokeratin—donkey anti-rabbit Alexa-647 (Jackson ImmunoResearch 711-605-152); 2) glypican-3—donkey anti-sheep Cy3 (Jackson ImmunoResearch 713-165-003); 3) CD45—donkey anti-mouse Alexa-488 (Jackson ImmunoResearch 715-545-150), which were. Cell nuclei were counterstained with DAPI (5 μg/mL in PBS, Life Technologies). Slides were mounted using ProLong Gold Antifade Reagent (Life Technologies). Stained cells were imaged by fluorescence microscopy (TiE or Eclipse 90i, Nikon) using the appropriate filter cubes for image acquisition and the BioView platform for automated image analysis. All candidate CECs detected were reviewed and scored based on intact morphology, localization of CEC markers (WS CK Alexa-647 and/or GPC3 Cy3) with DAPI nuclear counterstain, and absence of leukocyte markers (CD45 Alexa-488).
HepG2 Cell Spike-in
[0109] HepG2 cells were cultured following American Type Culture Collection-recommended culturing conditions. Individual cells were micropipetted using an Eppendorf TransferMan NK2 micromanipulator and introduced into 4 mL of blood from healthy donors, before processing through the iChip device.
RNA-Sequencing of CECs
[0110] The iChip device-processed blood sample aliquot was pelleted and flash frozen in RNAlater (Thermo-Fisher Scientific) at −80 deg C. RNA was extracted (RNEasy Micro, Qiagen) and processed as follows for RNA-seq. Amplified cDNA was generated from RNA from each sample using the SMARTer Ultra Low Input RNA Kit (v3 or v4) for Sequencing (Clontech Laboratories) according to the manufacturer's protocol. Briefly, 1 μl of a 1:50,000 dilution of ERCC RNA Spike-In Mix (Life Technologies) was added to each sample. First-strand synthesis of RNA molecules was performed using the poly-dT-based 3′-SMART CDS primer II A followed by extension and template switching by the reverse transcriptase. The second strand synthesis and amplification PCR were run for 18 cycles, and the amplified cDNA was purified with a 1× Agencourt AMPure XP bead cleanup (Beckman Coulter). The Nextera® XT DNA Library Preparation kit (Illumina) was used for sample barcoding and fragmentation according to the manufacturer's protocol. 1 ng of amplified cDNA was used for the enzymatic tagmentation followed by 12 cycles of amplification and unique dual-index barcoding of individual libraries. PCR product was purified with a 1.8× Agencourt AMPure XP bead cleanup. The eluted cDNA libraries did not undergo the bead-based library normalization step in the Nextera XT protocol. Library validation and quantification was performed by quantitative PCR using the KAPA SYBR® FAST Universal qPCR Kit (Kapa Biosystems). The individual libraries were pooled at equal concentrations, and the pool concentration was determined using the KAPA SYBR® FAST Universal qPCR Kit. The pool of libraries was subsequently sequenced in three replicates on a HiSeq 2500 in Rapid Run Mode using a 2×100 base pair kit and a dual flow cell. The paired-end reads from the three sequencing runs were combined and aligned to the hg38 genome from http://genome.ucsc.edu using the STAR v2.4.0h aligner with default settings. Reads that did not map or mapped to multiple locations were discarded. Duplicate reads were marked using the MarkDuplicates tool in picard-tools-1.8.4 and were removed. The uniquely aligned reads were counted using htseq-count in the intersection-strict mode against the publicly available Homo_sapiens.GRCh38.79.gtf annotation table. Data were then imported into the R statistical programming language for analysis. All RNA-seq raw data has been submitted to NCBI GEO: accession GSE117623.
Flow Sorting of White Blood Cells
[0111] For a subset of HCC patients, the iChip device-processed blood sample was divided into two equal aliquots: one aliquot was pelleted and flash frozen as above; the second was flow sorted to isolate subtypes of contaminating white blood cells (monocytes, granulocytes, NK cells, cytotoxic T cells, helper T cells, and B cells). Cells were fixed with Cytofix (BD Biosciences 554655). The following antibodies were used: CD45 (Beckman Coulter IM0782U), CD56 (Beckman Coulter IM2073U), CD16 (Biolegend 360712), CD14 (Biolegend 301808), CD3 (Biolegend 317330), CD19 (Biolegend 302216), CD4 (Biolegend 300556), CD8 (Biolegend 301016), CD66b (Biolegend 305112). As described above, flow sorted cells were pelleted, flash frozen in RNAlater, and subjected to RNA-seq.
