Patent Grant
11898209
This invention relates to blood sampling techniques, and more particularly to methods and systems for detecting and analyzing cells in blood samples. cl BACKGROUND
The ability to detect the presence of rare circulating tumor cells (CTCs) using a simple blood test, or “liquid biopsy,” has the potential to greatly enhance the monitoring of epithelial cancers, providing instant sampling of tumor cell numbers, genetic composition, and drug response parameters, without requiring invasive tumor biopsies. Thus, the detection of CTCs for early cancer detection has the potential to revolutionize the treatment of cancer, enabling the diagnosis of invasive cancer at a stage before it has metastasized, when curative treatment is expected.
However, CTCs are very rare, and identifying, visualizing, and scoring these tumor cells admixed with normal blood components remains a significant challenge, even after partial purification with known microfluidic devices or similar technologies. For example, per milliliter of whole blood, there are only 1-10 CTCs amongst more than 5 billion red blood cells (RBCs) and more than 5 million white blood cells (WBCs)(Plaks et al., “Cancer Circulating Tumor Cells,” Science, 341:1186; 2013). In addition, antibody staining of tumor cells is highly variable, due to high heterogeneity among cancer cells, even within an individual patient, as well as the poor physical condition of many tumor cells that circulate in the bloodstream, many of which have begun to undergo programmed cell death or anoikis. In addition, accurate scoring of antibody-stained tumor cells requires differentiation from large numbers of contaminating white blood cells, some of which bind to antibody reagents non-specifically. As such, only a subset of candidate tumor cells can be robustly identified by antibody staining, and as many as half of patients tested have no detectable cells, despite having widely metastatic cancer.
Thus, current protocols for imaging CTCs are seeking higher and higher levels of purity in the isolation of CTCs, especially from other nucleated blood cells, such as white blood cells (WBCs).
The present disclosure relates to methods, uses, and systems to obtain the highest possible sensitivity of data relating to rare CTCs in standard blood samples, while avoiding the need for extremely high levels of purity of the CTCs. In particular, the new methods do not need the CTCs to be completely isolated from contaminating WBCs, and instead can reliably detect as few as one CTC in products containing, e.g., up to 10,000 WBCs or more. The new assay methods and systems combine (1) an isolation system that can consistently obtain CTCs as intact, whole cells (with high quality ribonucleic acid (RNA)) from blood with (2) a droplet-based digital polymerase chain reaction (PCR) assay focused on ribonucleic acid RNA markers of specific cancer lineages for each tumor type that are absent in blood of healthy patients.
When combined as described herein, these two concepts provide a CTC digital droplet PCR assay method (“CTC ddPCR”) or simply stated a “digital-CTC” assay (“d-CTC”). In some embodiments, the isolation system is a microfluidic system, such as a negative depletion microfluidic system (e.g., a so-called “CTC-Chip,” that uses negative depletion of hematopoietic cells, e.g., red blood cells (RBCs), WBCs, and platelets, to reveal untagged non-hematopoietic cells such as CTCs in a blood sample).
In general, the disclosure relates to methods for early detection of cancer with ultra-high sensitivity and specificity, wherein the methods include the use of microfluidic isolation of circulating tumor cells (CTCs) and digital detection of RNA derived from the CTCs. In some embodiments, the CTC-derived RNA can be converted into cDNA and encapsulated into individual droplets for amplification in the presence of reporter groups that are configured to bind specifically to cDNA from CTCs and not to cDNA from other cells. The droplets positive for reporter groups can be counted to assess the presence of disease, e.g., various types of cancer.
In another aspect, the disclosure relates to methods of analyzing circulating tumor cells (CTCs) in a blood sample. The methods include or consist of isolating from the blood sample a product comprising CTCs and other cells present in blood; isolating ribonucleic acid (RNA) molecules from the product; generating cDNA molecules in solution from the isolated RNA; encapsulating cDNA molecules in individual droplets; amplifying cDNA molecules within each of the droplets in the presence of reporter groups configured to bind specifically to cDNA from CTCs and not to cDNA from other cells; detecting droplets that contain the reporter groups as an indicator of the presence of cDNA molecules from CTCs in the droplets; and analyzing CTCs in the detected droplets.
The methods described herein can further include reducing a volume of the product before isolating RNA and/or removing contaminants from the cDNA-containing solution before encapsulating the cDNA molecules.
In various implementations of the new methods, generating cDNA molecules from the isolated RNA can include conducting reverse transcription (RT) polymerase chain reaction (PCR) of the isolated RNA molecules and/or amplifying cDNA molecules within each of the droplets can include conducting PCR in each droplet. In the new methods, encapsulating individual cDNA molecules and PCR reagents in individual droplets can include forming at least 1000 droplets of a non-aqueous liquid, such as one or more fluorocarbons, hydrofluorocarbons, mineral oils, silicone oils, and hydrocarbon oils and/or one or more surfactants. Each droplet can contain, on average, one target cDNA molecule obtained from a CTC. In some embodiments, the reporter groups can be or include a fluorescent label.
The new methods can include removing contaminants from the cDNA-containing solution by use of Solid Phase Reversible Immobilization (SPRI), e.g., immobilizing cDNA in the solution, e.g., with magnetic beads that are configured to specifically bind to the cDNA; removing contaminants from the solution; and eluting purified cDNA.
In various implementations, the methods described herein include using probes and primers in amplifying the cDNA molecules within each of the droplets that correspond to one or more genes selected from the list of cancer-selective genes in
Table 1 herein. For example, the selected genes can include prostate cancer-selective genes, e.g., any one or more of AGR2, FOLH1, HOXDB13, KLK2, KLK3, SCHLAP1/SET4, SCHLAP1/SET5, AMACR, AR variants, UGT2B15/SET1, UGT2B15/SET5, and STEAP2 (as can be easily determined from Table 1). In another example, any one or more of ALDH1A3, CDH11, EGFR, FAT1, MET, PKP3, RND3, S100A2, and STEAP2 are selective for pancreatic cancer. Similar lists can be generated for the other types of cancers listed in Table 1.
In other examples, the selected genes include any one or more of the breast cancer-selective genes listed in Table 1. In other examples, the selected genes include genes selective for one or more of lung, liver, prostate, pancreatic, and melanoma cancer. For example, a multiplexed assay can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even all of the selected genes that are listed in Table 1 as being selective for a particular type of cancer, e.g., breast cancer, lung cancer, prostate cancer, pancreatic cancer, liver cancer, and melanoma. Typically a group of primers and probes for 5 to 12 cancer-selective genes from Table 1 are used for a particular type of cancer. Other specific combinations of selected genes (markers for those genes) are described in the Examples below.
The methods can also include using one or more genes selective for two or more, three or more, four or more, or five or more different types of cancer. For example, the genes can be selective for breast cancer and lung cancer; breast cancer, lung cancer, and liver cancer; breast cancer, lung cancer, and pancreatic cancer; breast cancer, lung cancer, and prostate cancer; breast cancer, liver cancer, and melanoma; breast cancer, lung cancer, and melanoma; breast cancer, lung cancer, liver cancer, and prostate cancer; breast cancer, lung cancer, liver cancer, and melanoma; breast cancer, lung cancer, liver cancer, and pancreatic cancer; breast cancer, lung cancer, prostate cancer, and pancreatic cancer; breast cancer, lung cancer, liver cancer, melanoma, and pancreatic cancer; or breast cancer, lung cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer.
In the methods described herein, the CTCs can arise from metastatic or primary/localized cancers. In the new methods, the step of analyzing the CTCs in the detected droplets cam include monitoring CTCs from a blood sample from a patient, e.g., with a known cancer, e.g., over time, and testing and/or imaging the CTCs (e.g., using standard techniques) to provide a prognosis for the patient. In other embodiments, the step of analyzing the CTCs in the detected droplets can include testing and/or imaging the CTCs (e.g., using standard techniques) from a blood sample from a patient to provide an indication of a response by the CTCs to a therapeutic intervention.
