Download Application and interpretation of FISH in biomarker studies Jane Bayani Mini-review

Cancer Letters 249 (2007) 97–109
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Mini-review
Application and interpretation of FISH in biomarker studies
Jane Bayani a, Jeremy A. Squire
a,b,*
a
b
Division of Applied Molecular Oncology, Princess Margaret Hospital, University Health Network, 610 University Avenue,
Room 9-717, Toronto, Ont., Canada M5G 2M9
Department of Laboratory Medicine and Pathobiology and the Department of Medical Biophysics, University of Toronto, Ontario
Cancer Institute, Princess Margaret Hospital, University Health Network, 610 University Avenue,
Room 9-717, Toronto, Ont., Canada M5G 2M9
Abstract
Emerging genomic and proteomic data is creating new opportunities to identify novel biomarkers that will have pathway-specific therapeutic impact on cancer progression. Molecular cytogenetic and fluorescence in situ hybridization
(FISH) methods have been primarily used in discovery genetic research laboratories until recently. New automated analytical platforms based on FISH technologies and tissue microarray methods are providing a rapid means to determine the
impact of consistent genomic aberrations in clinical trials, and in studies designed to investigate differential chemotherapeutic response.
Ó 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chromosomes; Cancer genes; Arrays
1. Cancer biology and biomarkers
The ideal biomarker is one that most closely
resembles the carcinogenic process it is modeling
[1], with the goal of molecular therapeutics to target
the underlying defects that lead to cancer initiation
and progression based on our current understanding of cancer biology [2]. The current view of cancer
biology has been reviewed by Hanahan and
Weinberg [3], and include, (1) the ability for selfsufficiency in growth signals; (2) the insensitivity
to anti-growth signals; (3) evading apoptosis; (4)
*
Corresponding author. Tel.: +1 416 946 4509; fax: +1 416 946
2840.
E-mail address: [email protected] (J.A. Squire).
sustained angiogenesis; (5) limitless replicative
potential; (6) tissue invasion and metastasis. These
capabilities are all believed be affected in all cancer
types in some way, though the mechanisms of their
actions may follow different paths. In addition,
because cancer is a disease of genetic progression
that is often associated with specific molecular,
genetic and histological changes [1], the ability to
develop biomarkers that can detect the critical components of these hallmarks of cancer together provides a powerful basis for diagnosing, monitoring
and predicting outcome and response to treatment.
In most tumours, a variety of chromosomal
abnormalities characterized by changes in chromosomal structure and number can be observed. Some
of these chromosomal abnormalities have provided
0304-3835/$ - see front matter Ó 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2006.12.030
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J. Bayani, J.A. Squire / Cancer Letters 249 (2007) 97–109
clues about basic genetic mechanisms and has led to
very specific molecular diagnostic testing in clinical
laboratories, while others have been found to have
prognostic significance. The purpose of this review
is to provide the reader with an appreciation of
the contribution that molecular cytogenetics, and
specifically Fluorescence in situ Hybridization
(FISH), is making in the assessment of biomarkers
in which chromosomal rearrangements are affecting
the molecular biology and clinical response of
tumours.
[9]. Gene amplification occurs frequently in solid
tumours but is seldom found in hematologic malignancies. In contrast, the cytogenetics of tumour suppressor genes are quite different to that associated
with oncogenes, because tumour suppressor genes
are subject to inactivation mechanisms [13]. When
they are functioning normally, the proteins encoded
by tumour suppressor genes inhibit cell proliferation. For these genes, loss or inactivation removes
the normal constraints on cellular growth. The loss
of such a gene often results in a tumour bearing a
genomic deletion detectable by FISH (see below).
