METHOD FOR DETERMINING TREATMENT OF INDIVIDUALS WITH

FRAGILE SITE

This application claims priority to U.S. provisional patent no. 60/843,657, filed September 11, 2006, the disclosure of which is incorporated herein in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of cancer treatment and more particularly to methods for analyzing chemotherapeutic reagents and treatment of cancer patients.

DESCRIPTION OF RELATED ART

Metastasis is the spread of cancer from a primary site and the formation of new tumors in distant organs. When cancer is detected at an early stage, before it has spread, it can often be treated successfully by surgery or local irradiation. However, when cancer is detected after it has metastasized, treatments are much less successful. Furthermore, for many patients in whom there is no evidence of metastasis at the time of their initial diagnosis, metastases can occur at a later time, even decades after apparently successful primary treatment.

Metastases can show an organ-specific pattern of spread that might occur years or even decades after apparently successful primary treatment. Breast cancer (BC), the most frequent cancer in the female population of industrialized countries, often metastasizes to bone. Metastases to bone occur in >70% of patients with advanced disease. Despite some advances in chemotherapeutic regimens, it is virtually impossible to cure breast cancer-induced metastasis and osteolysis. Metastasis of some other cancers is also considered to be organ specific. For example, prostate cancer is known to spread to bone.

A persistent clinical challenge that spans all types of cancer has been to predict which patients will actually progress from localized to metastatic disease and which will remain disease-free following initial therapy. While the mechanism of malignancy or

metastasis are not completely understood, chromosomal breakage is one mechanism by which functional loss of tumor suppressor gene activity may occur. Chromosomal locations in which breakage may be induced are known as fragile sites. Fragile site breakages may be induced, for example by certain chemicals, by hypoxia, or by physical force (such as a physical shock to the media containing the DNA). Fragile sites have been shown to be involved in some malignancies in which the fragile site lies within known genes, such as the FHIT gene (chromosome 3p) in lung cancer, and where small deletions are consistently observed on chromosome 3 (Mimori K, et al. Proc Natl Acad Sci U S A 1999 Jun 22;96(13):7456-61; Menin C, et al. Cancer Genet Cytogenet 2000 May;l 19(1):56-61). These fragile sites are inherited in a dominant Mendelian fashion. They are also known to contain specific motifs repeated more than 200 times. It has previously been shown that two fragile sites exist on the long arm of chromosome 12. FRAl 2B is located at 12q24.13 (Voiculescu I, et al. Hum Genet 1991 Feb;86(4):416-7) and FRA12E has been located at 12q24.2-3 (Sutherland GR. (1979) Am J Hum Genet 31: 136-148). It has also been previously estimated that approximately 5% of the human population is positive for one of these two fragile sites. However, this information has not been previous utilized as a factor in developing treatment modalities for such individuals. Moreover, the potential effect of chemotherapeutic agents on fragile sites has not been previously investigated or considered a factor when selecting chemotherapeutic regimens for treating cancer patients. Therefore, there is a need to determine whether particular chemotherapeutic agents adversely affect patients who harbor fragile sites, and to use such determinations for generating chemotherapeutic regimens for individual patients taking into account their fragile site status.

SUMMARY OF THE INVENTION

The present invention provides a method for assisting a health care provider in the development of a therapeutic plan for an individual at risk for, suspected of having, or diagnosed with cancer, comprising the steps of determining and communicating the FRA12E status of the individual to a health care provider who develops a therapeutic plan for the individual.

Also provided is a method for determining whether a chemotherapeutic agent induces breakage of human chromosome 12 in the FRAl 2E fragile site in the 12q24

region in the SMRT locus. The method comprises providing a cell known to contain a FRAl 2E fragile site, contacting the cell with a test chemotherapeutic agent and determining whether contacting the cell with the test chemotherapeutic agent induces breakage of the FRA12E fragile site. Breakage of the FRA12E fragile site can be determined by using any of a variety of assays that directly or indirectly analyze chromosome breakage.

The invention also utilizes the determination of whether a particular chemotherapeutic agent induces breakage of the FRA 12E fragile site in a method of recommending a course of chemotherapy for an individual. The method comprises obtaining a biological sample from an individual diagnosed with or suspected of having cancer and determining the presence or absence of a FRAl 2E fragile site in the sample. Depending on whether the individual is FRAl 2E positive or negative, the suitability of a chemotherapeutic agent for treating the individual can be determined. In particular, the presence of the FRA 12E fragile site is indicative for treatment of the individual with a chemotherapeutic agent that is known not to induce breakage of the FRAl 2E fragile site and is contraindicative for treatment of the individual with a chemotherapeutic agent that is known to induce breakage of the FRA 12E site fragile site. Conversely, the absence of the FRAl 2E fragile site is indicative for treatment of the individual with a chemotherapeutic agent that is known to induce a breakage within the FRA 12E fragile site and/or with a chemotherapeutic agent that is known not to induce breakage of the FRAl 2E fragile site.