Example 1: Overview of Classification of CLD Patients Using Random Forest Classifier
[0112] The RNA-seq raw data consisted of read counts for 59,074 transcripts on 64 CLD and 52 HCC samples. Of those, only samples with more than 250k total reads were kept, leaving 44 CLD and 39 HCC samples. In order to narrow the list of features in our data set to those with a higher likelihood of relevance for predicting HCC status, RNA-seq expression data was obtained from The Cancer Genome Atlas (TCGA) liver cancer project (LIHC), which contains expression counts for both normal liver and HCC tissue. A differential expression analysis was performed on this data set to identify transcripts overexpressed in HCC vs. normal liver tissue using the DESeq2 package (version 1.16.1) with Benjamini-Hochberg correction for multiple hypothesis testing in R. Using this analysis combined with RNA-seq data on bulk white blood cell (WBC) subsets obtained via flow sorting, a list of transcripts with adjusted p-value <0.05, log 2 fold change >2, WBCs <50 rpm in the summed WBC subsets, and a mean expression in healthy liver tissue >0.5 rpm was constructed. This list was used to narrow the 59,074 features in the raw data set to a set of 248 transcripts more likely to be predictive of HCC. The set of 248 transcripts were: ACTG2, ADM2, AFP, AGR2, AKR1B10, ALDH3A1, ALPK3, AMIGO3, ANKRD65, ANLN, AP1M2, APOBEC3B, ARHGAP11A, ARHGEF39, ASF1B, ASPHD1, ASPM, AURKA, AXIN2, BAIAP2L2, BEX2, C15orf48, C1orf106, C1QTNF3, C6orf223, CA12, CA9, CAMK2N2, CAP2, CBX2, CCDC170, CCDC28B, CCDC64, CCNA2, CCNB1, CCNE2, CCNF, CD109, CD34, CDC20, CDC25A, CDC6, CDC7, CDCA5, CDCA8, CDH13, CDK1, CDKN2A, CDKN2C, CDT1, CELF6, CENPF, CENPH, CENPL, CENPU, CENPW, CKB, CNNM1, COL15A1, COL4A5, COL7A1, COL9A2, CRIP3, CSPG4, CTNND2, CXorf36, CYP17A1, DLK1, DMKN, DSCC1, DTL, DUOX2, E2F1, ECT2, EEF1A2, EFNA3, EPHB2, EPPK1, ETV4, EZH2, F2RL3, FABP4, FAM111B, FAM3B, FAM83D, FANCD2, FANCI, FBXL18, FBXO32, FERMT1, FGF19, FLNC, FLVCR1, FMO1, FOXD2-AS1, FOXM1, FXYD2, GABRE, GAL3ST1, GCNT3, GINS1, GJC1, GMNN, GNAZ, GOLGA2P7, GPC3, GPR64, GPSM1, HRCT1, IGF2BP2, IGSF1, IGSF3, IQGAP3, ITGA2, ITPKA, KIAA0101, KIF11, KIFC1, KIFC2, KNTC1, KRT23, LAMA3, LEF1, LGR5, LINC00152, LINGO1, LPL, LRRC1, LYPD1, MAD2L1, MAGED4, MAGED4B, MAPK12, MAPK8IP2, MAPT, MCM2, MDGA1, MDK, MFAP2, MISP, MKI67, MMP11, MNS1, MPZ, MSC, MSH5, MTMR11, MUC13, MUC5B, MYBL2, MYH4, NAALADL1, NAV3, NCAPG, NDUFA4L2, NEB, NKD1, NMB, NOTCH3, NOTUM, NPM2, NQO1, NRCAM, NT5DC2, NTS, NUSAP1, OBSCN, OLFML2A, OLFML2B, OSBP2, PAQR4, PDZK1IP1, PEG10, PI3, PLCE1, PLCH2, PLK1, PLVAP, PLXDC1, PLXNB3, PODXL2, POLE2, PPAP2C, PRC1, PTGES, PTGFR, PTHLH, PTK7, PTP4A3, PTTG1, PYCR1, RACGAP1, RBM24, RECQL4, RHBG, RNF157, ROBO1, RP4-800G7.2, RPS6KL1, RRM2, S100A1, SCGN, 5-Sep, SERPINA12, SEZ6L2, SFN, SGOL2, SLC22A11, SLC51B, SLC6A2, SLC6A8, SLC6A9, SNCG, SOAT2, SP5, SPARCL1, SPINK1, SPP1, STIL, STK39, SULT1C2, TCF19, TDGF1, TESC, THY1, TK1, TMC5, TMEM132A, TMEM150B, TNFRSF19, TNFRSF25, TONSL, TOP2A, TPX2, TRIM16, TRIM16L, TRIM31, TRIM45, TTC39A, UBD, UBE2C, UBE2T, UGT2B11, USH1C, VSIG10L, WDR62, WDR76, ZWINT