2. Chromosomal aberrations and cancer genes
The molecular consequences of chromosome
aberrations influence the functioning of two broad
classes of cancer genes in human tumours: dominantly acting oncogenes and tumour suppressor
genes. When an oncogene becomes inappropriately
activated by a chromosomal mechanism, it can
stimulate cells to continue to proliferate, leading
to a tumour. There are at least three chromosomal
mechanisms for activating oncogenes: (1) Fusion
of the oncogene with a second gene at a site of translocation or inversion of chromosomes generating a
chimeric gene and a new protein [4]. This mechanism is found predominantly in leukemias, lymphomas, sarcomas, and more recently prostate cancer
[5–8]. (2) Juxtaposition of the oncogene to regulatory
elements in immunoglobulin or T-cell receptor
genes in B- and T-lymphocyte malignancies, respectively, leading to inappropriate expression of the
oncogene [6]. This mechanism is one of the commonest methods of oncogene activation in hematologic malignancies. (3) Gene amplification [9] arising
from an increase in the amount of DNA from a specific region of a chromosome. Such areas on chromosomes are referred to as homogeneously staining
regions (HSR) or double minutes (DMs). HSRs are
associated with extensive gene amplification, commonly associated with overexpression of oncogenes
that may impact on drug response [10]. Double minutes (DMs) on the other hand are a related form of
gene amplification that appear as paired extrachromosomal bodies of dark-staining material in a metaphase preparation. In some instances, the number of
DMs can be very large, approaching several hundred in a cell in some neuroblastomas [11] and
medulloblastomas [12]. Various mechanisms have
been proposed for the amplification process, based
on molecular analysis of the regions of amplified
DNA (known as amplicons) in different cell types
3. Molecular cytogenetics and biomarker detection in
tumours
Improvements in cloning technologies and antibody conjugation in the late 1970s and early 1980s
led to the introduction of FISH as early as 1977
[14]. By the late 1980s, FISH was being employed
to detect specific chromosomal regions and loci
[15] using recombinant libraries, enabling the chromosomal mapping of many genes [16–18] within
the human genome. Later in 1992, Kallioniemi
et al. [19] introduced Comparative Genomic
Hybridization (CGH), the predecessor to today’s
microarray CGH assays (aCGH) [20], which was a
FISH-based method that could assess the net genomic gains and losses in a given DNA sample without
the need for fresh (viable) material required for
short-term culture in traditional metaphase analysis.
The competitive hybridization strategy identified
regions of net genomic loss and gain, revealing
regions harboring potential tumour suppressor
genes (CGH regions of genomic deletion) and oncogenes (CGH regions of genomic gain/amplification).
The findings of both metaphase- and aCGH surveys
resulted in the discovery of genomic signatures characteristic of specific cancers, from which specific
genes/loci could be interrogated and validated by
other molecular methods. However, because of the
bulk extraction of the DNA, the information
regarding karyotypic clonality and genomic heterogeneity was lost. The advancement in FISH-based
technologies continued with improved flurochromes/dyes as well as in microscopy and imaging
in the late 1990s culminating in multi-fluorochrome
assays, termed MFISH [21] and in particular, Spectral Karyotyping (SKY) [22], which now allows the
entire metaphase spread to be analyzed in 24-colours, revealing the chromosomal origins of structural
rearrangements [23,24]. The NCBI hosts a public
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database containing SKY/M-FISH and CGH data
from various neoplasms (http://www.ncbi.nlm.nih.
gov/sky/) submitted by individuals. Together these
newer molecular cytogenetic techniques provide a
much more comprehensive survey of the genome
revealing the true karyotypic complexities of neoplastic cells (Fig. 1). For a more comprehensive
review of FISH-based techniques, the reader is
referred to Speicher and Carter [25].