BRIEF DESCRIPTION OF THE FIGURES

Figures IA and IB are schematic representations of mapping of the RPCIl 1 bacterial artificial chromosome (BAC) clones used to detect and map the FRA 12E fragile site within the SMRT gene. Figure IA shows BAC clones 339, 665 and 667 encompassing the SMRT locus; (BAC 469 is used as a control probe). Figure IB provides a schematic representation of the potential spreading of breakages over the SMRT locus. Figure 2 is a photographic representation of FISH analysis of a normal chromosome and a chromosome wherein a breakage was induced at the FRA 12E fragile site within the SMRT gene. To obtain these data, chromosomes were harvested after induction of breakage of the fragile sites with aphidicolin and hybridized with

the SMRT-specific RPCIl 1 BAC clones (green) and control BAC clone (red). One chromosome 12 shows a normal pattern of hybridization wherein the predominant signal is yellow (due to the overlapping/juxtaposition of the green and the red signals). The other signal shows a split green signal, next to a main yellow signal indicating that the BAC clones span a DNA double strand break due to activation of the fragile site. The SMRT specific probe hybridized on both sites of the breakage, giving rise to the split FISH signal.

Figure 3A and 3B provide photographic and graphical representations, respectively, of an RT-PCR time-course analysis of SMRT mRNA subsequent to incubation of 5256 FS + (cells with the FRA12E site) and EBV Lin FS- (cells without the FRA 12E site) cell lines with aphidicolin.

Figure 4 is a photographic representation of an RT-PCR time-course analysis of SMRT mRNA subsequent to incubation of 5256 FS + and EBV Lin FS- with doxorubicin. Figure 5 is a photographic representation of mmunofluorescence results performed by incubating the indicated cell lines with aphidicolin and detecting SMRT protein expression using a commercially available rabbit polyclonal anti-SMRT antibody (Affinity BioRreagents) followed by a secondary anti-rabbit-FITC. The counterstain is DAPI.

DETAILED DESCRIPTION OF THE INVENTION

According to standard fragile site nomenclature, FRAl 2E is the 5th potential fragile site on human chromosome 12 counting from the telomere of the p arm towards the q telomere. As described in U.S. Patent Publication No. 20050191674, the disclosure of which is incorporated herein by reference, when FRAl 2E is present, it is located within the SMRT gene locus at 12q24.2-3. This locus has also been described in Jiang et al. [21]. Therefore, a breakage at the FRA12E site results in breakage of the SMRT locus.

Our research in breast cancer, prostate and kidney cancer patients, as well as the involvement of SMRT down-regulation in several other cancers, such as melanoma, colorectal cancer, and endometrial cancer, demonstrate that breakage within FRAl 2E results in damage to the integrity of the SMRT gene which is correlated with increased metastasis and a poor prognosis for the individual in which

the breakage occurs. Therefore, it is desirable to mitigate breakages at the FRA 12E site, such as by reducing the exposure of a patient to agents that can induce breakage of the fragile site. Accordingly, the present invention provides a method for determining whether a chemotherapeutic agent can induce breakage of the FRAl 2E fragile site. The method comprises providing a cell known to contain a FRAl 2E fragile site, contacting the cell with one or more test agents and determining whether contacting the cell with the test agent(s) induces breakage of the FRA 12E fragile site. While any method for determining whether a breakage at the FRA 12E site has occurred can be used in the method of the invention, irrespective of the method used, if the agent is found to induce a breakage, the resulting characterization of the agent as an inducer of the breakage is useful in a method of determining a chemotherapy for an individual diagnosed with or suspected of having cancer. This method generally comprises the steps of obtaining a biological sample from the individual and determining the presence or absence of a FRA 12E fragile site in the sample, where the presence of the FRA 12E fragile site is indicative for treatment of the individual with an agent that has been determined not to induce breakage of the FRA 12E fragile site and is contraindicative for treatment of the individual with an agent that is known to induce breakage of the FRA12E site fragile site. Likewise, the absence of the FRA 12E fragile site is indicative that the individual may be treated with a chemotherapeutic agent that has been determined to induce breakage of the FRA 12E fragile site and/or with an agent that has been determined not to induce breakage of the FRAl 2E fragile site. In one embodiment, if an individual is determined to have the FRAl 2E site, the proposed chemotherapeutic agent(s) for treating that individual can be tested against a biological sample obtained from the individual using a method of the invention to verify that the agent(s) do or do not induce the break.

