WO2012070730A1 - Method for detecting alk gene rearrangement and method for diagnosing cancer using the same - Google Patents

Abstract

The present invention relates to a method for detecting ALK gene rearrangement, which is induced by ALK gene translocation, according to the overexpression of ALK protein after ALK antibody response, and a cancer diagnostic method using the same. The method for detecting ALK gene rearrangement according to the present invention provides a method for screening ALK gene rearrangement, which can primarily and efficiently identify targets for ALK inhibitors quickly and accurately by analyzing the overexpression of ALK protein, by suggesting a specific protocol for ALK antibody response and criteria for the analysis of IHC staining patterns as means of detection which makes up for the disadvantages of conventional FISH which is expensive and consumes long time. Accordingly, the present invention can broaden the range of therapies aimed at molecular targets.

Description

METHOD FOR DETECTING ALK GENE REARRANGEMENT AND METHOD FOR DIAGNOSING CANCER USING THE SAME

The present invention relates to a method for easily and efficiently detecting tissues with overexpressed ALK proteins resulting from ALK gene translocation in non-small cell lung cancer (NSCLC) patients by immunohistochemical staining.

With an increase in cancer incidence and mortality in the world, the proportion of lung cancer to all cancers in developed countries is steadily increasing. Lung cancer has the highest mortality rate of all cancers in Korea, and is surely a disease that will not be easily conquered despite much basic and clinical research. This creates an urgent need for research on lung cancer which is more organized and focused more on clinical applicability.

It is assumed that one of the reasons for the high mortality rate in lung cancer is because many cases of lung cancer are found in advanced stages of progression because of difficulties in early diagnosis and recurrence is common even after surgical resection. Accordingly, two main strategic goals can be set to conquer lung cancer: the first is the strategy of early surgery through the development of early diagnosis methods; and the second is the development of effective systemic cancer chemotherapy drugs which can be administered in addition to surgical treatment, or which itself can be primarily applied to patients with unresectable lung cancer. Particularly, while people who can benefit from the former strategy are limited to people who have a screening test regularly among people who are healthy now or who are diagnosed with early-stage lung cancer, the latter strategy gains more importance as an approach which provides direct benefit to a great number of potential patients whose disease has already progressed or will progress despite best medical treatment.

Lung cancers are generally divided into (SCLC) and non-small cell lung cancer (NSCLC). Non-small cell lung cancer accounts for approximately 80% of all lung cancers. Non-small cell lung cancer includes three subtypes: adenocarcinma and squamous cell carcinoma, which each account for 40% of lung cancers; and large cell carcinoma, which accounts for the remaining 20%. Such a TNM staging system is widely accepted in the management of lung cancer. The histological features of lung cancer vary with the aforementioned types of lung cancer, The clinical characteristics, including sites susceptible to the onset of disease, progression mode and rate and symptoms, and treatment methods vary with the different histological types of lung cancer (Brambilla et al., Eur Respir J.18(6):1059-68, 2001).

NSCLC is rarely treated by chemotherapy alone due to the lower effects of anticancer agents relative to SCLC, and thus, the only effective method involves completely removing tumors through surgery. However, fewer than 30% of lung cancer patients have tumors that cannot be entirely removed by surgical resection at the time of diagnosis, and less than one-third of them are alive five years after surgical resection, and the 10-year survival rate is as low as less than 10% despite recent advances in cancer therapy. Thus, to improve the efficacy of treatment of lung cancer, there is a great need for the development of a new diagnostic and therapeutic method for the each steps of the cancer progression, by elucidating the molecular biologic regulatory mechanism of carcinogenesis, tumor growth, and metastasis.

Moreover, there have been ongoing studies of the role of genetic changes in association with the carcinogenic mechanism of lung cancer. Like other solid tumors, chromosomal abnormalities have been classified as a very important molecular phenomenon in the incidence of lung cancer. Among them, many studies have been conducted on genetic changes of EGFR, p53, k-ras, etc. In particular, EGFR gene mutations in non-small cell lung cancer patients EGFR gene mutations were frequently observed in Asian ethnicities, female, non-smokers, and in patients with abdenocarcinomas. As a method for treating the EGFR gene mutations, Gefitinib (“Iressa”), which is an EGFR tyrosine kinase inhibitor, has been administered, but the problem of treatment failure due to drug resistance has emerged.

Therefore, the ALK gene as a oncogene is receiving attention from academia in recent years, which can serve as a target for novel drugs targeting lung cancer, and many multinational pharmaceutical companies are accelerating efforts to develop ALK inhibitors. Meanwhile, a treatment method targeting ALK has many difficulties in screening patients who require administration of ALK inhibitors because there is no adequate method to effectively detect ALK gene rearrangement.

Hence, there is an urgent need for a study of a new efficient and effective method to detect lung cancer with ALK gene rearrangement.

The present inventors have completed the present invention by developing a detection method for detecting ALK gene rearrangement by targeting the translocation of an ALK gene as a candidate target gene suitable for the administration of an ALK inhibitor recently receiving attention as a novel targeted drug, and a diagnostic method for diagnosing lung cancer based on the presence or absence of ALK gene rearrangement.

Accordingly, it is an object of the present invention to provide a method for screening lung cancer patients with ALK gene rearrangement, which can primarily and quickly identify targets for ALK inhibitors by analyzing the overexpression of ALK protein based on staining patterns of patient tissue samples stained with ALK antibody.

Therefore, the present invention provides ultimately a diagnostic method which can broaden the range of molecular targets for cancer therapy and swiftly screen the patients with the specific genetic alteration with relatively low incidence.

To accomplish the aforementioned objects, the present invention provides a method for detecting ALK gene rearrangement, the method comprising the steps of: reacting an ALK antibody with a pretreated specimen; staining the specimen reacted with the ALK antibody; and analyzing the staining pattern of the specimen.

Furthermore, the present invention provides a method for detecting ALK gene rearrangement, the method comprising the steps of: reacting an ALK antibody with a pretreated specimen; staining the specimen reacted with the ALK antibody; detecting a level of protein expression by analyzing the staining pattern of the specimen; and determining the presence of ALK gene rearrangement based on the level of protein expression.

In one embodiment of the present invention, the step of reacting the ALK antibody may be performed by a method selected from the group consisting of immunohistochemistry, immunoblot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), agglutination and radioimmunoassay.

In one embodiment of the present invention, the pretreatment of the specimen may comprise deparaffinizing the paraffin sections and heat-treating the deparaffinized paraffin sections in a CCL₄ solvent containing a tris-borate-EDTA (TBE) buffer at 95℃ to 105℃ for 15 to 25 minutes.

In one embodiment of the present invention, the step of reacting the ALK antibody may comprise treating an ALK antibody at the dilution of 1:25 to 1:35 and reacting the ALK antibody for 90 to 120 minutes at a temperature of 40 to 45℃.

In one embodiment of the present invention, the staining step may comprise the steps of: treating a secondary antibody binding to the ALK antibody; and treating a chromogenic substrate.

In one embodiment of the present invention, the chromogenic substrate may be selected from the group consisting of BCIP/NBT (5-bromo-4-chloro-3-indolyl- phosphate/nitroblue tetrazolium), DAB (diaminobenzidine), TMB (3, 3' , 5, 5' -tetramethyl benzidine), BCIP/INT (5-bromo-4-chloro-3- indolyl phosphate/iodonitrotetrazolium), NF (New fuchsin), FRT(FaSt Red TR Salt), ABTS[2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), 4-CN, AEC (3-amino-9-ethylcarbasole), X-Gal, and IPTG (isopropyl beta-D-thiogalactoside).