[0113] The final data set used in all analyses consisted of log.sub.2 (1+RPM) for the 248 transcripts and 83 samples identified as described above. Ten iterations of 10-fold cross-validation were implemented in order to evaluate the performance of the classification algorithm, which is described step-by-step below: [0114] 1. Feature selection. A one-sided t-test with alternative hypothesis H.sub.A: μ.sub.CLD<μ.sub.HCC was conducted on the training set for each of the 248 transcripts identified by the TCGA differential expression analysis using the R stats package (version 3.4.2). Only those with p-values less than 0.05 were retained. [0115] 2. Random forest classifier. All transcripts kept from the feature selection step were used to train the random forest, which was built using the randomForest package (version 4.6-12) in R. The parameter m.sub.try was left at its default value of sqrt(p), where p is the number of features in the data set, and n.sub.tree=500 trees were constructed. Sampling was stratified according to disease status. As a comparator classifier, a multivariable logistic regression model was created using the 10 most significant genes by p-value from the feature selection step. [0116] 3. Prediction. The proportion of trees in the random forest that voted for a classification of cancer for each sample in the test set were obtained from the random forest output and used to construct ROC curves with the pROC package (version 1.10.0).
Example 2: Detection of CECs by Immunofluorescence
[0117] CECs were first detected by immunofluorescence (IF). Blood samples were obtained from 10 healthy blood donors, 39 CLD patients undergoing routine clinical surveillance for but had no evidence of HCC, 54 patients with HCC, and 10 HCC patients who underwent curative treatment and had no clinical evidence of disease (NED) (See Tables 1-4). The iChip device performed size-based exclusion of red blood cells, platelets and plasma, followed by magnetophoresis of labelled white blood cells (WBCs) (as described in Ozkumur E, et al. Sci Transl Med 2013; 5:179ra47) (see
Example 3: Detection of CEC by RNA-Sequencing
[0118] RNA-sequencing (RNA-seq) was performed to detect CECs. To determine the sensitivity of this approach, 0, 1, 3, 5, 10, or 50 HepG2 HCC cells were spiked into 4 mL of healthy donor blood and processed through the iChip device for RNA-seq. HepG2 specific gene expression was detectable in whole blood from a single cell (see
Example 4: Generation of Classifier for Detecting HCC
[0119] To show that CECs may phenotypically differ depending on the underlying disease state, gene expression profiling was performed to identify qualitative rather than quantitative differences between CECs in the setting of CLD versus HCC (see
