EP1799851A1

EP1799851A1 - Methods of diagnosis or detection using three-dimensional analysis

Info

Publication number: EP1799851A1
Application number: EP05777871A
Authority: EP
Inventors: Sabine Mai; Yuval Garini; William Foulkes; Soumya Panigrahi; Bartholomeus QI/IST - TU Delft VERMOLEN; Tony Chuang; Alice Chuang; Gary Jones
Original assignee: University of Manitoba
Current assignee: University of Manitoba
Priority date: 2004-08-20
Filing date: 2005-08-22
Publication date: 2007-06-27
Also published as: EP1799851A4; WO2006017942A1; CA2577623A1

Abstract

The invention provides methods of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer. In particular, the invention relates to methods of diagnosing or detecting breast cancer cells that have mutant BRCA1 or BRCA2 genes as compared to wild type BRCA1 and BRCA2 genes. The invention also provides methods of detecting cancer using 3D analysis and genetic analysis. In particular, characterizing the telomeric organization of a cell and genetically analyzing the cell for the presence of a translocation, deletion or amplification. In addition, the invention provides methods of simultaneously analyzing protein and nuclear structures of a cell using 3D analysis.

Description

TITLE: Methods of Diagnosis or Detection Using Three-Dimensional Analysis FIELD OF THE INVENTION

The invention relates to methods of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer. In particular, the invention relates to methods of diagnosing or detecting breast cancer cells that have mutant BRCA1 or BRCA2 genes as compared to wild type BRCA1 and BRCA2 genes. The invention also relates to methods of detecting cancer using three-dimensional (3D) analysis and genetic analysis. In particular, characterizing the telomeric organization of a cell and genetically analyzing the cell for the presence of a translocation, deletion or amplification. In addition, the invention relates to methods of simultaneously analyzing protein and nuclear structures of a cell using 3D analysis. BACKGROUND OF THE INVENTION

Telomeres are the ends of chromosomes. By capping the chromosomes, they are responsible for chromosomal integrity to prevent genomic instability^1'3. Telomeres have been previously found at the nuclear edge⁴, at the nuclear periphery⁵, throughout the entire nucleus⁶, in non-Rabl association⁷, and in association with the nucleolus⁸. Similarly, the nuclear organization of chromosomes has been described as non-random^9'13 or random¹⁴, based on two-dimensional (2D) imaging, 3D reconstitution and mathematical modeling¹⁵. Organized territories of individual chromosomes have been observed in human^10"13, chicken¹⁶ and plant¹⁷. A regular nuclear organization has also been described for replication and transcription^18"21. While all this points to a well-defined nuclear organization, studies on radiation-induced aberrations suggested a random chromosomal organization¹⁴.

At present, there is inexact knowledge about the 3D organization of either telomeres or chromosomes in interphase nuclei of normal, immortalized or tumor cells. However, this knowledge is of fundamental importance to the structural and functional organization of the normal nucleus and to the puzzle of genomic instability in tumor cells. SUMMARY OF THE INVENTION

The inventors have identified that hereditary breast cancer cells and non-hereditary breast cancer cells have different three-dimensional (3D) organizations of their chromosomes. The differences in the 3D organization of the chromosomes of the hereditary breast cancer cells as compared to the non-hereditary breast cancer cells, includes differences in the size or volume of the aggregation of the telomeres, the number of telomeres, and the size or volume of the telomeres. The invention provides a method of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer in a subject using high- resolution deconvolution, or equivalent, 3D microscopy and imaging, and 3D quantitative analysis.

Accordingly, one aspect of the invention is a method of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer in a subject having breast cancer comprising the steps: a) characterizing the telomeric organization of cells in a test sample of breast cells or tissue from the subject using 3D analysis; and b) comparing the telomeric organization of cells in the test sample with the telomeric organization of a control, wherein a difference in telomeric organization between the sample and the control is indicative of hereditary versus non-hereditary breast cancer.

For instance, if the control comprises hereditary breast cancer cells and the test sample has a smaller telomeric aggregate (TA) size or volume than the control, then this indicates that the subject has non-hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a greater number of detectable telomeres as compared to the control, then this indicates that the subject has non-hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a larger telomeric size or volume than the control, then this indicates that the subject has non-hereditary breast cancer.

If the control comprises hereditary breast cancer cells and the test sample has a similar telomeric aggregate (TA) size or volume as compared to the control, then this indicates that the subject has hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a similar number of detectable telomeres as compared to the control, then this is indicates that the subject has hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a similar telomeric size or volume as compared to the control, then this indicates that the subject has hereditary breast cancer.

If the control comprises non-hereditary breast cancer cells and the test sample has a larger telomeric aggregate (TA) size or volume as compared to the control, then this indicates that the subject has hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a fewer number of detectable telomeres as compared to the control, then this indicates that the subject has hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a smaller telomeric size or volume than the control, then this indicates that the subject has hereditary breast cancer.

If the control comprises non-hereditary breast cancer cells and the test sample has a similar telomeric aggregate (TA) size or volume as compared to the control, then this indicates that the subject has non-hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a similar number of detectable telomeres as compared to the control, then this is indicates that the subject has non-hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a similar telomeric size or volume as compared to the control, then this indicates that the subject has non-hereditary breast cancer.

In one embodiment, the non-hereditary breast cancer comprises breast cancer cells that have wild type genes associated with hereditary breast cancer (such as wild type BRCA1, BRCA2, TP53, PTEN, CH K2, HER2 or ATM genes), while the hereditary breast cancer cells comprise breast cancer cells with mutated genes associated with hereditary breast cancer (such as mutated BRCA1, BRCA2, TP53, PTEN, CHK2, HER2 or ATM genes). -A -

Another aspect of the invention is a method of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer in a subject comprising the steps: a) characterizing the telomeric organization of cells in a test sample of breast cells or tissue from the subject using 3D analysis; and b) comparing the telomeric organization of the cells in the test sample with that of a control, wherein the control comprises a control sample of hereditary breast cancer cells, a control sample of non- hereditary breast cancer cells, and/or a normal control, wherein a smaller telomeric aggregate size or volume in the test sample as compared to the control sample of hereditary breast cancer cells and a larger telomeric aggregate size or volume in the test sample as compared to the normal control indicates that the subject has non-hereditary breast cancer; or wherein a similar telomeric aggregate size or volume in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a larger telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a larger telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer; or wherein morea greater number of detectable telomeres in the test sample as compared to the control sample of hereditary breast cancer cells and a fewer number of detectable telomeres as compared to the normal control indicates that the subject has non-hereditary breast cancer; or wherein a similar number of detectable telomeres in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a fewer number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a fewer number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer; or wherein a larger telomeric size or volume in the test sample as compared to the control sample of hereditary breast cancer cells and a smaller telomeric size or volume in the test sample as compared to the normal control indicates that the subject has non-hereditary breast cancer; or wherein a similar telomeric size or volume in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a smaller telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a smaller telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer.

In one embodiment, the non-hereditary breast cancer comprises breast cancer cells that have wild type genes associated with hereditary breast cancer (such as wild type BRCA1, BRCA2, TP53, PTEN, CHK2, HER2 or ATM genes), while the hereditary breast cancer cells comprise breast cancer cells with mutated genes associated with hereditary breast cancer (such as mutated BRCA1, BRCA2, TP53, PTEN, CHK2, HER2 or ATM genes). In a preferred example, the non-hereditary breast cancer cells comprise breast cancer cells with wild type BRCA1 and BRCA2 genes, while the hereditary breast cancer cells comprise breast cancer cells with mutated BRCA1 or BRCA2 genes. The inventors also provide a method of diagnosing or detecting cancer using 3D analysis and genetic analysis.

Accordingly, an additional aspect of the invention is a method of diagnosing or detecting cancer in a subject, comprising the steps: a) characterizing the telomeric organization of a cell in a test sample from the subject using 3D analysis; b) comparing the telomeric organization of the cell with that of a normal control, wherein a change in telomeric organization in the test cell as compared to the normal control indicates the presence of genomic instability in the test cell; and c) genetically analyzing the cell from step b) for a translocation, deletion or amplification. wherein the presence of genomic instability and a translocation, deletion or amplification in the test cell indicates a pre-cancerous cell or a cancerous cell. A further aspect of the invention is a method of diagnosing or detecting cancer in a subject, comprising the steps: a) genetically analyzing a cell in a test sample for a translocation, deletion or amplification, b) characterizing the telomeric organization of the cell from step a) using 3D analysis; and c) comparing the telomeric organization of the cell from step b) with that of a normal control, wherein a change in telomeric organization in the test cell as compared to the normal control indicates the presence of genomic instability in the test cell, wherein the presence of genomic instability and a translocation, deletion or amplification in the test cell indicates a pre-cancerous cell or a cancerous cell. In one embodiment, the genetic analysis is for a translocation involving the c-Myc genes and IgH genes. In another embodiment, the genetic analysis is for amplification involving HER2. In a further embodiment, the genetic analysis is for a deletion involving p53.

Another aspect of the invention is a method for simultaneously analyzing protein and nuclear structures of a cell, comprising the steps: a) allowing a protein-specific antibody to bind to a test sample of cells or tissue; b) allowing a telomeric probe to bind to the test sample; and c) detecting the binding of the protein-specific antibody and telomeric probe in the test sample using 3D analysis.

In one embodiment, a peptide nucleic acid (PNA) telomeric probe is used, and the protein-specific antibody is detected using a secondary antibody, which is specific for the protein-specific antibody. Preferably, the secondary antibody can directly or indirectly generate a detectable signal. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which: Figure 1 shows telomeric organization of normal breast cells (normal), breast cancer cells with wild type BRCA1 and BRCA2 genes (Non-BRCA1/2), breast cancer cells with mutated BRCA2 genes (BRCA2) and breast cancer cells with mutated BRCA1 genes (BRCA1). The following abbreviations are used: 2D means two-dimensional, 3DF means three-dimensional front view, 3DS means three-dimensional side view and 3DR means three-dimensional reconstruction.

Figure 2a-d shows the frequency distribution pattern of telomere signal intensities in cells with and without BRCA mutation. The X-axis is the telomere signal intensities and the Y-axis is the frequency. A total of 100 nuclei were studied per cell line, (a) MCF 7 cell line has wild type BRCA1 and BRCA2 (non-hereditary breast cancer); (b) HCC 1397 cell line has mutant BRCA1 (hereditary breast cancer); (c) Capan 1 cell line has mutant BRCA2; and (d) MCF 7, HCC 1397 and Capan 1 cell lines.

Figure 3 shows the average telomere signal intensities in the MCF 7 (wild type BRCA1 and BRCA2), HCC 1397 (mutant BRCA1) and Capan 1 (mutant BRCA2) cell lines. Figure 4 shows telomeric organization of pre-malignant plasmacytoma lesions. The 2D image shows an aggregate shared by 4 cells at the center of the image. An arrow points to the big aggregate in the 3DF and 3DS views. The following abbreviations are used: 2D means two-dimensional, 3DF means three-dimensional front view and 3DS means three-dimensional side view.

