JP2006242618A - Diagnosis of cancer using telomere amount by tissue fish method as index - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a superior diagnostic method for cancer that does not require a pathology physician who has received special training. <P>SOLUTION: Hybridizing is performed with a fluorescent label probe, specifically combined with respect to telomere and centromere using a tissue FISH method, relative fluorescent amount ratio (T/CR) of the both is determined, and normal tissue and cancerous tissue are discriminated, by comparing and analyzing the T/CR of the normal tissue and cancerous tissue. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for distinguishing between a normal tissue and a cancer tissue, and more particularly to a cancer diagnosis method for distinguishing between a normal tissue and a cancer tissue by measuring the relative value of the amount of telomeres in a specimen.

  Cancer diagnosis is performed by many methods, but is finally confirmed by pathological diagnosis by a pathologist (pathologist certified by the Japanese Pathological Society) (definite diagnosis). Pathological diagnosis is performed by a skilled clinical technologist making a tissue section for an optical microscope, and a pathologist observes cancer and normal cells under the microscope, based on the difference in morphology between the two. However, the histological image of cancer differs depending on the organ in which it develops, and because it is extremely diverse, it is often difficult to distinguish between the two. The number of pathologists themselves is also very small. Therefore, many diagnostic methods using biological markers have been carried out, but the method is insufficient, and no diagnostic method that ultimately surpasses the pathological diagnosis of pathologists has been considered.

  In addition, in morphological diagnosis by pathologists, in advanced cancer, the morphological findings from normal tissues are large and easy to diagnose (ie, distinguish between normal and cancer). Cancer is difficult to diagnose because of small histopathological differences from normal tissues.

  On the other hand, telomeres existing at the ends of chromosomes are shortened by 200 base pairs at every cell division. It is thought that telomeres maintain a certain length or more, thereby preventing the fusion and reconstruction of telomeres and maintaining the homeostasis of genes in the chromosome. Telomere shortening per cell division is explained as a replication of linear chromosomes presents a special problem. This is complementary to each cell division when a complementary base sequence (CCCTAA) is replicated from the 3 ′ end to the 3 ′ end of the single-stranded telomeric DNA (TTAGGG) serving as a replication template. It is thought to occur because the last 5 'end portion on the strand side is not replicated. Thus, in somatic cells, telomeres shorten each time they divide, but in germ cells and blood stem cells that require infinite cell division, telomeres that primarily elongate telomeres are not able to extend and maintain telomeres. It is.

  There are many unexplained aspects of how telomere length is regulated, and research is ongoing. As a new telomere length measurement method, quantitative fluorescent in situ hybridization (Q-FISH method) was developed in 1996 by Professor Lansdorp of BC Cancer Center, Canada (non-patent literature). 2, 3). When the Q-FISH method is used, telomere length measurement of individual chromosomes in the middle stage of cell division can be performed using cultured cells more accurately than the conventional Southern blot method.

  In the Q-FISH method, cells in the middle stage of cell division are forcibly produced using cultured cells, harvested, and then spread on the preparation. Next, this preparation was hybridized using a PNA (Peptide Nucleic Acid: peptide nucleic acid) probe labeled with a telomere, photographed with a fluorescence microscope, the light intensity of the telomere was applied to an image analyzer, and a telomere analysis software was used. Measure telomere length.

  Furthermore, since about 2002, a method for measuring the amount of telomeres in tissue sections (tissue FISH method) was developed by applying the Q-FISH method of Prof. Lansdorp et al. (Non-Patent Document 4). In this tissue FISH method, it is possible to measure the amount of telomeres for each cell on a paraffin section because it uses a PNA probe with high specific binding, which can be caused by a paraffin block that has already been prepared. The amount of each telomere in the cell is determined. The amount of telomeres is determined by the light intensity ratio between the telomere signal and the centromere signal in the middle cell division.

  In addition, many cancer tissues (about 90%) express telomerase, but it is known that the telomere length is shortened as compared with normal tissues because the number of cell division increases with cell proliferation. On the other hand, as one of the causes of aging, when telomeres become shorter than a certain length, they become senescent cells and cell proliferation stops. Studies using the Q-FISH method using cultured cells and cancer tissues have demonstrated that telomere shortening increases the instability of genes in the chromosome (Non-Patent Document 1).