4. Detection of specific biomarkers by interphase
FISH analyses
As outlined in Fig. 1, both metaphase and interphase FISH analysis possess advantages and disadvantages, and in an ideal situation should be
performed in parallel, but to do so is expensive,
labor-intensive and time-consuming. Fortunately,
the loss of information regarding the context of
the chromosomal aberration within the tumour
karyotype derived from metaphase analysis is less
important when the same information can be
derived through interphase cytogenetics. Due to
advances in the efficiency and stability of conjugat-
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ing fluorochromes/dyes into the DNA probe, interphase FISH has developed into a sensitive, stabile
and reproducible assay for the detection of genomic/gene deletion, amplification, specific translocations and global genomic instability by screening
nuclei of a tumour. Thus interphase FISH has the
compelling advantage for biomarker studies of
being capable of examining hundreds of nuclei to
survey numerical chromosomal content changes
such as gene amplification (Fig. 2). Since it can be
applied to both cytogenetic specimens and tissues
embedded in paraffin section, the technique has
been widely performed on Tissue Microarrays
(TMAs) [26] which afford the high-throughput
screening of many tumours within one experiment
and permits comparative analysis with histology
(Fig. 2). Interphase FISH-based methodologies
can now be automated [27] (also see Metasystems
http://www.metasystems.de/, Applied Imaging
http://www.aicorp.com/index.htm) which renders
then suitable for large-scale biomarker screens as
part of clinical trials investigating the influence of
chromosomal composition on therapeutic response
[10]. In the sections below we summarize the four
Fig. 1. Overview of metaphase and interphase cytogenetics. Metaphase cytogenetics requires the preparation of metaphase spreads from
the test specimen,which can then be interrogated with probes, either in a whole genome-fashion, such as SKY, MBand or CGH, or
through the use of locus-specific probes. Interphase Cytogenetics requires only interphase nuclei either from cytogenetic preparations or
from paraffin such as TMAs or frozen sections, as well as cytological smears or touch-preparations.
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Fig. 2. Interphase FISH analysis to archival material permits correlation to histological or immunological types. Shown is an example of
the type information that can be generated from interphase FISH analysis when applied to a histological section or Tissue Microarrays
(TMA). In one experiment, hundreds of tissue cores can be evaluated with specific probes to detect gene/loci deletions, amplifications,
specific translocations, changes in chromosomal ploidy or gauge chromosomal instability, as it relates to such aspects such as histological
subtype, tumour stage/grade, immunohistochemical makers; or to clinical parameters such as response to treatment, outcome, disease-free
interval, time to recurrence. A general sequence of events during the progression of carcinogenesis is depicted with the loss of a tumour
suppressor gene or formation of an oncogenic fusion/translocation event early in tumourigenesis, followed by chromosomal instability
(CIN) and events such as gene amplification occurring later; which can all be identified through interphase FISH analysis. An example of
the type of information generated by such analysis is shown at the bottom, where the distribution of centromere signals for chromosomes
2, 4, 7 and 8 were enumerated in 200 nuclei and graphically displayed. The distribution of centromere signals ranging from 2 to 9 per cells
for the various chromosomes implies a high level of chromosomal instability and karyotypic heterogeneity. An interphase FISH image
from this case shows signals for chromosomes 7 (red) and 8 (green). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this paper.)
main classes of genomic aberrations to which interphase FISH-based biomarker analyses has been
applied (Fig. 3).
5. Deletion studies
The deletion of specific chromosomal loci have
been associated with various cancers including oligodendrogliomas with deletions of 1p and 19q [28]
and neuroblastomas with deletions of 1p36 [29].
More specific gene deletions of known tumour suppressor genes include TP53 [30], RB1 [31], TP16 [32]
and have shown clinical correlation to parameters
including outcome and risk of metastasis. Typical
FISH-deletion studies employ the use of a locus-
specific probe for the gene/locus of interest as well
as the accompanying centromere which are labelled
for a two-colour strategy. The presence or absence
of the locus-specific gene in relation to the number
of copies of the centromere probe determines the
overall copy number status of that gene. Among
the tumour suppressor genes, PTEN has recently
shown the greatest promise as a biomarker with
strong correlation to therapeutic treatment. The
PTEN tumour suppressor gene is located at
10q23, whose function is impaired through somatic
mutation, deletion or by epigenetic modification in
many cancers [33]. PTEN dephosphorylates phosphotidylinositol 3,4,5-triphosphate (PIP3), a product of PI3K [34–36], leading to inactivation or
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Fig. 3. Examples of PTEN deletion in prostate cancer. Shown from left to right are normal prostate tissue, high-grade PIN and prostate
carcinoma tissue sections hybridized with a probe specific for the PTEN gene (red) and for centromere 10 (green). In each case, the inset is
an enlargement of a boxed nucleus. In the normal tissue, two signals for PTEN (red) and two signals for centromere 10 (green) are detected
in the typical pattern for a normal diploid cell. In high-grade PIN, two centromere 10 signals remain, but only one signal for PTEN was
detected indicating a loss of one homolog. In the prostate carcinoma, the same pattern for PTEN deletion was also detected. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
down-regulation of the PTEN/PI3K/AKT/PKB
signaling pathway. Lack of PTEN in a cell leads
to accumulation of PIP3 that activates the AKT/
PKB signaling oncoprotein [37–39]. Active AKT
modulates a number of downstream targets, which
have important roles in the regulation of apoptosis
and the cell cycle, including BAD [40], CASP3 and
CASP9 [41], MDM2 [42], p27 [43], and FOXO transcription factors [44]. Other AKT substrates include
the mammalian target of rapamycin (mTOR) [45]
and WNT/CTNNB1 [46], as well as other proteins
implicated in regulation of cellular proliferation,
differentiation and invasion. The loss of inhibition
of these pathways by PTEN gene inactivation is
associated with tumour progression in several types
of cancer, including prostate cancer [47,48].