[0120] The cross-validated classifier provided excellent separation between CLD and HCC samples, with a sensitivity (i.e., true positive rate) of 85% at a specificity (i.e., true negative rate) of 95% and with identification of both early and late stage HCC (by Milan criteria) (see
TABLE-US-00001 TABLE 1 Demographics and results for CLD patients undergoing surveillance for HCC. CECs are defined as cells expressing either CK or GPC3 by immunofluorescence. HCC Score is the vote fraction from the RF classifier. HBV, hepatitis B virus; HCV, hepatitis C virus; PSC, primary sclerosing cholangitis; NASH, non-alcoholic steatohepatitis; AIH (autoimmune hepatitis). CK+ GPC3+ CEC Advanced AFP (cells/ (cells/ (cells/ HCC Sample Age Sex Diagnosis Fibrosis (ng/mL) mL) mL) mL) Score CLD.001 61 F HCV Yes 2.7 0.0 10.1 10.1 0.39 CLD.002 64 M HBV No — 0.0 4.8 4.8 0.08 CLD.003 31 F HBV No 3.3 0.0 14.5 14.5 0.21 CLD.004 63 M Alcohol Yes 6.6 0.0 3.4 3.4 0.38 CLD.005 81 F HBV Yes 1.9 0.0 5.0 5.0 0.40 CLD.006 53 M HBV Yes 2.9 0.0 5.1 5.1 0.19 CLD.007 36 F HBV No 2 0.9 5.3 5.3 — CLD.008 64 M HBV No 1.9 0.9 0.9 0.9 0.37 CLD.009 59 F HBV No 3 0.8 3.2 3.2 — CLD.010 46 F Alcohol Yes 1.7 5.3 6.2 6.2 0.32 CLD.011 77 M HBV No 1.7 0.0 0.0 0.0 — CLD.012 85 F Alcohol Yes 1.4 0.0 8.9 8.9 — CLD.013 87 F HCV Yes 8.1 0.9 2.6 2.6 0.14 CLD.015_2 59 M HBV No 2.2 — — — 0.11 CLD.017 66 M HCV Yes 4.4 0.0 1.6 1.6 — CLD.019 67 M Alcohol Yes 1.9 5.8 5.8 5.8 — CLD.020 40 M HBV No 2.5 0.5 0.5 0.5 — CLD.022 42 M PSC Yes 3.3 5.3 0.0 5.3 0.27 CLD.023 72 M HCV Yes 1.7 0.8 1.6 1.6 — CLD.024 77 M HBV Yes 2.1 0.0 12.3 12.3 0.46 CLD.025 54 M Alcohol/ Yes 10.4 10.7 15.4 15.4 0.54 NASH CLD.026 55 F Alcohol Yes 4 0.0 5.3 5.3 — CLD.027 50 M HBV No 4.6 0.0 3.7 3.7 — CLD.028 70 M HBV No 3.2 0.0 0.0 0.0 — CLD.029 38 M PSC Yes 1.2 5.1 6.3 6.3 — CLD.030 59 F Crypto- Yes 5.5 0.9 16.0 16.0 — genic CLD.031 28 M HBV No 2.3 0.0 0.0 0.0 — CLD.032 70 M HCV Yes 3.2 0.0 0.6 0.6 0.33 CLD.033 54 M HCV Yes 2.8 — — — 0.26 CLD.034 73 F HBV Yes 2.8 1.8 3.6 3.6 0.46 CLD.037 60 F HBV No 3.3 0.0 0.0 0.0 — CLD.038 60 F HBV No 2.1 3.7 3.1 6.2 — CLD.039 54 F HBV No 3.5 0.0 0.0 0.0 0.80 CLD.040 39 M HBV No 4 0.0 0.0 0.0 0.22 CLD.041 51 M HBV No 4.8 0.0 0.0 0.0 — CLD.042 54 M HBV No 3.2 1.6 2.4 2.4 — CLD.043 65 F NASH Yes 3.1 0.8 0.8 0.8 — CLD.044 57 M NASH Yes 4 0.6 5.5 5.5 — CLD.045 68 M HCV/ Yes 12.2 0.0 4.9 4.9 — NASH CLD.046_2 60 M HBV Yes 3.2 — — — 0.35 CLD.048 66 F HCV Yes 3.1 2.3 5.1 5.1 — CLD.050 50 F AIH Yes 5 1.2 4.1 4.1 — CLD.055 37 F HBV No 1.4 — — — 0.30 CLD.056 47 M HBV No 4.4 — — — 0.25 CLD.057 44 F HBV No 1.6 — — — 0.09 CLD.058 31 M HBV No 1.7 — — — 0.18 CLD.060 61 F AIH Yes 10.5 — — — 0.08 CLD.061 69 F HCV Yes 3.2 — — — 0.04 CLD.062 42 F HBV No 4.8 — — — 0.05 CLD.065 54 M HCV Yes 2.4 — — — 0.07 CLD.070 45 F HBV No 4.8 — — — 0.25 CLD.071 66 M HBV No 4.8 — — — 0.32 CLD.072 48 F HBV No 1.3 — — — 0.18 CLD.073 69 F HCV Yes 4.6 — — — 0.20 CLD.077 44 M HBV Yes 5.1 — — — 0.10 CLD.078 40 M HCV No 1.3 — — — 0.16 CLD.079 54 M HBV No 1.6 — — — 0.28 CLD.081 74 M HBV No 2 — — — 0.29 CLD.082 74 M NASH Yes 1.9 — — — 0.47 CLD.083 60 F HBV No 4.4 — — — 0.25 CLD.084 69 M HBV Yes 3.2 — — — 0.11 CLD.085 64 M HBV Yes 2.6 — — — 0.20 CLD.087 64 M HBV Yes 2.6 — — — 0.08 CLD.088 69 M HCV Yes 4.4 — — — 0.09 CLD.089 43 M HBV No 6.6 — — — 0.13 CLD.090 69 M HCV Yes 4.4 — — — 0.20 CLD.091 43 M HBV No 6.6 — — — 0.03