In other embodiments, the step of analyzing the CTCs in the detected droplets includes determining a number or level of CTCs per unit volume of a blood sample from a patient to provide a measure of tumor burden in the patient. The methods can then further include using the measure of tumor burden in the patient to select a therapy or can further include determining the measure of tumor burden in the patient at a second time point to monitor the tumor burden over time, e.g., in response to a therapeutic intervention, e.g., for dynamic monitoring of tumor burden.
The methods and assays described herein can be used to amplify and detect CTCs in a wide variety of diagnostic, prognostic, and theranostic methods.
As used herein, the phrase “circulating tumor cells” (CTCs) refers to cancer cells derived from solid tumors (non-hematogenous cancers) that are present in very rare numbers in the blood stream of patients (e.g., about 1 CTC in about 10,000,000 WBCs in whole blood). CTCs can arise from both metastatic as well as primary/localized cancers.
As used herein, a “product” means a group of isolated rare cells and other contaminating blood cells, e.g., red blood cells, white blood cells (e.g., leukocytes), e.g., in some sort of liquid, e.g., a buffer, such as a pluronic buffer, that arise from processing in the methods described herein, e.g., using the systems described herein. A typical product may contain only about one to ten CTCs admixed with 500 to 2,500 or more WBCs, e.g., one to ten CTCs in a mixture of 1000 to 2000 WBCs. However, the limit of detection of the present methods can be about 1 CTC in 10,000 WBC. Thus, while the present methods can achieve a level of purity of about 1 CTC in 500 WBCs, the present methods do not require highly purified CTCs, as is required in some known methods of CTC analysis.
As used herein a Solid Phase Reversible Immobilization (SPRI) cleanup is a technique using coated magnetic beads to perform size selection on cDNA created from Reverse Transcription (RT)-PCR of a product. In the new assay methods described herein this accomplishes the two-fold purpose of (a) selecting only the cDNA of the correct size, and (b) removing harsh lysis detergents incompatible with the stability of the droplets.
The polymerase chain reaction (PCR) is a process of amplification of known DNA fragments by serial annealing and re-annealing of small oligonucleotide primers, resulting in a detectable molecular signal.
Reverse Transcription (RT)-PCR refers to the use of reverse transcription to generate a complementary c-DNA molecule from an RNA template, thereby enabling the DNA polymerase chain reaction to operate on RNA. An important aspect of the new methods disclosed herein is the availability of high quality RNA from whole cell CTCs that are not lysed or treated in such a way that might destroy or degrade the RNA.
As used herein, “positive droplets” are lipid-encapsulated molecules in which a PCR reaction performed with tagged primers allows visualization of the PCR amplified product. Thus, a droplet that contained a single template cDNA molecule of a particular targeted gene can become visible using fluorescence microscopy, while an “empty” or “negative” droplet is one that contains no targeted cDNA.
The new methods and systems provide numerous advantages and benefits. For example, the current methods and systems provide results that are far more accurate and robust than either of the prior known systems when used alone. By breaking down the signal from a single CTC into hundreds or thousands of brightly fluorescent droplets, each derived from a single cDNA molecule, the new digital-CTC assays enable dramatic signal amplification. Given the strict criteria in selecting and optimizing the biomarker genes described herein, the background signal from normal blood cells is negligible in d-CTC. Thus, d-CTC enables greatly amplified signal from patients with advanced cancer (nearly 100% of patients with prostate, lung, breast, and liver cancers). Not only is the fraction of patients with a positive score significantly increased, but the high level of signal enables dynamic measurements as tumor load declines following cancer therapy. In addition, the signal amplification permits detection of CTC-derived signatures even in patients with a very low tumor burden (something that is not readily accomplished with CTC cell imaging), thus enabling significantly earlier detection of cancer.
In sum, this novel microfluidics platform provides a streamlined, ultrahigh-throughput, rapid (e.g., 3 hours per run), and extremely high sensitivity method of enriching, detecting, and analyzing CTCs in patient blood samples. The platform provides rich, clinically actionable information, and with further optimization may enable early detection of cancer.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The present disclosure relates to methods and systems to obtain information from rare cancer cells in blood samples. These methods and systems combine the power of isolation techniques such as ultrahigh-throughput microfluidic techniques, for example, negative depletion techniques, e.g., those using negative depletion of hematopoietic cells to isolate untagged CTCs in a blood sample, with analysis techniques, such as droplet-based digital polymerase chain reaction (PCR) assays focused on ribonucleic acid (RNA) markers of specific cancer lineages. This strategy can also be applied to other CTC isolation technologies that provide partially purification of cells (e.g., filtration, positive tumor cell selection), although the quality of the RNA and hence the sensitivity of the assay will be inferior to the microfluidic technologies. Similarly, other digital PCR technologies applied to RNA are capable of detecting lineage-specific primers, although the sensitivity of the droplet-based assay is likely to be the highest.
The new methods described herein can be used not only for early detection of cancers based on the presence of the CTCs in the blood, but also for tumor burden quantification as well as to monitor CTCs from a particular tumor over time, e.g., to determine any potential changes in specific tumor marker genes present in the CTCs as well changes in the tumor as the result of specific therapies, e.g., in the context of a clinical trial or a particular therapy.
General Concepts of the Assay Methods
The isolation techniques are used to enrich CTCs from a blood sample, e.g., using ultrahigh-throughput microfluidic such as the so-called “CTC-iChip” described in, for example, International PCT Application WO 2015/058206 and in Ozkumur et al., “Inertial Focusing for Tumor Antigen-Dependent and -Independent Sorting of Rare Circulating Tumor Cells,” Sci. Transl. Med., 5:179ra47 (2013). The CTC-iChip uses a CTC antigen-independent approach in which WBCs in the blood sample are labeled with magnetic beads, and the sample is then processed through two enrichment stages. The first stage uses deterministic lateral displacement to remove small and flexible cells/particles (RBCs, platelets, unbound magnetic beads, and plasma) while retaining larger cells (CTCs and WBCs). The second stage moves all cells into a narrow fluid stream using inertial focusing and then uses a magnetic field to pull bead-labeled WBCs out of the focused stream, leaving highly enriched CTCs. The CTC-iChip product from 10 ml of whole blood typically contains <500,000 RBCs, <5,000 WBCs, and a variable number of CTCs.
Some analysis techniques further enrich and analyze the isolated CTCs, e.g., as obtained from the CTC-iChip, e.g., using droplet microfluidics. Some basic information on droplet microfluidics is described generally in Jeremy et al., “Ultrahigh-Throughput Screening in Drop-Based Microfluidics for Directed Evolution,” Proc. Natl. Acad. Sci. USA, 107:4004 (2010).
As used herein, the droplet microfluidic techniques can, in certain implementations, include encapsulation of single cells, RT-PCR reagents, and lysis buffer into droplets of typically non-aqueous liquids (e.g., fluorocarbons, hydrofluorocarbons, mineral oil, silicone oil, and hydrocarbon oil; surfactants can also be include in the non-aqueous liquid, e.g., Span80, Monolein/oleic acid, Tween20/80, SDS, n-butanol, ABIL EM90, and phospholipids), in the size range of, e.g., about 0.5 pL to 15 nL in volume and, e.g., 10 to 300 μm, e.g., 20 to 100 μm, e.g., 30 to 50 μm, e.g., 35 μm in diameter. As used in the new methods described in the present disclosure, these techniques further include amplification of cancer-specific transcripts within the droplets to produce a fluorescent signal, and sorting of amplification-positive drops. This approach results in isolation of pure CTCs that can be sequenced and analyzed for the purposes of diagnosis and individualized drug therapy. Due to the high heterogeneity of CTCs, it is useful to use multiplexed amplification to detect as many CTCs as possible. Thus, instead of using one pair of primers in the PCR mixture, one can increase the probability of detecting and sorting CTCs using a combination of tumor specific primers. For additional information on the use of PCR for sorting cancer cells, see, e.g., Eastburn et al., “Identification and genetic analysis of cancer cells with PCR-activated cell sorting,” Nucleic Acids Research, 2014, Vol. 42, No. 16 e128.