In recent years, CCI-779, a homolog of the macrolide antibiotic rapamycin, has been shown to
exhibit activity against proliferation in tumour cell
lines with defective PTEN both in vitro and in vivo
[reviewed by Mills in reference 2]. The availability
of such treatment has increased the practical important of determining PTEN status. Locus-specific
probes for the PTEN gene and centromere 10 permit FISH-based methods to do this, and when
applied to paraffin sections have the added advantage of being able to score many cells maintained
within their histological context. Because traditional
screening methods that rely on gene sequencing or
investigation of LOH require bulk extraction of
nucleic acids, small foci of cells containing the deletion may go undetected due to the ‘‘diluting effect’’
of contaminating surrounding normal cells. The
utility of FISH in detecting PTEN deletions in pros-
tate cancer has been demonstrated recently by our
group [49]. In prostate cancer, PTEN decreased
expression has been associated with high grade
and advanced stage [50]. We identified deletions of
PTEN in 23% of the high-grade prostatic intra-epithelial neoplasia (H-PIN) specimens examined and
in 66% of the prostate carcinomas. Results with fish
were consistent with immunohistochemical findings.
6. Amplification studies
The amplification of oncogenes is another feature
of many cancers, generally reflecting poor prognosis
and clinical outcome [51]. Amplification of this type
has included MYCC in osteosarcoma [52,53] and
prostate cancer [54–57], MYCN in neuroblastoma
[58,59] and medulloblastoma [60–62], EGFR in lung
cancer [10,63,64] and glioblastomas [65,66], and
HER2 in breast cancer [67–69]. The amplification
of these and other genes enhance their biological
roles, and lead to the six hallmarks of cancer outlined in the Introduction to this Review [3]. In many
cases, gene amplification occurs as a late event in
disease progression, often associated with advanced
stage, high grade, aggressive or metastatic disease,
with a poor clinical outcome [51]. Gene amplification when identified serves as an important prognostic marker. It may also serve to predict treatment
efficacy and thereby aid in treatment selection, particularly for breast and lung cancer patients. The
FISH strategy for detecting gene/locus-specific
amplification is identical to the strategy for detecting deletions, although it is also important to distinguish gains in copy number due to ploidy versus.
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Fig. 4. Interphase FISH patterns for gene amplification. Shown from left two right are the different patterns for gene amplification using a
locus-specific probe for EGFR (red) and centromere 7 (green). Low-copy number gains of EGFR are shown in the left panel where
anywhere from 2 to 3 copies of the gene may occur in the cell above the ploidy of the cell, established by the number of centromeres. When
gains or amplifications of EGFR are also associated with a gain of the resident chromosome 7, there are generally equal numbers red to
green signals, as shown in the middle panel. High-copy number gene amplification possesses the most distinctive interphase FISH pattern,
with large clusters or ‘‘domains’’ of signal within the nucleus as shown in the right panel. In this case, the EGFR signal intensity and signal
number greatly out-number the number of centromere 7 signals. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this paper.)
that due to locus/gene-specific amplification that is
independent of ploidy (Fig. 4).