Centromere (also called centromere) is a part of the chromosome that plays a key role in migrating to the pole during mitosis and is necessary for chromosomes to be distributed by mitosis It consists of a simple DNA sequence. In humans, an alphoid sequence of about 170 base pairs (also referred to as an alpha satellite sequence) forms a linear repetitive sequence from several hundred kilobase pairs to several megabase pairs to constitute a centromere DNA sequence. The proteins CENP-A, -B, -C and other proteins that specifically bind to the alphoid sequence form a complex and function as a human centromere.
O'Sullivan, J. N. et al, Nature Genet., 32: 280-284, 2002 Lansdorp, PM et al, Hum. Mol. Genet., 5: 685-691, 1996 “Measuring telomere length using Q-FISH” Chromatin / Chromosome Experiment Protocol, 186-193, Yodosha, Satoshi Kamimori, and Kai Takuma Minoru Kamimori, Kai Takubo, Recent advances in telomere research, Geriatrics, 41: 365-368, 2004

  The problem to be solved is to develop an excellent cancer diagnostic method that does not require specially trained pathologists.

  In order to solve the above problems, the present inventors focused on the relationship between cancer cells and telomere shortening, and thought that this could be applied to cancer diagnosis. However, the Southern blot method, which is the golden standard for measuring telomere length, has a large mix of DNA from different cells and errors in the measured values, which could not be applied to diagnosis.

  Therefore, the present inventors thought that cancer can be diagnosed by obtaining a telomere amount for each cell by applying a tissue FISH method suitable for measuring a telomere amount for each cell in the tissue. As described above, in the tissue FISH method, a fluorescent label-specific probe for telomere and centromere is bound to telomere and centromere on a tissue section, and the ratio of telomere and centromere is obtained from the fluorescence amount ratio. The telomere amount can be relatively determined by the ratio.

  The present inventors have found that there is a clear difference in the amount of telomere between normal cells and cancer cells by performing telomere measurement for each cell in many tissues, and a method of distinguishing both using telomere amount as an index. Further investigation was made.

  Specifically, using 4 to 6 μm thick tissue sections for pathological diagnosis, PNA probes for telomeres and centromeres labeled with fluorescent dyes (Cy3, FITC, Cy5, etc.) (specifically bind to telomeres and centromeres) And the relative light intensity of both, that is, T / CR (Telomere / Centromere Ration), the distribution of T / CR in cancerous tissue and non-cancerous tissue is completely different. I found out.

  That is, the distribution pattern of T / CR is widely distributed from 2 to 5 in normal cases, although T / CR of almost all cells (90% or more) in cancer is less than 1.0. The peak value of the T / CR distribution of cancer cells is about 0.5 and does not exceed 1.0, but is normally about 1.0. The average value of T / CR is mostly 1 or less in cancer cells, but 2 or more in normal cases.

  Thus, the present inventors have found that the distribution of T / CR between cancerous tissue and non-cancerous tissue is clearly different, so that normal tissue and cancerous tissue can be differentiated using T / CR as an indicator, and the present invention has been completed. It came to.