In another embodiment, the present invention provides a method for assisting a health care provider in the development of a therapeutic plan for an individual who is suspected of having, has been diagnosed with, or is at risk for developing cancer. The method comprises the steps of determining the FRA 12E status of the individual and communicating the FRA 12E status of the individual directly or indirectly to a health care provider who determines a therapeutic plan for the individual and/or treats the individual. In this manner, the health care provider, such as a physician, is able to determine prescription of a chemotherapeutic agent based upon the FRA 12E status of

the individual. Thus, prescribing a chemotherapeutic agent that is known to induce breakage of the FRA 12E site in a FRA 12E positive individual can be avoided by the health care provider. It is contemplated that in the development of the therapeutic plan, the health care provider will also rely on information characterizing chemotherapeutic agents as inducers or non-inducers of breakage of FRA12E as determined in accordance with the present invention. Communication of the FRAl 2E status to the health care provider can be performed by any method, such as verbally, by electronic correspondence, by providing paper copies of FRAl 2E status test records, or any other method by which FRA 12E status can be communicated. The FRA 12E status may be communicated to the health care provider by any individual or by any manually operated or automated process, machine or device. Determining whether an individual is at risk for developing cancer can be performed according to conventional techniques known to those skilled in the art, such as by analysis of family history and/or other risk factors, such as cigarette smoking or occupational risks.

In respect of the potential risks associated with treating a FRA 12E positive individual with a chemotherapeutic agent that induces breakages of the FRA 12E site, and without intending to be bound by any particular theory, it is considered that such chemotherapeutic agents are lethal to the majority of cancer cells, and therefore breakages of the FRAl 2E site in these cells prior to lethality due to administration of the agent will be of insignificant consequence. However, it is believed that in typical chemotherapies, a percentage of cancer cells are exposed to sub-lethal doses of the agent due to incomplete vascularization of the tumor tissue or because of other anatomical or physiological circumstances that prevent the cell from receiving a lethal dose. In these situations, it is plausible that the cancer treatment may induce a breakage at the FRAl 2E fragile site, thereby contributing to the survival, proliferation, drug resistance and motility advantages that are the characteristics of metastatic cells. Therefore, it is possible that by prescribing agents that are determined to not induce breakages of the FRA 12E site, circumstances whereby administration of a chemotherapeutic agent contributes to a worsening of the condition of a patient can be avoided.

In general, determining fragile site breakages entails incubation of cells known to contain the FRAl 2E fragile site with the test agent for a period of time,

after which a suitable analysis procedure is performed to ascertain whether or not a breakage at the FRA 12E site has been induced.

Breakage of the FRA 12E site can be detected either directly or indirectly using a variety of techniques, some of which utilize hybridization of polynucleotides or oligonucleotides (also referred to as "probes" herein) to FRA 12E sequences in the SMRT locus. Such polynucleotides can be provided alone, in phages, plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or any other suitable vector without limitation.

It is well known in the art that probes for nucleic acid hybridization based detections of complimentary sequences can be as small as 20 bases. Thus, for example when using BAC clones as probes, either the entire BAC clone can be used or portions thereof (20 bases or more) such that detection of a breakage of the fragile site can be effected. For FISH assays an example of a convenient length of probes which are made up of portions of BAC clones is about 1 kilo base. In other embodiments, breakage can be detected using probes designed based upon the genomic sequence of FRAl 2E and using the probes in PCR, QPCR, in situ PCR, primed in situ hybridization, or Southern Hybridization to elucidate the break.

In one embodiment, the invention provides a method for detecting the breakage of the FRA 12E site after incubation with a test agent by contacting the cells with one or more probes. The probes used in the present methods are polynucleotides or oligonucleotides which comprise sequences complementary to the 12q24 region at the SMRT gene locus.

The sequence of the oligonucleotide for hybridization assays or for PCR amplification will depend upon several factors known in the art. Primarily, the sequence of the oligonucleotide will be determined by its capacity to bind to the FRA 12E site.

Detection of the break can be performed by amplifying the SMRT gene locus using one of a variety of methods known in the art (such as PCR), then assaying the amplified DNA to determine a breakage at the FRA 12E site, for example by determining the average lengths of the polynucleotides before or after inducing FRAl 2E to break.

Indirect detection of a break can be performed, for example, by evaluating expression of mRNA for SMRT after inducing breakage at the FRA 12E site. This

can be performed, for example, using semi-quantitive RTPCR or quantitative RTPCR (QRTPCR). It can also be performed by northern blot or by using RNA probes for hybridization, for example in situ. The presence of a breakage at FRAl 2E can also be indirectly detected by evaluating expression of the SMRT protein, for example by immunodetection or immunoassay (such as Western Blot, ELISA, fluorescent labeling, radioimmunoassay, secretion assay, or immunostaining), nuclear staining (for example with visual or flow cytometry detection), or other non-immunoassays known to those skilled in the art.