In one embodiment of the present invention, the step of analyzing the staining pattern of the specimen may comprise the step of counting the number of cells with cytoplasmic staining or the step of determining the cytoplasmic staining intensity.

In one embodiment of the present invention, if the number of cells with cytoplasmic staining is less than 5% of the total number of cells of the target, ALK gene rearrangement is considered negative.

In one embodiment of the present invention, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells of the target and the cytoplasmic staining intensity is weak, ALK gene rearrangement is considered negative.

In one embodiment of the present invention, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells of the target and the cytoplasmic staining intensity is strong, ALK gene rearrangement is considered positive.

In one embodiment of the present invention, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells of the target and the cytoplasmic staining intensity is moderate, the method may further comprise the step of detecting ALK gene rearrangement by a method selected from the group consisting of FISH (fluorescence in situ hybridization), SISH (Silver In Situ Hybridization), and PCR (polymerase chain reaction).

Furthermore, the present invention provides a method for diagnosing cancer using the ALK gene rearrangement detection method.

In one embodiment of the present invention, the cancer may be lung cancer.

The method for detecting ALK gene rearrangement according to the present invention provides a method for screening ALK gene rearrangement, which can primarily identify targets for ALK inhibitors quickly and accurately by analyzing the overexpression of ALK protein, by suggesting a detection method which makes up for the disadvantages of conventional FISH which is expensive and consumes long time. Accordingly, the present invention can broaden the range of therapies aimed at molecular targets, and can be used as a cancer diganostic method.

The above and other objects and features of the present invention will become apparent from the following description of the preferred embodiments given in conjunction with the accompanying drawings, in which:

Fig. 1 shows FISH (Fluorescence in situ hybridization)-positive patterns using an LSI ALK Dual Color break-apart probe, in which (a) depicts two distinct red and green (break apart) signals and one intact fusion signal; and (b) depicts an isolated red signal (IRS) and one intact fusion signal.

Figs. 2a, 2b, and 2c are photographs showing immunohistochemical staining patterns, in which the number of cells with cytoplasmic staining is more than 5% of the total number of cells and the cytoplasmic staining intensity is strong (3+);

Figs. 3a, 3b, and 3c are photographs showing immunohistochemical staining patterns, in which the number of cells with cytoplasmic staining is more than 5% of the total number of cells and the cytoplasmic staining intensity is moderate (2+);

Figs. 4a and 4b are photographs showing immunohistochemical staining patterns, in which the number of cells with cytoplasmic staining is less than 5% of the total number of cells, or the number of cells with cytoplasmic staining is more than 5% of the total number of cells and the cytoplasmic staining intensity is weak (1+);

Figs. 5a and 5b are photographs showing immunohistochemical staining patterns, in which there is no cytoplasmic staining at all (0); and

Fig. 6 shows a diagnostic algorithm for accurate and cost-effective clinical application of ALK IHC and FISH in NSCLC.

The present invention provides a method for detecting ALK gene rearrangement by an immunohistochemical test, which can quickly and accurately screen patients with ALK gene translocation and rearrangement.

In general, many cancers are characterized by disruptions in cellular signaling pathways that lead to aberrant control of cellular processes, or to uncontrolled growth and proliferation of cells. Particularly, in solid tumors, like non-small cell lung carcinoma (NSCLC), it is known that gene deletions and/or translocations resulting in kinase fusion proteins with aberrant signaling activity can directly lead to certain cancers.

The identification of a novel kinase mutants, gene deletion and translocation mutations has strong relation for the potential diagnosis and treatment of solid tumors, such as NSCLC. NSCLC, for example, is often only detected after it has metastasized, and thus the mortality rate is 75% within two years after diagnosis. Accordingly, the ability to identify, as early as possible, patients having gene mutations that may lead to NSCLC, would be highly desirable.

Therefore, the present inventors have devised a new method for accurately and easily detecting the presence of ALK fusion proteins (short and long variants) resulting from gene translocations and mutations, which are expected to drive proliferation and survival of mammalian solid tumors, including lung cancers (such as NSCLC), as well as other cancers, in which an ALK fusion protein deriving from ALK gene rearrangement is expressed.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein fused with a protein called nucleophosmin (NPM), resulting from the t(2;5)(p23;q35) translocation in a certain malignant lymphoma type, namely, anaplastic large-cell lymphoma (ALCL). It is known that signal transduction through ALK is involved in biologically significant pathways such as cell proliferation, differentiation, and anti-apoptosis via several important signal transducers such as PLCγ, PI3K, RAS/MAPK, and JAK (Janus kinase)/STAT pathways (Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma kinase: signalling in development and disease. Biochem J. 2009;420(3):345-361). Particularly, the ALK gene contributes to the tumorigenic transformation of certain diseases by fusion with a partner gene other than NPM.

Moreover, genetic alteration or overexpression of the ALK gene has been reported in other types of lymphoma and other tumors, and has also been reported in a variety of tumors including diffuse large B cell lymphoma(DLBCL), inflammatory myofibroblastic tumors(IMT), non-small cell lung cancer, thyroid carcinoma, breast cancer, malignant melanoma, neuroblastoma, glioblastoma, astrocytoma, retinoblastoma, ewing’s sarcoma and rhabdomyosarcoma (Dirks W. G., Fahnrich S., Lis Y., Becker E., MacLeod R. A., Drexler H. G. Expression and functional analysis of the anaplastic lymphoma kinase (ALK) gene in tumor cell lines. Int. J. Cancer. 2002;100:49-56).

In particular, it was reported in 2007 that the ALK gene in non-small cell lung cancer is activated by fusion with the EML4 gene, and the frequency of ALK gene rearrangement is approximately 5% or so in the range of 3 to 7% non-small cell lung cancer patients, depending on detection methods and patient groups between the reports (Martelli M. P., Sozzi G., Hernandez L., Pettirossi V., Navarro A., Conte D., Gasparini P., Perrone F., Modena P., Pastorino U., et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. Am. J. Pathol. 2009;174:661-670).

Although not very frequent in all NSCLC, considering that NSCLC accounts for approximately 80% of all lung cancers, it is assumed that a considerable number of patients have ALK gene rearrangement, which is associated with tumorigenic transformation. At present, EGFR TKI, i.e., Iressa, serving as a drug targeting EGFR, is used for patients with advanced non-small cell lung cancer considering that no effective targeted therapy exists for patients who are not eligible for Irresa, ALK gene rearrangement is of great importance in that it can serve as novel targeted therapies.

Focusing attention on the importance of ALK translocations in lung cancer patients, a variety of ALK inhibitors have been tested in animal experiments and cell lines. Currently, five institutions in the U.S. including Harvard Medical School, one institution in Australia, and Seoul National University Medical School, the only Asian institution, are working on the Phase III clinical trial of an oral c-MET/ALK inhibitor (Crizotinib, PF-02341066) developed by Pfizer, a multinational pharmaceutical company.

According to the result of a phase I clinical trial targeted to patients with ALK-positive non-small cell lung cancer, which was presented by Prof. Young-Joo Bang at the 46^th American Society of Clinical Oncology (ASCO) in 2010, it was reported that an excellent result was achieved with 70% of a total of 79 patients with ALK gene rearrangement being reactive to the drug, which proved the effect of the ALK inhibitor. Given the successful result that this study has generated, it is highly likely that the ALK inhibitor will be approved by the U.S. FDA and introduced into the market in the future, and establishing a test method for quickly and accurately screening patients eligible for the drug is important than anything else.