Accordingly, the present inventors provide the following inventions.
(1) A method for distinguishing between normal tissue and cancer tissue, comprising measuring a relative value of a telomere amount in a sample collected from a subject with respect to a centromere amount.
(2) The method according to (1), wherein the relative value of the telomere amount to the centromere amount is measured as a relative light quantity (T / CR) of telomere and centromere fluorescently labeled signals by tissue FISH method.
(3) A method for differentiating between normal tissue and cancer tissue using the tissue FISH method, including the following steps:
(A) a step of hybridizing a specimen suspected of cancer collected from a tissue and a non-cancer specimen collected from the same tissue with a PNA probe for each of telomere and centromere labeled with a fluorescent dye;
(B) a step of photographing a specimen suspected of cancer and a non-cancer specimen under a fluorescence microscope;
(C) The photographed image is analyzed, and the relative light quantity (T / CR) of the telomere and centromere fluorescently labeled signals for each cell in each of the specimens suspected of cancer and non-cancer specimens is measured. Obtaining a T / CR distribution pattern, T / CR peak value, and T / CR average value in
(D) a step of comparing and analyzing a T / CR distribution pattern, a T / CR peak value, and a T / CR average value in each of a specimen suspected of cancer and a non-cancer specimen;
(E) A step of distinguishing normal tissue from cancer tissue from the result of the comparative analysis.
(4) Cancer detection method using tissue FISH method including the following steps:
(A) a step of hybridizing a specimen suspected of cancer collected from a tissue with respective PNA probes for telomere and centromere labeled with a fluorescent dye;
(B) a step of photographing the specimen under a fluorescence microscope;
(C) Analyzing the photographed image, measuring the relative light quantity (T / CR) of the telomere and centromere fluorescent labeling signal for each cell in the specimen, obtaining the T / CR distribution pattern in the specimen, 90% or more of the cells, wherein the T / CR of cells is less than 1.0.
(5) Cancer detection method using tissue FISH method including the following steps:
(A) a step of hybridizing a specimen suspected of cancer collected from a tissue with respective PNA probes for telomere and centromere labeled with a fluorescent dye;
(B) a step of photographing the specimen under a fluorescence microscope;
(C) Analyzing the photographed image, measuring the relative light quantity (T / CR) of the telomere and centromere fluorescently labeled signals for each cell in the specimen, determining the T / CR peak value in the specimen, A step of having cancer when the peak value of CR is less than 1.0.
(6) A method for detecting cancer using tissue FISH method, comprising the following steps:
(A) a step of hybridizing a specimen suspected of cancer collected from a tissue with respective PNA probes for telomere and centromere labeled with a fluorescent dye;
(B) a step of photographing the specimen under a fluorescence microscope;
(C) Analyzing the captured image, measuring the relative light quantity (T / CR) of the telomere and centromere fluorescently labeled signals for each cell in the specimen, obtaining the average value of T / CR in the specimen, A step of determining cancer when the average value of CR is less than 1.0.
(7) A kit for diagnosing cancer comprising each PNA probe for telomere and centromere labeled with a fluorescent dye, a hybrid solution for tissue FISH, and telomere analysis software.

  By using the present invention, normal tissue and cancer tissue can be differentiated without requiring a specially trained pathologist.

Hereinafter, the present invention will be described in detail.
In the method for diagnosing cancer according to the present invention, a sample collected from a subject using the fact that telomeres are shortened as canceration and cancer progress, that is, the telomere length in cancer cells is shorter than the telomere length in normal cells. Using the relative value of the telomere amount to the centromere amount as an index, the presence or absence of cancer cells in the sample is diagnosed.

  In the present invention, the tissue or organ to be diagnosed is not particularly limited, for example, brain, pituitary gland, spinal cord, salivary gland, thymus, thyroid, lung, breast, skin, skeletal muscle, heart, liver, Examples include spleen, adrenal gland, pancreas, esophagus, stomach, small intestine, large intestine, rectal bladder, prostate, testicle, ovary, placenta, uterus, bone marrow (leukemia, etc.), peripheral mononuclear cells and the like. In the case of diagnosis for the breast, papilloma and ductal ductal carcinoma are difficult to diagnose, but are easy with this method. In addition, in the case of targeting the esophagus, the determination of carcinoma in situ is controversial, but it can be distinguished even in very early carcinoma in situ.

  The degree of lesions of cancer to be diagnosed includes not only advanced cancers but also tissues of precancerous lesions. That is, in the present invention, cancer is a concept including precancerous lesions. In the present invention, the non-cancerous tissue (non-cancerous cell) or normal tissue (normal cell) means a tissue (cell) in which no sign of cancer is observed by a general diagnostic method conventionally used. .

  The inventors have found that telomeres tend to be shortened more in precancerous lesions and in situ cancer than in advanced cancers. Without being bound by any particular theory, the present inventors believe that the increase in cancer incidence associated with aging may be due to chromosomal instability due to telomere shortening. On the other hand, in cancer, the telomere length is slightly recovered by acquiring telomerase activity. Therefore, we believe that telomeres are shortened more in precancerous lesions than in advanced cancers.

  Examples of the type of cancer to be diagnosed in the present invention include esophageal carcinoma in situ, breast cancer, colon cancer and the like, including active ulcerative colitis as a precancerous lesion. Morphological diagnosis (pathological diagnosis) by a pathologist for very early cancer (carcinoma in situ) and early cancer is difficult because these early cancers are pathologically and morphologically different from normal. In this method, a difference is easily found in telomere length, and it can be said that this method is suitable for early and early cancer diagnosis. In early and early cancers, the histopathological difference from normal is small and pathological diagnosis is difficult, but this method can be distinguished (diagnosed) in the same way as advanced cancer.