In the new assay methods CTCs are lysed to release RNA molecules, which are representative of the genes expressed in a cancer cell. Most are “lineage” specific, rather than cancer specific, for example any prostate cell (whether cancerous or not) expresses these markers. However, normal blood cells do not, and the fact that the signal is derived from a cell circulating in the bloodstream defines it as an abnormal signal. By converting the RNA to a cDNA, we can now PCR amplify this lineage signal. We use droplet digital PCR, which is extraordinarily sensitive, allowing to convert the signal from a single cancer cell (i.e., one signal in an imaging assay) into thousands of positive immunofluorescent droplets. The combination of multiple, highly curated gene transcripts ensures high sensitivity and specificity for cancer, and also allows for functional insights (as in the status of hormone responsive pathways in prostate and breast cancers).
As noted, the new assay methods focus on the detection and analysis of high quality RNA rather than DNA. While there has been considerable work on DNA mutation detection in plasma and in CTCs, the present methods rely on RNA markers for the following reasons:
1. DNA mutations are not tumor specific, and the discovery that a healthy individual has some unidentified cancer cells in the blood is a very difficult clinical situation. In contrast, by selecting tumor-specific RNAs (e.g., prostate vs lung), the new methods can identify the source of cancer cells in the blood.
2. DNA mutations are very heterogeneous and besides a few recurrent mutations shared by many cancers, most blood-based mutation detection strategies require pre-existing knowledge of the mutations present in the primary tumor (i.e. not appropriate for screening for unknown cancers). In contrast, all tumor cells derived from specific organs express common lineage markers at the RNA level. Thus, a single cocktail of markers is used in the new methods for each individual type of cancer.
3. Low levels of CTCs are shed by invasive cancers before metastases are established (i.e., it is not too late for blood-based detection), but the presence of tumor cells in the blood connotes vascular invasion (i.e., invasive rather than indolent cancer). That is not the case for plasma DNA or plasma protein markers, which are leaked from dying cells in the primary tumor, and do not necessarily indicate vascular invasion. For example, serum PSA protein in the blood is shed by both benign prostate cells as well as primary prostate cancers. On the other hand, CTCs expressing PSA are shed only by invasive prostate cancers.
4. The analysis of RNA using the novel digital scoring technologies described herein is extraordinarily sensitive. However, free RNA is degraded in the bloodstream, and the use of isolation systems as described herein, such as microfluidic negative depletion systems (e.g., the CTC-Chip system) is unique in that the untagged tumor cells have high quality RNA which is extractable.
The choice of cDNA as a target molecule over DNA was made to not only to boost the signal originating from each tumor cell, but also to specifically target only tumor cell transcripts to the exclusion of white blood cell (WBC) transcripts. The boost in signal is a significant advantage, as it avoids the need for the isolation of CTCs to very high levels of purity. That is, it enables robust and repeatable results with products that contain one or more “isolated” CTCs that are still surrounded by hundreds or thousands of contaminating WBCs, e.g., leukocytes, in the same product. Nevertheless, the strategy of targeting cDNA made from RNA as used in the new methods allows the new assay methods to be exquisitely tailored for maximum specificity with minimal levels of CTC purity compared to prior approaches.
The CTC-iChip technology is highly efficient at isolating non-hematopoietic cells by microfluidic depletion of antibody tagged leukocytes. This feature of the CTC-iChip provides intact tumor-derived RNA (at levels far above those obtained using other technologies), and it is independent of tumor cell surface epitopes (which are highly heterogeneous among cancers and among epithelial vs mesenchymal cell subtypes within an individual cancer). Furthermore, even pre-apoptotic cancer cells whose antibody staining and selection is suboptimal for imaging analysis can provide a source of tumor-specific RNA that can be scored using the methods described herein. For all these reasons, an isolation technology or system that provides high quality RNA from intact CTCs with at least some reduction in the WBCs found in the sample along with the rare CTCs, such as a microfluidic negative depletion system, e.g., the CTC-iChip, is an important first step isolation before the tumor-specific digital readout is applied to the product.
The droplet-based digital detection of extremely rare molecules within a heterogeneous mixture was originally developed for PCR amplification of individual DNA molecules that are below detection levels when present within a heterogeneous mixture, but which are readily identified when sequestered within a lipid droplet before being subjected to PCR. The basic technology for droplet-based digital PCR (“Droplet Digital PCR (ddPCR)”) has been commercialized by RainDance and Bio-Rad, which provide equipment for lipid encapsulation of target molecules followed by PCR analysis. Important scientific advances that made this possible include work in the laboratory of David Weitz at Harvard and Bert Vogelstein at Johns Hopkins. For example, see U.S. Pat. Nos. 6,767,512; 7,074,367; 8,535, 889; 8,841,071; 9,074,242; and U.S. Published Application No. 2014/0303005. See also U.S. Pat. No. 9,068,181.
However, droplet digital PCR itself is not biologically significant unless coupled to a biological source of material, which is key to the new methods described herein. For instance detection of lineage-specific RNAs (the central focus of our detection strategy) does not distinguish between normal prostate epithelial cells and cancerous prostate cells. As such, detection of prostate-derived transcripts in the blood is not meaningful: they are present within debris from normal prostate cells or exosomes. It is only when coupled with the isolation of whole CTCs (i.e., intact CTCs in the blood) that the ddPCR assay achieves both extraordinary sensitivity and specificity. Hence these two technologies are ideally suited for each other, because the isolation systems provide high quality RNA, and the droplet-based digital PCR assays are focused on RNA markers in the new methods.
One additional aspect is important to the overall success of the new assay methods. As noted, the new assay methods described herein use cDNA made from total RNA, but key to this use is the identification of appropriate biomarkers that are tumor lineage-specific for each type cancer, yet are so unique as to be completely absent in normal blood cells (even with ddPCR sensitivity). The selection, testing, and validation of the multiple target RNA biomarkers for each type of cancer described herein enable the success of the new assay methods.
Assay Method Steps
The new assay methods start with the isolation of partially pure CTCs using an isolation system, such as a microfluidic negative depletion system, up to and including the analysis of data from a droplet digital PCR instrument. There are eight main assay steps, some of which are optional, though generally provide better results:
1. isolating from the blood sample a product including CTCs and other cells present in blood; e.g. from a patient or a subject;
2. reducing a volume of the rare cell-containing product (optional);
3. isolating ribonucleic acid (RNA) molecules from the product, e.g., by cell lysis, and generating cDNA molecules in solution from the isolated RNA; e.g., by RT-PCR of RNA released from cells contained in the product;
4. cleanup of cDNA synthesized during the RT-PCR step (optional);
5. pre-amplifying the cDNA using gene-specific targeted preamplification probes, e.g., using the Fluidigm BioMark™ Nested PCR approach, or non-specific whole-transcriptome amplification, e.g., using the Clontech SMARTer™ approach (optional);
6. encapsulating cDNA molecules in individual droplets, e.g., along with PCR reagents;
7. amplifying cDNA molecules within each of the droplets in the presence of reporter groups configured to bind specifically to cDNA from CTCs and not to cDNA from other cells, e.g., using PCR;
8. detecting droplets that contain the reporter groups (e.g., “positive” droplets) as an indicator of the presence of cDNA molecules from CTCs in the droplets; and
9. analyzing CTCs in the detected droplets, e.g., to determine the presence of a particular disease in a patient or subject.