One of the standard methods currently used to detect ALK gene rearrangement is ALK “FISH(fluorescence in situ hybridization)” testing for patients who are negative resulting from EGFR mutation test in lung cancer patients.

The FISH test is the most reliable method of gene testing in that the expression of a particular gene in tissue can be observed as it is using fluorescent probes.

However, the FISH test has side effects that are time delay(at least three weeks) and increase in non-insurance covered medical expenses (the cost of EGFR mutation testing is 300,000 to 400,000 won; and the cost of FISH testing is around 400,000 won) due to use of expensive equipment and reagents and these add a double burden to patients suffering from disease.

Moreover, an excessive physical burden is placed on pathologists checking the pattern and number of fluorescent probes and reading ALK FISH results of samples under a fluorescent microscope in a dark room. This is because it is difficult and complicated to determine an ALK gene alternation status since many fusion points exist between EML4 and ALK genes, and because a considerable amount of time and high concentration are required to distinguish between the original normal status and the fusion gene status (ALK gene rearrangement) induced by translocation since the ALK gene and the EML4 gene are located relatively close to each other in the same chromosome. Accordingly, there is an urgent need to establish a technique for detecting ALK gene translocation easily and at low cost.

Therefore, the present inventors intend to provide a method for detecting ALK gene rearrangement by a simple immunohistochemical(IHC) technique, which can quickly detect ALK translocation, is economical, and can relieve the pathologists’ burden.

That is to say, the present inventors have established a concrete staining method using ALK immunohistochemical staining and criteria for the detection of translocation which can be used practically in pathology laboratories by overcoming the drawbacks and problems of the existing ALK immunochemical staining method, for which it has been necessary so far to use the FISH method to identify ALK gene mutations although immunohistochemical staining is the most common method used in pathology laboratories to identify gene mutations.

The existing immunohistochemical staining method is not used in the ALK gene rearrangement detection method because it is considered that, unlike ALK positive lymphoma, there is a limitation in detecting gene translocations by protein expression since an ALK gene translocation, if any, occurring in lung cancer has a low transcriptional activity in protein expression. Moreover, as immunohistochemical staining is widely used, the types of antibodies using clones and the types of staining methods become diverse. Thus, there have been no guidelines or standardized test guides for ALK immunohistochemical staining (Palmer RH et al., J Natl Cancer Inst 2005; 97:339-346., Inamura K et al., Mod Pathol 2009;22:508-515).

Actually, from the results of studies in other countries, the concordance between results of immunohistochemical staining for ALK and FISH results was very low due to low sensitivity of immunohistochemical staining. Also, Mano et al. who first discovered ALK acknowledged the need for further research on the method of quickly and accurately finding ALK gene translocation by immunohistochemistry (Han SW et al., J ClinOncol 2005;23:2493-2501., Shepherd FA et al., N Engl J Med 2005;353:123-132., Dirks WG et al., Int J Cancer 2002;100:49-56., Martelli MP et al., Am J Pathol 2009;174:661-670., Perner S et al., Neoplasia 2008;10:298-302).

Therefore, the present inventors intend to provide a concrete protocol of immunohistochemistry and a diagnostic algorithm, which can primarily screen patients with ALK gene rearrangement who are known to be approximately 5% of those with non-small cell lung cancer (NSCLC).

As a result of one embodiment of the present invention, the ALK immunohistochemistry established by the present inventors showed high concordance with FISH results.

Accordingly, the present invention provides a method for detecting ALK gene rearrangement, the method comprising the steps of: obtaining a specimen from a subject; pretreating the specimen; reacting the pretreated specimen with an ALK antibody; staining the specimen reacted with the ALK antibody; and analyzing the staining pattern of the specimen.

Furthermore, the present inventors provide a method for detecting ALK gene rearrangement, the method further comprising the steps of: detecting a level of protein expression by analyzing the staining pattern of the specimen; and determining the presence of ALK gene translocation based on the level of protein expression.

In the “ALK gene rearrangement” of the present invention, the term “gene rearrangement” generally refers to a structural change which causes a change in the position and order of genes on a chromosome by recombination between somatic gene segments. Also, the term “translocation”, which is a chromosomal abnormality, refers to an abnormality in which a part of a chromosome is broken and reattached to another part of the same chromosome or another chromosome and hence a change occurs in the shape of the chromosome. Specifically, translocation occurred in the same chromosome is called intrachromosomal translocation or simple translocation, and translocation between different chromosomes is called interchromosomal translocation. Further, the attachment of a part of one chromosome to another chromosome is called simple translocation, and the breakage of two chromosomes with exchange of broken parts to the opposite chromosome is called reciprocal translocation.

Accordingly, “ALK gene rearrangement” according to the present invention refers to an abnormality in which a part of the ALK gene is moved into another gene due to translocation of the ALK gene and then inserted into that gene and rearranged. As a result of the ALK gene rearrangement caused by the translocation, not a normal ALK protein but an ALK fusion protein comprising different proteins is created in a cell. Therefore, in one embodiment of the present invention, a test was carried out for a method of detecting an ALK-EML4 fusion gene formed by the translocation and rearrangement of the ALK gene into the EML4 gene.

An ALK antibody response required to detect the ALK gene rearrangement of the present invention is not limited, but preferably selected from the group consisting of immunohistochemistry, immunoblot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), agglutination and radioimmunoassay. Immunohistochemical techniques are particularly preferred.

As used herein, the term "specimen" indicates cells or a biopsy isolated from a human body. The specimen may be isolated from a subject with cancer (e.g., breast cancer tissue) or a healthy person. The specimen may be processed into, for example, paraffin-embedded tissues, frozen tissues, formalin fixed tissues, or cell smear. Of them, paraffin-embedded tissues are most widely used.

The fixation of a specimen is the most important for morphological observation of cells or tissues. In suitably fixed tissues, cell organelles are well maintained. Insufficient fixation makes cell organelles unstable in morphological structure. Excessive fixation causes antigen loss and non-specific responses.

Tissue fixatives may be divided into coagulation-type fixatives and non-coagulation (denaturation) type fixatives. Formalin, a most widely used fixative, is of non-coagulation type. While infiltrating tissues at a rate of 1 mm per hour, formalin fixes the tissues. Formalin destroys epitopes only to a small extent and crosslinks proteins and peptides, thereby maintaining the morphology of cells. In one embodiment of the present invention, tissue specimens obtained from NSCLC patients were fixed in formalin and embedded in paraffin in order to carry out ALK IHC.

After embedding the tissues fixed with tissue fixatives in paraffin and sectioning them into very thin sections, it is preferred to carry out a pretreatment step including deparaffinization. The pretreatment step may comprise a step of deparaffinizing the paraffin sections and heat-treating them in a CCL₄ solvent containing a tris-borate-EDTA (TBE) buffer at 95℃ to 105℃ for 15 to 25 minutes. In one embodiment of the present invention, the paraffin sections are deparaffinized by EZ prep for 4 minutes at 75℃, and then preheated in a CCL₄ solvent containing a TBE buffer at 100℃ for 20 minutes.