  In the present invention, the telomere amount is preferably measured by applying the tissue FISH method. Therefore, the specimen is collected so that the chromosomes of the cells of the tissue or organ to be diagnosed can be observed under a microscope, and the collected specimen is processed so as to be capable of staining telomeres by the FISH method. Use. In the tissue FISH method, it is possible to use a sample generated from a paraffin block that has already been prepared. Therefore, when actually used in clinical practice, a normal tissue section used for pathological examination (mostly cells in the interphase cell division) is used. On the other hand, it may be convenient to apply the method of the present invention. In this case, the fixation is preferably performed with, for example, 10% buffered formalin and 4% formaldehyde, but is not limited thereto.

  In the present invention, the amount of telomere is determined from the fluorescence amount ratio (T / CR) of a specific label for telomere and centromere using a tissue FISH method in which hybridization is performed with a fluorescently labeled probe for each of telomere and centromere on a tissue section. Measure.

  The telomere and centromere probes are not particularly limited as long as they are specifically bound probes. For example, as a telomere probe, a commercially available fluorescent dye-labeled telo-Cy3 (complementing TTAGGG, 5'-terminal-CCTAAA-3'-terminal 6-base sequence three-fold telo Cy3, Mac DNA Synthesis Division) can be used. As the centromere probe, cenp1 (Cenp1 FITC, manufactured by Fasmac DNA Synthesis Division) labeled with another fluorescent dye is used. In normal cells, the fluorescence amount of the centromere portion where cenp1 is molecularly stained is considered to be almost constant for the total amount of chromosome 46, and the telomere length can be known relatively by T / CR. The conditions for hybridization in the tissue FISH method can be applied by appropriately modifying the conditions of the in situ hybridization method as described in Non-Patent Documents 3 and 4, and in principle, telomere Q- Application of FISH method is easy.

  After hybridization, the T / CR for each cell can be obtained by observing and photographing the slide glass with a fluorescence microscope equipped with a CCD camera and analyzing the photographed image. Specifically, the fluorescence amount of the probe bound to the telomere and centromere in the cell nucleus is captured in the field of view of the fluorescence microscope with different filters, and both fluorescence amounts are photographed using a CCD camera that can measure light separately under the microscope. The image is taken into a computer with image analysis software, and the fluorescence signal is quantified using the image analysis software.

  The analysis device can be individually devised by those skilled in the art. For example, for example, image analysis using a Nikon 80i fluorescence microscope equipped with a high-sensitivity digitally cooled CCD camera (Hamamatsu Photonics / ORCA-ER-1394) The software can be processed into 8-bit images with image-Pro Plus (Planetron) and analyzed as T / CR with telomere analysis software TFL2004.

  In the present invention, normal tissue and cancer tissue can be differentiated by comparing T / CR in normal tissue and cancer tissue. That is, in a sample collected from normal tissue and cancer tissue, the relative light quantity (T / CR) of the telomere and centromere fluorescent labeling signal for each cell is measured, and the distribution pattern of T / CR in each sample, T / CR, Normal tissue and cancer tissue can be differentiated by obtaining a peak value of CR and an average value of T / CR for comparative analysis. The analysis index will generally be as follows.

The pattern of T / CR distribution is different between cancer cells and normal cells, and in cancer, T / CR of almost all cells is less than 1.0, but in normal cases, it is widely distributed from 2 to 5.
The peak value of the T / CR distribution of cancer cells is about 0.5 and does not exceed 1.0, but is normally about 1.0.

The average value of T / CR is mostly 1 or less in cancer cells, but 2 or more in normal cases. (See attached diagram)
EXAMPLES Next, although an Example demonstrates this invention further in detail, the scope of the present invention is not limited to these Examples at all.

1. Preparation of Specimen Similar to normal pathological examination, after fixing tissue material by biopsy and surgery, a paraffin block is prepared, sliced into 3-6 microns with a microtome, and attached to a slide glass. After this, in normal pathological examination, hematoxylin and eosin staining is performed, and the shape and size of the cell itself (from the normal sag) are determined to determine cancer cells and non-cancer cells. In this method, the following is performed. .
2. Tissue FISH (probe, hybridize)
As the telomeric probe, a PNA probe labeled with telo-Cy3 labeled with Cy3 (complementing (TTAGGG), 5 'end-CCTAAA-3' end 6 base sequences 3 times) was used. As the centromere probe, a PNA probe labeled with cenp1 labeled with FITC was used.
3. Observation and photography of a slide glass A high-sensitivity CCD camera is used to take a fluorescent stained image of a group of cells whose cancer or non-cancerous cells you want to know.
4). Image Analysis FIG. 1 shows the T / CR distribution pattern of each constituent cell (epithelial basal cell, spine cell, stromal fibroblast) of the esophageal mucosa, and squamous cell carcinoma cell. Differentiation between cancer and normal cells is possible. Also, the T / CR peak value is about 0.5 for cancer and about 1.0 for non-cancer cells, and can be distinguished.

