Patent Description:
Cancer has high incidence rate and fatality rate around the world, which has seriously threatened human health. Liquid biopsy is a new technique for non-invasive tumor detection appeared in recent years. High metastasis capacity of tumor cells causes them to abscise from in situ tumor focus, and further disseminate and migrate towards body fluid including blood, hydrothorax, cerebrospinal fluid, and even urine. Detecting free rare tumor cells and molecular characteristics thereof from these liquid sample can be an alternative to biopsy on in situ tumor focus, thus it is referred to as liquid biopsy. Traditional biopsy causes trauma and is often difficult to conduct, the liquid biopsy, especially the liquid biopsy against blood is therefore welcomed by the clinic, because it is non-invasive and easy to obtain a sample. However, its technical difficulty primarily lies in how to confirm that a certain free cell in the liquid sample is really a tumor cell. At present, a gold standard for identifying a malignant tissue in clinic is based on morphological characteristics in pathology. This method can also be used in identifying exfoliated tumor cells in a liquid sample (such as hydrothorax, cerebrospinal fluid, urine and the like). However, the cellular constituents in the liquid sample are complex, and there exists a large amount of cells which are similar in morphology to and easily confusable with the tumor cells (such as reactive mesothelial cell in hydrothorax). Meanwhile, the number of the tumor cells maybe small, therefore it is often difficult to draw a definite conclusion, resulting in low sensitivity of detection for the malignant cells in the liquid sample. In blood, the number of the circulating tumor cells is very few, while the number of other cells is very large (up to <NUM><NUM>), thus it is more difficult to accurately detect by a morphological method.

At present, various techniques and equipment are available, commercially or academically, for detecting circulating tumor cells (CTC) in blood, but the core CTC identification standard essentially adopts the method used by Cell Search system of Johnson & Johnson Company approved by US FDA, in which an epithelial marker is combined with a leucocyte marker, namely the cells which are EpCAM+/CK+/CD45-/DAPI+ and meet a certain morphological standard are defined as CTC, wherein EpCAM and CK (cytokeratin) are both epithelial markers, CD45 is a leucocyte marker, and DAPI is a nuclear marker. Therefore, the above-mentioned definition mainly depends on the epithelial marker, because most of the tumors are of epithelial origin. However, this standard cannot detect CTC of a tumor having interstitial origin (such as osteosarcoma, melanoma and the like) and CTC having epithelial-interstitial transformation in a tumor having epithelial origin. According to the existing biological theory of cancer, EMT is an important step in tumor cell metastasis, thus the tumor cells transformed from epithelial type to interstitial type are closely related to the tumor metastasis, and this part of tumor cells are difficult to be detected by the epithelial marker. Accordingly, this promotes us to develop a new CTC marker capable of more sensitively detecting CTC, especially CTC of a tumor having interstitial origin and CTC of a tumor having epithelial origin which does not express the epithelial marker (such as CK). We primarily search for a new CTC marker on the basis of general characteristics of cancer.

Cancer is harmful and hard to conquer, because a malignant tumor cell has several characteristics different from a normal cell, including self-sufficiency in growth signals, insensitivity to antigrowth signals, resisting cell death, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis, avoiding immune destruction, tumor promotion inflammation, deregulating cellular energetics as well as genome instability and mutation etc. (See <NPL>). Abnormal energy metabolism of the tumor cell, as one of main indicators for cancer, attracts more and more attentions. It provides tumor detection and therapy with new method and target. The tumor cells use glycolysis as their main source of energy metabolism, and even under an aerobic circumstance they also uptake a large amount of glucose, which phenomenon is referred to as Warburg effect, one of the basic characteristics of tumor. Otto Warburg accordingly won the Nobel prize of <NUM>.