Figure 5 shows the same area of interest of the cells in Figure 4 after the fluorescent in situ hybridization (FISH) experiment with c-Myc and IgH probes. Panel A shows 2D, 3DF, and 3DS images of IgH gene alone. Panel B shows 2D, 3DF, and 3DS images of c-Myc gene alone. Panel C shows 2D, 3DF, and 3DS images of IgH and c-Myc genes. The IgH gene is FITC- labeled (green). The c-Myc gene is Cy3-labelled (red). The orange signals appear in the 2D image, which is the mixture of both signals and indicates translocation/juxtaposition. The green color shows IgH gene alone without combining with c-Myc gene. The translocation/juxtaposition of two genes are clearly shown in the 3DS image. The following abbreviations are used: 2D means two-dimensional, 3DF means three-dimensional front view and 3DS means three-dimensional side view.

Figure 6: MycER™ activation scheme. Overview of experimental design for time-dependent analysis of c-Myc deregulation. 4-hydroxy- tamoxifen (4HT) was added at various time points and for various durations. It was administered for 2 hours or 12 hours and thereafter removed from the culture medium. In parallel, 4HT was added every 12 hours or just once and not removed. The effects of 4HT are cell type and species dependent and last 15-24 hours in cell lines^62'64. This is indicated by dashed lines. Cells were harvested every 6 hours over a time period of 120 hours. Mock-treated control cells were processed in parallel. Figure 7: Telomere organization in nuclei of primary mouse B cells, in

Pre B and Ba/F3 lymphocytes. Images showing the telomeric organization in interphase nuclei of the primary cells and immortalized lines examined in this study. Note that there is no overlap in telomere positions, a: Primary mouse B cell nucleus; b: nucleus of near diploid Pre B cell; c: nucleus of tetraploid Ba/F3 cell. Telomeres are shown in red. Nuclei are shown in blue. 2D: two- dimensional; 3DF: three-dimensional front view; 3DS: three-dimensional side view.

Figure 8: Experimental activation of MycER™ with 4HT in Pre B and Ba/F3 nuclei respectively. Representative immunohistochemistry images from both cell lines at 0 hours and 4 hours after MycER™ activation. After c-Myc activation, nuclear Myc staining is visible in all nuclei⁶¹, (a) Pre-B cells without 4HT; (b) Pre-B cells with 4HT; (c) Ba/F3 cells without 4HT; (d) Ba/F3 cells with 4HT.

Figure 9: c-Myc deregulation induces telomeric aggregates (TAs) in interphase nuclei of Pre-B and Ba/F3 cells, (a) Mock-treated Pre-B cells show non-overlapping telomeres (red) (see also Figure 7). (b) As a result of c-Myc deregulation in Pre-B cells, telomeric aggregates form (green arrow heads), (c) Mock-treated Ba/F3 cells show non-overlapping telomeres (see also Figure 7), (d) Ba/F3 cells with c-Myc deregulation show the formation of telomeric aggregates (green arrow heads).

Figure 10a: Conditional c-Myc deregulation in Pre-B cells as examined by fluorescent immunohistochemistry. Relative fluorescent nuclear c-Myc staining as determined by immunohistochemistry and quantified with Northern Eclipse software. The graph illustrates the different levels of nuclear c-Myc protein that are observed as a result of different lengths of MycER™ activation.

Figure 10b: Conditional c-Myc deregulation causes telomeric aggregates (TAs) in Pre-B nuclei, (a) Negative control: non-Myc-deregulated Pre-B nucleus. This figure is consistent with the data summarized in Figure 7 that telomeres occupy non-overlapping 3D nuclear positions, (b-d) TAs of various sizes and numbers are present after conditional c-Myc expression. Telomeres are shown in green. Red arrows point to TAs. (e) Positive control. A plasmacytoma cell line, MOPC460D, with constitutive c-Myc deregulation due to T12;15, shows telomeric aggregates. Similar results were obtained with primary plasmacytoma cells.

Figure 10c: c-Myc induces cycles of telomeric aggregates (TAs). Graph summarizing the fold increase in TAs over control levels and during a period of 120 hours. TA frequencies were analyzed every 6 hours. During the experimental period, c-Myc had been up regulated for different lengths of time (see Figure 6). Black: 4HT was given for 2 hours and subsequently removed; red: 4HT was administered for 12 hours and subsequently removed; green: 4HT was added once and was not removed; blue: 4HT was added at 0, 12 and 24 hours. All cells that had received consecutive inductions of c-Myc died after 30 hours. Arrows point to the highest levels of TA formation, an arrow head shows the single peak of TAs observed following consecutive activations of MycER™. Figure 11 : Spectral karyotype (SKY) and telomere FISH of MycER™- activated Pre B cells. Panel A: spectral karyotype for control cells. Mock- treated Pre-B cells are near diploid⁷¹ and did not show chromosomal rearrangements, such as translocations or fusions. Panel B: a: SKY analysis revealed telomeric fusions and chromosome breakage. Top three images: raw image of the metaphase (left), classified image of the same metaphase (middle) and inverted DAPI image of the same metaphase (right). Bottom image: spectral karyotype of the same metaphase. A red arrow points to the end-to-end fusion of chromosomes 18 and 4, a green arrow points to the fusion of chromosome 1 with a broken piece of chromosome 1. A broken chromosome 1 is circled. Note additional broken chromosomes 1 ,2,3, and 7. b: End-to-end fusions of chromosomes revealed by telomere FISH. Two representative metaphases are shown. Top: centromeric fusion (see arrow and insert), bottom: telomeric fusion (see arrow and insert), c: Anaphase bridges. Left image: short exposure of DAPI stained nucleus (100 msec); right image: longer exposure (500 msec) of same image makes anaphase bridge visible (white arrow) but overexposes nuclei, d: SKY analysis illustrating both chromosomal fusions (red arrow) and non-reciprocal translocations (white arrows). Broken chromosomes are present as well (chromosomes 4,6,12,17).

Figure 12: Chromosome positions in c-Myc-activated nuclei, a.

Representative images of interphase nuclei painted with selected chromosome pairs over a period of 96 hours after administration of 4HT alone (Figure 6). Top: chromosomes 5 and 13 are stained, middle: hybridization with chromosomes 7 and 10, bottom: painting with chromosomes 7 and 17. b. Measurements of chromosomal overlaps in interphase nuclei of c-Myc deregulated cells for chromosomes 5 and 13, 7 and 10, 7 and 17 over a 96 hour period. Figure 13 is a block diagram of a system for characterizing the three- dimensional organization of telomeres and/or chromosomes in cells. DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified that hereditary breast cancer cells and non-hereditary breast cancer cells have different three-dimensional organizations of their chromosomes. The differences in the three-dimensional organization of the chromosomes of the hereditary breast cancer cells as compared to the non-hereditary breast cancer cells, includes differences in the number of telomeres, the size or volume of the telomeres and aggregation of the telomeres. The invention provides a method of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer in a subject using high- resolution deconvolution, or equivalent, 3D microscopy and imaging, and 3D quantitative analysis.

The invention provides a method of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer in a subject known to have breast cancer.

The term "hereditary breast cancer" as used herein means a type of breast cancer that is genetically transmitted or transmittable from parent to offspring. Hereditary breast cancers include, without limitation, breast cancer cells with mutations in the BRCA1, BRCA2, TP53, PTEN, CHK2, HER2 or

ATM genes, preferably mutations in the BRCA1 and/or BRCA2 genes.

The term "non-hereditary breast cancer" as used herein means a type of breast cancer that is not genetically transmitted or transmittable from parent to offspring. Non-hereditary breast cancers include, without limitation, breast cancer cells with wild type BRCA1, BRCA2, TP53, PTEN, CHK2, HER2 or

ATM genes, preferably wild type BRCA1 and BRCA2 genes.

The term "method of diagnosing or detecting" refers to a process of determining whether an individual has or does not have a certain indication or characteristic. One embodiment of the invention is a "method of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer" which refers to a method or process of determining if an individual has hereditary or non-hereditary breast cancer. The term "subject" as used herein means any member of the animal kingdom, particularly humans, and includes individuals having breast cancer, suspected of having breast cancer or at risk of having breast cancer. Thus the methods of the invention can be used in a general screening of individuals for hereditary or non-hereditary breast cancer. In some embodiments of the invention the phrase "subject having breast cancer" is used and refers to individuals known to have breast cancer. Individuals known to have breast cancer can be diagnosed using known techniques and methods, including the method disclosed in PCT/C2004/000241.

The term "test sample" as used herein means a sample of cells or tissue from any animal. The sample may be biological fluids (such as blood, serum, saliva, cerebrospinal fluid, rectal mucus, lung sputum, breast nipple aspirate, cervical mucus, seminal fluid, plasma, and lymphatic fluid), tissue, hair or organ. The term "cell" includes more than one cell or a plurality of cells or portions of cells. The term "test sample of breast cells" as used herein refers to a sample of breast cells from any animal, in particular from humans. The sample may be taken from the subject using techniques known to one skilled in the art, for example a test sample of breast cells may be obtained by biopsy.

The term "telomeric organization" as used herein refers to the three- dimensional arrangement of the telomeres during any phase of a cell cycle and includes parameters such as telomere number, size or volume, alignment, state of aggregation and the size or volume of the three- dimensional space that the telomeres occupy. Telomere organization also refers to the size and shape of the telomeric disk which is the organized structure formed when the telomeres condense and align during the late G2 phase of the cell cycle. The term "state of aggregation" refers to the size and shape of the aggregates of telomeres.

The term "three-dimensional (3D) analysis" as used herein refers to any technique that allows the three-dimensional visualization of cells, for example high resolution deconvolution microscopy.

The phrase "characterizing the telomeric organization of cells in the test sample using three-dimensional analysis" as used herein refers to determining the telomeric organization of the cells. For example, by counting the number of telomeres in the cell, measuring the size or volume of any telomere or telomere aggregate, and measuring the alignment of the telomeres. The "difference in telomeric organization between the sample and the control" may be determined for example by counting the number of telomeres in the cells, measuring the size or volume of any telomere or telomere aggregate, or measuring the alignment of the telomeres, and comparing the difference between the cells in the sample and the cells in the control. The differences in telomeric organization between the sample and the control can be measured and compared using individual cells or average values from a population of cells.

In one embodiment, if the control comprises hereditary breast cancer cells and the test sample has a smaller telomeric aggregate (TA) size or volume than the control, then this indicates that the subject has non-hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a greater number of detectable telomeres as compared to the control, then this indicates that the subject has non-hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a larger telomeric size or volume than the control, then this indicates that the subject has non-hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a similar telomeric aggregate (TA) size or volume as compared to the control, then this indicates that the subject has hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a similar number of detectable telomeres as compared to the control, then this is indicates that the subject has hereditary breast cancer. If the control comprises hereditary breast cancer cells and the test sample has a similar telomeric size or volume as compared to the control, then this indicates that the subject has hereditary breast cancer.