Furthermore, the present inventors have established a specific condition of ALK antibody response in order to detect ALK overexpression caused by ALK translocation more accurately. The present invention intends to provide an ALK IHC method for quick and economical primary screening in order to detect ALK gene rearrangement. After many comparative experiments to provide correct immunohistochemistry results consistent with results of ALK FISH conventionally used to detect ALK gene rearrangement, the optimum condition of ALK antibody response according to the present invention was achieved.

An ALK antibody response according to the present invention can be obtained by treating an ALK antibody at the dilution of 1:25 to 1:35 and reacting it for 110 to 130 minutes at a temperature of 40 to 45℃. In one embodiment of the present invention, an ALK antibody was treated at a 1:30 dilution, and then incubated for 2 hours at 42℃.

To measure the accuracy of the ALK IHC results depending on the above-mentioned condition of antibody response, an ALK IHC test was carried out by diluting the ALK antibody at a ratio of 1:20, 1:25, 1:30, 1:35, and 1:40, and a comparison of IHC results and FISH results about the presence of ALK translocation was conducted (see Example 8).

As a result, a strong correlation between IHC and FISH was observed when the ALK antibody was treated at a dilution of 1:25 and 1:35, as compared to when the ALK antibody was treated at a dilution of 1:20 and 1:40. Moreover, an IHC test carried out at varying incubation times proved that the results of the IHC test involving reaction for 110 minutes and 130 minutes had a strong correlation with FISH, as opposed to the IHC test involving incubation for 60 minutes, 90 minutes, 150 minutes, and 180 minutes. In addition, in a test carried out by diluting the ALK antibody at a ratio of 1:30 and incubating it for 120 minutes at varying reaction temperatures, there was a strong correlation between IHC test results obtained at 40℃ and 45℃ and FISH results as compared to the IHC results obtained at 35℃ and 50℃.

The staining step following the ALK antibody response comprises the step of treating a secondary antibody binding to the ALK antibody and the step of treating a chromogenic substrate. The step of treating the secondary antibody may be performed using, although not limited to, either an Avidin-Biotinylated enzyme complex (ABC) method or an enzyme-labeled secondary antibody. In one embodiment of the present invention, the secondary antibody was treated by the former ABC method (see Example 2).

Moreover, the chromogenic substrate used after the treatment of the secondary antibody is preferably selected from, but not limited to, the group consisting of BCIP/NBT (5-bromo-4-chloro-3-indolyl- phosphate/nitroblue tetrazolium), DAB (diaminobenzidine), TMB (3, 3' , 5, 5' -tetramethyl benzidine), BCIP/INT (5-bromo-4-chloro-3- indolyl phosphate/iodonitrotetrazolium), NF (New fuchsin), FRT(FaSt Red TR Salt), ABTS[2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), 4-CN, AEC (3-amino-9-ethylcarbasole), X-Gal, and IPTG (isopropyl beta-D-thiogalactoside). In one embodiment of the present invention, a detection system provided with HRP (Horseradish peroxidase) was used in combination with DAB (diaminobenzidine) as a chromogenic substrate (see Example 2).

The step of analyzing the specimen pattern stained by a specimen staining method according to the present invention comprises the step of counting the number of cells with cytoplasmic staining or the step of determining the cytoplasmic staining intensity. That is to say, the present invention provides a method for detecting ALK gene rearrangement through the steps of determining the number of cells with cytoplasmic staining and the staining intensity of the cytoplasm, determining the level of protein expression, and determining ALK gene translocation based on the determined level of protein expression. Therefore, the present inventors demonstrated the result of the staining pattern analysis by scores on a scoring system.

First, in the analysis of the staining pattern of the specimen, the number of cells with cytoplasmic staining is determined. If the number of cells with cytoplasmic staining is less than 5% of the total number of cells of the target, ALK gene rearrangement is considered negative. In this case, the cytoplasmic staining of 0 to 5% of the number of cells is scored as 1+, and cells with no cytoplasmic staining (no staining, 0%) is scored as 0.

Moreover, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells of the target and the cytoplasmic staining intensity is weak, ALK gene rearrangement is considered negative and scored as 1+ as is with the previous case.

In contrast, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells of the target and the cytoplasmic staining intensity is strong, ALK gene rearrangement is considered positive and scored as 3+.

By the way, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells and the cytoplasmic staining intensity is moderate, the step of detecting ALK gene translocation has to be implemented additionally by a method selected from the group consisting of FISH (fluorescence in situ hybridization), SISH (Silver In Situ Hybridization), and PCR (polymerase chain reaction) and ALK gene translocation is scored as 2+. In one embodiment of the present invention, a FISH test was used in addition to the above case to determine ALK gene rearrangement.

In the determination of staining intensity, specifically, “strong” staining intensity denotes an luminosity of more than 6, “moderate” staining intensity denotes an luminosity ranging from 4 to 6, and “weak” staining intensity denotes an luminosity ranging from 0 to 4.

The term “luminosity” refers to a property representing the lightness and darkness of colors or the brightness of colors, which is a measure of brightness sensation. The brightness of both achromatic and chromatic colors is called intensity. The whiter the color, the higher the intensity, and, the blacker the color, the lower the intensity.

The range of luminosity variations with scores according to the present invention refers to differences in color intensity depending on the type of chromogenic substrate. Although not limited to the luminosity of a specific color, the grades of color luminosity variations with scores depending on the type of preferably used chromogenic substrate are as follows. The codes in parentheses represent colors by a color system using the Munsell Color System created by A.H. Munsell in 1905.

The Munsell Color System was revised by the Colorimetry Committee of The Optical Society of America in 1943 and has been widely accepted as an international standard for specifying colors. In the Munsell Color System, a single color is represented by three dimensions of color: hue(H), value(V)(luminosity) and chroma(C) and numerically expressed in the form H V/C. Accordingly, the term “luminosity” used as a measure of staining pattern in the present invention is based on the Munsell Color System, and therefore was expressed in numbers ranging from 0 (white) to 10 (black).

First, for a chromogenic substrate which is originally brown, such as DAB (diaminobenzidine) and BCIP/INT (5-bromo-4-chloro-3-indolyl phosphate/iodonitrotetrazolium), colors with an luminosity of 1+(in scoring system) refer to light yellow orange (10YR 8/7) and pale yellowish brown (10YR 6/4), and colors with an luminosity of 2+(in scoring system) refer to yellowish brown (10YR 5/9) and dark yellowish brown (10YR 5/5). Colors with an luminosity of 3+(in scoring system) refer to brown (10YR 4/7) and dark brown (10YR 4/4).

For a chromogenic substrate which is originally red, such as NF (New fuchsin), FRT(FaSt Red TR Salt), and AEC (3-amino-9- ethylcarbasole), colors with an luminosity of 1+ refer to pale pink (7.5R 8/4) and dark pink (7.5R 6/6), colors with an luminosity of 2+ refer to pale red (7.5R 4/6) and dark red (7.5R 5/8), and colors with an luminosity of 3+ refer to red (7.5R 4/14), vivid red (7.5R 4/16), and deep red (7.5 3/12).

For a chromogenic substrate which is originally purple, such as BCIP/NBT (5-bromo-4-chloro-3-indolyl- phosphate/nitroblue tetrazolium), 4-CN, and IPTG (isotropyl beta-D-thiogalactoside), colors with an luminosity of 1+ refer to pale purple (2.5P 6.5/4) and light purple (2.5P 7/7), and a color with an luminosity of 2+ refers to dark purple (2.5P 5/5). Colors with an luminosity of 3+ refer to purple (2.5P 3/9) and deep purple (2.5P 2/9).