HER2 is one of the best known genes associated
with gene amplification and cancer. It is a member
of the receptor tyrosine kinase 1 (RTK1) family,
shows both gene amplification and increased expression in breast cancer, and is associated with poor
clinical outcome [70]. In 1998, treatment with trastuzumab became available and was found to be beneficial to those women with amplified Her2 [71]. In
addition the combination of paclitaxel with trastuzumab, following adjuvant chemotherapy was
found to significantly improve the outcome for
women positive for Her2 amplification, implicating
a role for trastuzumab for early stage breast cancer
[72]. More recently, our group [10] contributed in a
clinical trial that compared erlotinib with a placebo
for non-small-cell lung cancer. The trial demonstrated a survival benefit for erlotinib. Tumour-biopsy samples from participants in this trial were
used to investigate whether responsiveness to erlotinib and its impact on survival were associated with
expression by the tumour of epidermal growth factor receptor (EGFR) and EGFR gene amplification
and mutations. EGFR expression was evaluated by
immunohistochemical, mutation and paraffin interphase-FISH analysis in non-small-cell lung cancer
specimens. Using univariate analyses, survival was
longer in the erlotinib group than in the placebo
group when EGFR was expressed (hazard ratio
for death, 0.68; P = 0.02) or where there were a high
number of copies of EGFR (hazard ratio, 0.44;
P = 0.008). Using multivariate analyses, adenocarcinoma (P = 0.01), never having smoked (P < 0.001),
and expression of EGFR (P = 0.03) were associated
with an objective response. In multivariate analysis,
survival after treatment with erlotinib was not
influenced by the status of EGFR expression, the
number of EGFR copies, or EGFR mutation, suggesting that among patients with non-small-cell lung
cancer who receive erlotinib, the presence of an
EGFR mutation may increase responsiveness to
the agent, but it is not indicative of a survival
benefit.
7. Translocation studies
One of the landmark discoveries of cytogenetics
was the identification of the Ph chromosome
[73,74] as a recurrent aberration in CML. Later,
other recurrent translocations were identified, particularly among the hematologicial malignancies
and sarcomas (http://atlasgeneticsoncology.org//
index.html) and more recently in prostate carcinoma
[8,75]. The Ph chromosome results from the translocation between chromosomes 9 and 22 that causes
the fusion of the ABL (9q34) and BCR (22q11)
genes [76]. Imatinib therapy leads to major cytogenetic remission in a majority of CML patients as
assessed by FISH as well as RT-PCR methods
[77]. Translocation FISH employs a two-colour
approach, whereby the genes involved in the translocation event are differentially labelled so that
when hybridized together, the co-localization of
both probes indicates the presence of the translocation. This is typically referred to as ‘‘fusion’’ FISH.
Another way of identifying translocations is
through the disruption of the gene. By this
J. Bayani, J.A. Squire / Cancer Letters 249 (2007) 97–109
approach, a FISH probe spanning the entire gene of
interest or spanning the known breakpoint, is
hybridized to the target specimen. The appearance
of a ‘‘split’’ signal indicates the disruption of the
gene. Although a single colour FISH approach
can be used, typically these ‘‘break-apart’’ assays
uses a two-colour probe set with one probe on the
5 0 side of the breakpoint and the other probe on
the 3 0 side of the breakpoint. The BCR/ABL translocation makes use of the fusion-FISH approach
which is routinely used in cancer cytogenetics laboratories for diagnosis and residual disease monitoring. Interestingly, atypical FISH patterns using the
dual-colour probe strategy led to the identification
of a submicroscopic deletion of the 5 0 region of
ABL and the 3 0 region of the BCR genes on the
9q(+) chromosome by our group [78]. It was determined that the CML patients with deletions had a
shorter survival time and a high relapse rate following bone marrow transplant. In addition, because
deletions are associated with both Ph(+) CML
and ALL, it seemed probable that other leukemiaassociated genomic rearrangements may also have
submicroscopic deletions. This was confirmed by
the detection of deletions of the 3 0 regions of the
CBFB and the MLL genes in AML M4 patients
with inv(16) and in patients with ALL and AML
associated with MLL gene translocations,
respectively.