If the control comprises non-hereditary breast cancer cells and the test sample has a larger telomeric aggregate (TA) size or volume as compared to the control, then this indicates that the subject has hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a fewer number of detectable telomeres as compared to the control, then this indicates that the subject has hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a smaller telomeric size or volume than the control, then this indicates that the subject has hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a similar telomeric aggregate (TA) size or volume as compared to the control, then this indicates that the subject has non-hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a similar number of detectable telomeres as compared to the control, then this is indicates that the subject has non-hereditary breast cancer. If the control comprises non-hereditary breast cancer cells and the test sample has a similar telomeric size or volume as compared to the control, then this indicates that the subject has non-hereditary breast cancer. A "control" as used herein refers to one or more tissue or cell samples that are either classified as "normal", "non-hereditary breast cancer", "hereditary breast cancer" or "cancer". The term "normal control" as used herein refers to a sample comprising normal, disease-free cells. The term "non-hereditary breast cancer control" as used herein means a sample comprising cells that are known to be non-hereditary breast cancer cells. The term "hereditary breast cancer control" as used herein means a sample comprising cells that are known to be hereditary breast cancer cells. The term "cancer control" as used herein means a sample comprising cells that are known to be cancerous cells, but it might not been known whether the cancer cells are hereditary or non-hereditary cancer cells. The control cells may be obtained from a subject who is known to be disease free, have hereditary or non-hereditary breast cancer, or cancer. The control cells may also be obtained from cell lines, which are classified as disease free cell lines, hereditary or non-hereditary breast cancer cell lines, or cancer cell lines. The term "telomeric aggregate size or volume" as used herein refers to the three-dimensional size or volume occupied by the telomeric aggregate.

The term "telomeric size or volume" as used herein refers to the three- dimensional size or volume occupied by individual telomeres.

The phrase "similar telomeric size or volume" as used herein means the telomeric size or volume of the cells in the control sample as compared to the cells in the test sample is not statistically different.

The phrase "similar telomeric aggregate size or volume" as used herein means the telomeric aggregate size or volume of the cells in the control sample as compared to the cells in the test sample is not statistically different.

The phrase "similar number of detectable telomeres" as used herein means the number of telomeres that are detectable in the cells of the control sample as compared to the cells of the test sample is not statistically different.

In one embodiment, the non-hereditary breast cancer comprises breast cancer cells that have wild type genes associated with hereditary breast cancer (such as wild type BRCA1. BRCA2. TP53, PTEN, CHK2, HER2 or

ATM genes), while the hereditary breast cancer cells comprise breast cancer cells with mutated genes associated with hereditary breast cancer (such as mutated BRCA1. BRCA2. TP53, PTEN, CHK2, HER2 or A TM genes).

The invention provides a method of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer in a subject using 3D analysis, whether or not it is known that the subject has breast cancer. This method utilizes a normal control, in addition to the non-hereditary and hereditary breast cancer cell controls. The 3D analysis can include the use of high-resolution deconvolution, or equivalent, 3D microscopy and imaging, and 3D quantitative analysis.

Accordingly, another aspect of the invention is a method of diagnosing or detecting hereditary breast cancer versus non-hereditary breast cancer in a subject comprising the steps: a) characterizing the telomeric organization of cells in a test sample of breast cells or tissue from the subject using 3D analysis; and b) comparing the telomeric organization of the cells in the test sample with that of a control, wherein the control comprises a control sample of hereditary breast cancer cells, a control sample of non- hereditary breast cancer cells, and/or a normal control wherein a smaller telomeric aggregate size or volume in the test sample as compared to the control sample of hereditary breast cancer cells and a larger telomeric aggregate size or volume in the test sample as compared to the normal control indicates that the subject has non-hereditary breast cancer; or wherein a similar telomeric aggregate size or volume in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a larger telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a larger telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer; or wherein morea greater number of detectable telomeres in the test sample as compared to the control sample of hereditary breast cancer cells and a fewer number of detectable telomeres as compared to the normal control indicates that the subject has non-hereditary breast cancer; or wherein a similar number of detectable telomeres in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a fewer number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a fewer number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer; or wherein a larger telomeric size or volume in the test sample as compared to the control sample of hereditary breast cancer cells and a smaller telomeric size or volume in the test sample as compared to the normal control indicates that the subject has non-hereditary breast cancer; or wherein a similar telomeric size or volume in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a smaller telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a smaller telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer.

In one embodiment, the non-hereditary breast cancer comprises breast cancer cells that have wild type genes associated with hereditary breast cancer (such as wild type BRCA1, BRCA2, TP53, PTEN, CHK2, HER2 or ATM genes), while the hereditary breast cancer cells comprise breast cancer cells with mutated genes associated with hereditary breast cancer (such as mutated BRCA1, BRCA2, TP53, PTEN, CHK2, HER2 or ATM genes).

An additional aspect of the invention is a method of diagnosing or detecting cancer in a subject, comprising the steps: a) characterizing the telomeric organization of a cell in a test sample from the subject using 3D analysis; b) comparing the telomeric organization of the cell with that of a normal control, wherein a change in telomeric organization in the test cell as compared to the normal control indicates the presence of genomic instability in the test cell; and c) genetically analyzing the cell from step b) for a translocation, deletion or amplification, wherein the presence of genomic instability and a translocation, deletion or amplification in the test cell indicates a pre-cancerous cell or a cancerous cell. Cancers that can be detected using this method include, without limitation, plasmacytoma, Burkitt's Lymphoma, Chronic myeloid leukemia (CML) and other leukemias or lymphomas associated with translocations. In a preferred embodiment, the cancer is plasmacytoma and the cells are genetically analyzed for translocation of the c-Myc gene and IgH gene.

The phrase "change in telomeric organization in the test cell as compared to the control" as used herein means determining the telomeric organization of the test cell and comparing it to the telomeric organization of a control cell.

The phrase "genetically analyzing the cell for a translocation" as used here refers to using genetic means, such as FISH (fluorescent in situ hybridization), to localize specific genes on the chromosomes of the cells. For example, FISH can be used to localize the c-Myc gene and IgH gene on the chromosomes of a cell. In mice, the c-Myc gene is normally located on chromosome 15, while the IgH is located on chromosome 12. However, plasmacytoma is associated with a chromosomal translocation that juxtaposes the c-Myc gene on chromosome 15 to the IgH gene on chromosome 12.

In one embodiment, the genetic analysis is for a translocation involving the c-Myc genes and IgH genes. In another embodiment, the genetic analysis is for amplification involving HER2. In a further embodiment, the genetic analysis is for a deletion involving p53.

One advantage of this method is the ability to detect and diagnose different stages of cancer progression. For example, the method can be used to detect and diagnose cancer cells at an early stage, such as pre-cancerous cells. It also can be used to detect transition stages and the progression of cancer, such as normal cells to dysplastic cells, dysplastic cells to invasive cells, and invasive cells to metastatic cells.

The method of diagnosing or detecting cancer in a subject is not limited to the order of steps of characterizing the telomeric organization and then genetically analyzing the cell. Accordingly, a further aspect of the invention is a method of diagnosing or detecting cancer in a subject, comprising the steps: a) genetically analyzing a cell in a test sample for a translocation, deletion or amplification, b) characterizing the telomeric organization of the cell from step a) using 3D analysis; and c) comparing the telomeric organization of the cell from step b) with that of a normal control, wherein a change in telomeric organization in the test cell as compared to the normal control indicates the presence of genomic instability in the test cell, wherein the presence of genomic instability and a translocation, deletion or amplification in the test cell indicates a pre-cancerous cell or a cancerous cell. Another aspect of the invention is a method for simultaneously analyzing protein and nuclear structures of a cell, comprising the steps: a) allowing a protein-specific antibody to bind to a test sample of cells or tissue; b) allowing a telomeric probe to bind to the test sample; and c) detecting the binding of the protein-specific antibody and telomeric probe in the test sample using 3D analysis.

The 3D analysis can include the use of high-resolution deconvolution, or equivalent, 3D microscopy and imaging, and 3D quantitative analysis.

In one example, a peptide nucleic acid (PNA) telomeric probe is used, and the protein-specific antibody is detected using a secondary antibody, which is specific for the protein-specific antibody. Preferably, the secondary antibody can directly or indirectly generate a detectable signal. In another embodiment, the protein-specific antibody may be able to generate a detectable signal directly or indirectly. The binding of the protein-specific antibody in the cell can be detected using a secondary antibody that is specific for the protein-specific antibody and that can generate a detectable signal directly or indirectly, such as a fluorescent tag or an enzyme.

The term "protein-specific antibody" as used herein means an antibody that specifically binds to a protein of interest in a cell. For example, the protein of interest could be a nuclear protein structure, such as histones, and the "protein-specific antibody" can be H3P antibody. The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include Fab, Fab¹, F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab')₂ fragments can be generated by treating the antibody with pepsin. The resulting F(ab')₂ fragment can be treated to reduce disulfide bridges to produce Fab¹ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')₂, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

The term "telomeric probe" as used herein means a substance, such as peptide nucleic acid (PNA), that can bind to telomeres and be detected.

The methods of the invention have a number of applications in clinical medicine and oncology. For example, the methods that use both genetic and telomere information allow for better detection, sensitivity and specificity of specific cancers or disease. These methods can be used for: • Assessing the effects of environmental exposure to hazards

(smoking / EtOH / radiation exposure / toxins / heavy metals).

• Early detection of preneoplastic lesions: dysplasia -> severe dysplasia -> CIS (carcinoma in-situ) -> microinvasion.

• Monitoring treatment response. • Selection of optimal treatment regimens for patients with hematologic malignancies.

• Selection of patients who may benefit more from combined chemo- radiation therapy versus surgery for head and neck cancers.

• Intraoperative molecular margin to assess completeness of tumor resection during surgery.

• Investigation of effects and side effects of drug.

• Prognosticate disease outcome. ^• Assessment of biologic aggressiveness of tumors.

^• Detection of minimal residual diseases.

^• Assessment of metastatic potential of tumors.

• Screening tests for family members who are at risk of developing inheritable or progressive diseases such as cancers, metabolic disorders, cardiovascular disorders (e.g. hyperlipidemia, hypertension etc).

• Discovery of new targets for treatment using molecular / gene therapy. • Screening tool for major drug trials to provide insight on biologic effects of the drug.

The methods of the invention that use both telomeric analysis and genetic analysis also have applications in prenatal care. They can be used in prenatal genetic testing to rapidly assess fetal chromosomal abnormalities such as trisomy 13 (Patau's syndrome), 18 (Edward's syndrome), and 21 (Down's syndrome) using interphase fetal cells instead of using cells in metaphase. The methods of the invention can also be used to detect mental retardation.⁹⁸ Patients will appreciate receiving genetic testing results as soon as possible so that decisions can be made regarding abortion/counseling. The present invention utilizes methods and systems for characterizing the 3D organization of telomeres and/or chromosomes.

Figure 13 is an example of a block diagram of a system 100 for characterizing a 3D organization of telomeres and/or chromosomes. The system 100 includes an input module 102, an image data processor 104, an optimizer 106 and a characteristic module 108.

The input module 102 inputs image data of the 3D organization of telomeres and/or chromosomes. The input module 102 includes appropriate hardware and/or software, such as a CD-ROM and CD-ROM reader, or other data storage and reading means. The inputting performed by the input module 102 need not be from outside the system 100 to inside the system 100. Rather, in some embodiments, the inputting of data may describe the transfer of data from a permanent storage medium within the system 100, such as a hard disk of the system 100, to a volatile storage medium of the system 100, such as RAM.

