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"Method and System for Detecting And/Or Classifying Cancerous Cells in a Cell Sample" in Patent Application Approval Process

June 12, 2014

By a News Reporter-Staff News Editor at Politics & Government Week -- A patent application by the inventors Mathuis, Philip (Brussels, BE); Jooris, Serge (Brussels, BE); Magniette, Olivier (Deurle, BE), filed on July 16, 2012, was made available online on May 29, 2014, according to news reporting originating from Washington, D.C., by VerticalNews correspondents.

This patent application is assigned to Ovizio Imaging Systems Nv/sa.

The following quote was obtained by the news editors from the background information supplied by the inventors: "To diagnose whether a patient is suffering from cancer or has a predisposition, cells need to be sampled from a patient and a thorough analysis of the cell sample is required in order to evaluate whether abnormal or aberrant cells are present. Mainly, a pathologist or other skilled medical personnel will base the diagnosis on specific characteristics of the cells in the sample, such as cell morphology, the presence of certain types of cells or proteins and more. These cytological tests are based on a two-dimensional presentation of the cells present in the sample and mostly require the fixation of cells on a substratum and the use of dyes or stainings to visualize specific features of the cells. This is a time consuming and cumbersome work, and requires well-trained specialists. Moreover, as many of the solutions used to fix and stain the cells, this approach will inevitably lead to loss of cell structures and information stored therein. This might thus interfere with the possibility of a reliable interpretation and diagnosis from the sample. Inadequate processing of a sample may lead to an increased number of false negatives diagnoses. For instance, of the over 50 million cervical cytological PAP smears, which are performed in the USA each year, a high false-negative interpretation rate of 20-40% has been described (Williams et al., 1998), frequently leading to fatal consequences. Most of these false negatives are the result of inadequate sample processing.

"Since 1990 many advanced technologies focusing on sampling, smear preparation, or screening quality control have been developed and introduced into the practical work to prevent the false negative rate in screening. These commercial devices can be divided into the following categories based on their approaches: (1) for a better slide preparation to reduce sampling error, such as thin-layered liquid based preparation (ThinPrep.TM., SurePath, Tripath); (2) for reducing workload and screening error, such as autoscreening system (ThinPrep Imaging System, Cytyc, Boxborough, Mass.) and FocalPoint System (Tripath Imaging, Burlington, N.C.); (3) for laboratory quality control, such as rescreening (Papnet); and (4) for quality assurance, such as proficiency test. However, most of these devices are not designed to assist diagnosis by supplying the calculable parameters to eliminate interpretation errors and inter-observer discrepancy. In addition, it is not applicable for general cytological laboratory because of high cost and technical or linguistic gaps. Thus, without a reproducible and quantitative tool, it is still an unsolved problem for a routine cytological laboratory to improve the diagnostic divergence caused by visual observation.

"Therefore, the field of cancer diagnosis is in need for methods and devices that analyses cell samples in a non-destructive, non-detrimental and objective manner, or at least provide information of the status of the sample and the cells present prior to its further processing by a specialist. Preferably, the gathered information is obtained by a three-dimensional analysis method in order to perturb the sampled cells to a minimum prior to analysis. Moreover, three-dimensional information will store substantially more cellular data than conventional two-dimensional information. This will undeniably lead to a more reliable diagnosis method as more accurate information will be obtained from the analyzing sample.

"US 2010 006 089 7 discloses a method and device for non-destructive analysis and characterization of a cell sample. The invention makes use of a digital holographic microscope for analyzing certain parameters of a cell and to determine the number of cells in the sample. US 2010 006 089 7 does not disclose specific parameters to be measured in order to classify a cell as healthy or aberrant. Therefore, the method as disclosed in US 2010 006 089 7 can be implemented in a diagnostic system, but can serve there merely as an extra tool for gathering information on a sample, and not as the main determining factor whether a sample contains aberrant cells or not.

"Choi et al. (2007) from the Massachusetts Institute of Technology (MIT) describes a method based on tomographic phase microscopy to map 3D structures of suspended or substrate-attached cells and to quantify refractive index measurements. By creating overlapping tomograms, a 3D image of a cell could be reconstructed. The authors state that the refractive index data obtained by their described technique can be used to characterize cell sample aberrations. Specific parameters which are suitable to be used in a diagnostic setting are however not disclosed. Moreover, the technique does not generate a real time three-dimensional image, but rather artificially creates a 3D image by superimposing several two-dimensional images captured by the microscope.

"Reshetov et al. (2010) present a method to visualize thyroid cancer cells and to study their morphology by atomic force microscopy (AFM). The authors showed a difference in height of the nucleus, height of cytoplasm and ratio thereof of thyroid cancer cells when compared to benign colloidal goiter cells. Disadvantage of the system is the slow scanning speed of the AFM technique, requiring several minutes for one scan. Other disadvantage is the limited area which can be scanned (only micrometer scale, 100.times.100 .mu.m in X and Y direction, and 10 .mu.m in Z direction) by an AFM as well as the poor image resolution. Moreover, imaging of liquids, for instance cells in solutions, have been proven to be challenging with conventional AFM. These disadvantages make it unlikely that AFM will be widely implemented in oncocytological diagnostic devices.