Glycolysis is known as a cascade reaction process catalyzed by a series of enzymes. A first key rate-limiting enzyme which catalyzes glycolysis is hexokinases (HKs). HK catalyzes phosphorylation of glucose entering into the cell to generate glucose-<NUM>-phosphate (G-<NUM>-P), and consumes one molecule of ATP. Now it has been found that human HK has four subtypes, which are respectively encoded by HK1, HK2, HK3 and HK4 genes. HK1 is widely expressed in almost all mammalian tissues, HK2 is generally expressed in an insulin sensitive tissue such as fat, skeleton and myocardium. HK3 tends to be expressed at a low level. The expression of HK4 is limited to pancreas and liver. Reports and studies using a kinase (such as HK) as the marker for identifying rare tumor cells in a human liquid sample have not been seen.

<NPL>) discloses a method for detecting rare tumor cells based on the detection of <NUM>-NBDG.

The present invention provides a method for detecting rare tumor cells in a cancer patient's liquid sample, and use of an anti-HK2 antibody, to solve the problem of the epithelial-marker-based method namely it cannot detect CTC of the tumor having interstitial origin and CTC having epithelial-interstitial transformation of the tumor having epithelial origin, and to further increase the detection sensitivity of CTC.

A first aspect of the present invention provides a method for detecting rare tumor cells in a human body fluid sample, comprising: conducting a staining treatment on cells from the human body fluid sample with a fluorescein labeled anti-hexokinase-<NUM> (anti-HK2) antibody substance, conducting a fluorescence detection on the cells after the staining treatment, and confirming HK2 positive cell according to a fluorescence signal and cells with high HK2 levels cell are identified as the rare tumor cells.

In the above-mentioned method for detecting rare tumor cells in the human body fluid sample, preferably, mean value of HK2 fluorescence values of all leucocytes at the time of fluorescence detection plus five folds of a standard deviation is regarded as a threshold for determining cells with high HK2 levels.

In the above-mentioned method for detecting rare tumor cells in the human body fluid sample, preferably, it also comprises conducting a staining treatment on the cells from the body fluid sample with a fluorescein labeled antibody targeting a leukocyte marker, and conducting a staining treatment on the cells from the human body fluid sample with a cell nucleus stain, and cells with high HK2 levels/leucocyte marker negative/cell nucleus staining positive are identified as rare tumor cells.

In the above-mentioned method for detecting rare tumor cells in the human body fluid sample, preferably, the fluorescein labeled anti-HK2 antibody substance is a fluorescein labeled Anti-HK2 antibody, or it is a combination of an Anti-HK2 antibody with a fluorescein labeled secondary antibody targeting HK2 primary antibody.

In the above-mentioned method for detecting rare tumor cells in the human body fluid sample, preferably, said leucocyte marker antibody is an antibody against cell membrane surface protein CD45, said nuclear stain is a fluorescent dye targeting cell nucleus; mean value of HK2 fluorescence value plus five times of standard deviation of CD45 positive cells is defined as a threshold for determining cells with high HK2 levels.

In the above-mentioned method for detecting rare tumor cells in the human body fluid sample, preferably, said human body fluid sample is blood, hydrothorax, cerebrospinal fluid or urine samples, after conducting an selective enrichment treatment on said body fluid sample, the cells are prepared into a cell suspension, and spread in a form of single cell onto an addressable microporous array chip or a glass sheet to conduct a staining treatment, said enrichment treatment comprises decreasing the number of erythrocyte and/or leucocyte in the human body fluid sample.

In the above-mentioned method for detecting rare tumor cells in the human body fluid sample, preferably, it further comprises the following steps: (a) conducting a pre-treatment and an enrichment on the human body fluid sample; (b) dispersing all the cells in the sample after enrichment onto the addressable microporous array chip or the glass sheet; (c) conducting a fluorescent staining and an imaging based on the marker for all the cells on the microporous array chip or the glass sheet; (d) identifying a suspected tumor cell according to the threshold of the fluorescence signal or in a manner of artificial intelligence, and counting the number after a manual review; (e) recycling the tumor cells identified above by a micromanipulation technique, conducting a single cell sequencing to verify and optionally detecting the drug target thereof.

A second aspect of the present invention provides use of the anti-HK2 antibody substance for.