For a chromogenic substrate which is originally blue, such as TMB (3, 3', 5, 5' -tetramethyl benzidine) and X-Gal, a color with an luminosity of 1+ refers to light blue (5PB 8/7), and a color with an luminosity of 2+ refers to pale blue (5PB 5.5/5). Colors with an luminosity of 3+ refer to blue (5PB 3/9), dark blue (5PB 4/5), and light navy (5PB 3/5).

For a chromogenic substrate which is originally green, such as ABTS [2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], colors with an luminosity of 1+ refer to light yellowish green (2.5G 8/8) and pale yellowish green (2.5G 7/5), and colors with an luminosity of 2+ refer to pale green (2.5G 5/5) and dark yellowish green (2.5G 5.5/5). Colors with an luminosity of 3+ refer to green (2.5G 3/9), dark green (2.5G 3.5/5), and deep green (2.5G 3/6).

To determine the best criteria for the analysis of IHC staining patterns to predict ALK genetic conditions, the present inventors compared the results of an ALK translocation test using FISH, which is currently used to detect ALK gene rearrangement in pathology laboratories with the results of an ALK translocation test using IHC according to the present invention (see Example 6). The ALK FISH test performed by the present inventors was conducted by the method as in Example 3, and the clinicopathologic characteristics of NSCLC tissues determined to be ALK FISH-positive and ALK FISH-negative, which are derived from the results of the ALK FISH test, are as shown in Tables 1 to 3.

The ALK FISH results and the results of analysis of ALK protein expression using the IHC method according to the present invention demonstrated that all the cases with IHC scores of 3+ were ALK FISH-positive and all the cases with IHC scores of 0 or 1+ were ALK FISH-negative. For cases with scores of 2+, 30% (3/10) were ALK FISH-positive and 70% (7/10) were ALK FISH-negative.

Assuming that ALK IHC scores of 0 and 1+ were biologically negative, an ALK IHC score of 3+ was biologically positive, and an ALK IHC score of 2+ was equivocal (grey zone), the results of biological ALK IHC assay and FISH were strongly correlated (p=0.000 by Pearson’s chi-square test, Pearson’s R-0.933, p=0.000). After excluding cases with ALK IHC scores of 2+ (equivocal), the ALK IHC scores (0 and 1+ versus 3) and ALK FISH (negative versus positive) were completely correlated. These results indicate that an ALK IHC score of 3+ could strongly predict FISH-positivity and an IHC score of 0 or 1+ could predict FISH-negativity (see Table 4).

As a validation set for validating these results, specimens obtained from 187 patients with suspected ADC were examined, and, as a result, there was a consistent relationship between ALK IHC and FISH as shown in Table 4. Therefore, the present inventors derived a diagnostic algorithm of clinical application of ALK IHC and ALK FISH (see FIG. 6).

Accordingly, the present invention provides a method for detecting ALK gene rearrangement comprising the step of reacting a specimen obtained from a subject with an ALK antibody and the step of staining the specimen reacted with the ALK antibody. As the ALK gene is known to form fusion genes with other genes because of translocation and contribute to tumorigenic transformation of cells, it is also possible to provide a cancer diagnostic method comprising the ALK gene rearrangement detection method. In particular, the aforementioned cancer may be lung cancer since the report says that ALK gene rearrangement is found in about 5% of the patients with NSCLC.

Like ALK, ALK gene rearrangements according to the present invention are mutually exclusive to EGFR or KRAS mutations, which are known to cause tumorigenesis (see Example 4 and Tables 1 to 3), and groups with EGFR and ALK gene alterations have similarities in their patient profiles excluding sex and preference. Taking this into consideration, patients not responding to EGFR-targeting agents are suspected to have ALK gene translocations. Therefore, the present invention provides a method for detecting ALK gene rearrangement, which can quickly identify targets for ALK inhibitors from among cancer patients who have not benefited from the existing EGFR-targeting agents. Therefore, the range of therapies aimed at molecular targets can be broadened.

Hereinafter, the present invention will be described in detail with respect to Examples. However, these Examples are intended to describe the present invention in further detail and should not be construed as limiting the scope of the invention.

<Reference Example>

Patients and Samples

465 consecutive patients with NSCLC underwent surgical resection at the Seoul National University Bundang Hospital in Korea from May 2003 to May 2008 and 465 patient samples were obtained and used for tissue microarray (TMA). All the cases were diagnosed as initial NSCLC originating from the lungs, and diagnoses of NSCLC were confirmed through review of pathological slides and medical records. The 465 NSCLC cases consisted of 269 cases of adenocarcinoma (ADC), 169 cases of squamous cell carcinoma, 10 cases of adenosquamous carcinoma, five cases of pleomorphic carcinoma, two cases of large cell carcinoma, eight cases of large cell neuroendocrine carcinoma, one case of carcinosarcoma, and one case of lymphoepithelioma-like carcinoma. The mean age of the 465 patients was 63.8 years and ranged from 21 to 84. In the 453 cases, FISH results were examined due to the problem of tissue attachment or poor quality.

As a validation test, the present inventors examined whole sections of surgically resected or biopsied samples from 187 patients with ADC at the Seoul National University Bundang Hospital in Korea from September 2009 to May 2010 using IHC. All the cases corresponding to IHC 1+, 2+, and 3+ were evaluated again through FISH. This study was approved by the institutional review board of the Seoul National University Bundang Hospital.

Statistical Analysis

SPSS version 12.0 (Systat, Chicago, IL, USA) was used for the statistical analysis of the results of the tests conducted in the embodiment of the present invention, which included Mann-Whitney test, Pearson’s chi-square test, and Pearson’s R test. Statistical significance was defined as p<0.05.

<Example 1>

Construction of Tissue Microarray

One representative core tissue (2 mm in diameter) was embedded in a paraffin block and arranged in new TMA blocks using a trephine apparatus (Superbiochips Laboratories, Seoul, Korea), as described previously (Lee HJ, Xu X, Choe G, et al, 2010).

<Example 2>

Immunohistochemistry

Formalin-fixed and paraffin-embedded (FFPE) tissues were sectioned at a thickness of 4μm and stained using a Ventana automated immunostainer (Ventana Medical Systems, Tucson, AZ) according to the manufacturer’s protocol.

Specifically, the sectioned slides were dried at 60°C for 1 hour and deparaffinized using EZ Prep (Ventana Medical Systems) at 75°C for 4 minutes. The cells were conditioned (heat pretreatment) using a CC1 solution containing Tris/borate/ethylenediaminetetraacetic acid at 100°C for 20 minutes.

The antibody for ALK (mouse monoclonal, clone 5A4, Novocastra, Newcastle, United Kingdom) was diluted to 1:30, treated, and incubated at 42°C for 2 hours. Signals were detected using an i-view detection kit (Ventana Medical Systems) based on the labeled streptavidin-biotin (LSAB) method. The steps used with the kit included treatment with an inhibitor (1% H2O2, 4 minutes), biotinylated immunoglobulin (8 minutes), streptavidin-Horseradish peroxidase (8 minutes), diaminobenzidine (chromogen+substrate, 8 minutes), and copper (4 minutes) at 37°C. Counterstaining was performed with Mayer’s hematoxylin (ScyTek, Logan, UT) for 2 minutes at room temperature.