"There remains a need in the art for an improved, non-destructive method for measuring and obtaining specific cellular parameters of cell samples in a three-dimensional manner, which can be used to diagnose the status of the analyzed cell sample. The method should be readily implemented in a cytological screening and diagnosis system and provide a fast, objective and correct analysis of cell samples thereby limiting the requirement of highly trained personnel and man hours which are currently needed to process and analyze each cell sample."

In addition to the background information obtained for this patent application, VerticalNews journalists also obtained the inventors' summary information for this patent application: "The present invention provides for a method and system for analyzing cell samples in a non-destructive, fast, inexpensive and objective manner and to detect cancerous cells present in the sample. In the current invention, said cell sample will be analyzed by a digital holographic microscope (DHM) and the practitioner will be provided with a digital report, comprising a set of cellular parameters related to cells present in the cell sample as well as with diagnostic information on the cell sample. This will give the practitioner or pathologist the chance to evaluate the raw sample in an unbiased manner, by taking the provided cell sample parameters into account. Diagnosis can be solely based on the report provided by the system, or if desirable, the practitioner or pathologist can proceed by more conventional means of diagnosing. DHM provides both a highly specific and sensitive method for analyzing cell samples, which is often a problem for other analytical methods currently known. For instance, the PAP smear test, a well-known test for analyzing cervical cell samples, although highly specific, lacks sensitivity. This increases the risk in false negative results, which is to be avoided at all cost.

"In a first aspect, the current invention discloses a method for detecting cancerous cells and/or classifying cells in a cell sample as disclosed in claim 1. Preferably, said cell sample is a liquid cell sample.

"Digital holographic microscopy enables the study of living cells without the need for markers or dyes, and enables quantitative analysis of the studied cells as well as various sub-sections of said cells by obtaining a three-dimensional image. The possibilities of digital holographic microscopy (DHM) have increased during the last years due to an increase in the development of digital sensors and computers. The methods visualizes cells without any staining up to a degree of cellular and compartment distinguishability which allows efficiently segmenting the cells, counting their number and reliably classify them according to their histological provenience.

"In a preferred embodiment of the method according the current invention, at least one cellular parameter is obtained derived from holographic information. A Scoring Factor is appointed to the cells of said cell sample, based on said cellular parameters. Said Scoring factor determines the classification of said cells. Digital holographic microscopy enables a quantitative multifocus phase contrast imaging that has been found suitable for technical inspection and quantitative, three dimensional cell imaging. The holographic information obtained by DHM holds sufficient information in order to classify the cells for diagnostic purpose.

"In a more preferred embodiment, at least one cellular parameter derived from obtained holographic information comprises the optical nuclear height. The optical nuclear height, derived from the holographic information, has been found by the inventors to be a highly reliable parameter for detecting cancerous cells. It was found that the optical nuclear height can be correlated to the status of malignancy of said cells.

"In a further more preferred embodiment, at least one cellular parameter derived from obtained holographic information comprises cell nucleus diameter, chromatin texture, cell size, cell form and cell morphology. These parameters all will lead to an adequate classification of the cells present in the cell sample.

"In a preferred embodiment, after deriving at least one cellular parameter, a Scoring Factor Sc is appointed to each cell, cell type and/or cell sample whereby said Scoring Factor Sc determines the classification of said cells, cell type and/or cell sample. By doing so, each cell is objectively evaluated, ensuring furthermore that all cells essential for diagnosis have been evaluated in the same, objective manner. This is a huge benefit when compared to the analysis of cell samples by a practitioner, as these analysis are often more subjective, and are dependent on the skills and knowledge of the practitioner, as well as to the employed method of analysis and the handling the sample underwent prior to this analysis. Preferably, a practitioner will be provided with a digital report on the classification of said cells in cell sample. After receiving said digital report and diagnostic information stated therein, said practitioner can decide whether it is necessary or not to perform extra analyses, for instance to screen for the presence of a viral infection, e.g. a HPV detection. The extra analysis techniques are preferably based on the detection of a member or combination of members of the following group: Cyclin Dependent Kinase p14Arf, p15INK4b, p16INK4a, p18INKc, p19INK4d, p21WAF1/CIP1 and p27Kip1; cell proliferation marker Ki67, Ki-S5, Ki-S2, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, Pomfil2, Unc-53, a kinase or phosphatase engaged in the replication process, CDC6, CDC7, CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45, MCM10, a protein engaged in the processive replication fork, a topoisomerase, topoisomerase 2 alpha, PCNA, a DNA polymerase, DNA polymerase delta, replication protein A (RPA), replication factor C (RFC) or FEN 1; HPV genotypes such as HPV genotype 6, HPV genotype 11, HPV genotype 16, HPV genotype 18, HPV genotype 31, HPV genotype 40, HPV genotype 58, HPV genotype 58, HPV genotype c*31, HPV genotype 33, HPV genotype 54, HPV genotype c*33, HPV genotype 35, HPV genotype 39, HPV genotype 40, HPV genotype 42, HPV genotype 43, HPV genotype 44, HPV genotype 45, HPV genotype 51, HPV genotype 52, HPV genotype 53, HPV genotype 56, HPV genotype 74, HPV genotype c*56, HPV genotype 58, HPV genotype c*58, HPV genotype 59, HPV genotype 66, HPV genotype 68, HPV genotype 70, HPV c*68; HPV viral proteins E1-E7, L1-L2. The detection might either imply detection of the presence of a protein or peptide, or detection of DNA, cDNA or RNA.