In the use of the above-mentioned anti-HK2 antibody substance, preferably, identifying the cells with high HK2 levels/leucocyte marker negative/cell nucleus staining positive cell in the human body fluid sample as rare tumor cells; identifying said rare tumor cells of epithelial cell marker negative in the human body fluid sample as the tumor cell having epithelial-interstitial transformation or the tumor cells having interstitial origin; cells with high HK2 levels refers to cells in which the fluorescence signal value after the HK2 fluorescent staining treatment reaches a predetermined threshold.

A third aspect of the present invention provides a combinational use of an anti-HK2 antibody substance and an antibody substance targeting an epithelial cell marker for the preparation a detection kit for identifying characteristics of CTC, wherein said anti-HK2 antibody substance is a fluorescein labeled Anti-HK2 antibody, or a combination of a primary Anti-HK2 antibody with a fluorescein labeled secondary antibody targeting the primary anti-HK2 antibody, said epithelial cell marker antibody substance is a fluorescein labeled CK antibody, said characteristics of CTC refers to distinguishing CTC cell as a tumor cell having epithelial origin, a tumor cell having epithelial-interstitial transformation, or a tumor cell having interstitial origin; said kit further includes a fluorescein labeled antibody targeting the leukocyte marker, a nuclear stain, and a instruction, said instruction describing identifying the epithelial cell marker positive cell of the CTC cells as the tumor cell having epithelial origin, identifying the epithelial cell marker negative cell as the tumor cell having epithelial-interstitial transformation, or the tumor cell having interstitial origin.

The present invention uses HK2 as a marker, and identify the rare tumor cells with a marker combination of HK2, CD45 (leucocyte marker), DAPI (nuclear dye). The method disclosed herein has increased detection sensibility in that it can detect CTC of the tumor having interstitial origin which cannot be detected by a traditional epithelial marker and CTC having epithelial-interstitial transformation in the tumor having epithelial origin. On the other hand, HK2 is related to the abnormal energy metabolism which is common in tumor, namely high glycolysis, thus it can be used in identifying malignant degree of the cells, so as to ensure specificity of the detection. We further validate reliability of detecting the suspected tumor cell with this new marker by mean of a single cell sequencing.

The method described in the present invention can be suitable for various liquid biopsy samples of cancer patients, including blood, hydrothorax, cerebrospinal fluid, urine and the like. However, when practicing the method, because the number of total cells contained in different human body fluid samples are different, it is required to adopt different sample enrichment methods. For instance, the number of the cells contained in cerebrospinal fluid is smallest (less than <NUM> thousand), thus it is not required to conduct any sample enrichment steps, all the cells in the sample can be directly dispersed onto the addressable microporous array chip or the glass sheet to conduct staining and imaging. The number of the cells in urine is slightly more, but generally it is less than one million, thus it is not required to adopt any enrichment steps either. The number of erythrocytes in hydrothorax, especially in bloody hydrothorax is larger, it is required to conduct a simple cell enrichment, namely lysis and removal of erythrocyte, without the need of further enrichment steps. The number of the cells in blood is largest (every milliliter of blood contains <NUM> billion erythrocytes, and several million leucocytes), therefore it is often required to conduct one step or multiple steps of enrichment, for instance a positive selection against a target cell, or removing a non-target cell (such as erythrocyte, leucocyte) by a negative selection, or combining the positive selection with the negative selection. The aforementioned treatment and enrichment on different human body fluid samples as well as staining and imaging against the marker and single cell sequencing and validation are all exemplified in the examples.

The present invention is further explained below in conjunction with the specific embodiments. It should be understood that, these examples are merely used to illustrate the present invention but not to limit the scope of the present invention. In addition, it should be understood that, after reading the contents recorded in the present invention, one skilled in the art can make various alterations or modifications in the present invention, these equivalent forms also fall into the scope defined by the appended claims of the present application.

When the present invention is not particularly illustrated, technical measures adopted in the following examples are conventional technical measure well known to one skilled in the art, and the raw materials and the reagents used in the invention are all also commercially available products.