The image data can be obtained using regular or confocal microscopy and can include the intensities of one or more colors at pixels (totaling, for example, 300x300 or 500x500) that comprise an image of a nucleus. The image data can also be grey level image data of a nucleus that has been appropriately stained to highlight telomeres and/or chromosomes. Several images (on the order of 100) are obtained corresponding to slices along a particular axis. Thus, the image data may correspond to a total of about 2.5 x 10⁷ pixels. In one embodiment, the slices may be on the order of 100 nanometers apart. In this manner, the image data accounts for the 3D quality of the organization of telomeres and/or chromosomes. In addition, the deconvolution, confocal or similar three-dimensional imaging approaches is able to obtain the intensity of two colors, for example blue and green, of the nucleus at every pixel imaged, thereby doubling the amount of data points.

To obtain an image of telomeres and/or chromosomes, a stain such as DAPI can be used to preferentially mark the heterochromatin material that comprises DNA. A second stain, such as Cy3, together with an appropriate label, such as PNA telomere probe, can be used to mark the telomeric portion of the heterochromatin material.

To improve the quality of the image data, various techniques can be brought to bear as known to those of ordinary skill, such as constrained iterative deconvolution of the image data to improve resolution. Constrained iterative deconvolution may not be required if 3D image data of high enough resolution and free of any blurring are obtained (i.e. by ApoTome technology, enhanced confocal or any other novel 3D imaging technology).

The image data processor 104 processes the image data to find a set of coordinates i = l,...,N_t where is a position of the ith telomere. For this purpose, the image data processor 104 identifies "blobs" within the image data that can be identified as a telomere and/or chromosome using a segmentation process. Each blob identified as a telomere and/or chromosome has a non-negligible volume (for example, a small telomere may have a volume of 4x4x4 pixels, a large one a volume of 10x10x10, where the size of the nucleus may be approximately 200x200x100 pixels). There is some freedom, therefore, in choosing "the position" of the telomere and/or chromosome. One possibility is to choose for this position the center of gravity of the telomere and/or chromosome, or more generally, the telomere and/or chromosome organization.

The optimizer 106 finds a plane P^mn that is closest to the set of coordinates. To find the closest plane, the distance A between the location of the ith telomere, CWiO₁ and the plane given by cu + by + cz = 0 j_s considered:

The optimizer 106 finds the parameters a,b,c,d that minimize the

N

∑D,(a,b,c,d) function <=i

The characteristic module 108 proceeds to find at least one parameter that can be used to characterize the 3D organization of telomeres and/or chromosomes. "Parameters used to characterize the organization of telomeres and/or chromosomes" include:

1) A set of distances h--,N_t where ^d, is the distance between

C*..?,.*,) and the plane P^mD .

2) d and σ, the average distance and standard deviation of the set of distances Wh

- 1

N and

$ JL ( \dd, - - d ^Λ)²

Jl -I ^N , respectively. 3) A three-dimensional geometrical shape that best encompasses the 3D organization. For example, the geometrical shape can be the ellipsoid, having principal axes Wv ^and «₃, that best encompasses the 3D organization of the telomeres and/or chromosomes. Several definitions of "best encompasses" can be used. For example, the ellipsoid that best encompasses the telomeres can be defined as the ellipsoid of smallest volume that encloses a certain fraction (e.g., 100%) of the telomeres. If a set of more than one ellipsoid fulfills this condition, other restrictions can be used to reduce the set to just one ellipsoid, such as further requiring the ellipsoid to have the smallest largest ratio of principle axes (i.e., the "most circle-like" ellipsoid). It should be understood that other definitions of "best encompasses" the telomeres and/or chromosomes can be used.

It has been observed that the ellipsoid that best encompasses the telomeres often approximates an oblate spheroid with °i approximately equal to ^a2. In such case, it is sufficient to specify just ^a2 and ^α3. Alternatively, an oblateness ratio, ^ ^ or ^<V^α3, can be used to characterize the oblate spheroid describing the organization of the telomeres.

4) A set of volumes vw , where ^v> is the volume of the ith telomere.

5) A set of three dimensions {(^Dx,^>Dy.'^Dz,)} , i - WV, _wnere are principle axes of an ellipsoid describing the ith telomere.

6) A set of intensities Iv, ' = !.->#, where ^ is the total intensity of the ith telomere. (In other embodiments, instead of the total intensity, the average intensity of each telomere can be computed.) That is, if the ith telomere is associated with K pixels, then

where ¹^ is the intensity of the jth pixel of the ith telomere. In the last three cases, the sets can be used to calculate statistical measures such as an average, a median or a standard deviation. The parameters 1-5 outlined above characterize the 3D organization of the telomeres and/or chromosomes by focusing on the geometrical structure of the telomeres and/or chromosomes. Parameters 1 and 2 are motivated by the finding that, especially during the late G2 phase of the cell cycle, telomeres tend to lie on a plane. Parameters 1 and 2 measure deviations of telomeres from a planar arrangement.

Parameter 3 attempts to describe, with features, such as the three principal axes of an ellipsoid or the oblateness ratio, the overall shape of the 3D organization. While parameters 1-3 are global geometric characteristics, dealing with the overall shape of the organization, parameters 4 and 5 are local geometric characteristics in the sense that they involve the geometry of each individual telomere.

The final parameter is also local, involving the intensity of each individual telomere. As stated above, the characterizations of the 3D organization of telomeres and/or chromosomes can be used for a number of purposes. For example, by comparing a parameter to a standard value, diseases may be monitored or diagnosed, as described above. The standard (control) values can arise from population studies, theoretical models, or the characterization of control cells. In addition, the characterizations of the 3D organization of telomeres and/or chromosomes can be used to monitor or detect genomic instability in a cell.

The following non-limiting examples are illustrative of the present invention: EXAMPLES

Example 1 : Detecting Hereditary versus Non-hereditary Breast Cancer

Quantitative-fluorescent in situ hybridization (Q-FISH) analysis was performed on breast cancer tissues with and without BRCA1/2 gene mutations in order to determine the telomeric organization in hereditary breast cancers. The results show a distinctive telomeric organization in hereditary breast cancers as compared to non-hereditary breast cancers and controls. Methods Cells

The following cells were used in this study:

^• normal breast cells obtained from normal area of breast cancer tissue;

• non-BRCA1/2 breast cancer cells, which are breast cancer cells having wild type BRCA1 and BRCA2 genes (non-hereditary breast cancer);

• BRCA2 breast cancer cells, which are breast cancer cells with mutated BRCA2 genes (hereditary breast cancer); and

• BRCA1 breast cancer cells, which are breast cancer cells with mutated BRCA1 genes (hereditary breast cancer).

The non-BRCA1/2 breast cancer cells are non-hereditary, while the BRCA1 and BRCA2 breast cancer cells are a hereditary form of breast cancer.

Q FISH (Telomere Hybridization)

The following protocol was used to perform the telomere hybridizations. 1. slides were incubated at room temperature (RT) in IxPBS for 5 minutes; not shaken 2. slides were transferred into 3.7% formaldehyde/1 xPBS (freshly prepared). Incubated for 10 minutes (if using cytospins or single cell suspension or cells grown on slides) or incubated for 30 minutes if using tissue sections (5 μm). The incubation was done in the hood. 3. slides were shaken on a platform 3 x 5 minutes in IxPBS at RT 4. 10' 37⁰C pepsin treatment was done (in 0.01 N HCI, final concentration of pepsin: 50 μg/ml)

5. slides were shaken on a platform 1x5 minutes in IxPBS at RT

6. 3.7% formaldehyde /IxPBS was applied for 10 minutes at RT, not shaken (incubated in the hood) 7. slides were shaken on a platform 3 x 5 minutes in IxPBS at RT

8. slides were washed with 70%, 90%, 100% EtOH at RT for 2 minutes each, not shaken 9. slides were air dried

10.5 μl of PNA-telomere probe was applied (in the dark), 25x25 mm cover slip was added, rubber cement was applied to seal

11. denatured for 3 minutes 8O⁰C, hybridized 2 hours at 3O⁰C (Hybrite™, Vysis)

12. rubber cement was removed carefully and the rest of the protocol was worked in the dark

13. slides including cover slips were placed in 70% formamide/10mM Tris (pH 7.4) at RT, the cover slip was let to float off 14. slides were washed 2 x 15 minutes in 70% formamide/10 mM Tris (pH

7.4) shaking

15. washed in IxPBS at RT for one minute

16. washed 5 minutes at 55⁰C in 0. IxSSC, shaking

17. washed 2 x 5 minutes in 2xSSC/0.05% Tween 20, shaking 18. stained with DAPI (0.1 mg/ml stock), 50 μl was applied, covered with cover slip, incubated for 3 minutes in the dark at RT

19. rinsed with ddH2O

20. dehydrated with 70%, 90%, 100% EtOH (RT), 2 minutes each

21. air dried slides 22. mounted with a drop of anti-bleach

23. imaged Image acquisition and 3D analysis.

The cells were analyzed by 3D imaging using Axioplan 2 (Zeiss) with 100W fluorescence, a cooled AxioCam HR B&W with 1x adaptor (Zeiss). DAPI, FITC and Cy3 filters (Zeiss) were used in combination with Planapo 63x/1.4 oil (Zeiss). Axiovision 3.1 software with deconvolution module and rendering module were used (Zeiss). 80 and 160 sections were acquired at 100 and 200 nm respectively. The constrained iterative algorithm option was employed²² and surfaces rendered in x, z and x, y axes. The total nuclear volume (sphere) was calculated using the following formula; v=(4π/3)r³=(π/6)d³. The volume of the segment of the 3D nuclear sphere was calculated using the following formula: v=(π/6)(3r-ι² + 3r₂ ² +h²)h, with radius of the sphere: r, radii of bases: T₁₁T₂, height: h, volume: v, diameter: d (PCT/CA2004/000241).

Results As can be seen in Figure 1 , breast cancer tissues with mutated BRCA1 or BRCA2 genes (hereditary breast cancers) have telomere aggregates that are much larger in volume and a fewer number of detectable telomeres than non-hereditary breast cancer cells with wild type BRCA1 and BRCA2 genes. The following abbreviations are used in Figure 1 : 2D means two-dimensional, 3DF means three-dimensional front view, 3DS means three-dimensional side view and 3DR means three-dimensional reconstruction.

The results show distinct telomeric organizations for normal breast cells, breast cancer cells with wild type BRCA1 and 2 genes (non-hereditary), and breast cancer cells with mutant BRCA1 or 2 genes (hereditary). On examination of breast cancer tissues containing wild type BRCA1 and 2 genes (non-hereditary), telomere aggregation is encountered as expected (PCT/CA2004/000241). Specifically, the telomeres show aggregates, and fail to properly align as compared to telomeres of normal breast cells. On the other hand, breast cancer tissues harboring mutated BRCA1 or 2 genes (hereditary) have telomere aggregates that are distinctively much larger in volume and a fewer number of telomeres as confirmed by geometric analysis as compared to breast cancer cells with wild type BRCA1 and 2 genes (non- hereditary). In addition, there also are cases of telomeres being much smaller in volume, which may reflect heterogeneity among breast cancer patients. Initial results have allowed differentiation of mutant versus non-mutant breast cancer. The current data suggests that telomeric organization in inherited diseases has distinctive parameters including volume and number when compared to their wild type counterpart. Example 2: Detecting Hereditary versus Non-hereditary Breast Cancer

3D image analysis was performed on cancer cells with BRCA1 mutation (HCC 1397), BRCA2 mutation (Capan 1), and wild type BRCA1 and

BRCA2 (MCF 7), and analyzed using TeloView™ software as disclosed in PCT/CA2004/00241 and herein. A total of 100 nuclei were studied per cell line.