"By providing the practitioner with diagnostic information prior to any handling of the cell sample, time-consuming procedures may be avoided, moreover saving costly man hours for a diagnostic laboratory or service.

"Preferably, said appointed Scoring Factor is based upon comparison of said at least one cellular parameter and a threshold database. This threshold database comprises threshold values linked to each derived cellular parameter, whereby these threshold values are indicative of the status of the cells of said cell sample. In a preferred embodiment, said threshold database is stored on an external server. In an even more preferred embodiment, said Scoring factors are appointed by use of queries on said external server.

"In a further preferred step of the method according to the current invention, a practitioner will be provided with a digital report comprising Scoring Factors and classification of said cells in cell sample. More preferably, two- and three-dimensional images of said cell sample are provided in said digital report. Based on the holographic information obtained by DHM, three-dimensional and two-dimensional images from the cell sample may be reconstructed. This is again an advantage over the currently known techniques where firstly an image is obtained from a cell sample, mostly a cell sample on a carrier such as a microscope slide, after which quantitative information is calculated. The method and system according to the current invention reconstructs the image after obtaining all necessary quantitative information, being the holographic information, providing a more reliable source of data.

"Preferably, the method according the current invention implements an identification step, identifying the cellular type of said cells in the sample, prior to said classifying cells. Identification of the cellular type of the cells in the cell sample occurs based on said cellular parameters derived from holographic information, more preferably based on the cell size. Preferably, this occurs prior to classifying said cells. More preferably, only subsets of cells are classified, whereby said subsets of cells are associated to specific cell types identified in said cell sample. By identifying the cell types prior to classification, only those subsets of cells which are essential to come to a reliable diagnosis of the cell sample can be subsequently classified. This will provide the practitioner afterwards with a digital report which only comprises the most fundamental data, and leaves out redundant information. Furthermore, by only classifying the essential cells, a considerable amount of time is saved during analysis of each sample.

"In a preferred embodiment, said cell sample is a cervical sample, preferably a liquid cell sample. In a more preferred embodiment, said cells in said cell sample comprise superficial squamous cells, intermediate squamous cells, basal cells, parabasal cells, red blood cells, macrophages, lymphocytes and micro-organisms. In a further preferred embodiment of the current invention, only said superficial squamous cells, intermediate squamous cells, basal cells and parabasal cells are appointed a Scoring Factor.

"In another aspect, current invention provides a system for the detection of cancerous cells and/or classification of cells in a cell sample employing the method according to the current invention, as described in claim 16.

"In a preferred embodiment, said system comprises a server, preferably an external server. Said server provides algorithms for the comparison of said cellular parameters with a threshold database.

"In another preferred embodiment, said system comprises an exchangeable sample vial comprising identifying indicia. Preferably, said indicia comprise an RFID. Information on RFID tags is stored electronically and is reprogrammable. This way, the practitioner can add or change the information stored on the RFID according to his preferences and according to the procedures utilized in the laboratory where the samples are analyzed.

"In a third aspect, the current invention relates to a method for updating and/or improving a database comprising thresholds linked to holographic information, as claimed in claim 21.

"In a final aspect, the current invention equally discloses a database of objects comprising holographic information according to claim 28.


"FIG. 1 depicts a schematic overview of a one embodiment of the current invention, whereby cells of a cell sample, in the current example a cervical sample, are identified by a cellular parameter.

"FIG. 2A-C depicts an exemplary decision tree according to embodiments of the current invention, used to classify cells in a cell sample.

"FIG. 3 depicts a three-dimensional image of cells in a cell sample, obtained by DHM. FIG. 3A depicts the phase-contrast image of the cell, while FIG. 2B shows the three-dimensional image from the same field of cells, obtained by DHM. FIG. 3C is a top-view of the cells, obtained by DHM.

"FIG. 4A depicts a graphical overview of results obtained by the method according to the current invention, for cervical cells with diagnostic status equal to or higher than CIN1.

"FIG. 4B depicts a graphical overview of results obtained by the method according to the current invention, for cervical cells with diagnostic status equal to or higher than CIN2."

URL and more information on this patent application, see: Mathuis, Philip; Jooris, Serge; Magniette, Olivier. Method and System for Detecting And/Or Classifying Cancerous Cells in a Cell Sample. Filed July 16, 2012 and posted May 29, 2014. Patent URL:

Keywords for this news article include: Ovizio Imaging Systems Nv/sa.

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Source: Politics & Government Week

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