As shown in <FIG>, a detecting method for rare tumor cells in a human body fluid (such as blood, cerebrospinal fluid, hydrothorax, urine and the like) sample mainly comprises the following specific steps:.

The fluorescein labeled HK2/CD45/CK antibody substance is an HK2/CD45/CK antibody which is directly labeled with fluorescein or other fluorescent substance (such as quantum dot), or a combination of a non-labeled HK2/CD45/CK primary antibody and a secondary antibody labeled with fluorescein or other fluorescent substance.

Further, in order to reduce or eliminate interference to fluorescent analysis by some non-cellular impurities having adsorption on fluorescein labeled antibody substance of HK2 in the detection sample or being introduced in the treatment process (such as cell debris, bubble, non-cellular particle and the like), the specific example of the present invention method also comprises conducting staining on the sample cells by using a nuclear dye e.g. fluorescent dye targeting cell nucleus (DAPI or Hoechst series staining) simultaneously with, before or after HK2 staining. In this case, the HK2 positive cells which are also cell nucleus staining positive are preliminarily judged as rare tumor cells.

In conclusion, these results suggest that the method has high sensitivity and reliability, it can be used in detecting the rare tumor cells having high glycolysis activity in a lung adenocarcinoma patient's hydrothorax sample, especially it can be used in detecting the rare tumor cells having epithelial-interstitial transformation in an tumor having epithelial origin, and provide the liquid biopsy of cancer with new detection means and important biological information.

A lot of tests shown hereinafter indicates that, for a purpose of detecting the rare tumor cells, it is not required to implement the present invention by using the epithelial cell marker (e.g. CK), or the present invention is not required to determine whether the sample cell is epithelial cell marker positive or not. The present invention can simultaneously detect the tumor cell having epithelial origin, the tumor cell of the epithelial origin tumor having epithelial-interstitial transformation, and the circulating tumor cells of interstitial origin tumor in the sample as the tumor cell. It of course does not rule out that a part of tumor cell with apoptosis or very low metabolic activity cannot be detected by the present invention.

On the other hand, if there is an actual need for distinguishing CTC characteristics, for instance what proportion of the tumor cells in CTC have epithelial-interstitial transformation, in preferred examples of the present invention, HK2 can be used together with CK. In the present invention, the detected epithelial cell marker positive cell in the tumor cells can be confirmed as the tumor cell of epithelial origin, and the epithelial cell marker negative cell can be confirmed as the tumor cell having epithelial-interstitial transformation, or the tumor cell of interstitial origin.

The following examples particularly discuss the whole process and the analysis result which adopts the above-mentioned detection method to conduct a specific identification on the rare tumor cells in the human body fluid, in order to show validity and superiority of the present invention.

In this example, the specific method comprises the following steps:.

<FIG> shows a CTC detection in <NUM> of lung adenocarcinoma initial patient's blood sample, wherein CD45 negative/DAPI positive/HK2 or CK positive cells are defined as CTC. The figure marks the number of CTC in four quadrants partitioned by four HK2 and CK positive thresholds.

<FIG> shows a fluorescent sub-channel diagram of CTC in a lung adenocarcinoma patient related to <FIG>, according to expression of HK2 and CK, the cells can be further divided into three cell subgroups such as HK2 positive/CK positive/CD45 negative/DAPI positive, HK2 positive/CK negative/CD45 negative/DAPI positive and HK2 negative/CK positive/CD45 negative/DAPI positive.

(<NUM>) Target tumor single cell were accurately recycled by means of a micromanipulation platform, and single cell genome-wide amplification was carried out by using commercial kit MALBAC (Yikon Genomics, China). Because there is a mature targeted therapy regime in clinic, BRAF, EGFR and KRAS target gene mutation detection were firstly conducted on the tumor single cell genome-wide amplification product, after the PCR amplification product was confirmed by agarose gel electrophoresis, gene mutation was detected by means of first generation sequencing; the remaining tumor single cell amplification products were used in genome-wide sequencing library construction. The remaining single cell amplification products after the recycling were purified by using <NUM>×Agencourt® AMPure XP beads (Beckman Coulter, USA), the purified nucleic acids were used in genome-wide sequencing library construction, the genome-wide sequencing library construction was carried out by using NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs, UK), concentration and mass of the sequencing library were evaluated respectively by using Qubite <NUM> (Thermo Fisher Scientific, USA) and Agilent <NUM> Bioanalyzer (Agilent, USA), in the genome-wide sequencing HiseqXten (Illumina, USA) sequencing platform was used, and PE150 sequencing strategy was adopted.