As described in further detail below, one of the important features of the new d-CTC assay methods is the careful selection of a number of target gene biomarkers (and corresponding primers) that deliver excellent sensitivity, while simultaneously maintaining nearly perfect specificity. A unique list of target gene biomarkers described herein (Table 1, below) was determined using bioinformatics analyses of publicly available datasets and proprietary RNAseq CTC data. Great care was taken to select markers that are not expressed in any subpopulations of leukocytes, but are expressed at a high enough frequency and intensity in CTCs to provide a reliable signal in a reasonably wide array of different and distinct patients. A specific set of markers was selected for each cancer type (e.g. prostate cancer, breast cancer, melanoma, lung cancer, pancreatic cancer, among others.)
The separate steps of the assay methods will now be described in more detail.
1. CTC Isolation
Patient blood is run through the CTC-iChip, e.g., version 1.3M or 1.4.5T and a sample is collected in a 15 mL conical tube on ice. CTC-iChips were designed and fabricated as previously described (Ozkumur et al., “Inertial Focusing for Tumor Antigen-Dependent and -Independent Sorting of Rare Circulating Tumor Cells,” Science Translational Medicine, 5(179):179ra47 (DOI: 10.1126/scitranslmed.3005616) (2013)).
The blood samples (˜20 mls per cancer patient) are collected in EDTA tubes using approved protocols. These samples are then incubated with biotinylated antibodies against CD45 (R&D Systems) and CD66b (AbD Serotec, biotinylated in house) and followed by incubation with Dynabeads® MyOne® Streptavidin T1 (Invitrogen) to achieve magnetic labeling of white blood cells (Ozkumur et al., 2013).
The sample is then processed through the CTC-iChip, which separates the blood components (red and white blood cells and platelets) as well as unconjugated beads away from the CTCs. The CTCs are collected in solution while the red blood cells, platelets, unconjugated beads and the tagged white blood cells are collected in a waste chamber. The process is automated and 10 ml of blood is processed in 1 hour.
2. Volume Reduction and Storage of the Rare Cell-Containing Product
To fully lyse all cells isolated in the product, it is preferable to reduce the product volume from a typical starting point of several milliliters to a final volume of about 100 μl. This can be achieved, for example, by centrifuging the product, and resuspending in pluronic buffer in preparation for cell lysis and generation of cDNA. At this point samples can be processed for long-term storage by adding RNAlater™ (ThermoFisher), followed by flash-freezing in liquid nitrogen and storage at −80C.
3. Isolating RNA and Generation of cDNA from Cells in the Product
The RNA isolation step is important to the process to fully release all RNA molecules from cells in preparation for RT-PCR. A one-step, in-tube reaction can be used to minimize the risk of cell and RNA loss likely to be incurred during standard transfer steps. For example, one can use the lnvitrogen SuperScript III® First-Strand Synthesis Supermix® for qRT-PCR kit, by adding the RT-PCR mastermix directly to the pelleted product, pipetting to lyse fully, and performing the reaction according to the kit protocol targeting a 1:1 RNA:cDNA ratio. Once cDNA has been synthesized, RNase H is applied to the reaction to remove any remaining RNA. Alternatively, if one wants to perform whole transcriptome pre-amplification of the sample in a later step, cDNA can be synthesized using the SMARTer™ Ultra Low Input RNA Kit protocol, which uses proprietary oligonucleotides and reverse transcriptase enzyme.
4. Cleanup of cDNA Synthesized During RT-PCR
Another useful, yet optional, step in the process involves the removal of lysis reagents from the cDNA-containing solution. The presence of harsh detergents can lead to the destabilization of the droplets used in the ddPCR method, once the cDNA-containing solution is transferred to the ddPCR instrument. Detergent removal can be accomplished, e.g., through the use of Solid Phase Reversible Immobilization (SPRI). This technique uses coated magnetic beads to first bind cDNA of a specific size range, then allows removal of detergent-containing supernatant, and finally elution of pure cDNA for input into the ddPCR instrument. In addition to the cleanup of the RT-PCR, the SPRI process also accomplishes a size selection of cDNA, which reduces the number of non-target cDNA molecules that enter the ddPCR phase of the process, which in turn reduces background and noise.
5. Pre-Amplification
Pre-amplification of the cDNA is an optional step that increases the number of template molecules that can be detected in the droplet PCR step thus improving signal-to-noise ratio and boosting the confidence in a positive read-out. It can be a very powerful approach for the detection of markers that are expressed at low levels in CTCs, and for analyzing samples that contain very small numbers of possibly apoptotic CTCs, such as in the context of early detection of pre-metastatic disease. These two approaches have been modified to be applied in the workflow of d-CTC assay. Specific Targeted Amplification (STA), based on the Fluidigm BioMark™ Nested PCR protocol, relies on the use of primers specifically designed to amplify the region targeted by the probes used in the droplet PCR step (see Table 2). These primers were carefully designed and tested in conjuncture with their respective fluorescent probes to ensure efficient and specific amplification without increase in noise in healthy controls. Alternatively, whole transcriptome amplification, based on the SMARTer™ Ultra Low Input RNA Kit protocol, relies on the amplification of every transcript in the product, including both those found in WBCs and those found in CTCs, using random primers.
6. Encapsulation of cDNA Plus PCR Reagents in Droplets
Once cDNA has been synthesized and purified of contaminating detergents, the entire aggregate of cDNA molecules in solution plus qPCR reagents is divided into many tiny compartmentalized reactions, for example, by a droplet making instrument, e.g., a droplet generator such as the Biorad Automated Droplet Generator, which generates 20,000 droplets per sample. Each reaction consists of an extremely small droplet of non-aqueous fluid, e.g., oil (PCR stable, e.g., proprietary formulation from vendor), which contains Taqman-type PCR reagents with gene-specific primers and an oligonucleotide probe, and a small amount of sample. Once droplet generation is complete, the sample consists of an emulsion containing a vast number of individual PCR-ready reactions.
For this step, one can use the PCR probes and related primers for any one or two or more different target genes listed in Table 1 below for overall determination of tumor load, e.g., to determine tumor progression or response to therapy, in single or multiplex reactions. Thus, although in some cases a single set of PCR primers and probes for a particular gene from Table 1 can be included in each droplet, it is also possible to multiplex PCR primers and probes for two or more different genes in each droplet using different fluorescent probes for each primer/probe set, to maximize the detection of tumor cells, given the heterogeneity of gene expression in CTCs. It is also possible to multiplex PCR primers and probes for multiple genes targeting different cancer types in each droplet, thus enabling the broad yet specific detection of multiple tumor types in a single assay.
7. PCR of Droplet Encapsulated cDNA Molecules
Standard PCR cycling is performed on the entire emulsion sample using qPCR cycling conditions. The reaction is carried to 45 cycles to ensure that the vast majority of individual droplet-PCR volumes are brought to endpoint. This is important because, although the reaction is performed with Taqman-type qPCR reagents and cycled under qPCR conditions, the fluorescent intensity of the sample will not be measured during the PCR cycling, but rather in the next step.
8. Detection of Positive Droplets
Since each individual partitioned PCR is brought fully to endpoint before any measurement of fluorescence is performed, each individual droplet will be either a fully fluorescent droplet or will contain virtually no fluorescence at all. This enables the simple enumeration of all positive (fluorescent) and negative (non-fluorescent) droplets.
9. Analysis
Because the upstream RT-PCR targeted a 1:1 RNA:cDNA ratio, each positive droplet should represent a single originating RNA transcript. This interpretation depends on the number of individual droplets far exceeding the number of target cDNA molecules. In the new process, at one extreme we consider the possibility of a single CTC being isolated and lysed, releasing some number of RNA transcripts which are then reverse-transcribed 1:1 into cDNA, partitioned, PCR-amplified, and enumerated.