Recently, Tomlins et al. [8] made the exciting discovery of recurrent translocations in prostate cancer
involving the TMPRSS2 gene on chromosome
21q21.2 to members of the ETS transcription factor
genes ERG on chromosome 21q21.2 or ETV1 on
103
chromosome 7p21.2. The significance of such a discovery is the fact that this is the first example of a
specific recurrent translocation in carcinomas. To
date, specific translocations have been identified
for the hematological malignancies [6] and for a
handful of sarcomas [5]. The finding of a recurrent
translocation in a carcinoma leads to the speculation that there may be others. This initial finding
by Tomlins was confirmed by our laboratory [75]
using a three-colour FISH strategy that confirmed
the fusion event, and that the TMPRSS2/ERG
fusion may be accompanied by a small hemizygous
sequence deletion on chromosome 21 between ERG
and TMPRSS2 genes (Fig. 5). Since these initial
findings, other ETS family genes have been identified as translocation partners [7,79] The specific biological role of this translocation and its variants is
still unclear, however studies have already been conducted to determine the relationship between the
presence of the translocation, and its specific isoform and expression level, to clinical outcome [80].
Wang et al. [80] found that a significant variation
in the alternatively spliced isoforms was expressed
in different cancers and that expression of an isoform, in which the native ATG in exon 2 of the
TMPRSS2 gene is in frame with exon 4 of the
ERG gene, was associated with clinical and pathologic variables of aggressive disease. Expression of
other isoforms, in which the native ERG ATG in
exon 3 was the first in-frame ATG, was associated
with seminal vesicle invasion, which is correlated
with poor outcome following radical prostatectomy.
Cancers not expressing these isoforms tended to
express higher levels of fusion mRNAs, and in this
Fig. 5. The TMPRSS2/ERG translocation in prostate cancer. Shown is an example of interphase FISH analysis on paraffin section using
a three-colour FISH approach detecting the deletion of the 3 0 TMPRSS2 gene, resulting in the fusion of ERG to the 5 0 TMPRSS2 as
described previously by Yoshimoto et al. [75]. In this strategy, the ERG gene is labelled in red, the 3 0 end of TMPRSS2 is labelled in green,
and the 5 0 TMPRSS2 is labelled in aqua. In the normal chromosome 21, the genes are oriented as shown in the left ideogram from
centromere to telomere and the resulting FISH pattern shows all three signals in close proximity to each other. The fusion results from the
deletion of the green probe, bringing the red ERG gene adjacent to the blue 5 0 TMPRSS2. In this particular case, two fusions were
detected in this cell, with one normal.
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group, higher expression levels of fusion mRNA
were present in cancers with early prostate-specific
antigen recurrence.
8. Chromosomal instability (CIN) studies
Chromosomal instability (CIN) refers to the rate
of chromosomal gain and loss as well as the presence of structural rearrangements [81]. The presence
of CIN is believed to be the driving force of carcinogenesis, although this is the subject of continuing
debate [82–87]. However, it is evident that CIN provides the necessary genomic variation for cell selection and for cancer cells to develop a growth
advantage [83]. The mechanisms that drive the formation of complex chromosomal aberrations or
copy number changes are influenced by sequence
structure, DNA conformation, aberrations in
DNA damage repair as well as telomere and mitotic
segregation dysfunction (reviewed by Bayani et al.
[88]). Thus, the detection of CIN in cancer may be
an important tool for assessing disease progression,
aggressiveness and disease resistance (Figs. 2 and 6).
In neuroblastoma for example, it has been demonstrated that the presence of a di-tetraploid was associated with a significantly worse prognosis and
overall survival at 4 years in comparison with near
triploid tumours [89]. In ovarian cancer it has been
demonstrated that carcinoma develops apparently
through 2 pathways – one characterized by +7,
+8q, and +12, and one by 6q- and 1q-. Moreover,
at least three phases of karyotypic evolution were
also identified [90]. At the early stages, Phase I,
the karyotypic evolution seems to proceed though
step-wise acquisition of changes. The transition to
Phase II showed signs of an increased chromosomal
instability, most probably caused by extensive telomere crisis and the onset of breakage-fusion-bridge
(BFB) cycles. This process was linked to the presence of imbalances characteristic for the 6q-/1qpathway. The transition to Phase III involved
triploidization and was also linked to the presence
of the 6q-/1q-pathway. Because of the metaphaseand array-CGH based findings in various neoplasms
(http://www.progenetix.de/~pgscripts/progenetix/
Aboutprogenetix.html), genomic signatures with
characteristic gains and losses of chromosomes/
chromosomal regions have enabled investigators
to use these combinations of gain/loss/amplification
to screen specimens for aspects such as cancer risk
[91,92] prognosis [93], progression and metastasis[94–96], as well as residual disease [97].
