Affiliations of authors: P. C. Galipeau, L. J. Prevo, C. A. Sanchez (Programs in Cancer Biology and Gastrointestinal Oncology, Divisions of Human Biology and Public Health Sciences), G. M. Longton (Department of Biostatistics, Division of Public Health Sciences), Fred Hutchinson Cancer Research Center, Seattle, WA; B. J. Reid, Programs in Cancer Biology and Gastrointestinal Oncology, Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, and Departments of Medicine and Genetics, University of Washington, Seattle.
Correspondence to: Brian J. Reid, M.D., Ph.D., C1-015 Fred Hutchinson Cancer Research Center, Seattle, WA 98109 (e-mail: pgal{at}fhcrc.org).
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ABSTRACT |
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INTRODUCTION |
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A systematic protocol of endoscopic surveillance in Barrett's esophagus can detect esophageal adenocarcinomas when they are early and curable (7,8). Therefore, current recommendations for patients with Barrett's esophagus include periodic endoscopic biopsy surveillance for early detection of cancer by use of a five-tiered histologic classification of dysplasia and cancer (9). However, the value of endoscopic surveillance has been questioned because multiple studies (10-15) have shown that most patients with Barrett's esophagus do not progress to cancer. Most patients will not benefit from endoscopic surveillance in terms of increased life expectancy because they will not progress to cancer during their lifetime (14,16,17). Furthermore, the five-tiered histologic classification of dysplasia in Barrett's esophagus has not been shown to be reproducible in formal, blinded studies (18). High-grade dysplasia is a risk factor for subsequent development of cancer, but the disease in many patients with high-grade dysplasia does not progress and in some it may even regress (19,20). These observations indicate the need for objective, intermediate biomarkers of neoplastic progression in Barrett's esophagus that can be used alone or in combination with histologic staging to stratify patients' risk of progressing to cancer. Such diagnostic markers could be used to identify patients at increased risk of progression to cancer so that they could be placed in more frequent surveillance, while patients at lower risk could be counseled, reassured, and undergo less frequent surveillance. In addition, such biomarkers could serve as intermediate end points in clinical and population-based prevention trials.
Barrett's specialized metaplastic epithelium is hyperproliferative relative to other tissues of the upper gastrointestinal tract (21-24). Flow-cytometric techniques have been used to identify proliferating epithelial cells, abnormal cell cycle fractions, and cell populations with abnormal DNA contents in Barrett's esophagus (24-27). These methods are based on cell sorting by either staining DNA alone or dual staining involving proliferation-associated antigen identified by Ki67 antibody and DNA. Prospective studies (28,29) have shown that an increased number of cells with 4N fractions (cells with double the number of chromosomes or an increase in cells in the G2/M fraction of the cell cycle) and/or aneuploidy are risk factors for subsequent neoplastic progression in Barrett's esophagus. Thus, Ki67 antibody-staining/DNA-content multiparameter flow-cytometric cell sorting can be used to purify populations of cells with 2N, 4N, and aneuploid DNA contents, as well as proliferating cells in the G1 phase of the cell cycle, for subsequent molecular analyses (28,30,31).
Abnormalities involving the p16 (also known as cyclin-dependent kinase N2 [CDKN2], p16 [INK4a], or MTS1) and p53 (also known as TP53) tumor suppressor genes, located on chromosomes 9p21 and 17p13, respectively, are among the most common somatic genetic lesions in human cancers. Loss of heterozygosity (LOH) is the predominant mechanism for inactivating one of the two alleles of each gene, and 9p21 and 17p13 are the two most common regions of LOH in esophageal adenocarcinomas, occurring in approximately 75% and 95% of cases, respectively (31-35). In Barrett's esophagus, the remaining p16 allele is inactivated in the majority of cases by either CpG island methylation or mutation, and the remaining p53 allele is typically inactivated by mutation (31,36-40). Retrospective investigations (30,31,41) of patients who had already progressed to cancer suggest that p16 and p53 can be inactivated as early events in neoplastic progression before the development of aneuploidy and cancer in Barrett's esophagus. Furthermore, LOH at 17p is associated with the development of increased 4N fractions that precede the development of aneuploidy in Barrett's esophagus, probably as a consequence of inactivation of p53's cell cycle checkpoint functions (28). LOH at 9p and LOH at 17p have also been shown to be precursors of clonal progression in the bladder, lung, and other cancers (42,43). Thus, 9p LOH and 17p LOH are potential candidates for objective molecular markers that can be used in combination with flow-cytometric and histologic staging to stratify patients' risk of progressing to esophageal adenocarcinoma.