All the cases were evaluated and scored using the following evaluation system by three pathologists (J.H.P., G.C. and J.H.C.): 1) no staining was scored as 0; 2) positive staining in less than 5% of tumor cells or staining with a weak and faint intensity of 6 to 10 regardless of the percentage of positively stained cells was scored as 1+; 3) positive staining in more than 5% of tumor cells with an moderate staining intensity of 4 to 6 was scored as 2+; and 4) positive staining in more than 5% of tumor cells with a strong staining intensity of 0 to 4 was scored 3+.

<Example 3>

FISH (Fluorescence in situ hybridization)

FISH for ALK was performed on all the cases in the test set using TMA, and 14 cases were found to be ALK-positive through IHC in the validation set as previously reported (Boland JM, Erdogan S, Vasmatzis G, et al, Hum Pathol 2009;40:1152-1158, 2009).

3μm thick sections from FFPE tissue blocks fixed in formalin were deparaffinized, dehydrated, immersed in 0.2 N HCl, and washed. The sections were immersed in 0.01 M citrate buffer, boiled in a microwave for 5 minutes, treated with pretreatment reagent (Abbott Molecular Inc., Abbott Park, IL) at 80°C for 30 minutes, and reacted with protease mixed with a protease buffer (Abbott Molecular Inc.).

Dual-probe hybridization was performed using the LSI ALK dual-color probe, which hybridizes to the 2p23 band with SpectrumRed and SpectrumGreen on either side of the ALK gene breakpoint (Vysis, Downers Grove, IL). After applying the probe mixture, they were treated with protease, incubated in a humidified atmosphere with HybriteTM (Vysis) at 75°C for 5 minutes to denature the probe and the target DNA, and incubated at 37°C for 16 hours to allow hybridization. They were then immersed in 0.3% NP-40 (Abbott Molecular Inc.)/2X saline sodium citrate for washing the samples.

For the nuclei counterstaining, DAPI (4,6-diamidino-2-phenylindole) II (Vysis) and an antifade compound (p-phenylenediamine) were applied. Signals for each probe were evaluated under a microscope equipped with a triple-pass filter (diamidino-2-phenylindole/Green/Orange, Vysis) and an oil immersion objective lens.

All the FISH tests were performed without knowledge of the IHC results for ALK.

FISH for ALK locus rearrangement was considered positive if 5% of 200 cells analyzed showed splitting apart of the florescent probes flanking the ALK locus. This distance was estimated using the signal size as a reference.

Unlike normal interphase nuclei showing two fusion (orange and green adjacent or fused yellow) signals, all of the three signals were shown in more than 5% of tumor cells. That is, one fusion signal (native ALK) and two separate red and green (break-apart) signals (translocated ALK) were assessed as ALK gene translocation. Moreover, one fusion signal (native ALK) and one red signal with no corresponding green signal were found in more than 15% of tumor cells, and labeled “isolated red signal (IRSP)” pattern (see FIG. 1).

<Example 4>

EGFR and K-RAS Mutation Study

All the cases that were determined to be ALK FISH-positive were analyzed for EGFR mutations at exons 18 to 21 and K-ras mutations at codons 12, 13, and 61 using a direct DNA sequencing method and the polymerase chain reaction, as described previously (Chung JH, Choe G, Jheon S, et al. J Thorac Oncol 2009). DNA was extracted from paraffin-embedded tissue fixed in formalin, and PCR amplification was performed in a solvent with a total volume of 20 μL containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 2.5 mM deoxynucleotide triphosphates, 0.5 μM of each primer, 0.9 units Taq DNA polymerase (Takara Bio, Shiga, Japan). After preincubation at 94°C for 2 minutes, DNA was amplified for 35 cycles under the following conditions: denaturation for 30 seconds at 94°C, annealing for 30 seconds at 55℃ and elongation for 30 seconds at 72℃.

The PCR products were processed with BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster, CA), and sequence data were generated with the ABI PRISM 3100 DNA Analyzer (Applied Biosystems). All sequence variants were confirmed by sequencing the products of independent PCR amplifications in both directions.

<Example 5>

Analysis of Clinicopathologic Characteristics of ALK FISH-Positive and -Negative NSCLC

A total of 453 NSCLC cases were properly evaluated by FISH on tissue microarray (TMA) sections, and 12 TMA cores were excluded due to poor hybridization quality. As in the following Table 1, comparison of the clinicopathologic characteristics of ALK FISH-positive and -negative cases was carried out. 4.2% (19/453) of the total number of NSCLC patients was ALK FISH-positive, and they were strongly correlated with ADC histology, as compared to ALK FISH-negative NSCLC patients (p=0.001, by Mann-Whitney test, two-sided). Given clinicopathologic differences between ALK FISH-positive patients and ALK FISH-negative patients in a patient group test set and a validation set, ALK FISH-positive patients out of the total number of non-small cell lung cancer patients, which is the sum of the two groups, were younger than ALK FISH-negative patients, and this tendency was observed in the validation patient set (p=0.014, by Mann-Whitney test, two-sided) while no significant difference was observed in the test set (p>0.05). Thus, it was observed that age was significantly associated with lung cancer showing abnormality involving ALK gene rearrangement. Moreover, it was found that tumor size and sex were not significantly different between the two groups.

The Relationship between ALK FISH and Clinicopathologic and Genetic Features in the Test Set

Variables		No. (%)	ALK FISH-positive	ALK FISH-negative	P-value* (*=p<0.05)
Total		453 (100)	19 (4.2)	434 (95.8)
sex	Male	309 (68.2)	11 (57.9)	298 (68.7)	0.324
Female	144 (31.8)	8 (42.1)	136 (31.3)
Age (yr)	>65	230 (50.8)	6 (31.6)	224 (51.6)	0.103
≥65	223 (49.2)	13 (68.4)	210 (48.4)
Smoking habit	Never	171 (37.7)	10 (52.6)	161 (37.1)	0.226
Smoker	282 (62.3)	9 (47.4)	273 (62.9)
Tumor size (cm)	>3	210 (46.4)	6 (31.6)	204 (47.0)	0.242
≥3	243 (53.6)	13 (68.4)	230 (53.0)
Histology	ADC	263 (58.1)	18 (94.7)	245 (56.5)	<0.001*
SCC	163 (36.0)	0	163 (37.5)
Others	27 (5.9)	1 (5.3)	26 (6.0)
EGFR	Mutation (+)	41 (9.1)	0	41 (9.4)	<0.001*
Mutation (-)	49 (10.8)	19(100)	30 (6.9)
NA	363 (80.1)	0	363 (83.6)
KRAS	Mutation (+)	7 (1.5)	0	7 (1.6)	<0.001*
Mutation (-)	57 (12.6)	19(100)	38 (8.8)
NA	389 (85.9)	0	389 (89.6)
Pathological stage	I	198 (43.7)	8 (42.1)	190 (43.8)	1.000
II-IV	255 (56.3)	11(57.9)	244 (56.2)

The Relationship between ALK FISH and Clinicopathologic and Genetic Features in the Validation Set