As can be seen in Figures 2 and 3, there is a significant difference in telomere signal intensities between cell lines with and without BRCA mutations. The p values were highly significant (<0.0001), when HCC 1397 and Capan 1 are compared to MCF 7 by analysis of variance (ANOVA) and unpaired t test (Figure 2). The average telomere signal intensities of the three cell lines are shown in Figure 3 (p value O.001).

Example 3: Translocation and Telomeric Organization of Plasmacytoma Genomic instability was investigated by incorporating information of individual genes. A novel technique called the three-dimensional (3D) combined telomere-gene analysis was used to analyze translocations of chronic inflammation (e.g. pristine)-induced BALB/c murine plasmacytomas that harbor c-Myc (chromosome 15)//gH (chromosome 12) translocations. This technique begins with three-dimensional spatial analysis of the nucleus by performing Q-FISH to assess telomeric organization, and then performing c-MycllgH FISH on the identical nucleus to localize the translocation. Methods

1. Q-FISH (telomere hybridization) was performed. See Q-FISH protocol above.

2. Areas of interest (AOI) were imaged. The AOI was recorded using a position slide or other means to track the same position.

3. FISH (c-Myc/lgH FISH) was performed. See FISH protocol below.

4. The SAME AOI was imaged using the position slide. FISH Protocol

1. equilibrate slides in 2xSSC, RT, 10 minutes

2. RNAase A treatment (100μg/ml in 2xSSC), 1 hour, 37⁰C 3. wash slides three times for 5 minutes each in 2xSSC, RT, shaking

4. pepsin treatment (50μg/ml in 0.01 M HCI), 37⁰C, 10 minutes

5. washes: 2x5 minutes in IxPBS at RT, shaking

6. 1x5 minutes in 1xPBS+50mM MgCl2 at RT, shaking 7. postfixation: (fresh solution) 1 % formaldehyde in 1xPBS+50mM MgCl2, RT, 10 minutes

8. 1x5 minutes wash in IxPBS at RT, shaking

9. dehydrations: in 70%, 90%, and 100% ethanol, 3 minutes each, RT, then air dry (5 minutes) 10.denaturation of slides in 70% formamide/2xSSC (pH=7.0, critical, formamide must be deionized), prewarm slides for 5 minutes at 70⁰C, transfer them immediately to denaturation solution, incubate 2 minutes, transfer immediately to cold ethanol (-20⁰C): 11. dehydration in cold ethanol: 70%, 90%, 100%, 3 minutes each, -20⁰C 12. denature probe: 5 minutes 85⁰C, add approximately 100ng probe per slide in hybridization solution, 50% formamide, deionized, 2xSSC, 10% dextransulfate, 50 mM phosphate

13. add cover slip, seal, incubate in humidified atmosphere, 37⁰C, 12-16 hours 14. wash: 3x5 minutes in 50% formamide/2xSSC, 42⁰C, shaking

15. wash 5x2 minutes in 2xSSC, shaking

16. add counterstain (i.e. DAPI: 1 μg/ml for 2D studies; 0.1 μg/ml for 3D studies) and anti-bleach if no additional detection steps are required

17. If indirect labeled probes are used, continue the detection of probe by the use of specific antibodies:

18. block in 3% BSAMxSSC for 10 minutes, RT

19. add antibody in 3% BSA/4xSSC for 30 minutes, 37⁰C

20. wash 2x5 minutes in 4xSSC/0.1% Tween 20, 42⁰C, shaking.

21. Note: additional washes may be needed. 22. add counterstain (i.e. DAPI: 1 μg/ml for 2D studies; 0.1 μg/ml for 3D studies) and anti-bleach. Results

Figure 4 shows telomeric organization of pre-malignant plasmacytoma lesions. The 2D image shows an aggregate shared by 4 cells at the center of the image. An arrow points to the big aggregate in the 3DF and 3DS views. The following abbreviations are used: 2D means two-dimensional, 3DF means three-dimensional front view and 3DS means three-dimensional side view.

Figure 5 shows the same AOI imaged after the FISH experiment. Panel A shows 2D, 3DF, and 3DS images of IgH gene alone. Panel B shows 2D, 3DF, and 3DS images of c-Myc gene alone. Panel C shows 2D, 3DF, and 3DS images of IgH AND c-Myc genes. The IgH gene is FITC-labeled (green). The c-Myc gene is Cy3-labelled (red). The orange signals appear in the 2D image, which is the mixture of both signals and indicates translocation. The green color shows IgH gene alone without combining with c-Myc gene. The translocation/juxtaposition of two genes are clearly shown in the 3DS image. Note there is polarity of translocation. IgH gene (green color) is always surrounded by c-Myc gene (red color). The following abbreviations are used: 2D means two-dimensional, 3DF means three-dimensional front view and 3DS means three-dimensional side view. Table 1 shows that juxtaposition/translocation of the c-Myc gene and

IgH gene is observed in pre-neoplastic lesion (plasmacytotic focus or atypical focus) in inflammation-induced BALB/c mice as early as 14 days. Note the telomeric changes (telomere aggregation) occur even earlier than translocation. In focus 1919 areas 1 and 2, focus 1937 areas 4 and 5, and focus 1938 area 3, telomere aggregations are seen before the occurrence of translocation. The only exception is focus 1938 area 1 , translocation is observed before telomere aggregation (no telomere aggregation is seen). Alternatively, telomeric aggregates are resolved once translocations have formed. Translocations observed in these 3 cases are statistically significant as the p value = 0.0102.

In this study, 3D combined telomere-gene analysis was successfully used to relate spatial organization of genes and telomeres. Juxtaposition of c- Myc/lgH in 3D intranuclear space was clearly demonstrated. Moreover, telomere aggregates are seen in cells with the translocation. Results suggest that more translocations are seen as early pre-neoplastic lesions advance into late pre-neoplastic lesions.

Example 4: Translocations and Telomeric Organization in Immortalized

Murine Cells

Methods

Cells and conditional Myc activation Ba/F3⁵⁷ (pro-B lymphocyte) and Pre-B⁵⁸ (pre-B lymphocyte) cell lines and the plasmacytoma cell line MOPC460D were used. Cells were maintained at a density of 10⁵ to 10⁶cells/ml. Cell viability was determined by hemacytometer counts using trypan blue (Sigma-Aldrich, Oakville ON, Canada). The primary mouse plasmacytoma DCPC21 was isolated from a BALB/c mouse⁵⁹. v-ab//myc-induced plasmacytomas⁶⁰ and primary lymphocytes were collected from BALB/c mice (Central Animal Care protocol 02-039).

To activate MycER™⁶¹ in Ba/F3 (pro-B lymphocyte) or Pre-B (pre-B lymphocyte) cells, 10⁵cells/ml were treated with 100 nM 4-hydroxytamoxifen (4HT) (Sigma-Aldrich, Oakville ON, Canada). Cells were split 24 hours prior to 4HT treatment. Non-4HT treated control cells were cultivated in the presence of ethanol that is used to dissolve 4HT⁴⁷⁴⁸⁶¹. Two different MycER™ activation schemes were performed. First, analyses of c-Myc-induced changes in telomere organization after 4HT were carried out over a 10 day time course where nuclei were examined every 24 hours. A second time course was performed every six hours over a time period of 120 hours (Figure 6). To enable a time-dependent analysis of Myc activation, 4HT was given for 2 or 12 hours respectively and was subsequently removed. Alternatively, 4HT was added every 12 hours or was given once but left in the culture. The effects of 4HT are cell type and species specific and last for 15-24 hours in cell lines^62"64. MycER™ activation was determined by fluorescent immunohistochemistry. lmmunohistochemistry

Cytospin preparations were used for fluorescent immunohistochemistry of c-Myc protein. Immunohistochemistry was performed in three independent experiments⁶⁵. A polyclonal anti-c-Myc antibody (N262; Santa Cruz, California, USA) at a dilution of 1 :100 was used. The antibody was visualized with a goat anti-rabbit IgG FITC antibody (Sigma-Aldrich, Oakville ON, Canada) at a dilution of 1 :100. Analysis of all samples was performed using a Carl Zeiss Axiophot 2 microscope and a 63x oil objective. Images were acquired with a Hamamatsu CCD SensiCam Camera and the Northern Eclipse v 6.0 software. Cell death

Apoptotic bodies for control and MycER™-activated cells were assessed by two independent observers who scored 300 DAPI-stained interphase nuclei per time point in the presence or absence of MycER™ activation.

Telomere fluorescent in situ hybridization (FISH)

Ba/F3 (pro-B lymphocytes), Pre-B (pre-B lymphocytes) and plasmacytoma cells were fixed in suspension with 3.7% formaldehyde. Cells were collected (200xg for 10 minutes), resuspended in phosphate buffered saline containing 3.7% formaldehyde (Fluka, Basel, Switzerland) and incubated for 20 minutes. Thereafter, the telomere FISH protocol was performed²³'⁶⁶ using Cy3- or FITC-labeled PNA probes (DAKO, Glostrup, Denmark). Three independent experiments were performed for each data set. The number of interphase nuclei examined was >30 per experiment; the number of metaphases examined by telomere FISH was 20. 3D image acquisition of nuclei was performed as described below and in Chang TC et al.²³. Imaging of metaphases after telomere FISH was performed using Axioplan 2 (Carl Zeiss Inc., Canada) with 100W fluorescence, a cooled AxioCam HR B&W with 1x adaptor (Carl Zeiss Inc., Canada), DAPI, Cy3 or FITC filters (Carl Zeiss Inc., Canada) in combination with Planapo 63x/1.4 oil (Carl Zeiss Inc., Canada). Metaphase images were acquired using the Axiovision 3.1 software (Carl Zeiss Inc., Canada) in Multichannel mode. Due to the presence of multiple variables, the General linear Modeling (GLM) procedure was used. To test average aggregates among different groups and two-way ANOVA test was performed for normality and robustness of the data (≥ 30 nuclei per time point).

3D image acquisition

Thirty or more nuclei were analyzed by 3D imaging using Axioplan 2 (Carl Zeiss Inc., Canada), a cooled AxioCam HR B&W. DAPI, FITC and Cy3 filters (Carl Zeiss Inc., Canada) were used in combination with Planapo 63x/1.4 oil (Carl Zeiss Inc., Canada), which gives a sampling distance of 107 nm in the xy-direction. Axiovision 3.1 software with deconvolution module and rendering module were used (Carl Zeiss Inc., Canada). For every fluorochrome the 3D image consists of a stack of 100 images with a sampling distance of 200 nm in the z-direction. The constrained iterative algorithm option was employed⁶⁷.