LOH is a common lesion found in most human cancers, but it has been difficult for a variety of reasons to use LOH as a biomarker of neoplastic progression in large clinical or population-based studies. Recently, we developed a method for investigating LOH in large-scale studies using small clinical samples (44). Here, we use this approach to determine the prevalence, distribution, and relationships among LOH at 9p and 17p chromosomes and flow-cytometric abnormalities in flow cytometrically sorted or purified samples from mapped endoscopic biopsy specimens in 61 consecutive patients who had a diagnosis of high-grade dysplasia without cancer in Barrett's esophagus.
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MATERIALS AND METHODS |
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Biopsy specimen collection. Endoscopic biopsy specimens were obtained, as described previously (7,45). Biopsy specimens were taken at 2-cm intervals in the Barrett's segment. The cell populations in the biopsy specimen were sorted and purified by flow cytometry. In most instances, purified DNA was available for LOH analysis from tissue taken at sequential 2-cm intervals. We evaluated 404 flow cytometrically purified DNA samples. An average of seven (range, 2-20) flow-purified DNA samples, depending on the Barrett's segment length, plus a constitutive control were evaluated for each patient.
Flow cytometry. Biopsy specimens were frozen in dimethyl sulfoxide and were
stored at
-70 °C. Each frozen biopsy specimen was minced, and the homogenates were
processed
and then sorted by flow cytometry on the basis of cellular DNA content alone or sorted by
dual-marker
selection involving fluorescent labeling of proliferation-associated antigen by Ki67 antibody and
DNA
content, as described previously (30,44). Ki67 is an antibody that
identifies a
proliferation-associated antigen expressed in G1, S, and G2/M but not in G0 phases of the cell cycle (46,47). With the use of Ki67
staining/DNA
content flow cytometry, a minimum of two flow cytometrically purified fractions were generated
per
biopsy specimen, yielding Ki67-negative cells with a 2N DNA content, Ki67-positive cells in G1 phase, cells with a 4N DNA content, or aneuploid fractions. In Fig. 1,
these cell cycle fractions will be denoted as 2N (Ki67-negative diploid) and G1
(Ki67-positive diploid). A 4N cell fraction greater than 6% was classified as having an
abnormal
increased 4N fraction, as described previously (25). The prevalence of
abnormalities as determined by flow cytometry in these 61 patients has been reported previously (48). We used these flow cytometric techniques, in addition to LOH at
chromosomes 9 and 17, to characterize cell populations that have evolved into apparent clones in
vivo as part of the natural disease process. No in vitro culturing or expansion of
clones in
the laboratory was performed.
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Statistical analysis. Exact binomial 95% confidence intervals (CIs) are
provided,
with observed LOH prevalence rates shown in Table 1. The Pearson
chi-squared test was used to test for the association between 9p LOH or 17p LOH with
flow-cytometric abnormalities in Table 2.
We used the Mantel-Haenszel
chi-squared test for stratified tables and the corresponding Mantel-Haenszel common odds ratio
estimator (ORMH) to examine the relationship between each LOH and flow
cytometric
abnormalities, with stratification on presence or absence of the other LOH (49). Statistical analysis for Fig. 1
considers the data as matched
pairs of 9p LOH
and 17p LOH
measurements arising from the flow cytometrically purified samples and uses McNemar's
test (49). Logistic regression models were used to model and test for the
extent to
which a particular genotype was found in additional 2-cm intervals of the Barrett's segment
given
that it was present in at least one 2-cm interval (see Fig. 4
). The
robust sandwich variance
estimator was used to allow for nonindependence of data arising from multiple 2-cm intervals per
patient (50). Statistical tests were two-sided.