We estimate that in the case of a moderately expressed gene, such as the KLK3 gene in prostate cancer cells, each cell contains approximately 80-120 copies of KLK3 mRNA. The Biorad QX200 ddPCR System generates 20,000 droplets, which ensures that for small numbers of isolated CTCs and moderately-expressed target genes there will never be more than one target cDNA molecule per droplet. On the other hand, in cases where the numbers of CTCs reach dozens or hundreds, for moderately-expressing genes there will likely be multiple copies of target cDNA per droplet. In such cases, approximate numbers of originating transcript can be estimated using Poisson statistics.
Novel Gene Panels to Enable Lineage-Specific Identification of CTCs
As discussed above, the identification of gene transcripts that are highly specific for cancer cells within the context of surrounding normal blood cells is central to the new methods. While many genes are known to be more highly expressed in cancer cells, the vast majority of these genes also typically have at least limited expression in normal tissues, including blood. Given the extraordinary sensitivity required for this assay, complete absence of signal in normal blood cells is essential for high confidence identification of tumor cells in the bloodstream.
Candidate tumor-specific transcripts used to detect CTCs in blood are first selected by analyzing publicly available gene expression data sets derived from breast, prostate, lung, pancreas, and liver cancers and melanoma, as well as our lab-generated single cell RNASeq data from CTCs isolated from breast, prostate and pancreatic cancer patients and mouse models of these cancers. Transcripts whose expression is restricted to tumors and absent or undetectable in blood components are chosen for further downstream analysis. Demonstrating and validating total absence of expression (with the highest level of sensitivity, i.e., Digital PCR assays) in normal blood cells is important. In general, we found that only ˜10% of candidate genes predicted based on computational models or RNA Seq data are truly negative in human blood samples.
In particular, candidate tumor-specific mRNA transcripts for the detection of CTCs were initially identified through the analysis of gene expression data sets (microarray and RNA-Seq) derived previously for human breast, prostate, lung, pancreas, hepatocellular, and melanoma cancers. Specific publically available data sets used for this analysis include The Cancer Genome Atlas (TCGA) (The Cancer Genome Atlas, available online at tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) and the Cancer Cell Line Encyclopedia (CCLE) (available online at broadinstitute.org/ccle/home; see also, Barretina et al., The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity, Nature 483:603-607 (2012)). In addition, single-cell RNA-seq gene expression data from
CTCs isolated from human patients with breast, prostate, and pancreatic cancers were analyzed (GEO accession numbers GSE51827, GSE60407, and GSE67980) (Aceto et al., Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis, Cell, 158:1110-1122 (2014); Ting et al., Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells, Cell Rep, 8:1905-1918 (2014); and Miyamoto et al., RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance, Science 349:1351-1356 (2015). Tumor specific transcripts identified through these databases were then compared to human leukocyte RNA-Seq gene expression data (GEO accession numbers GSE30811, GSE24759, GSE51808, GSE48060, GSE54514, and GSE67980). Transcripts that displayed significant differential expression, with high expression in tumors and low or undetectable expression in leukocytes, were then selected for further downstream analysis. Moreover, a literature search was performed to select additional candidate tumor-specific transcripts. Between 50 and 100 candidate genes were selected for each type of human cancer.
For each candidate gene within each specific cancer type, two to four sets of PCR primers were designed to span regions across the target transcript. Primers are synthesized by IDT (Integrated DNA Technologies), probes are labeled with FAM or HEX, ZEN, and IABkFQ to create a probe targeting the middle of the amplicon. Unique features of our PCR primer design methodology necessary for the successful application of digital PCR-based mRNA transcript detection in human CTCs include the following: 1) the specific targeting of the 3′ end of each mRNA transcript, given the proclivity of cellular mRNA transcripts to degrade from the 5′-end, particularly in unfixed, fragile cells such as CTCs; 2) the design of primers to generate amplicons that span introns in order to exclude the unintentional amplification of contaminating genomic DNA, for example from excess contaminating leukocytes in the enriched CTC mixture; and 3) the design of primers to inclusively amplify multiple splice variants of a given gene, given the uncertainty in some cases regarding the clinical relevance of specific splice variants.
The specificity of the primers was first tested by qRT-PCR using cDNA derived from cancer cell lines (representing breast, prostate, lung, pancreas, and liver cancers and melanoma). For each type of human cancer, 2 to 5 established cancer cell lines were cultured and used for initial testing to evaluate PCR primer performance and assess for expression of the target transcript in the specified cancer. To provide an initial test of specificity, the same primers were used to evaluate expression of the target transcript in leukocytes from healthy individuals who do not have a diagnosis of cancer. Leukocytes from a minimum of five different healthy individuals were tested in this phase of testing (mixture of male and female individuals—this was dependent on the type of cancer; i.e. candidate prostate cancer and breast cancer genes required the use of male or female healthy donors only, respectively).
Leukocytes from healthy individuals were isolated from whole blood using Cell Preparation Tubes with Sodium Heparin (CPT) (Becton, Dickinson, and Co., NJ) following product insert instructions. RNA extraction and first-strand cDNA synthesis was performed for cancer cell lines and isolated leukocytes using standard methods. The specificity of expression of each gene (using 2 to 4 distinct sets of primers for each gene) was tested using qRT-PCR (cell line cDNA as positive controls, leukocyte cDNA from healthy donors as negative controls, and water as an additional negative control). Transcripts present in cancer cell lines, but absent in leukocytes based on qRT-PCR testing were then selected for further validation by droplet digital PCR. The selection criteria to pass this stage of testing were highly stringent, and required qRT-PCR signal to be present in at least one cancer cell line and absent in all healthy donor leukocyte samples tested.
Target transcripts and specific primer pairs that passed the qRT-PCR stage of testing were further validated using droplet digital PCR. For this stage of testing, the CTC-iChip (see, e.g., Ozkumur et al., “Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells,” Sci Transl Med, 5, 179ra147 (2013) was used to process whole blood samples donated by healthy individuals. The CTC-iChip performs negative depletion of red blood cells, platelets, and leukocytes from whole blood, and generates a sample product that is enriched for cells in the blood that do not express leukocyte markers, including CTCs (which should not be present in healthy individuals). For each blood sample, the product from the CTC-iChip was supplemented with an RNA stabilization solution (RNAlater®, Life Technologies) and processed for RNA extraction and cDNA synthesis using standard methods. Droplet digital PCR (Biorad, CA) was then used to quantitate the number of transcripts present in each sample based on the specific primer pairs being tested. Samples assessed by droplet digital PCR during this phase of testing included cDNA from cancer cell lines, leukocyte cDNA from healthy donors processed through the CTC-iChip (at least four healthy individuals per primer pair being tested), and water as a negative control.
Criteria for passing droplet digital PCR testing were stringent, and included: 1) the presence of transcript signal in cancer cell lines (at least one cell line with >10 positive droplets); 2) excellent signal-to-noise ratio represented by separation of signal between positive and negative (empty) droplets; 3) minimal or absent droplet signal in healthy donors (<3 droplets per healthy donor); and 4) absent droplet signal in water (0 positive droplets).
Primers that amplified transcripts specifically in cell lines and not in leukocytes in the above droplet digital PCR testing were then subjected to detailed testing of sensitivity of signal. Using single cell micromanipulation, precise numbers of cancer cells (1, 5, 10, 25, and 50 cells) were spiked into whole blood donated by healthy individuals, and then processed through the CTC-iChip. Each sample was then processed as above for testing with droplet digital PCR, and evaluated for sensitivity to ensure the signal was sufficient for the desired clinical application.