TABLE-US-00002 TABLE 2 Demographics and results for HCC patients with active disease (with or without treatment prior to blood draw). CECs are defined as cells expressing either CK or GPC3 by immunofluorescence. HCC Score is the vote fraction from the RF classifier. NASH, non-alcoholic steatohepatitis; PSC, primary sclerosing cholangitis; HBV, hepatitis B virus; HCV, hepatitis C virus; A1AT alpha-1-antitrypsin deficiency; RT, radiotherapy (external); TACE, transarterial chemoembolization; SIRT, selective internal radiation therapy. CK+ GPC3+ CECs Risk AFP Milan (cells/ (cells/ (cells/ HCC Sample Age Sex Factor Cirrhosis (ng/mL) Treatment Criteria mL) mL) mL) Score HCC.008 43 M Hemo- No 9534 RT, No 57 103.5 103.5 — chromatosis sorafenib, chemo, checkpoint inhibitor, resection HCC.011 74 M NA No 939.2 Ablation, No 2.4 18.4 18.4 0.32 resection HCC.013_0 63 F Budd Yes 6.1 None Yes 31.1 90.4 90.4 — Chiari HCC.013 63 F Budd Yes 3.4 Ablation, RT Yes — — — 0.87 Chiari HCC.014 69 F NASH Yes 218 TACE Yes 0.7 0.7 0.7 0.24 HCC.015 70 F PSC Yes 3.8 None Yes 3 0 3 0.78 HCC.016 70 M HBV No 101.5 Thymalfasin Yes 0 0 0 — HCC.018 82 M NASH No 132367 None Yes 40.6 61.8 61.8 0.86 HCC.019 64 M Alcohol Yes 10.4 None No 44 45.1 55.4 — HCC.019_2 64 M Alcohol Yes 21 RT, sorafenib No — — — 0.85 HCC.021 68 M NASH, Yes 4891 Ablation No 0 4.2 4.2 — alcohol HCC.025 68 M Alcohol Yes 4.3 None Yes 0.9 1.7 2.6 — HCC.026 85 M Crypto- Yes 1.3 None Yes 0 15.4 15.4 — genic HCC.027 55 M HBV No 731.8 None No 5.4 6.6 11.4 0.83 HCC.029 70 M NASH Yes 4800 Ablation No 15.2 23.6 34.2 0.80 HCC.030_0 78 M HBV Yes 151.6 None No 3 9 9 — HCC.030 78 M HBV Yes 26.9 TACE Yes — — — 0.42 HCC.031_0 63 M HCV Yes 64.9 Ablation, NED 15 34.5 35.3 — TACE, transplant HCC.034 71 F NA No 3.8 None No 0.6 3.6 3.6 — HCC.035 82 F NA No 2043.5 SIRT, RT, No 1.1 9.1 9.1 — sorafenib HCC.037 54 M Alcohol Yes 5947 Ablation, No 0 3.3 3.3 — SIRT, sorafenib HCC.040_0 70 M NA No 2.2 Resection No 1.3 14.5 15.8 — HCC.040 70 M NA No 5.6 RT, No NA NA NA 0.77 checkpoint inhibitor, resection HCC.041 72 M Alcohol Yes 338 RT No 0 1.2 1.2 — HCC.041_3 72 M Alcohol Yes 1092 RT, sorafenib No — — — 0.52 HCC.042 58 M HCV, Yes 5.4 None No 0.5 0.5 0.5 0.43 alcohol HCC.044 79 F NA No 19598 None No 0.5 0 0.5 — HCC.046 66 M HBV Yes 5.5 Ablation Yes 1.4 10.3 10.3 0.05 HCC.047 62 M Alcohol Yes 12.7 Ablation Yes 0 0 0 — HCC.050 76 M Alcohol Yes 5.4 Ablation, Yes 0 2.3 2.3 — TACE, RT HCC.052 23 M Biliary Yes 1.5 RT No 0 2.5 2.5 — atresia HCC.059 66 M HCV, Yes 4847 Ablation No 43.6 48.7 49.5 