3D image analysis for telomeres

Telomere measurements were done using TeloView™ ²³'⁶⁸ choosing a simple threshold for the telomeres. Based on the resulting binary image, the center of gravity of intensities was calculated for every object. This resulted in a set of coordinates (x, y, z) denoted by crosses on the screen. The integrated intensity of each telomere was calculated since it is proportional to the telomere length⁶⁹. The integration region was determined by growing a sphere on top of the found coordinate. After every step of growth (iteration) the sum under this volume (the telomere) was subtracted by the sum just surrounding it (its background level). When the growth of the sphere did not contribute to an integrated intensity increase, the algorithm stopped and the integrated intensity of the telomere with an automatic background correction was obtained. Chromosome painting and measurements of chromosomal overlap(s) in interphase nuclei

Chromosome painting was carried out in three independent experiments according to standard protocols⁷⁰ using chromosome paints for mouse chromosomes 5 (Cy3), 13 (FITC), 7 (Cy3), 10 (FITC) and 17 (FITC) purchased from Applied Spectral Imaging (Vista, CA, USA). 3D image acquisition of nuclei painted with the above chromosome paints was performed as described above and in Chuang TC et al.²³. Measurements of chromosomal overlaps in interphase nuclei were performed after 3D image acquisition and constrained iterative deconvolution. The percentage of overlaps was calculated using three steps: (1) based on the DAPI counter- stain image the 3D boundary of the nuclear volume was determined. Data outside that volume were ignored. (2) For each one of the chromosomes, an intensity threshold was determined and referred only to voxels that were above the threshold that belonged to the specific chromosomes. The total volume occupied by each one of the chromosome pairs was measured (\Λ and V₂). (3) The volume occupied by both chromosome pairs was measured, V₀. By dividing this value by Vi and by V₂, the level of overlap relative to the total volume of each chromosome pair was measured, Vo/V-i, VoAZ₂. Spectral karyotyping (SKY)

Mouse spectral karyotyping (SKY) was carried out as described⁵⁹. SKY was performed over a 10 day period. Significant values for chromosomal rearrangements per individual chromosome were found after MycER™ activation. Mean total chromosomes, as well as numbers of each specific chromosome observed for control and c-Myc-activated cells were compared over time by two-way analysis of variance. For other chromosomal aberrations the common (at least 10%) incidences in specific chromosomes were also compared for both groups of cells over time with the Mantel- Haenzsel stratified analysis. In addition, statistical analyses were performed for the occurrence of translocations, breakages and fusions over the experimental period of 120 hours, p values of less then 0.05 were considered significant. Only the Frequency procedure was used followed by Fisher's exact test. The p value of the overall study was < 0.0001. Results

The three-dimensional organization of telomeres prior to c-Myc activation

It was examined whether c-Myc deregulation affected the 3D organization of telomeres in the interphase nucleus over a time period of 10 days. The effect of conditional c-Myc expression was analyzed in two independent immortalized mouse B lymphocyte lines, Ba/F3⁵⁷ (pro-B lymphocytes) and Pre-B⁵⁸ (pre-B lymphocytes), stably transfected with MycER™⁶¹. For both Ba/F3 (pro-B lymphocytes) and Pre-B (pre-B lymphocytes) cells, the 3D organization of telomeres in nuclei of non- MycER™-activated cells were first evaluated. Primary BALB/c B lymphocytes served as a control. Telomeres of normal primary BALB/c B nuclei showed non-overlapping telomere positions as determined by 3D imaging (Figure 7a). In the absence of MycER™ activation, both Pre-B and Ba/F3 interphase nuclei also displayed non-overlapping telomere positions (Figures. 7b and c respectively). Therefore, the above cell lines were appropriate to study the effects of conditional c-Myc activation on the 3D telomeric organization. c-Myc-dependent disruption of the 3D telomeric organization: formation of telomeric aggregates (TAs) in interphase nuclei To test c-Myc's potential impact on the 3D nuclear organization of telomeres, the effect of conditional c-Myc expression on their 3D organization in Pre-B (pre-B lymphocyte) and Ba/F3 (pro-B lymphocyte) interphase nuclei was analyzed. After a transient MycER™ activation with 4-hydroxytamoxifen (4HT), nuclear c-Myc signals were observed in both Pre-B (pre-B lymphocytes) and Ba/F3 (pro-B lymphocytes) cells (Figures 8b and d). Non- 4HT treated cells were used as controls and showed only cell cycle dependent fluctuations in their c-Myc protein expression. Most of their nuclei therefore were devoid of protein staining, and MycER™ was found in the cytoplasm (Figures 8a and c, see also Littlewood TD et al.⁶¹). To determine whether the conditional expression of c-Myc affected the

3D organization of telomeres, a time course of 10 days was performed. In these first sets of experiments, c-Myc deregulation and 3D telomeric organization were investigated in both Pre-B (pre-B lymphocytes) and Ba/F3 (pro-B lymphocytes) cells. Nuclei were analyzed following c-Myc deregulation at 0, 24, 48, 72 and 96 hours as well as at 10 days. They were compared at all times to nuclei from mock-treated control cells. Analysis of the 3D organization of telomeres in nuclei of these cells revealed that c-Myc deregulation induced the formation of telomeric aggregates (TAs) in both cell lines. TAs are group(s) of telomeres that are found in clusters and thus in close association in the interphase nucleus. This 3D telomeric organization is distinct from the normal 3D organization seen in both non-c-MycER™- activated Pre-B (pre-B lymphocytes), Ba/F3 (pro-B lymphocytes) cells and primary mouse lymphocytes (Figure 7). Figure 9 illustrates the presence of TAs in interphase nuclei of c-MycER™-activated Pre-B and Ba/F3 cells (Figure 9b and d respectively). The presence of TAs in conditional c-Myc expressing cells is a novel finding. Statistical analyses showed that the significance of TA formation per nucleus was time-dependent as was the induction of TAs. For example in Pre- B cells, TA numbers increased to significance at 48 hours (p=0.01) and reached their highest significant values at 72 hours (p=0.001). At 96 hours, the number of TAs per nucleus declined. Similar results were obtained for Ba/F3 cells. c-Myc induces cycles of telomeric aggregates (TAs) in interphase nuclei In subsequent experiments, the time relationship between c-Myc deregulation and the formation of TAs was investigated more closely. To this end, cells were harvested every six hours over a time period of 120 hours (Figure 6). In addition, the duration of conditional expression of c-Myc was experimentally varied (Figure 6) and nuclear c-Myc staining was confirmed as above (Figures 8 and 10a). Next, the 3D organization of telomeres was determined (Figs. 8b and c). At this point of the study, near diploid Pre-B cells were only focused on⁷¹. The positive controls were cells constitutively overexpressing c-Myc (mouse plasmacytomas⁴⁹ and a mouse plasmacytoma cell line (Figure 10b (e)). Negative controls were mock-treated Pre-B cells (Figure 10b (a)). This detailed time course confirmed that c-Myc deregulation induces TAs. As shown in representative images, TAs vary in size and numbers per MycER™-activated Pre-B cell nucleus (Figure 10b (b-d), red arrows). High induction levels of TAs were observed at 30, 48, 72 and 96 hours and declined after 96 hours (Figure 10c, arrows). The highest levels of TA formation, hereafter referred to as peaks, corresponded to the first set of experiments. Additionally, this detailed time course revealed a more complex picture. TAs formed in a c-Myc-dependent manner and showed a cyclic appearance. The number of TA cycles was directly linked to the duration of c- Myc deregulation. For example, 2 hours of 4HT-mediated c-Myc activation induced three such cycles, while 12 hours of 4HT administration led to five such cycles. In this context, repeated consecutive activations of MycER™ given every 12 hours (Figure 6) caused TAs in 96% of all nuclei. These cells died after 30 hours (Figure 10c, blue curve) due to repeated cycles of c-Myc deregulation and not due to toxicity exerted by 4HT⁷². Thus only a single TA cycle is observed in this experimental setting (Figure 10c, arrow head). The c-Myc induced TA cycles represent breakage-bridge-fusion (BBF) cycles and chromosomal rearrangements

The cycles of c-Myc induced TAs in Pre-B interphase nuclei showed similar periodicity for all c-Myc activation periods (Figure 10c). It was hypothesized that these cycles might reflect both ongoing associations and dissociations of telomeres or breakage-bridge-fusion (BBF) cycles. The BBF cycle could be induced by the breakage of dicentric chromosomes during anaphase inducing apoptosis of cells having high numbers or large sizes of TAs per nucleus. To address these possibilities, metaphase chromosomes at different times prior, during and after the peaks of nuclear TA formation period for a 120 hour period were examined. Both whole genome analysis by mouse spectral karyotyping (SKY) as well as telomeric FISH of metaphase chromosomes were used. A significant level of dicentric chromosomes was noted. Figure 11 shows representative examples.

Control cells had normal karyotypes (Figure 11 , Panel A). In MycER™- activated Pre-B cells, however, fusions had occurred at the telomeric ends of chromosomes 18 and 4 (Figure 11 Panel B (a), red and green arrows) as well as between two chromosomes 1 (Figure 11 Panel B (a), green arrow). Chromosome 1 was most probably was broken in the previous anaphase (Figure 11 Panel B (a), green circle). An additional broken chromosome 1 is in the center of the same metaphase, and chromosomes 2, 3, and 7 show terminal deletions (Figure 11 Panel B (a)). Telomeric fusions involving both ends of mouse chromosomes as well as sister chromatids were also confirmed by telomeric FISH (Figure 11 , Panel B (b)). Anaphase bridges and ring chromosomes were present as well (Figure 11 , Panel B (c)). Careful analysis allowed the sequence of events to be determined:

Initially, at peaks of TA formation, high levels of end-to-end fusions were observed. These were followed by increased numbers of broken chromosomes and non-reciprocal translocations (Figure 11 , Panel B (d)). Statistical analyses confirmed the significance of these chromosomal aberrations over time (Table 2). It was concluded that the TA cycles reveal BBF cycles, namely the fusions of two chromosomes, consequently the formation of dicentrics, and their subsequent breakage in anaphase (Figure 11 , Panel B (a-d)). The cycles are induced by conditional c-Myc deregulation and lead to the onset of genomic instability as demonstrated by the chromosomal rearrangements that result from these BBF cycles.

The contribution of cell death to the decrease in the number of cells with TAs during the time course of the experiments was then investigated. If this was the case, a correlation of cell death in c-Myc activated cells and the peak of TA formation or shortly thereafter would be observed. Apoptotic bodies were scored by two independent observers. The level of apoptosis was about twofold higher in c-Myc-activated cells than in control cells (Table 3). There was no preference in apoptotic cell death for any specific time point during the time course 120 hours. Thus, breakage-bridge-fusion cycles and not apoptotic cell death contributed to the cycling of TA formation. Table 2 shows chromosomal rearrangements in MycER™-activated

Pre-B cells over a period of 120 hours. 20 metaphases were analyzed per time point using SKY and after a single administration of 4HT (see Figure 6). End-to-end fusions predominate at early TA cycles (40% of the metaphases show fusions, p<0.0001). The frequency of chromosome breakage was significant throughout this analysis period (p=0.001). Non-reciprocal translocation were also found in the first TA cycle (in 20% of the metaphases) and reached significance albeit at lower levels. As time progresses, fusions decreased and translocations and breakages increased. M: metaphase; numbers following M indicate metaphase numbers; h: hours.