<FIG> shows single cell copy number variation detection result of CTC in the lung adenocarcinoma patient related to <FIG>. This result confirms that the CTC identified based on HK2 marker are indeed tumor cells.

In all examples on the lung adenocarcinoma patient's blood sample, in about <NUM>% of the samples the rare tumor cells were detected. As shown in <FIG> and <FIG>, according to phenotypes of HK2 and CK, these rare tumor cells can be divided into three subgroups: HK2 positive/CK positive/CD45 negative/DAPI positive cell subgroup, HK2 positive/CK negative/CD45 negative/DAPI positive cell subgroup and HK2 negative/CK positive/CD45 negative/DAPI positive cell subgroup.

The target single cell was accurately recycled by means of a micromanipulation platform and single cell amplification and sequencing study was conducted. The result was shown in <FIG>. The HK2 positive/CK positive/CD45 negative/DAPI positive cell subgroup was confirmed as the tumor cell of epithelial origin; the HK2 positive/CK negative/CD45 negative/DAPI positive cell subgroup was the tumor cell which may have epithelial-interstitial transformation; HK2 negative/CK positive/CD45 negative/DAPI positive cells may include the cells of normal epithelial origin and the mesothelial cells, but they are not tumor cells. <FIG> shows detection result of CTC in blood of <NUM> initial lung adenocarcinoma patients and typing result based on CK expression, it is thus proved that this detection method has high sensitivity and accuracy.

In conclusion, a certain number of target cells were detected in about <NUM>% of the lung adenocarcinoma blood samples, suggesting that sensitivity of this method is high. A validation experiment in which single cell sequencing is conducted on the target cell can show this method also has high accuracy. The method can be used in detecting high glycolysis activity of the circulating tumor cells in a lung adenocarcinoma patient blood, especially it can be used in detecting the circulating tumor cells having epithelial-interstitial transformation in the tumor of epithelial origin, and provide the liquid biopsy of cancer with new detection means and important biological information.

In this example, the method comprises the following steps:.

<FIG> shows single cell copy number variation detection result of CTC detected in multiple osteosarcoma patients, it is confirmed that CTC identified based on HK2/CD45/DAPI marker combination are indeed tumor cells, which indicates that HK2/CD45/DAPI as a marker combination can be used in detecting CTC of sarcoma having interstitial origin.

For <NUM> osteosarcoma patient's peripheral blood samples, a certain amount of HK2 positive/CD45 negative/DAPI positive rare tumor cells were found in <NUM> samples. The target single cells were accurately recycled by means of the micromanipulation platform and a single cell amplification and sequencing study was conducted. The results are as shown in <FIG>, wherein HK2 positive/CD45 negative/DAPI positive cells are confirmed as CTC, suggesting that this method has high sensitivity and accuracy, being suitable in detecting the circulating tumor cells of the tumor having interstitial origin.

<FIG> shows single cell copy number variation detection result of the rare tumor cells in the lung adenocarcinoma patient's cerebrospinal fluid related to <FIG>. It is proved that the rare tumor cells identified based on HK2/CD45/DAPI marker combination are all tumor cells.

In a lung cancer patient's cerebrospinal fluid sample experiment, rare tumor cells were detected in all the samples, according to HK2 and CK of phenotype, these rare tumor cells can be divided into three subgroups: HK2 positive/CK positive/CD45 negative/DAPI positive cell subgroup, HK2 positive/CK negative/CD45 negative/DAPI positive cell subgroup and HK2 negative/CK positive/CD45 negative/DAPI positive cell subgroup.