In addition to changes in chromosome number
and ploidy, the assessment of telomere length by
FISH has become an increasingly useful tool,
although there are currently few studies applied to
large patient cohorts. Telomeres are complex nucleoprotein structures located at the ends of linear
chromosomes and are critical for maintaining genome integrity [98]. In the absence of telomerase,
telomeres progressively shorten. The loss of telomere capping function resulting in dysfunctional
telomeres is one of the telomere-mediated mechanisms promoting genomic instability. Excessive telomere shortening has been shown to lead to BFB
cycles and, eventually to generalized genome instability, leading to either cell death or crisis. Only cells
that have acquired a telomere maintenance
Fig. 6. Detecting CIN through interphase FISH. Shown in the left panel is an example of multi-colour interphase FISH analysis using a 4colour probe cocktail for centromere 3 (red), centromere 7 (green), centromere 17 (aqua) and a locus-specific probe for the gene p16
(yellow/gold), to detect levels of aneuploidy and intratumoural heterogenetity. The right panel shown the hybridization of a tissue section
with telomere-specific probes (red), from which the signal intensities can be quantified (QFISH). The shortening or stabling of telomere
length has been correlated to various stages of tumour progression and may have some bearing on response to treatment and outcome.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
J. Bayani, J.A. Squire / Cancer Letters 249 (2007) 97–109
mechanism can escape from this crisis. Telomere
length analysis studies [99–101] have revealed a relationship between telomere length and karyotypic
complexity, such that cells with shorter telomeres
possessed more structurally complex chromosomal
aberrations and chromosomal instability. Telomere
FISH studies, also known as QFISH (Fig. 6),
involve the use of telomere specific probes, which
can be quantified for both average signal intensity
within an interphase cell, or for individual signal
intensity on a chromosome in a metaphase spread
[102]. In a study of spontaneous tumour regression
of pediatric low-grade gliomas (PLGG) [103], it
was determined that younger PLGG patients, who
exhibit more aggressive and frequently recurrent
tumours, had significantly longer telomeres than
older ones (P = .00014). Tumours with a terminal
restriction fragment length <7.5 did not recur,
whereas the presence of longer telomeres (>8.0) conferred a high likelihood of late recurrences in
PLGG, implicating a plausible biologic mechanism
to explain the tendency of PLGG to exhibit
growth arrest and spontaneous regression. Telomere studies by our laboratory in prostate cancer
[104] showed that a significant decrease in telomere length was shown in both HPIN and CaP
in comparison with normal epithelium, and that
elevated rates of aneusomy suggested that
increased levels of chromosomal aberrations were
associated with decreased telomere length. In
addition, it was revealed that multiple foci of
HPIN exhibited a heterogeneous overall reduction
of telomere length, which was more evident in the
histologic regions of the prostate containing CaP.
This implicates telomere erosion as a consistent
feature of Cap oncogenesis and a marker for
disease progression and increasing genomic
instability.
9. Conclusions and perspectives
Treatment failure is often associated with
tumours acquiring new genomic alterations that
lead to the evolution of more aggressive malignant
clones. Systematic molecular cytogenetic and
FISH-based analyses can now contribute to the
improved assessment of cell-to-cell variation and
the complexity of numerical and structural alterations in clinical trials by using TMA and automated
imaging platforms. The FISH technologies we have
outlined above are highly specific and can be used to
determine the clinical impact of a genomic change
105
that may influence a therapeutic pathway of
response. Understanding the chromosomal mechanisms driving tumour progression, and relating
these findings to the observed dynamic behaviour
of cancer cells may enable a better prediction of
patient outcome and response to treatment.
Acknowledgements
The authors wish to acknowledge Paula Marrano, Maisa Yoshimoto, Olga Ludkovski, Maria Zielenska, Anthony Joshua, Elena Kolomietz, Jana
Paderova, and Shamini Selvarajah for their technical and conceptual contributions. This work was
supported by the Ontario Cancer Biomarker Network with funds from the Ontario Cancer Research
Network.
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