Table 3 shows apoptosis levels in non-MycER™ and in MycER™- activated Pre-B cells. Two independent observers scored 300 nuclei per time point and determined the percentage of apoptotic bodies. The numbers given are percent apoptosis per time point and fold increase in apoptosis per time point.

3D organization of chromosomes in c-Myc activated interphase nuclei TAs and the initiation of BBF cycles with subsequent chromosomal rearrangements prompted the investigation of whether or not chromosomes were affected in their 3D nuclear positions during MycER™ activation. To this end, the overlap of specific chromosomes over the time period of 120 hours was examined. SKY analyses of Pre-B MycER™ cells suggested chromosomal rearrangements involving chromosomes 7, 13, and 17. Other chromosomes were involved in rearrangements as well but did not reach significant levels. Three different combinations of chromosomes over a period of 96 hours were examined. This period covered all peaks of TA formation (Figure 10c). As shown in Figure 12, a change in chromosomal overlaps between chromosomes 5 (red) and 13 (green) was observed over the time period examined (Figures. 12a and b). Both chromosomes were found in closer vicinity as the cells entered into the first TA cycle. Chromosomes 10 (green) and 7 (red) also showed an increase in the percentage of overlap (Figures 12a and b). The overlap patterns for chromosomes 7 (red) and 17 (green) also changed (Figures 12a and b). Discussion c-Myc induces telomeric aggregates, fusions, and BBF cycles

Previous studies have unequivocally established that c-Myc triggers a complex network of genomic instability at the level of single genes^35"3741 and whole chromosomes^38"39 (for review, see Parada LA et al.²⁸). In addition, c- Myc induces illegitimate replication initiation⁴¹'⁴², long-ranging chromosomal rearrangements⁴¹, DNA breakage, alterations of DNA repair^43"45 and a low level of point mutations^46"47. A novel mechanism underlying c-Myc-dependent genomic instability at the chromosomal level directly affects the integrity of the telomeres and is revealed here for the first time.

The clear periodicity of the TA cycles that was found with four different c-Myc activating treatments suggested a biological relevant c-Myc-dependent process. Theoretically, cycles of c-Myc-induced TAs could reflect; i) nuclear remodeling with the transient association and subsequent dissociation of telomeres; (ii) end-to-end fusions of chromosomes that initiate breakage- bridge-fusion (BBF) cycles as first described for metaphase chromosomes by Muller⁷³ and B. McClintock⁷⁴; (iii) c-Myc induced cell death; and (iv) a combination of all of the above. The data obtained are most consistent with BBF cycles and exclude apoptosis as a direct contributor to the TA cycles. Apoptosis occurred at equal levels throughout the study and consistently reached about twice the levels seen in the control cells and at all times. The loss of cells was compensated by a twofold increase in proliferation in MycERTM-activated Pre-B⁷⁵. Thus, apoptosis did not directly contribute to the TA cycles. These data also indicate that there is genetic separation of genomic instability and apoptosis as had been previously reported for Ba/F3 cells⁷⁶. Whether telomere associations and dissociations as imaged in live cells over a period of twenty minutes⁷⁷ contributed to the TA cycles is presently unknown.

Direct evidence for the role of BBF cycles in the periodicity of TAs came from a detailed analysis of chromosomal fusions, breakage and rearrangements observed over the time course of five TA cycles. The occurrence of end-to-end fusions of chromosomes that generated dicentric chromosomes was demonstrated. Such dicentric chromosomes are known to break during anaphase, leaving one chromosome or chromatid with a piece from the chromosome or chromatid it was fused to. The resulting chromosomes or chromatids then carry broken ends that will often undergo fusions with other chromosomes, a cycle of events termed breakage-bridge- fusion (BBF) cycle. Experimental data support the time frame of these cytogenetic events; first fusions, then breakage, then non-reciprocal translocations. Moreover, the periodicity of these events is consistent with a ~12 hour population doubling time of the Pre-B cells⁷⁵. Thus each peak of TAs observed in interphase nuclei after conditional c-Myc expression was consistent with the repeated formation of TAs. Each time point after the peak that was devoid of TAs is in agreement with the breakage of dicentric chromosomes. The telomere-free ends would then fuse again and begin new cycle(s) until no more free chromosome ends persisted. From telomeres to chromosomal rearrangements: A new pathway of c- Myc-dependent genomic instability

BBF cycles have been first reported by Muller⁷³ and McClintock⁷⁴. The inventors have shown that c-Myc is one key factor that is able to initiate genomic instability through BBF cycles that begin with the aggregation and/or fusion of telomeres in the interphase nucleus and are confirmed by the occurrence of end-to-end fusions of chromosomes in metaphase chromosomes. Such BBF cycles in telomerase-positive immortalized mouse Pre-B cells with long telomeres are distinct from BBF cycles reported for critically short telomeres⁸³⁸⁶. Some TAs represent fusions as evident by the analysis of metaphase chromosomes. TAs and end-to-end fusions depended on the time and levels of c-Myc activation. The analysis of frequencies of both events showed that they are closely linked: TAs and fusions precede translocations. As the fusions initiate BBF cycles, the frequencies of breakage and non-reciprocal translocations increase over time. A novel pathway of c-Myc-dependent genomic instability thus starts at the telomeric ends of the chromosomes. Both TAs and BBF cycles are the manifestation of deregulated c-Myc expression leading to chromosomal rearrangements and subsequently to genomic instability.

Local chromosome movements that increase the overlap of chromosomes in the interphase nucleus may also contribute to this phenotype since this temporal change in local positioning may permit the direct contact of chromosomal ends and may thus facilitate recombinations and/or fusions. These movements were observed after c-Myc deregulation and suggested an impact of the oncogene on local nuclear positioning of chromosomes. Chromosome movements studied by others were of three types. One, they led to repositioning of sub-chromosomal regions within stable chromosome territories, second, there were selective movements of single territories, and third they represented small scale refolding events within sub-chromosomal regions⁸⁷. Positional changes of chromosomes during the cell cycle or during quiescence or senescence have been reported as well^88"91.

Several regulatory pathways that involve oncogene deregulation may affect the 3D nuclear organization. Oncoproteins, including c-Myc, can alter the 3D nuclear organization and the organization of chromatin^92'94. Also, they can have an effect on the nuclear matrix. High mobility group protein I(Y) (HMGI(Y)) is a c-Myc-dependent nuclear matrix protein⁹⁵ with increased expression during neoplasia²⁶. Also, the analysis of myc binding sites in the human genome suggests c-Myc binds to genes encoding nucleoskeletal components⁹⁶. Furthermore, constitutive c-Myc expression was shown to be associated with down regulation of the telomere repeat binding protein TRF2³², a protein required for telomere capping and genome stability⁹⁷. Myc is also involved in the regulation of DNA repair⁴⁴'⁴⁵ and has been shown to induce DNA breakage⁴³. Thus taken together, many different c-Myc- dependent mechanisms could potentially affect the nuclear organization and, as shown here for the first time, converge at the telomeres.

Example 5: 3D Simultaneous Protein-FISH Analysis A novel approach was developed termed the 3D simultaneous protein-

FISH analysis to reduce the amount of time and labor involved in studying proteins and nuclear structures. This analysis allows the expression of protein and nuclear structures of interest in the identical cells to be concurrently assessed. As an illustration of the application, pre-B cells are stained with primary H3P antibody, followed by Q-FISH and then stained with a secondary antibody. The following method was used:

1. Cells were harvested 24 hours after splitting.

2. Cells were fixed in 3.7% formaldehyde/1 xPBS.

3. 3D fixation - cells were dropped on the slide, air dried, fixed in 3.7% formaldehyde for 30 minutes. 4. Slides were washed in 1xPBS/50mM MgCI₂ (2x5minutes).

5. Permeablized in 0.2% Triton X-100 for 12 minutes.

6. Washed in 1X PBS/50mM MgCI₂ (3x5minutes).

7. Blocked in lamb serum for 5 minutes.

8. Primary antibody was added and incubated in room temperature (RT) for 45 minutes.^*

9. Washed in 1XPBS/ 50mM MgCI₂.

10. Fixed in 3.7% formaldehyde/1 X PBS for 20 minutes RT.

11. Dehydrated in ethanol (70%, 90%, 100%) 2 minutes each and allowed to air dry. 12.5 μl of PNA telomere probe was applied to each slide.

13. Denatured for 3min at 80⁰C

14. Hybridized for 2 hours at 30⁰C (humidified atmosphere)

15. Washed in 70%formamide/0.5MTris (2x15minutes) at RT.

16. Washed in IxPBS for 1 minute 17. Washed in 0.1 xSSC (55C) for 5 minutes.

18. Washed in 2xSSC/Tween 20 (2x5minutes).

19. Washed in 1XPBS/50mM MgCI₂ for 5 minutes.

20. Blocked in lamb serum for 5 minutes

21. Secondary antibody was added. Incubated in RT for 30 minutes^* 22. Washed slides in 1XPBS/50mM MgCI₂

23.Added DAPI (0.1μg/mL), incubated for 5 minutes 24. Dipped slides in ddH20 several times 25. Dehydrated slides in ethanol (70%, 90%, 100%). Air dry.

26. Applied anti-bleach.

27. Imaged

*May be necessary to incubate primary and secondary antibody overnight depending on antibody-antigen affinity.

Using 3D simultaneous protein-FISH analysis, the inventors have visualized higher levels of H3P expression in pre-B cells that are in G2 phase. In the same nucleus we also confirmed the presence of the telomeric disk which is formed during G2 phase. This method will significantly expedite the study involving protein and nuclear organization. In the future, this technique will be valuable in clinical laboratories that demand acquiring results with least amount of time.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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Table 1

p value of telomere aggregates: 0.8031 p value of translocations: 0.0102

Table 2

Time Translocations Fusions Insertions Breakage

M8:T3;15, T15;10, M2:5:16, M5:9:1:11, M4:ls9;Y, M8:lsX;11, M2:1,M5:X, M10:3,16, M10:TX;16, TX;5, M6:6:16, M8:X:X, M11:ls3;16, M17:1;14 M11:7, M16:13, M20:7. 12h M14:TX;1, M10:12:6, M11:15:Y:12,

M16:T1;5, T1;8. M17:7:13, M18:17:14 20%,p:0.0138 40%, p: <Q.0Q01 20%, p: 0.1084 25%

M5:T6;19, T13;4, M4:15:19, M7:14:X, none M1:15,6, M2:18, M5:4, T16;1;12, M9:T11;1, M9:4:6, M10:8:3, M8:X, M12:13, M13: 24h T9;19 M13:4:18 4,12,13,X, M15:13,

M16:13,15, M19:7 10%,p.0.1468 25%, p: 0.0168 45%

M4:T18;4, M10:15:10, M20:9:19 M12:ls13;1^*,ls13;1;14, M1:18, M2:3,6,13, M13:T16;3, M13:ls3;16, ls12;M20:1 M4:16, M7:19,4,11, M17:T2;17 M9:7,8, M10:13,