Variables		No. (%)	ALK FISH-positive	ALK FISH-negative	P-value* (*=p<0.05)
Total		187 (100)	9 (4.8)	178 (95.2)
sex	Male	105 (56.1)	3 (33.3)	102 (57.3)	0.324
Female	82 (43.9)	6 (66.7)	76 (42.7)
Age (yr)	>65	71 (38.0)	0	71 (39.9)	0.103
≥65	116 (62.0)	9 (100)	107 (60.1)
Smoking habit	Never	104 (55.6)	6 (66.7)	98 (55.1)	0.226
Smoker	83 (44.4)	3 (33.3)	80 (44.9)
Tumor size (cm)	>3	77 (41.2)	5 (55.6)	72 (40.4)	0.242
≥3	110 (58.8)	4 (44.4)	106 (59.6)
Histology	ADC	187 (100)	9 (100)	178 (100)	<0.001*
SCC	0	0	0
Others	0	0	0
EGFR	Mutation (+)	62 (33.2)	0	62 (34.8)	<0.001*
Mutation (-)	56 (29.9)	9(100)	47 (26.4)
NA	69 (36.9)	0	69 (38.8)
KRAS	Mutation (+)	6 (3.2)	0	6 (3.4)	<0.001*
Mutation (-)	54 (28.9)	9(100)	45 (25.3)
NA	127 (67.9)	0	127 (71.3)
Pathological stage	I	64 (34.2)	3 (33.3)	61 (34.3)	1.000
II-IV	123 (65.8)	6(66.7)	117 (65.7)

The Relationship between ALK FISH and Clinicopathologic and Genetic Features in all NSCLC cases

Variables		No. (%)	ALK FISH-positive	ALK FISH-negative	P-value* (*=p<0.05)
Total		640 (100)	28 (4.4)	612 (95.6)
sex	Male	414 (64.7)	14 (50.0)	400 (65.4)	0.324
Female	226 (35.3)	14 (50.0)	212 (34.6)
Age (yr)	>65	301 (47.0)	6 (21.4)	295 (48.2)	0.103
≥65	339 (53.0)	22 (78.6)	317 (51.8)
Smoking habit	Never	275 (43.0)	16 (57.1)	259 (42.3)	0.226
Smoker	365 (57.0)	12 (42.9)	353 (57.7)
Tumor size (cm)	>3	287 (44.8)	11 (39.3)	276 (45.1)	0.242
≥3	353 (55.2)	17 (60.7)	336 (54.9)
Histology	ADC	450 (70.3)	27 (96.4)	423 (69.1)	<0.001*
SCC	163 (25.5)	0	163 (26.6)
Others	27 (4.2)	1 (3.8)	26 (4.2)
EGFR	Mutation (+)	103 (16.1)	0	103 (16.8)	<0.001*
Mutation (-)	105 (16.4)	28(100)	77 (12.6)
NA	432 (67.5)	0	432 (70.6)
KRAS	Mutation (+)	13 (2.0)	0	13 (2.1)	<0.001*
Mutation (-)	111 (17.3)	28(100)	83 (13.6)
NA	516 (80.6)	0	516 (84.3)
Pathological stage	I	262 (40.9)	11 (39.3)	251 (41.0)	1.000
II-IV	378 (59.1)	17(60.7)	261 (59.0)

<Example 6>

Correlation between ALK Protein Expression as Determined by IHC and FISH results

ALK protein expression was observed in tumor cells with a predominantly cytoplasmic staining pattern. Nonneoplastic bronchial epithelium, alveolar type I and type II pneumocytes, mesenchymal tissue, and inflammatory cells in the adjacent lung tissue were IHC-negative for ALK in all of the 465 cases.

The percentage of ALK immunostained tumor cells is substantially higher than 5% of the total number of tumor cells in most cases of ALK protein expression. The staining intensity ranges from weak and fain cytoplasmic staining to dark brown cytoplasmic staining. ALK protein expression was detected in 8.6% (40/465) of the NSCLC cases, consisting of IHC scores of 1+(14/40, 35%), 2+(10/40, 25%), and 3+(16/40, 40%) (see FIGs. 2 to 5).

To determine the best criteria for IHC assessment to predict gene rearrangements, we compared the ALK results using IHC and FISH as shown in Table 4. All the cases with IHC scores of 3+ were FISH-positive, and all the cases with IHC scores of 0 or 1+ were FISH-negative. For cases with scores of 2+, 30%(3/10) were FISH-positive and 70%(7/10) were FISH-negative.

Accordingly, the present inventors analyzed the predictability of ALK IHC for ALK FISH status. Assuming that ALK IHC scores of 0 and 1+ were biologically negative, an ALK IHC score of 3+ was biologically positive, and an ALK IHC score of 2+ was equivocal, the results of biological ALK IHC assay and FISH were strongly correlated (p=0.000 by Pearson’s chi-square test, Pearson’s R-0.933, p=0.000). After excluding cases with ALK IHC scores of 2+ (equivocal), the ALK IHC scores (0 and 1+ versus 3) and ALK FISH (negative versus positive) were completely correlated. These results indicate that an ALK IHC score of 3+ could strongly predict FISH-positivity and an IHC score of 0 or 1+ could predict FISH-negativity (see Table 4). Therefore, the present inventors derived a diagnostic algorithm of clinical application of ALK IHC and ALK FISH (see FIG. 6).

The Relationship between ALK IHC and FISH in the Test Set

ALK IHC	ALK FISH-positive NSCLC	ALK FISH-negative NSCLC	Total (%)
0	0	413 (91.2)	413 (91.2)
1+	0	14 (3.1)	14 (3.1)
2+	3 (0.7)	7 (1.5)	10 (2.2)
3+	16 (3.5)	0	16 (3.5)
Total (0%)	19 (4.2)	434 (95.8)	453 (100)

<Example 7>

Validation of the IHC Interpretation Criteria to Detect ALK FISH-Positive NSCLC

As a validation set, the present inventors performed IHC and FISH tests on surgically resected or biopsied specimens from 187 patients with suspected ADC. Among these specimens, 14 were positively stained for ALK, including two cases of ALK IHC score of 1+, six with a score of 2+, and six with a score of 3+. The 14 cases included 7 surgically resected specimens and 7 biopsied specimens that were taken from 9 females and 5 males. The age of the patients ranged from 28 to 84 years with a mean age of 55.1 years.

The FISH results were as follows: all the IHC-negative patients were FISH-negative, two cases of IHC 1+ (biologically negative) were FISH-negative, 6 cases of IHC 3+ (biologically positive) were FISH-negative (see Tables 5 and 6), and the cases of IHC 2+ were 50% (3/6) FISH-positive. The FISH-positive cases included eight break-apart (split) patterns and two IRS patterns.

As predicted, all the ALK FISH-positive cases were negative for EGRF and K-RAS mutations, while two ALK IHC 1+ cases had EGFR exon 19 deletion mutations. Also, ALK IHC 1+ (biologically negative) and 3+ (biologically positive) cases maintained complete predictability for FISH in the validation set. Moreover, the majority of the ALK IHC 2+ cases was ALK FISH-positive, which was consistent in the results of the test set using TMA.