0.91 alcohol HCC.060 63 F NASH Yes 13629 None No 0 3.2 3.2 0.77 HCC.061 67 F NA No 60.6 Reection No 0 1.3 1.3 — HCC.062 63 M NASH Yes 185.2 TACE Yes 0 3.7 3.7 0.71 HCC.064 53 M HCV Yes 1.6 None Yes 0 0.8 0.8 0.85 HCC.065 59 M HBV No 63.3 None Yes 0 0 0 0.80 HCC.067 74 F NA No 2.5 Sorafenib No 0.7 1.3 1.3 0.89 HCC.068 83 M HBV Yes 4.7 RT No NA NA NA 0.79 HCC.069 62 M NASH Yes 20.8 TACE Yes NA NA NA 0.88 HCC.074_0 64 M NASH, Yes 167580 None No 0 0 0 — alcohol HCC.075 81 M HBV Yes 7.7 Ablation, No 0 1 1 0.76 soracenib, chemo, checkpoint inhibitor, resection HCC.076 79 M HBV Yes 13322 None No 0 1.5 1.5 — HCC.078 60 M HBV No 4.7 Ablation, No 2.2 2.2 2.2 0.83 resection HCC.079 71 M NA No 5.2 None No — — — 0.73 HCC.082 62 M HCV, Yes 5.9 None Yes 6.7 7.3 7.3 0.80 alcohol HCC.083 59 M HCV, Yes 8.5 TACE, SIRT, No 2.1 2.1 2.1 0.89 alcohol RT, sorafenib HCC.084 81 M NA No 16.8 Sorafenib No 0 0 0 — HCC.087 57 M Alcohol Yes 16.7 Ablation, Yes 0 1.2 1.2 0.80 TACE HCC.090 69 M Alcohol Yes 19960 None No 4.7 5.3 5.3 0.92 HCC.091 72 M HBV No 3.2 None Yes 0 0 0 0.91 HCC.093 77 M NA No 3.2 RT, resection No 0 0 0 0.80 HCC.094 70 M NA No 156.4 SIRT, RT No 4 6 6 — HCC.095 64 M HCV, Yes 7.6 TACE Yes 1.3 2.7 2.7 0.66 alcohol HCC.097 52 F NA No 1.3 Ablation, No 0 6.4 6.4 0.66 chemo, transplant, resection, everolimus/ leuprolide, sitravatinib HCC.098 66 M Alcohol Yes 356 None No 0 0 0 0.65 HCC.099 58 M Alcohol Yes 254.9 TACE Yes 1.5 1.5 1.5 0.51 HCC.101 78 M NASH Yes NA None Yes 1.2 2.5 2.5 0.54 HCC.102 61 M Alcohol, Yes 9.6 None Yes 0 5.6 5.6 0.87 Hemo- chromatosis HCC.103 68 M A1AT Yes 286.8 Ablation Yes 0 2.7 2.7 0.59 HCC.104 74 M NA No 15.4 None No 3.2 6.4 6.4 0.49 HCC.105 56 M HCV Yes 3.7 None No 1.6 1.6 1.6 0.89
TABLE-US-00003 TABLE 3 Demographics and results for HCC patients with no evidence of disease after treatment. CECs are defined as cells expressing either CK or GPC3 by immunofluorescence. NASH, non-alcoholic steatohepatitis; HBV, hepatitis B virus; HCV, hepatitis C virus. CK+ GPC3+ CECs Risk AFP (cells/ (cells/ (cells/ Sample Age Sex Factor Cirrhosis (ng/mL) Treatment mL) mL) mL) HCC.033_0 85 M Hemo- No 1.3 TACE 2.0 3.3 3.3 chromatosis HCC.051 68 M NASH Yes 2.5 Ablation, 0.9 10.4 10.4 TACE, transplant HCC.053 63 M HCV, Yes 2.3 Resection 0.0 6.4 6.4 alcohol HCC.055 51 M Alcohol Yes 2.6 Ablation, 2.4 5.6 8.0 transplant HCC.058_2 54 M HBV Yes 3.6 Resection 0.0 0.0 0.0 HCC.063 68 M HBV Yes 22.3 TACE 8.0 9.8 9.8 HCC.077 77 M Budd No 16.7 Ablation 8.0 9.8 9.8 Chiari HCC.085 71 M NASH Yes 7.4 TACE 1.2 4.9 4.9 HCC.086 75 M HBV Yes 2.9 Ablation 0.5 1.1 1.1 HCC.088 81 M HCV No 1.6 Ablation 1.5 5.1 5.1
TABLE-US-00004 TABLE 4 Healthy blood donor demographics. CK+ GPC3+ CECs Sample Gender Age (cells/mL) (cells/mL) (cells/mL) HD.01 F 28 0 0.4 0.4 HD.02 M 34 0 0.4 0.4 HD.03 M 40 0 0.4 0.4 HD.04 M 29 0 2.4 2.4 HD.05 M 23 0 0 0 HD.06 M 38 0 0 0 HD.07 F 24 0 0.4 0.4 HD.08 F 26 0 0.4 0.4 HD.09 F 35 1.2 0 1.2 HD.10 F 27 0 0 0