  Even in the average value of T / CR shown in FIG. 2, the non-cancerous cells are clearly larger than the cancer, and both can be distinguished (3-10 times). In addition, the T / CR of cancer cells falls within the range of approximately 1, but the distribution is different in non-cancer cells, and there are many cells in the range of 2 or more.

  According to the method of the present invention, normal tissue and cancer tissue can be easily distinguished from each other using a normal pathological section without requiring a specially trained pathologist. In addition, samples accumulated so far can be used in the method of the present invention, and other clinical medical data, immunohistological staining, presence or absence of hTERT (human telomerase) expression by in situ hybridization method, etc. It is possible to comprehensively judge malignancy determination and prognosis estimation by utilizing information so far such as collation with data.

It is a distribution map of T / CR of each constituent cell (cancer, epithelial base, epithelial spine, stromal fibroblast) of esophageal mucosa. Example 1 It is an average value of T / CR of each constituent cell (cancer, epithelial base, epithelial spine, stromal fibroblast) of esophageal mucosa. Example 1

Claims (7)

  A method for distinguishing between normal tissue and cancer tissue, comprising measuring a relative value of a telomere amount in a sample collected from a subject with respect to a centromere amount.   The method according to claim 1, wherein the relative value of the telomere amount to the centromere amount is measured as a relative light amount (T / CR) of fluorescently labeled signals of telomeres and centromeres by a tissue FISH method. A method for distinguishing between normal tissue and cancer tissue using tissue FISH method,
(A) a step of hybridizing a specimen suspected of cancer collected from a tissue and a non-cancer specimen collected from the same tissue with a PNA probe for each of telomere and centromere labeled with a fluorescent dye;
(B) a step of photographing a specimen suspected of cancer and a non-cancer specimen under a fluorescence microscope;
(C) Analyzing the captured image, measuring the relative light quantity (T / CR) of the telomere and centromere fluorescently labeled signals for each cell in each of the specimens suspected of cancer and non-cancer specimens. A step of obtaining a T / CR distribution pattern, a T / CR peak value, and an average value of T / CR in (d) a T / CR distribution pattern in each specimen of a specimen suspected of cancer and a non-cancer specimen, The method of comparing and analyzing the peak value of T / CR and the average value of T / CR (e) The method of distinguishing normal tissue from cancer tissue from the result of the comparative analysis. A method for detecting cancer using a tissue FISH method, wherein (a) a specimen suspected of cancer collected from a certain tissue is hybridized with respective PNA probes for telomere and centromere labeled with a fluorescent dye,
(B) a step of photographing the specimen under a fluorescence microscope;
(C) Analyzing the photographed image, measuring the relative light quantity (T / CR) of the telomere and centromere fluorescently labeled signals for each cell in the specimen, obtaining the T / CR distribution pattern in the specimen, 90% or more of the above, wherein the T / CR of cells is less than 1.0, the method is said to be cancerous. A method for detecting cancer using a tissue FISH method, wherein (a) a specimen suspected of cancer collected from a certain tissue is hybridized with respective PNA probes for telomere and centromere labeled with a fluorescent dye,
(B) a step of photographing the specimen under a fluorescence microscope;
(C) Analyzing the captured image, measuring the relative light quantity (T / CR) of the telomere and centromere fluorescently labeled signals for each cell in the specimen, obtaining the T / CR peak value in the specimen, The method as described above, wherein the cancer is found when the peak value of CR is less than 1.0. A method for detecting cancer using a tissue FISH method,
(A) a step of hybridizing a specimen suspected of cancer collected from a tissue with respective PNA probes for telomere and centromere labeled with a fluorescent dye;
(B) a step of photographing the specimen under a fluorescence microscope;
(C) Analyzing the photographed image, measuring the relative light quantity (T / CR) of the telomere and centromere fluorescently labeled signals for each cell in the specimen, obtaining the average value of T / CR in the specimen, The method as described above, wherein the cancer is found when the average value of CR is less than 1.0.   A cancer diagnostic kit comprising a PNA probe for telomere and centromere labeled with a fluorescent dye, a hybrid solution for tissue FISH, and telomere analysis software.

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