Breakage of the FRA 12E fragile site could also be indirectly detected by assaying the levels of metabolic byproducts which result when there is alteration to SMRT expression as a result of the breakage of FRAl 2E. For example, differences in cell secretions due to altered biochemical pathways could be detected by a tissue, blood, or urine test. Alternatively, alterations of cell surface expressed proteins could be detected, for example by antibody labeling and flow cytometry. In one embodiment, breakages of FRAl 2E are detected by in situ hybridization techniques. In the performance of such techniques typically, prior to harvesting the cells (such as 24 hours prior to harvesting), the cells are incubated with the chemotherapeutic test agent and preferably a control test agent known to induce breakage at the FRAl 2E site, and subsequently exposed to a microtubule blocker (such as Colcemid) to block the cells in metaphase, where chromosomes are highly condensed and can be visualized. Chromosome preparations are then fixed (such as with a solution of methanol/acetic acid (3:1 vol/vol) and then spread onto slides. Pretreatments (such as RNase and pepsin) can be applied to the preparations to potentially lower the background and to facilitate the penetration of the probes in the nuclei, respectively.

The cell suspension is applied to slides such that the cells are preferably present as a single layer. Cell density can be measured by a light or phase contrast microscope. Prior to in situ hybridization, chromosomal probes and chromosomal DNA are denatured. Denaturation typically is performed by incubating in the presence of high pH, heat (e.g., temperatures from about 70°C to about 95°C), organic solvents such as formamide and tetraalkylammonium halides, or combinations thereof. For example, chromosomal DNA can be denatured by a combination of temperatures above 70° C (e.g., about 73°C) and a denaturation buffer containing 70% formamide

and 2XSSC (0.3M sodium chloride and 0.03 M sodium citrate). Denaturation conditions typically are established such that cell morphology is preserved. Chromosomal probes can be denatured by heat. For example, probes can be heated to about 73 ⁰ C for about five minutes. After denaturation, hybridization is carried out. This hybridization can be performed using a variety of laboratory techniques known to those skilled in the art, for example, in situ hybridization, florescent in situ hybridization (FISH), Southern Hybridization, liquid hybridization, or micro arrays (for example using re-sequencing arrays). As is known to those skilled in the art, nucleic acid hybridization conditions vary, depending on the concentrations, base compositions, complexities, and lengths of the probes, as well as salt concentrations, temperatures, and length of incubation. The higher the concentration of probe, the higher the probability of forming a hybrid. For example, in situ hybridizations are typically performed in hybridization buffer containing 1-2XSSC, 50% formamide and blocking DNA to suppress non-specific hybridization. In general, hybridization conditions include temperatures of about 25°C to about 55 ⁰ C, and incubation lengths of about 0.5 hours to about 96 hours. More particularly, hybridization can be performed at about 32°C to about 40 ⁰ C for about 2 to about 16 hours. Non-specific binding of chromosomal probes to DNA outside of the FRA 12E region can be reduced by a series of washes. Temperature and concentration of salt in each wash depend on the desired stringency. For example, for high stringency conditions, washes can be carried out at about 65°C to about 80 ⁰ C, using 0.2X to about 2XSSC, and about 0.1% to about 1% of a nonionic detergent such as Nonidet P- 40 (NP40). Stringency can be lowered by decreasing the temperature of the washes or by increasing the concentration of salt in the washes.

Chromosomal probes typically are chosen for maximal sensitivity and specificity. The probes generally range from about 50 to about IXlO ⁵ nucleotides in length. Chromosomal probes typically are directly labeled with a fiuorophore, an organic molecule that fluoresces after absorbing light of lower wavelength/higher energy, but could also be labeled radioactively, chromogenically, or with a tag (such as biotin or digoxygenin) which requires a secondary molecule for detection. In a preferred embodiment, the fiuorophore allows the probe to be visualized without a

secondary detection molecule. After covalently attaching a fluorophore to a nucleotide, the nucleotide can be directly incorporated into the probe with standard techniques such as nick translation, random priming, and PCR labeling. Alternatively, deoxycytidine nucleotides within the probe can be transaminated with a linker. The fluorophore then is covalently attached to the transaminated deoxycytidine nucleotides. Fluorophores of different colors are chosen such that each chromosomal probe in the set can be distinctly visualized. Suitable fluorophores include: 7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas Red™ (Molecular Probes, Inc., Eugene, Oreg.), 5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC), 7- diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5— (and-6)- isothiocyanate, 5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3- carboxylic acid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid, N-(4,4- difluoro-5,7-dimethyl-4-bora— 3a,4a diaza-3-indacenepropionic acid, eosin-5- isothiocyanate, erythrosin-5-isothiocyanate, and Cascade,™ blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.). Probes are viewed with a fluorescence microscope and an appropriate filter for each fluorophore.