The above stringent procedure of evaluating candidate genes and primers using qRT-PCR and droplet digital PCR resulted in a final primer list consisting of approximately 10% of the initial list of 50-100 candidate genes for each type of cancer (total of approximately 400 initial candidate genes). These primers are then further evaluated for signal in patient CTCs using blood samples donated by cancer patients undergoing cancer treatment at the MGH Cancer Center, collected under an IRB-approved clinical protocol. Key to this portion of the evaluation is a comparison with blood collected from healthy individuals without a diagnosis of cancer. The following Table 1 lists the primers and probes for that have been developed thus far using these methods for the specific detection of CTCs from patients with prostate, breast, hepatocellular, pancreatic, lung, and melanoma cancers using droplet digital PCR.
While a single gene for each cancer type could be used, the presence of multiple genes within each panel is useful both for sensitivity (CTCs are heterogeneous even within individual patients in their expression patterns) and specificity (detection of multiple gene signals confers added confidence that this represents a true cancer cell signature).
The gene list provided below in Table 1 includes transcripts that are unique to specific types of cancer (e.g., highly specific markers of prostate or breast or liver cancers), as well as genes that are shared by several cancer types, e.g., all epithelial cancer types (and thus may serve as pan-cancer markers), and genes that are induced in certain conditions (e.g., active androgen signaling in prostate cancer or active estrogen signaling in breast cancer). Thus, each type of cancer was assigned a specific panel of genes that is designed for optimal sensitivity, specificity, and clinically actionable information for the given cancer type.
In addition, primers described in Table 2 are designed to pre-amplify some of the genes listed in Table 1, while maintaining their high specificity. If STA is a method of choice, these nested primers become additional components of each cancer panel.
Gene Lists for Different Types of Cancers
The following Table 1 provides a list of names of genes (with (Genbank ID) and Sequence Identification numbers (SEQ ID NO)), along with cancer types for which they are selective (Br: breast, Lu: lung, Li: liver, Pr: prostate, Panc: pancreatic, Mel: melanoma). In addition, optimized primer sets are listed for each gene (primers 1 and 2), along with the composition of the fluorescent primer probes (e.g., 6-FAM™ (blue fluorescent label) or HEX™ (green fluorescent label) for tagged probes, and ZEN-31ABkFQ quencher) for optimal visualization of the digital PCR product.
The following Table 2 lists nested primers designed to specifically pre-amplify the regions targeted by primers listed in Table 1.
Multiplex Digital Analysis of Gene Transcripts from CTC-Chip Products
To improve the detection of tumor-specific mRNA from minimal amounts of RNA derived from CTCs, we established a multiplex assay capable of testing many different gene transcripts from a minute amount of CTC-Chip product. This combines the higher sensitivity/specificity of using multiple independent genes, with the fact that the amount of input template is limited (and hence should not be diluted into multiple reactions). Our assay includes 4 genes per reaction, with each gene being resolved uniquely in 2-dimensional space by selecting different ratios of fluorescent conjugated primers. Thus, in a single reaction, we can independently measure 4 gene transcripts without having to dilute the template. For different cancers, we have gone as far as up to 4 different reactions (i.e., up to 20 different gene transcripts), and with application of nested RT-PCR digital assays, there is no limit to the number of reactions that can be performed.
This multiplex strategy achieves the ideal balance between analyzing multiple transcripts (and hence ensuring against heterogeneous variation in cancer cell expression patterns), but not diluting the input material by performing multiple independent PCR reactions. Depending on tumor types and the number of genes required for optimal signal, we have developed assays ranging from 2-4 multiplex reactions (each multiplex reaction testing for 4-genes). Thus, without undue dilution of input template, we can interrogate the product of a single CTC for expression of anywhere from 8 to 16 different genes. It is important to the assay to be able to add the signal from all of these genes (i.e. cumulative signal), while also having individual gene results (to optimize signal/noise at the individual gene level, and also gather information from specific signaling pathways that each gene interrogates—for example androgen signaling in prostate CTCs).
To display the results of the multiplex reaction in a single view (and hence differentiate amplification of each gene is isolation), we varied the concentrations of the two fluorescent probes (FAM (blue) and HEX (green)). By doing this, each individual gene amplification reaction has a unique combination of FAM/HEX signal that reflects the composition of the gene-specific primers, and hence identifies the gene-specific PCR product. In 2-dimensional space, we can illustrate the signal position of 4 different gene amplification products produced from a single multiplex reaction. As applied to digital PCR using droplets to encapsulate each PCR reaction, this method separates the targets into individual clusters by modifying the binary signal amplitude of positive droplets, which are displayed quantitatively. As predicted, this method allows both cumulative scoring of total signal for multiple genes (e.g., 16 markers in a total of 4 reactions), while also retaining the ability to quantify the signal from each individual gene target.
Specific results of testing are detailed in the examples below.
Applications of the d-CTC Assay Methods
The early detection of epithelial cancers at a time when they can be surgically resected or irradiated provides the best chance of cure, and the administration of adjuvant chemotherapy in the setting of minimal cancer dissemination is far more effective in achieving cure than the treatment of established metastatic disease. However, current efforts at early cancer detection suffer from lack of specificity. For instance widespread screening of men for prostate cancer, using serum PSA measurements is effective in uncovering early cancers, but it also identifies a much larger number of non-malignant prostate conditions (e.g., benign hypertrophy of the gland) or even cancers that are indolent and never destined to become invasive. As such, broad PSA screening is not recommended by public health organizations, because the number of complications (including deaths) from over-diagnosis match or even outweigh the calculated benefit in early cancer detection.
For other cancers, such as breast cancer, mammography is considered effective, but even then a large number of breast biopsies are performed to diagnose each true malignancy. For lung cancer, the recently recommended low dose CT scanning of individuals with a heavy cigarette smoking history is also likely to detect hundreds of innocent radiographic abnormalities for each true malignancy.
It is in this context that the addition of a blood-based ultra-sensitive readout for the presence of cancer cell-derived signatures would provide the required specificity. The d-CTC assays described herein can be used for both initial screening and as a confirmation of earlier screenings at a later time. For example, in some cases the assays can be used as a second-line test to validate a highly sensitive, but nonspecific screening test (e.g., PSA in prostate cancer). In other settings for which a cancer is highly lethal, but no screening approach currently exists (e.g., pancreatic cancer), routine periodic blood screening using the assays described herein may become the norm to monitor a patient's status or condition over time.
The new d-CTC readouts are also highly relevant to the serial monitoring of patients, e.g., seemingly healthy patients with a family history and/or genetic markers of a specific type of cancer, or patients with advanced or metastatic cancer. Imaging of CTCs is expensive and relatively insensitive, in that intact cells that stain appropriately for all required markers produce a single signal. The use of the new d-CTC assays described herein, in which each CTC (no matter how intact or pre-apoptotic) can give rise to hundreds of molecular signals, dramatically enhances the ability to detect and monitor CTCs in patients with known cancer, and to quantitatively monitor and analyze their response to therapeutic interventions. Beyond scoring for cell numbers through molecular markers, specific interrogation of mutations or cancer-associated rearrangements (e.g., EML4-ALK in lung cancer) can be achieved with comparable sensitivity.
In addition to providing a digital (quantitative) measure of CTCs present within a blood sample, the new d-CTC assay also allows analysis of specific signaling pathways that are unique to the tumor cells in the blood. For instance, a subset of prostate lineage-specific genes are driven by androgen signaling (such as PSA), while another subset are repressed by androgen signaling (such as PSMA). By analyzing these genes together, we can ascertain the status of androgen signaling within CTCs. Similarly, in breast cancer, expression of estrogen-responsive genes (such as progesterone receptor) provides a measure of the status of the estrogen-responsive pathway within CTCs. These measurements are particularly important in that therapeutic interventions in both prostate and breast cancers are derived to target the androgen and estrogen receptors, respectively. Thus, defining the total number of CTC signal in the blood, simultaneously with information about the effectiveness of the therapeutic agent in targeting and shutting off the critical pathway is important for therapeutic monitoring.