M11:3,13, M12:1,

3Oh M13:5, M14:15,

M15:6,8,X, M16:1,

M17:5,7, M18:18,

15%, p: 0.0717 M19:2,M20:15

20%, p: 0.035 10%, p: 0.1468 75%

M1:T11;3,T12;6, M11:ls11;19, M12:ls5;X, M1:18, M2:4,7, M3:3,7, M4:T16;3, M15:ls1;X, M17:ls9;X M4:17, M5:2, M10:T2;19, M7:3,17,18, M8:3,6,11, M15:T8;16, M9:11,M11:1,4,9,

42h M17:T11;2, M12:2,M13:2, M18:T15;14, M14:2,7,8,12, M20:T13;19 M17:2,7,19,X, M18:15,

M19:10,13, M21:1,13

35%, p: 0.0036 20%, p: 0.035 80%

M2:T4;15, M16:10:X, M18:6:13, M4:ls13;15, ND

M3:T11;19, M4:T8;2, M19:14:3 M7:ls11;Y;8,

48h M7:T1;16 M11:ls3;Y,

M12:ls12;11;19, M17:ln10;2

20%, p: 0.035 15%, p: 0.0717 25%,p:0.0168

M1:T3;12, M15:2:9, 3:16 M1:ls3;Y, M17:ls11;19 ND M15:T13;15, 66h M7:T10;12, T2;12,

M10:T5;X, 20%, p: 0.035 5%, p: 0.3112 10%, p: 0.1468

M4:TY;15;4, M3:6:17,8:18, M4:7:4, M2:ls5;14*, M3:ls16;X, ND M10:T15;4, M9:5:4 M7:ls15;16, M8:ls3;19,

72h M14:T19;8 M9:ls4;7, M10:ls15;13, M11:ls13;Y,M20:lsX;2, In3;4

15%, p: 0.0717 15%, p: 0.0717 40%, p: 0.0016

M4:T15;6, M9:T14;5, M13:2:15 M11:ls4;16,ls11;19, ND

M15:T4;8,T6;19,

96h M8:ls3;16, M15:ls14;3, T16;4 M20:ls3;16,ls14;Y 15%, p: 0.0717 5%, p: 0.3112 20%, p: 0.035

M1:T16;3, ND M5:ls4;19

102h M2:T15;11 ND 10%, p: 0.1468 5%, p: 0.3112

ND M5:ls14;3

12Oh ND 5%, p: 0.3112 Table 3

Claims

WE CLAIM:

1. A method of diagnosing or detecting hereditary breast cancer versus non- hereditary breast cancer in a subject having breast cancer comprising the steps: a) characterizing the telomeric organization of cells in a test sample of breast cells or tissue from the subject using three-dimensional (3D) analysis; and b) comparing the telomeric organization of cells in the test sample with the telomeric organization of a control, wherein a difference in telomeric organization between the sample and the control is indicative of hereditary versus non-hereditary breast cancer.

2. The method according to claim 1 , wherein the control comprises hereditary breast cancer cells and wherein a smaller telomeric aggregate size or volume in the test sample as compared to the control indicates that the subject has non-hereditary breast cancer.

3. The method according to claim 1 , wherein the control comprises hereditary breast cancer cells and wherein a greater number of detectable telomeres as compared to the control indicates that the subject has non-hereditary breast cancer.

4. The method according to claim 1 , wherein the control comprises hereditary breast cancer cells and wherein a larger telomeric size or volume in the test sample as compared to the control indicates that the subject has non- hereditary breast cancer.

5. The method according to claim 1 , wherein the control comprises hereditary breast cancer cells and wherein a similar telomeric aggregate size or volume as compared to the control indicates that the subject has hereditary breast cancer.

6. The method according to claim 1 , wherein the control comprises hereditary breast cancer cells and wherein a similar number of detectable telomeres as compared to the control indicates that the subject has hereditary breast cancer.

7. The method according to claim 1 , wherein the control comprises hereditary breast cancer cells and wherein a similar telomeric size or volume as compared to the control indicates that the subject has hereditary breast cancer.

8. The method according to claim 1, wherein the control comprises non- hereditary breast cancer cells and wherein a larger telomeric aggregate size or volume in the test sample as compared to the control indicates that the subject has hereditary breast cancer.

9. The method according to claim 1, wherein the control comprises non- hereditary breast cancer cells and wherein a fewer number of detectable telomeres as compared to the control indicates that the subject has hereditary breast cancer.

10. The method according to claim 1 , wherein the control comprises non- hereditary breast cancer cells and wherein a smaller telomeric size or volume in the test sample as compared to the control indicates that the subject has hereditary breast cancer.

11. The method according to claim 1, wherein the control comprises non- hereditary breast cancer cells and wherein a similar telomeric aggregate size or volume as compared to the control indicates that the subject has non-hereditary breast cancer.

12. The method according to claim 1, wherein the control comprises non- hereditary breast cancer cells and wherein a similar number of detectable telomeres as compared to the control indicates that the subject has non- hereditary breast cancer.

13. The method according to claim 1 , wherein the control comprises non- hereditary breast cancer cells and wherein a similar telomeric size or volume as compared to the control indicates that the subject has non- hereditary breast cancer.

14. The method according to any one of claims 1 to 13, wherein the non- hereditary breast cancer comprises breast cancer cells that have wild type BRCA1 and wild type BRCA2 genes.

15. The method according to any one of claims 1 to 13, wherein the hereditary breast cancer comprises breast cancer cells that have mutated BRCA1 genes.

16. The method according to any one of claims 1 to 13, wherein the hereditary breast cancer comprises breast cancer cells that have mutated BRCA2 genes.

17. The method according to any one of claims 1 to 13, wherein the hereditary breast cancer comprises breast cancer cells that have mutated BRCA1 and BRCA2 genes.

18. The method according to any one of claims 1 to 13, wherein the hereditary breast cancer comprises breast cancer cells that have mutated BRCA1, BRCA2, TP53, PTEN, CHK2, HER2 or ATM genes.

19. A method of diagnosing or detecting hereditary breast cancer versus non- hereditary breast cancer in a subject comprising the steps: a) characterizing the telomeric organization of cells in a test sample of breast cells or tissue from the subject using 3D analysis; and b) comparing the telomeric organization of the cells in the test sample with that of a control, wherein the control comprises a control sample of hereditary breast cancer cells, a control sample of non- hereditary breast cancer cells, and/or a normal control, wherein a smaller telomeric aggregate size or volume in the test sample as compared to the control sample of hereditary breast cancer cells and a larger telomeric aggregate size or volume in the test sample as compared to the normal control indicates that the subject has non-hereditary breast cancer; or wherein a similar telomeric aggregate size or volume in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a larger telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a larger telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar telomeric aggregate size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer; or wherein morea greater number of detectable telomeres in the test sample as compared to the control sample of hereditary breast cancer cells and a fewer number of detectable telomeres as compared to the normal control indicates that the subject has non-hereditary breast cancer; or wherein a similar number of detectable telomeres in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a fewer number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a fewer number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar number of detectable telomeres in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer; or wherein a larger telomeric size or volume in the test sample as compared to the control sample of hereditary breast cancer cells and a smaller telomeric size or volume in the test sample as compared to the normaf control indicates that the subject has non-hereditary breast cancer; or wherein a similar telomeric size or volume in the test sample as compared to the control sample of hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a smaller telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells and the normal control indicates that the subject has hereditary breast cancer; or wherein a smaller telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has hereditary breast cancer; or wherein a similar telomeric size or volume in the test sample as compared to the control sample of non-hereditary breast cancer cells indicates that the subject has non-hereditary breast cancer.

20. The method according to claim 19, wherein the non-hereditary breast cancer comprises breast cancer cells that have wild type BRCA1 and wild type BRCA2 genes.

21. The method according to claim 19, wherein the hereditary breast cancer comprises breast cancer cells that have mutated BRCA1 genes.

22. The method according to claim 19, wherein the hereditary breast cancer comprises breast cancer cells that have mutated BRCA2 genes.

23. The method according to claim 19, wherein the hereditary breast cancer comprises breast cancer cells that have mutated BRCA1, BRCA2, TP53,

PTEN, CHK2, HER2 and/or ATM genes.

24.A method of diagnosing or detecting cancer in a subject, comprising the steps: a) characterizing the telomeric organization of a cell in a test sample from the subject using 3D analysis; b) comparing the telomeric organization of the cell with that of a normal control, wherein a change in telomeric organization in the test cell as compared to the normal control indicates the presence of genomic instability in the test cell; and c) genetically analyzing the cell from step b) for a translocation, deletion or amplification, wherein the presence of genomic instability and a translocation, deletion or amplification in the test cell indicates a pre-cancerous cell or a cancerous cell.

25. The method according to claim 24, wherein the telomeric organization is characterized by one or more of telomere size, telomere alignment, telomere state of aggregation and size of 3D space occupied by the telomeres.

26. The method according to claim 24, wherein the genetic analysis is for translocation involving the c-Myc gene and IgH gene.

27. The method according to claim 24, wherein the cancer is a lymphoma or leukemia.

28. The method according to claim 24, wherein the cancer is plasmacytoma or

Burkitt's Lymphoma.

29. The method according to claim 24, wherein the genetic analysis is for amplification involving the HER2 gene.

30. The method according to claim 24, wherein the genetic analysis is for deletion involving the p53 gene.

31. A method of diagnosing or detecting cancer in a subject, comprising the steps: a) genetically analyzing a cell in a test sample for a translocation, deletion or amplification, b) characterizing the telomeric organization of the cell from step a) using 3D analysis; and c) comparing the telomeric organization of the cell from step b) with that of a normal control, wherein a change in telomeric organization in the test cell as compared to the normal control indicates the presence of genomic instability in the test cell, wherein the presence of genomic instability and a translocation, deletion or amplification in the test cell indicates a pre-cancerous cell or a cancerous cell.

32. The method according to claim 31 , wherein the telomeric organization is characterized by one or more of telomere size, telomere alignment, telomere state of aggregation and size of 3D space occupied by the telomeres.

33. The method according to claim 31 , wherein the genetic analysis is for translocation involving the c-Myc gene and IgH gene.

34. The method according to claim 31 , wherein the cancer is a lymphoma or leukemia.

35. The method according to claim 31 , wherein the cancer is plasmacytoma or

Burkitt's Lymphoma.

36. The method according to claim 31 , wherein the genetic analysis is for amplification involving the HER2 gene.

37. The method according to claim 31 , wherein the genetic analysis is for deletion involving the p53 gene.

38.A method for simultaneously analyzing protein and nuclear structures of a cell, comprising the steps: a) allowing a protein-specific antibody to bind to a test sample of cells or tissue; b) allowing a telomeric probe to bind to the test sample; and c) detecting the binding of the protein-specific antibody and telomeric probe in the test sample using 3D analysis.

39. The method according to claim 38, wherein the telomeric probe is a peptide nucleic acid (PNA).

40. The method according to claim 38, wherein the antibody is detected using a secondary antibody which binds to the antibody of step b) and generates a detectable signal directly or indirectly.

41. The method according to claim 38, wherein the antibody generates a detectable signal directly or indirectly.