Probes also can be indirectly labeled with, for example, biotin or digoxygenin, or labeled with radioactive isotopes such as ³² P and ³ H, although secondary detection molecules or further processing then is required to visualize the probes. For example, a probe indirectly labeled with biotin can be detected by avidin conjugated to a detectable marker. For example, avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase. Enzymatic markers can be detected in standard colorimetric reactions using a substrate and/or a catalyst for the enzyme. Catalysts for alkaline phosphatase include 5-bromo-4-chloro-3- indolylphosphate and nitro blue tetrazolium. Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Examples of probes useful for detecting breakages of FRAl 2E include portions of the SMRT gene of sufficient length to hybridize to the SMRT gene locus, or the entire SMRT gene locus. In one embedment, such probes are provides as BAC clones (such as commercially available clones from the Roswell Park Cancer Institute (RPCI) BAC library). The sequences of suitable clones are easily accessed if necessary, for example, through GenBank at the National Center for Biotechnology

Information (NCBI). Suitable examples of probes for FISH analysis include BAC constructs BAC-RPl 1-339Bl 9, BAC-RPl 1-665C13, BAC-RP 11-677L6, BAC-RPI l- 408118 and BAC-RPl 1-30G17. The GenBank references for these sequences are: AC068837 (July 13, 2000 entry; BAC-RPl 1-339Bl 9); AC027706 (May 2, 2000 entry; BAC-RPl 1-665C13); AC025685 (May 26, 2000 entry; BAC-RPl 1-677L6); AC073916 (March 27, 2003 entry; BAC-RPl 1-408118) and AC069261 (March 27, 2003 entry; BAC-RPl 1-30G17). Each of these BAC clones are available to the public from, for example, the RPCI, Buffalo, New York. While any one of these probes or portions thereof of sufficient complementarity to the SMRT locus such that it can hybridize to the locus can be employed, it is preferable to use more than one probe such that a majority of the SMRT gene locus is encompassed.

In one embodiment, three overlapping probes which span the entire SMRT gene locus are used. For example, by using a panel of probes such as BAC-RPl 1 - 339Bl 9, BAC-RP 11-665C 13 and BAC-RPl 1-677L6, any breakage of chromosome 12 in the sequence of the entire SMRT gene locus can be assessed. Alternatively, two probes which bind to sequences located at the 3' and 5' ends of the SMRT gene respectively, may be employed. For one embodiment, we have determined that RPl 1-677L6 and RPl 1-665C13 alone encompass a sufficient amount of the SMRT locus such that they can be employed in a FISH assay to determine whether a chromosomal breakage has occurred within the SMRT locus from patient samples which comprise the FRA12E fragile site. Negative control probes can also be used in the hybridization assays to improve accuracy of the results. The negative control probes comprise nucleotide sequences corresponding to regions of chromosome 12 outside the SMRT locus, such as BAC RPl 1-469 A24 (also available to the public from the RPCI BAC library).

As an alternative to visualization of chromosomal breakage of the fragile site using in situ visualization techniques such as FISH, a chromosomal break induced by a test agent can be determined by detecting alterations in SMRT gene expression. For example, we demonstrate that a reduction in SMRT gene expression as evidenced by a reduction SMRT mRNA and/or SMRT protein expression is indicative that a test agent induces breakage of the FRA 12E fragile site. In particular, when cells that contain the FRA 12E fragile site are for alterations in SMRT gene expression, a transient reduction in SMRT mRNA expression occurs between approximately 2 and