The target single cell was accurately recycled by means of the micromanipulation platform and single cell amplification and sequencing study was conducted. The results are as shown in <FIG>, the HK2 positive/CK positive/CD45 negative/DAPI positive cell subgroup are tumor cells having epithelial origin; HK2 positive/CK negative/CD45 negative/DAPI positive cell subgroup may be tumor cells having epithelial-interstitial transformation; HK2 negative/CK positive/CD45 negative/DAPI positive cell subgroup may be tumor cells having low glycolysis of epithelial origin, suggesting that this method has high sensitivity and reliability, and can be used in detecting the rare tumor cells having high glycolysis activity in the lung adenocarcinoma patient's cerebrospinal fluid sample, especially it can be used in detecting the rare tumor cells having epithelial-interstitial transformation in the tumor of epithelial origin.

<FIG> shows detection of the rare tumor cells in one lung adenocarcinoma patient's hydrothorax sample, wherein CD45 negative/DAPI positive/HK2 or CK positive cells are defined as rare tumor cells.

<FIG> shows a size comparison of HK2 positive/CK positive rare tumor cells in the lung adenocarcinoma patient's hydrothorax related to <FIG> with HK2 positive/CK negative rare tumor cells, wherein the size is measured by using absolute area of the cells on a fluorescent scanning picture.

(<NUM>) The target tumor single cell was accurately recycled by means of the micromanipulation platform. A single cell genome-wide amplification was carried out by using commercial kit MALBAC (Yikon Genomics, China). Because there is mature targeted therapy regime in clinic, BRAF, EGFR and KRAS target gene mutation detections were firstly conducted on the tumor single cell genome-wide amplification product, after the PCR amplification product was confirmed by agarose gel electrophoresis, the gene mutation was detected by means of the first generation sequencing; the remaining tumor single cell amplification products were used in the genome-wide sequencing library construction. The remaining single cell amplification products after recycling were purified by using <NUM>×Agencourt® AMPure XP beads (Beckman Coulter, USA), the purified nucleic acids were used in of genome-wide sequencing library construction, the genome-wide sequencing library construction was carried out by using NEBNext® UltraTM DNA Library Prep Kit (New England Biolabs, UK), the concentration and mass of the sequencing library were evaluated respectively by using Qubite <NUM> (Thermo Fisher Scientific, USA) and Agilent <NUM> Bioanalyzer (Agilent, USA), a genome-wide sequencing was conducted by using Hiseq Xten (Illumina, USA) sequencing platform, and PE150 sequencing strategy was adopted.

<FIG> shows single cell copy number variation detection result of the rare tumor cells in the lung adenocarcinoma patient's hydrothorax related to <FIG>, on the basis of the single cell sequencing. The cells shown in the figure all have EGFR L858R driver mutation; thus, they are all tumor cells. However, the single cell copy number variation characteristics of CK positive and CK negative tumor cells are different.

<FIG> shows a Sanger sequencing diagram of six asterisked cells in <FIG>, showing that the six cells all carry the EGFR L858R mutation. They are therefore in fact tumor cells.

<FIG> shows single cell copy number variation detection result of rare tumor cells in a bladder cancer patient's urine. It can be confirmed that the rare tumor cells identified based on HK2 marker high expression are tumor cells.

In a bladder cancer patient's urine sample experiment, the rare tumor cells were detected in all the samples. The target single cells were accurately recycled by means of the micromanipulation platform and a single cell amplification and sequencing study was conducted. The results are as shown in <FIG>, in which HK2 positive/CD45 negative/DAPI positive cell was confirmed as tumor cells.

Claim 1:
A method for detecting rare tumor cells in a human body fluid sample, comprising:
conducting a staining treatment on cells from a human body fluid sample with fluorescein labeled anti-hexokinase-<NUM> (anti-HK2) antibody substance,
conducting a fluorescence detection on the cells after the staining treatment for identifying HK2 positive cells based on fluorescence signal of HK2, and
identifying cells with high HK2 levels as rare tumor cells.