As discussed in the examples below, the new methods described herein are illustrated in prostate cancer, where the anti-androgenic agent abiratorone (e.g.,) ZYTIGA®) is effective in suppressing cancer progression, particularly in tumors that are still dependent on the androgen pathway.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
To test the feasibility of CTC-Chip-Droplet assay, we first selected several transcripts that are specifically expressed in prostate tumor cells, but are absent in contaminating leukocytes. These were the prostate lineage specific markers KLK3 (kallikrein-related peptidase; aka Prostate Specific Antigen, or PSA), FOLH1 (Folate Hydrolase; aka Prostate Specific Membrane Antigen, or PSMA) and AMACR (alpha-methylacyl-CoA racemase), as well as EpCAM (Epithelial Cell Adhesion Molecule). PCR conditions were optimized using intron-spanning primers and ZEN double-quenched FAM-labelled probes from Integrated DNA Technologies (Coralville, Iowa) following standard qPCR protocols. These conditions were first tested with encapsulated cDNA from admixtures of cancer cells and leukocytes in order to explore the dynamic range of the system. Next, using manual isolation techniques for individually selecting cells, 0, 3, 6, 12, 25, and 125 prostate cancer LNCaP cells were progressively spiked into individual 5 ml aliquots of HD blood, followed by CTC-iChip processing, RT-PCR and droplet encapsulation using the RainDrop system. We chose KLK3 as the target transcript for this experiment as it is predicted to be modestly abundant. Using an intensity threshold of 5,000, we found that as few as 3 cells worth of KLK3 transcript were readily detected at approximately 250 droplets.
Based on these preliminary data, we tested the CTC-Chip Droplet assay in patients with metastatic and localized prostate cancer versus healthy controls. Each sample was run through the iChip, then CTC-containing product was run through droplet RT-PCR using the four prostate markers mentioned above: KLK3, AMACR, FOLH1 and EpCAM. Patients with either local or metastatic prostate cancer produced significantly higher positive droplet counts as compared to HD controls.
These results suggest that the application of a droplet-based PCR readout to the CTC-iChip greatly enhances its sensitivity in detecting virtually all CTCs present in a biological specimen. Taken together, the CTC-iChip and Droplet-PCR represent two powerful microfluidic technologies that are highly compatible with each other and can be integrated in-line to create a new and highly sensitive and accurate biological assay.
This example provides a general digital CTC assay protocol that can be used for the methods described herein. Different aspects of this general protocol were used in some of the Examples described herein. For example, Approach 1 of Step 3 of the protocol described below (relating to RNA purification to cDNA synthesis), was used to generate data for
1. Patient blood is run through I-Chip, version 1.3M or 1.4.5T. Sample is collected in a 15 mL conical tube on ice.
2. Sample is spun down at 4C. Supernatant is decanted and SUPERase™ In (DTT independent RNAse inhibitor)+RNALater® Stabilization Solution (prevents RNA degradation by inhibiting RNAses) is added to the pellet. Sample is flash frozen and placed at −80 until further processing. Samples are stable at −80.
3. There are two different processing protocols for RNA purification to cDNA synthesis that were used in the examples described below.
Approach 1
Approach 2
4. cDNA (synthesized from Approach 1 or 2) can be processed in two different ways:
5. cDNA (from step 4a or 4b), Biorad Supermix™ for probes, primer or primers (for gene of interest; up to 4 different primers (FAM and HEX) can be multiplexed) were added in a total volume of 22 pl.
6. Droplets were generated (˜15,000-18,000 droplets per well).
7. Droplet Sample were put in a PCR machine. The PCR conditions were different than Biorad recommendations. We used a step-down rather than a slow ramp to ensure that all droplets reach the same temperature. This is different than what both RainDance and Biorad uses. Better results (i.e., more signal and more separation between positive and negative droplets) can be obtained with the step-down rather than the gradient.
8. After the PCR, positive droplets were counted in a ddPCR machine.
9. Data is collected and analyzed using TIBCO® Spotfire® analysis software.
The reagents, reagent concentrations, and reaction volumes are provided below:
Reagents:
Testing Relevant Cell Lines
Per single reaction:
A master-mix containing ddPCR supermix, cDNA, and water were aliquoted into wells and 1.1 μl of each the primer was added to each well and mixed well.
Patient Samples
Per single reaction for Individual Genes
Per single multiplexed reaction for Multiple Genes
When testing multiple patients against a gene-specific primer or multiplexing primers against multiple genes, a master-mix, which includes the ddPCR supermix and primers, was aliquoted into wells followed by addition of patient cDNA to each well and mixed well.
The following protocol was used for selecting the specific marker genes listed in Table 1.
The validity of this strategy is shown below in a spiked cell experiment, in which a carefully measured number of tumor cells (from the LNCAP prostate cancer cell line) are individually micro-manipulated, added to control blood specimens, passed through the CTC-iChip and then analyzed by d-CTC assay as above. Increasing numbers of spiked cells show increasing numbers of digital signal as shown in
This assay is both highly sensitive and reproducible. As shown in
We established a multiplex assay capable of testing many different gene transcripts from a minute amount of CTC-Chip product. This combined the higher sensitivity and specificity of using multiple independent genes, with the fact that the amount of input template is limited (and hence should not be diluted into multiple reactions). The new assays include multiple genes, e.g., 2, 3, 4, 6, 8, 10, or more genes per reaction, with each gene being resolved uniquely in 2-dimensional space by selecting different ratios of fluorescent conjugated primers. Thus, in a single reaction, one can independently measure 2, 3, 4, or more gene transcripts without having to dilute the template. For different cancers, one can run and analyze multiple different reactions (e.g., up to 20 different gene transcripts in four runs), and with application of nested RT-PCR digital assays, there is no limit to the number of reactions that can be performed.
To display the results of the multiplex reaction in a single view (and hence differentiate amplification of each gene is isolation), we varied the concentrations of the two fluorescent probes (FAM and HEX). By doing this, each individual gene amplification reaction has a unique combination of FAM/HEX signal that reflects the composition of the gene-specific primers, and hence identifies the gene-specific PCR product. In 2-dimensional space, we can illustrate the signal position of 4 different gene amplification products produced from a single multiplex reaction. As applied to digital PCR using droplets to encapsulate each PCR reaction, this method separates the targets into individual clusters by modifying the binary signal amplitude of positive droplets, which are displayed quantitatively. As predicted, this method allows both cumulative scoring of total signal for multiple genes (e.g., 16 markers in a total of 4 reactions), while also retaining the ability to quantify the signal from each individual gene target.
Probe 1: 100% FAM
Probe 2: 100% HEX
Probe 3: Mixture of FAM and HEX—sum up to 100%
Probe 4: Mixture of FAM and HEX—sum up to 100%
As shown in Tables 3 to 7, the following probe mixtures were used in the multiplex reactions:
Validation and Testing
To validate and demonstrate the effectiveness of this multiplex strategy, we illustrated both the concept (using spiked cell experiments) and patient-derived samples.
On the other hand,
The strategy of representing four different genes within each reaction was applicable to multiple different cancers, with specific lineage markers substituted for each tumor type. For instance, in prostate cancer, we predicted (theoretical model) a multiplex reaction with four quadrants (one gene per quadrant) for each of 2 reactions (total of 8 gene markers). The spiked cell experiment (prostate cancer cells introduced into control blood and processed through the CTC-iChip) precisely recapitulated the predicted results.
Furthermore,
These figures show the results when the same approach of testing multiple gene transcripts in multiplex fashion (4 genes per reaction) was applied to breast cancer. Six different reactions were performed of the same CTC chip product (enabling a total of 24 gene transcripts to be tested independently), with each one having a designated signal position (predicted in upper panel) and observed in spiked cell validation experiments (observed in lower panel).