5 hours after treating cells with an agent known to induce fragile site breakage (aphidocholin), and that a test agent (doxorubicin) heretofore unknown to have an adverse effect on chromosome integrity also induces this effect. In contrast, cells which do not contain the FRA 12E fragile site do not exhibit such transient reduction in SMRT mRNA expression after incubation with the agent. (The sequence of the SMRT cDNA is presented in SEQ ID NO:1.) Likewise, it is also demonstrated that when alterations in SMRT protein expression from cells that contain the FRA 12E fragile site are evaluated, a reduction in SMRT protein expression occurs approximately 18 hours after treating cells with an agent known to induce fragile site breakage (aphidocholin), while cells that do not contain the FRA 12E fragile site do not exhibit this reduction in SMRT protein expression. Accordingly, the same technique for analyzing alterations in SMRT protein expression can be applied to evaluate test chemotherapeutic agents, and aphidocholin is one example of an agent that can be employed as a positive control in such an assay. Determining whether the FRAl 2E is present in an individual can be performed by analyzing any of a variety of biological samples obtained from the individual. Importantly, since FRAl 2E is a somatic trait, its presence could be detected in a sample taken from a tumor or any other part of the body. For example, it could be detected from a blood sample of the patient, from lymphocytes taken from the patient, or from an endothelial or other tissue sample taken from the patient using any of the methods which are known in the art for taking tissue or other biological samples from a patient. The sample could also be taken from a culture of tissue which has been grown from a biological sample taken from a patient. For example, the culture could be an in vitro culture or an in vivo propagation of the tissue either in the patient or in some other individual or animal. The culture could also be of an immortalized cell line derived from a tissue taken from the patient. Thus, it will be apparent to those skilled in the art that this invention encompasses virtually every method by which a genetic sample might be acquired and/or amplified from a patient. The present invention is also useful for determining a chemotherapeutic treatment for individuals with a variety of malignancies. For example, such malignancies might include solid tumors (such as prostate, breast, colorectal, lung (small cell and non-small cell), ovarian, melanoma, urinary system, uterine, endometrial, pancreatic, oral cavity, thyroid, stomach, brain and other nervous system,

liver, and esophagial, to give a few examples) or hematological cancers (such as Hodgkins Lymphoma, Non-Hodgkins Lymphoma (NHL), chronic and other leukemias, and myeloma, to give a few examples).

The presence or absence of the FRA 12E site can be detected using any of the methods set forth herein for determining whether breakages are induced by test chemotherapeutic agents, with the exception that when a breakage of the FRA 12E site is induced in a sample obtained from the individual , it is preferred that the breakage is induced using an agent that is known to induce breakage of the fragile site. In another aspect, kits for performing tests to determine whether a chemotherapeutic agent induces a breakage at the FRAl 2E site and/or whether an individual has the FRAl 2E fragile site are provided. For example, the kits could comprise test probes and optionally negative control probes. In one embodiment, the kit comprises probes comprising sequences identical to BAC clones which can hybridize to the SMRT gene locus. For example, suitable test probes include polynucleotides corresponding to human chromosome 12 sequences of BAC-RPl 1- 339B19, BAC-RPl 1-665C13, and BAC-RPl 1-677L6 (all available to the public from, for example, the Roswell Park Cancer Institute, Buffalo, New York). The test probes could have fluorescent labels thereon (such as fluorescein or rhodamine and the like). The negative control probes could have a fluorescent label which is different from the label on the test probes. Optionally, for determining whether an individual has the FRA 12E site, the kits could also include a DNA polymerase inhibitor (such as aphidicolin), either in a solution form or as a powder, or other chemical which could be used to facilitate the visualization of fragile sites. Further, cell lines that have been tested and identified to be either positive or negative for the fragile site could also be included in the kits for use as controls and/or for determining whether a particular chemotherapeutic test agent induces breakage of the FRAl 2E site.

The invention is further described by the Examples presented below. These Examples are illustrative and are not intended to be restrictive in any way.

EXAMPLE l

This Example demonstrates detection of breakage of the FRA 12E site.

We conducted a study where lymphocytes obtained from healthy individuals (as normal controls) and from cancer patients were separately cultured in the presence of aphidicolin (an inducer of fragile sites) and metaphase chromosomes were subsequently prepared as described below. These preparations were then subjected to fluorescence in situ hybridization using a set of SMRT-specific RPCI BAC clones (Figure IA).

The three RPCIl 1 BAC clones encompassing the SMRT locus used in our experiments and are depicted as green bars in Figure IA. BAC DNA labeling for the FISH analysis was performed as follows. The BAC DNAs were extracted using a DNA extraction kit (Qiagen). DNA was then subject to nick translation labeling with fluorochrome-conjugated nucleotides using a commercially available kit (Vysis, Downers Grove, IL). The SMRT-specific clones were labeled in green whereas the control was labeled in red.

For chromosome preparation, the following procedure was used. One tube of peripheral blood was obtained from cancer patients and controls. The blood was set for a 3 -days culture in 10 ml of RPMI medium, supplemented by 10% of fetal calf serum and antibiotics, with 135 μg/ml of PHA. Twenty- four hours before harvesting chromosomes, aphidicolin (0.2 μg/ml) was added to the culture in order to induce fragile sites. Chromosome harvesting was then carried out according to classical cytogenetic techniques for chromosome preparations. Microscope slides were then prepared for hybridization with the labeled BAC clones.