TABLE-US-00005 TABLE 5 Gene signature for blood-based biomarker to diagnose HCC. Gene weight is the mean decrease in Gini, as a metric for the contribution of the gene to the classifier. Involvement in Gene Weight Gene Function Cancer Publication TESC 5.170 Functions as an integral Metastasis in Kang J, et al. cofactor in cell pH Colorectal Cancer Tumour Biol 2016 regulation by controlling plasma membrane-type Na+/H+ exchange activity. OSBP2 4.203 Lipid binding protein Carcinogenesis via Du X, et al. Semin ERK pathway Cell Dev Biol 2018 SLC6A8 3.937 Required for the uptake Increases survival Loo J M, et al. Cell of creatine in muscles of metastases 2015 and brain. SEPT5 2.504 Cytokinesis and vesicle Carcinogenesis Russell S, et al. Br trafficking J Cancer 2005 F2RL3 1.502 Protease-activated Methylation status Zhang Y, et al. Int receptor involved in associated with J Cancer 2015 transmembrane lung cancer risk and signaling morality E2F1 1.378 Cell cycle control Proliferation Zhan L, et al. Cell Signal 2014 EZH2 1.079 Regulates Altered Kim K, et al. Nat transcriptional transcriptional Med 2016 repression programming CDC20 0.924 Cell cycle control Proliferation Kidokoro T, et al. Oncogene 2008 CCNA2 0.894 Cell cycle control Proliferation Gao T, et al. PLOS One 2014 CCNB1 0.876 Cell cycle control Proliferation, Patil M, et al. hepatocarcinognesis Cancer Res 2009 PLXNB3 0.766 Cell migration Unclear CDC6 0.754 Cell cycle control Carcinogenesis Yao Z, et al. Cancer Biol Ther 2009 MYBL2 0.689 Cell cycle control Proliferation, Musa J, et al. Cell survival Death Dis 2017 APOBEC3B 0.653 Cytadine deaminase Mutagenesis Kuong K, et al. Nat Genet 2014 SPP1 0.648 ECM protein important Involved with Sangaletti S, et al. for tissue remodeling. metastasis Cancer Research Also acts as cytokine. 2014 AKR1B10 0.639 Aldo-keto reductase Mediates liver Jin J, et al. cancer cell Scientific Reports proliferation 2016 TOP2A 0.606 Topoisomerase Carcinogenesis Wong N, et al. Int J Cancer 2009 ASPM 0.600 Mitotic spindle Marker of Lin S Y, et al. Clin regulation invasiveness in Cancer Res 2008 HCC SLC6A9 0.579 Sodium-dependent Unclear reuptake of glycine RECQL4 0.554 Human DNA helicases Upregulation poor Li J, et al. involved in genomic prognosis in HCC Oncology Letters instability 2017 NUSAP1 0.554 Spindle microtubule Involved with Gordon C, et al. organization invasion and Oncotarget 2017 metastasis PLVAP 0.540 Involved in the Induced in Carson-Walter E B, formation of stomatai endothelium of et al. Clin Cancer and fenestral cancers with Research 2005 diaphragms of caveolae. enhanced metastasis and angiogenesis FMO1 0.523 Oxidative metabolism Unclear of xenobiotics PDZK1IP1 0.520 Intracellular protein Regulation of Garcia-Heredia trafficking immune J M, et al. microenvironment Oncotarget 2017 FBXO32 0.510 Substrate recognition Unclear for ubiquitination
OTHER EMBODIMENTS
[0121] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.