To improve the detection of tumor specific RNAs, a nested PCR strategy was optimized for each of the gene-specific amplifications. To achieve this, cDNA derived from the CTCs was first amplified with gene-specific primers which are situated a few base pairs external to the gene-specific primers used for d-CTC assay. For each gene, two to three primer sets were tested, and the primer set that is compatible with the gene-specific d-CTC assay primer and tests negative in HD blood was chosen for analysis of patient samples.
As described above, the target specific amplification protocol was first tested in cell lines derived from the different cancers. The primer combinations that are specific for tumor cells (and absent in leukocytes) were then tested with a mixture of cancer cell lines mixed into blood and enriched through the CTC-iChip. HD blood processed through the CTC-iChip was used as control. Key to this strategy is the design of the nested PCR conditions to enhance the signal from minute amounts of CTC-derived cDNAs, without increasing the minimal baseline signal from normal blood cells. This selectivity was achieved by careful optimizing of PCR primer sequences and assay conditions, as well as balancing the cycle number for the external and internal PCRs. All conditions are validated first with purified nucleic acids, then with individual tumor cells that are spiked into control blood samples and processed through the CTC-iChip, then with large panels (>10) of different healthy blood donors, and ultimately with patient-derived blood samples from patients who have either metastatic or localized cancers of the prostate, breast, melanoma, liver, lung or pancreas.
Reagents
1 μl of the pre-amplified product is loaded in each droplet PCR reaction.
The assays described herein have been validated using actual patients samples from clinical studies. These include patients with metastatic cancer (lung, breast, prostate and melanoma), as well as patients with localized cancer (prostate). The assays are conducted as described in Examples 2 through 5.
As illustrated above, one can also break down the individual gene markers for independent validation and quantitation, using the multiplex fluorescence color scheme described above. In this example below, a patient with metastatic prostate cancer had multiple positive markers, a patient with localized prostate cancer has a smaller number of positive scores within fewer markers, and a healthy control is negative for all markers.
In addition to providing a digital (quantitative) measure of CTCs present within a blood sample, our d-CTC assay also allowed analysis of specific signaling pathways that are unique to the tumor cells in the blood. For instance, a subset of prostate lineage-specific genes were driven by androgen signaling (such as PSA), while another subset was repressed by androgen signaling (such as PSMA). By analyzing these genes together, we can ascertain the status of androgen signaling within CTCs. Defining the total number of CTC signal in the blood, simultaneously with information about the effectiveness of the therapeutic agent in targeting and shutting off the critical pathway is important for therapeutic monitoring.
We have illustrated this concept in prostate cancer, where the anti-androgenic agent abiratorone is effective in suppressing cancer progression, particularly in tumors that are still dependent on the androgen pathway. Below, we showed the results of a patient with “Castrate Resistant Prostate Cancer (CRPC)” who is no longer responding to first line leuprolide and was treated with abiratorone. The androgen response markers (green) were initially suppressed by the therapy as it shows initial efficacy, but subsequently returned as the tumor becomes resistant and the patient experiences disease progression on this drug.
Similar to Example 5, non-specific whole transcriptome amplification (WTA) can be used to increase the detection rate of CTC-specific transcripts. This method relies on the use of random primers that amplify not only the targets of interest but all messages found in the product. In this example, the SMARTer™ Ultra Low RNA kit protocol (Clontech) was used as described below:
Transfer RNA to PCR Tubes or Plate
Transfer PCR product to lo-bind 1.5 mL Eppendorfs and label a second set of tubes with sample IDs; run the SPRI protocol at RT until the final elution
This whole transcriptome amplification (WTA) approach was first tested in cell lines derived from different cancers.
For each sample, 10-20 mL of blood was collected from each patient. Blood was processed within 3 hours of arrival on a CTC-iCHIP running in negative depletion mode. RNA was extracted from the product using a Qiagen RNeasy™ plus Micro kit, and 5 uL of the available 17 uL amplified using ClonTech's v3 SMARTer™ whole-transcriptome amplification (WTA) strategy. 1% of the WTA product was then loaded into each well of a digital PCR plate, and 500 nM Taqman™ primer/probe combinations used to determine the transcript concentration for each gene of interest. Transcript counts were normalized to blood volume and compared between HCC, HD, and CLD patients. HCC patients are defined as biopsy-confirmed non-resected hepatocellular carcinoma, CLD patients are patients with liver disease of varying etiologies (alcohol-mediated, HBV, HCV) who have negative ultrasound/MRI. HD are healthy donors external to the lab who donate 10-20 mL of blood.
The sensitivity and specificity of each assay are dependent on the threshold values chosen to define “diseased” vs. “non-diseased,” but using 20 ug/L, the AFP gene marker has a sensitivity between 50-80% and a specificity between 80-90%. In a study using 20 ng/ml as the cut-off point, the sensitivity rose to 78.9%, although the specificity declined to 78.1% (Taketa, Alpha-fetoprotein, J. Med. Technol., 1989;33:1380). On the other hand, the overall detection rate of the present assay was 76% when taking into account the clinical history of the patients and correcting for the ones that received curative resection or liver transplant with 100% specificity.
In addition, while all 11 markers of the liver cancer assay used herein contributed to the 76% sensitivity, the top 5 markers (AHSG, ALB, APOH, FGB and FGG) by themselves have 70% sensitivity, while the top 3 markers alone (ALB, FGB, FGG) result in 67% sensitivity. ALB alone detected 56% of the cases.
Blood samples from 8 metastatic lung patients and 8 healthy donors were processed through the CTC-chip as previously described. Samples were spun down, treated with RNAlater™ and stored at −80C. RNA was purified and cDNA was synthesized as described. STA was performed on each sample using 6 μl cDNA and the nested primers corresponding to the probes listed in the figure. 1 μl of STA product was loaded per each droplet PCR reaction.
Droplet numbers were normalized to blood volume. As shown in
Assays for SERPINA3 and SFRP2 indicated that SFRP2 is effective for both lung and breast cancer detection, whereas the former seems more specific for breast cancer detection, but also detects some lung cancer samples.
Blood samples from 9 metastatic breast cancer patient, 5 localized breast cancer patients, and 15 healthy donors were processed though the CTC-Chip. Products were pelleted, treated with RNAlater™ and stored at −80C. RNA and cDNA from each sample were prepared as previously described. 6 μl cDNA from each sample was STA amplified using nested primers corresponding to the probes listed in
Blood samples from 10 metastatic breast cancer patient and 7 healthy donors were processed though the CTC-Chip. Products were pelleted, treated with RNAlater™ and stored at −80C. RNA and cDNA from each sample were prepared as previously described. 6 μl of non-amplified cDNA were loaded into each droplet PCR reaction. The samples were analyzed with probes against the v7 isoform of the androgen receptor (ARv7, sequence in Table 1). Droplet number was normalized to blood volume.
As shown in
Blood samples from 34 metastatic or unresectable melanoma patients, each with multiple draw points (total draw points: 182), and 15 healthy donors were processed though the CTC-Chip. Products were pelleted, treated with RNAlater™ and flash frozen at −80C. RNA and cDNA from each sample were prepared as previously described. 12 μl cDNA from each sample was amplified by specific target amplification (10 cycles) using nested primers corresponding to the probes listed along the bottom of the graph in
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.
This application is a continuation of U.S. application Ser. No. 15/560,324, filed on Sep. 21, 2017, which is a 371 U.S. National of PCT Application No. PCT/US2016/024367, filed on Mar. 25, 2016, which claims priority from U.S. Provisional Application Ser. No. 62/253,619, filed on Nov. 10, 2015, U.S. Provisional Application Ser. No. 62/219,339, filed on Sep. 16, 2015, and U.S. Provisional Application Ser. No. 62/137,891, filed on Mar. 25, 2015, the contents of which are incorporated herein by reference in their entireties.
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| Number | Date | Country | |
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| Parent | 15560324 | US | |
| Child | 17065889 | US |