Fluorescence in situ hybridization was carried out as follows. Hybridization was carried out according to established protocols (Q Jiang, et al. Cytogenet Cell Genet, (2001) 97: 217-220). A mixture containing one hundred nanogram of each labeled BAC DNA was applied to the chromosome preparation after denaturation. Hybridization was carried out overnight and slides were washed the next morning. Slides were mounted with antifade solution prior to be observed under a UV microscope with the appropriate filters for the assessment of the signals.

As shown in Figures IA and IB, overlapping clones that encompass the entire SMRT locus in order to detect any breakages in that region may be used, but it is not necessary to use probes encompassing the entire region, since we have demonstrated that RPl 1-677L6 and RPl 1-665C13 alone encompass enough of the SMRT locus such that they can be used in FISH assays to determine whether the breakage has

occurred within the SMRT locus from patient samples which contain the FRAl 2E fragile site. Specifically, five blood cell samples from five different patients with or without metastatic breast cancer were analyzed by FISH using RPl 1-677L6 and RPl 1-665C13. This method successfully identified chromosomal breakage at the SMRT locus to within 5% accuracy of using all three probes.

Using the three BAC clone approach, we were able to demonstrate that the FRA 12E fragile site is localized within the SMRT gene by using a FISH analysis, the results of which are presented in Figure 2, which depicts a FISH analysis of lymphocytes obtained from a cancer patient. This figure shows two signals. The upper right hand corner signal (identified by the short arrow) is characterized by a large signal in yellow due to the SMRT-specific green signal mixed with the control probe red signal. In addition, split green signals can be observed just to the right of this first yellow signal. The split greet signals represent the presence of the fragile site. The FISH signal present at the bottom left hand corner shows mostly yellow color signal, due to the overlapping/juxtaposition of the SMRT-specific probes green signals with the red signal from the control probe. No split green signal is observed in this case indicating the absence of the fragile site. Therefore, this Example demonstrates a method for determination of the presence or absence of of a FRAl 2E site by inducing and visualizing its breakage.

EXAMPLE 2

This Example demonstrates determining whether certain commonly utilized chemotherapeutic agents are able to induce breakage of the FRA12E sties. We performed FISH assays essentially as described in Example 1 using our lymphoblastoid cell lines that have been demonstrated to be positive or negative for the FRA 12E fragile site. However, we replaced the aphidicolin in the cell cultures by escalating sub-optimal doses of the test chemotherapeutic agents listed in Table 1.

As used herein, "optimal dose" refers to the concentration of chemotherapeutic agent that kills greater than 90% of the cells in vitro. "Sub-optimal dose" refers to a fraction of the optimal dose that will kill less than 10% of cells after a 24 hour exposure. Optimal and sub-optimal doses for chemotherapeutic agents can be determined according to standard techniques well know to those skilled in the art.

We used sub-optimal concentrations to mimic the predicted in vivo environment wherein cancer cells are exposed to an amount of chemotherapeutic agent insufficient to cause lethality, but where the amount of chemotherapeutic agent is nonetheless able to induce breakage of the FRA 12E site. This strategy also maintains a sufficient number of viable cells in the chemotherapeutic agent assay to perform FISH analysis. However, it will be recognized by those skilled in the art that breakages induced by the sub-optimal dosing of the cells can be determined using any suitable technique. It will also be recognized that, irrespective of the optimal and sub- optimal dosages, the determination that a chemotherapeutic agent is capable of inducing breakage of the FRAl 2E site is contraindicative for its prescription to a FRA12E positive individual.

In this Example, we demonstrate that a dose of 0.0IuM of Adriamycin is able to induce the fragile site as visualized through the detection of disrupted FISH signals. Similar results were obtained with other drugs used in cancer treatment (Table 1). Of interest is the observation that certain chemotherapeutic agents (Taxol and

Dexamethasone) do not induce breakages, indicating that they can be given to patients who have the FRA 12E site without a likelihood of inducing deleterious breakages.

Table 1.

Thus, this Example demonstrates that the method of the invention is useful for determining whether a test chemotherapeutic agent induces breakages of the FRA 12E site.

EXAMPLE 3

This Example demonstrates a method for determining whether an agent induces breakage of the FRAl 2E fragile site at the SMRT locus by detecting alterations in SMRT gene expression. Distinct cell lines were used for the purpose of studying the effect that particular agents have on SMRT mRNA levels. EBV-LIN was used as the control cell line because we have determined by FISH analysis that it does not contain the FRAl 2E fragile. The EBV-LIN cell line is derived from lymphocytes taken from an individual without cancer, and the cell line has been immortalized with the Epstein- Barr virus.