The Relationship between ALK IHC and FISH in the Validation Set

ALK IHC	ALK FISH-positive NSCLC	ALK FISH-negative NSCLC	Total (%)
0	0	173 (92.5)	173 (92.5)
1+	0	2 (1.1)	2 (1.1)
2+	3 (1.6)	3 (1.6)	6 (3.2)
3+	6 (3.2)	0	6 (3.2)
Total (0%)	9 (4.8)	178 (95.2)	187 (100)

The Relationship between ALK IHC and FISH in All NSCLC Cases

ALK IHC	ALK FISH-positive NSCLC	ALK FISH-negative NSCLC	Total (%)
0	0	586 (91.6)	586 (91.6)
1+	0	16 (2.5)	16 (2.5)
2+	6 (0.9)	10 (1.6)	16 (2.5)
3+	22 (3.4)	0	22 (3.4)
Total (0%)	28 (4.4)	612 (95.6)	640 (100)

<Example 8>

Analysis of Correlation between ALK IHC and FISH According To Antibody Response Conditions

To verify the accuracy of ALK IHC test results through ALK antibody response according to the present invention, the correlation between ALK IHC results of specimen and FISH results thereof according to various conditions of antibody response was analyzed. If IHC results showing ALK protein overexpression is highly correlated with FISH results showing ALK gene rearrangement, it will be possible to detect ALK gene rearrangement by an economical and quick IHC method, instead of expensive and long time-consuming FISH.

Accordingly, the present inventors compared and analyzed the correlation between IHC and FISH according to various response conditions in order to determine whether the ALK antibody response conditions established in the present invention can ensure high correlation between ALK IHC and FISH. That is, IHC results of specimens according to various ALK antibody conditions were classified by scores on a scoring system based on criteria for the analysis of ALK IHC staining patterns. Among them, the concordance for a group with scores of 0 and 1+ determined to be “biologically negative” with the FISH result was compared the concordance for a group with a score of 3+ determined to be “biologically positive “ with the FISH result. For the “biologically positive group”, it was determined that the two results (IHC and FISH) were concordant with each other if the FISH result was negative for ALK translocation, i.e., no ALK gene rearrangement occurred, and for the “biologically negative group”, it was determined that the IHC result and the FISH result were concordant with each other if the FISH result showed ALK translocation and confirmed the presence of ALK gene rearrangement.

To measure the accuracy of ALK and IHC results according to antibody response conditions, an ALK IHC test was performed on an ALK antibody at a ratio of 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, and 1:40. The result showed, as shown in Table 7, high concordance, i.e., correlation between IHC results and FISH when the ALK antibody was treated with a dilution of 1:25 and 1:35, as compared to the treatment of the ALK antibody with a dilution of 1:20 or 1:40.

Concordance between ALK IHC results and FISH According to Dilution Ratio of ALK Antibody

	1:10	1:15	1:20	1:25	1:30	1:35	1:40
IHC 0, 1+ (biologically negative)	48%	55%	68%	98%	100%	97%	60%
IHC
3+ (biologically positive)	42%	50%	60%	98%	100%	98%	55%

Moreover, the present inventors conducted a comparative test on incubation time, which is another condition of ALK antibody response. After conducting an IHC test for various incubation times as shown in Table 8, the results of the IHC test for ALK antibody response for 110 minutes and 130 minutes showed high concordance with FISH, as opposed to the incubation of the ALK antibody with specimen samples for 60 minutes, 90 minutes, 150 minutes, and 180 minutes.

Concordance between ALK IHC results and FISH According to Incubation Time of ALK Antibody

	60 mins	90 mins	110 mins	120 mins	130 mins	150 mins	180 mins
IHC
0, 1+ (biologically negative)	68%	75%	98%	100%	98%	80%	70%
IHC
3+ (biologically positive)	70%	72%	98%	100%	99%	70%	68%

In addition, the present inventors conducted a comparative test on antibody response temperature. Under the optimum ALK response condition found in the aforementioned comparative test, the ALK antibody was diluted at a ratio of 1:30 and incubated for 120 minutes at different reaction temperatures. As shown in Table 9, the results of the IHC test performed at 40℃ and 45℃ showed high correlation with the FISH results as compared to the IHC results performed at 35℃ and 50℃.

Concordance between ALK IHC results and FISH According to Temperature of ALK Antibody Response

	35℃	40℃	42℃	45℃	50 ℃
IHC
0, 1+ (biologically negative)	65%	97%	100%	98%	60%
IHC
3+ (biologically positive)	62%	99%	100%	98%	55%

Accordingly, the present inventors confirmed that excellent ALK IHC results showing the highest concordance with FISH results could be derived from an ALK antibody response in which the ALK antibody was diluted at a ratio of 1:25 to 1:35 and incubated for 90 to 120 minutes at a temperature of 40 to 45℃.

Although the invention has been described focusing on the preferred embodiments, those skilled in the art will appreciate that the invention may be carried out in modified forms without departing from the essential characteristics of the present invention. Therefore, the above embodiments should be construed in all aspects as illustrative and not restrictive. The scope of the invention should be determined by the appended claims and their legal equivalents, not by the above description, and all changes coming within the equivalency range of the appended claims should be construed as being embraced in the invention.

Claims (13)

1. A method for detecting ALK gene rearrangement, the method comprising the steps of:
   reacting an ALK antibody with a pretreated specimen;
   staining the specimen reacted with the ALK antibody; and
   analyzing the staining pattern of the specimen.
2. The method of claim 1, wherein the reaction of the ALK antibody is performed by a method selected from the group consisting of immunohistochemistry, immunoblot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), agglutination and radioimmunoassay.
3. The method of claim 1, wherein the pretreatment of the specimen comprises deparaffinizing the paraffin sections and heat-treating the deparaffinized paraffin sections in a CCL₄ solvent containing a tris-borate-EDTA (TBE) buffer at 95℃ to 105℃ for 15 to 25 minutes.
4. The method of claim 1, wherein the reaction of the ALK antibody comprises treating an ALK antibody at the dilution of 1:25 to 1:35 and reacting the ALK antibody for 90 to 120 minutes at a temperature of 40 to 45℃.
5. The method of claim 1, wherein the staining step comprises the steps of:
   treating a secondary antibody binding to the ALK antibody; and
   treating a chromogenic substrate.
   
   
6. The method of claim 5, wherein the chromogenic substrate is selected from the group consisting of BCIP/NBT (5-bromo-4-chloro-3-indolyl- phosphate/nitroblue tetrazolium), DAB (diaminobenzidine), TMB (3, 3' , 5, 5' -tetramethyl benzidine), BCIP/INT (5-bromo-4-chloro-3- indolyl phosphate/iodonitrotetrazolium), NF (New fuchsin), FRT(FaSt Red TR Salt), ABTS[2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), 4-CN, AEC (3-amino-9-ethylcarbasole), X-Gal, and IPTG (isopropyl beta-D-thiogalactoside).
   
   
7. The method of claim 1, wherein the step of analyzing the staining pattern of the specimen comprises the step of counting the number of cells with cytoplasmic staining or the step of determining the cytoplasmic staining intensity.
8. The method of claim 7, wherein, if the number of cells with cytoplasmic staining is less than 5% of the total number of cells of the target, ALK gene rearrangement is considered negative.
9. The method of claim 7, wherein, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells of the target and the cytoplasmic staining intensity is weak, ALK gene rearrangement is considered negative.
10. The method of claim 7, wherein, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells of the target and the cytoplasmic staining intensity is strong, ALK gene rearrangement is considered positive.
11. The method of claim 7, wherein, if the number of cells with cytoplasmic staining is more than 5% of the total number of cells and the cytoplasmic staining intensity is moderate, the method may further comprise the step of detecting ALK gene translocation by a method selected from the group consisting of FISH (fluorescence in situ hybridization), SISH (Silver In Situ Hybridization), and PCR (polymerase chain reaction).
12. A method for diagnosing cancer using the ALK gene rearrangement detection method according to any of claims 1 to 11.
13. The method of claim 12, wherein the cancer is lung cancer.

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