Cell lines determined to possess the FRA12E fragile site, labeled "5256 and 5278" where shown in Figures 3A, 3B, 4 and 5 are normal lymphocytes taken from a patient with glioblastoma, an aggressive form of brain cancer. SMRT mRNA levels from these cell lines were compared after they had been treated with agents for specified periods of time, as follows.

The cells were seeded at a concentration of 5 xlO ⁵ cells per mL for a total of 1 x 10 ⁶ cells for 2 ML's per well. This concentration was determined to be sufficient for extracting RNA using standard RT-PCR procedures. The EBV-LESf cells were cultured in RPMI 1640 + 10% FBS and the 5256/5278 cells were cultured in RPMI 1640 + 20% FBS. Both media were supplemented with 5 mL of penicillin/Sstreptomycin and .5 mL of fungizone. We have determined that a volume of 300ul ml of aphidicolin per 10 mi's of cell culture is efficient in inducing fragile site breakage without killing the cells. Therefore, when cells were treated with aphidicolin, a volume of 60 ul of aphidicolin was added to each 2 mL well, equivalent to a 0.56 micromolar concentration of aphidicolin per sample. Doxorubicin was also analyzed and we determined that a concentration of .01 micromolar in each 2 ml sample.

RNA was extracted from cultured cells by means of an RNA isolation kit (Stratagene, Absolutely RNA Miniprep Kit). The cDNA synthesis reaction is based on the Moloney Murine Leukemia

Virus (M-MuLV) Reverse Transcriptase enzyme. The enzyme was purchased from New England Biolabs, along with the rest of the products used in preparing first strand cDNA synthesis. The M-MuIV Reverse Transcriptase is an RNA directed DNA

polymerase. This enzyme can synthesize a complementary DNA strand initiating from a primer using either RNA or single-stranded DNA as a template. Also, the M- MuIV Reverse Transcriptase lacks 3'->5' activity.

The gene specific primers used were SMRT (exons 26-29), forward 5'-ACA GTG GCT GAG TGC GTC CTC T-3 ¹ (SEQ ID NO:2) and reverse 5'-ACG TGG AGC TGG ACC GAC ATT C-3' (SEQ ID NO:3). These primers amplify Exons 26 through 29. Amplification of SMRT cDNA was performed using the parameters set forth in Table 2; amplification of GAPDH was performed using the parameters set forth in Table 3.

Table 2.

At 0, 2, 4, 5, 6, and 7 hours of treatment with aphidicolin, SMRT mRNA from the 5256 cell line was consistently shown to decrease specifically after 4 hours of treatment with aphidicoline. (Figures 3A and 3B). In contrast, EBV-LEN SMRT mRNA was shown to remain steady. The housekeeping gene GAPDH was used as a control for cDNA integrity and allows for semi-quantification.

We also analyzed the effect of a test chemotherapuetic agent (doxorubicin) on SMRT mRNA experssion in 5256 cells. Doxorubicin is a cancer chemotheraputic agent that has a biological mechanism of action similar to that of aphidicolin

(inhibition of DNA polymerase activity). The results from this analysis, which were performed as described in this Example for aphidocolin, are depicted in Figure 4. The results demonstrate that there is a loss of SMRT expression in the cell line 5256 when treated with doxorubicin (Figure 4). The loss of SMRT expression begins at three hours of exposure to .01 microcolar of doxorubicin, and after 21 hours of treatment the expression returns to the levels seen in the 0, untreated sample. In comparison, SMRT mRNA levels remained essentially unchanged in the EBV-LIN cell line throughout the time point treatments with doxorubicin.

As compared to the treatment of the 5256 cell line with aphidicoline, treatment with doxorubicin of 5256 cells results in a similar loss of SMRT expression at a specific time point, after which the expression levels return to the level of their respective 0 hour, untreated sample. Thus, the present Example demonstrates that determining whether a test agent induces a breakage of the FRAl 2E site can be performed by determining alterations in SMRT mRNA expression after administration of the test agent.

We also tested the effect of aphidicolin on SMRT protein expression in cells harboring the FRA12E site (5256 cells). The results are presented in Figure 5 and demonstrate a reduction in SMRT protein expression after approximately 18 hours in the 5256 cells. However, addition of aphidicolin to cells which do not contain the FRA 12E site (EBV-LIN) does not cause a reduction in SMRT protein expression.

Thus, this Example demonstrates that determining whether a test chemotherapeutic agent induces breakage of the FRA 12E fragile site at the SMRT locus can be performed by measuring alterations in SMRT gene expression induced by the test chemotherapeutic agent.

While the present invention has been described using the above examples, routine modifications to this invention will be apparent to those skilled in the art and are intended to be within the scope of the invention.