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RESULTS |
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We determined the prevalence of LOH at chromosomal regions
encompassing p16 and p53 genes by use of six and eight short-tandem
repeats (STRs) on chromosomes 9p and 17p, respectively (Table 1). A
high prevalence of LOH was detected; 44 (73%) of 60 patients (95% CI
= 60%-84%) had LOH at one or more loci on 9p, 17p, or both
chromosomes.
Association of Aneuploidy and Increased 4N With 9p LOH and 17p LOH
Aneuploid cell populations and increased 4N fractions in esophageal
biopsy specimens have been shown to identify a subset of patients at
increased risk of neoplastic progression in Barrett's esophagus
(28,29). Because we flow cytometrically purified diploid (2N),
diploid Ki67-positive cell fraction in G1 phase, 4N, and
aneuploid fractions for LOH analysis, we were able to evaluate
relationships among ploidy, cell cycle abnormalities, and LOH involving
9p and 17p chromosomes throughout the Barrett's segment of each
patient. Fifty-six patients were informative for one or more loci at or
flanking both the p16 and p53 genes. Thirty-three of the 56 patients
had increased 4N fractions and/or aneuploidy, and 23 had only diploid
samples (Table 2) (48). In patients with increased
4N
and/or
aneuploidy, 30 (91%) of 33 (95% CI = 76%-98%) had 17p
LOH
compared
with only four (17%) of 23 (95% CI = 5%-39%) who had
diploid
samples only (P<.001; odds ratio [OR] = 48; 95% CI
=
10-225). 9p LOH was also more common in patients with increased 4N
and/or aneuploidy; 24 (73%) of 33 (95% CI = 54%-87%) had
9p
LOH
compared with 10 (43%) of 23 (95% CI = 23%-66%) with
diploid
samples only (P = .03; OR = 3.5; 95% CI = 1.1-10.5). We
recognize that there may be a biologic basis for the association
between 9p LOH and 17p LOH and thus took into account the possible
confounding effects by looking at the association between
flow-cytometric abnormalities and each LOH, with stratification on the
other LOH. The association between patients with 17p LOH and
flow-cytometric abnormalities remains equally strong with or without
adjusting for a confounding effect of 9p LOH (ORMH = 40; 95%
CI = 8.0-200; P<.001). However, when accounting for 17p
LOH status, there is no evidence for an association between patients
with 9p LOH and aneuploidy and/or increased 4N fractions
(ORMH = 1.7; 95% CI = 0.32-8.9; P = .54).
We determined the presence or absence of 9p LOH and 17p LOH in 404 flow cytometrically
purified samples from all patients, separated into 2N (diploid), Ki67-positive diploid G1,
normal 4N (G2/M), increased 4N (>6% of the cells with 4N in the cell
cycle),
and aneuploid cell populations (Fig. 1). 9p LOH was detected in
42%
of normal-sorted diploid
fractions (2N, G1, and normal 4N G2/M fractions) in contrast to 17p
LOH,
which was detected in only 20% of the same samples [P<.001]). 9p
LOH and 17p LOH were detected in 73% and 78% of increased 4N fractions,
respectively. 9p LOH was detected in 58% of aneuploid populations, and 17p LOH was
detected in 73% of aneuploid populations. The difference between 9p LOH and 17p LOH
in
increased 4N and aneuploid populations was not statistically significant (P = .10).
We
re-evaluated these results using only patients with LOH and excluding patients without LOH and
found
similar results to those in which all patients were analyzed together.
Ordering LOH at Chromosomes 9p and 17p
We used the method of clonal ordering (comparing genetic changes
within a clone two by two to determine whether the events are dependent
or independent and whether they occur in a specific order relative to
each other) to determine the relative order of occurrence of the two
LOH events in 21 patients who had both 9p LOH and 17p LOH in a single
clonal population (Table 3) (30,31,41,51).
For LOH on one chromosome arm to be classified as occurring
"before" the other, a patient must have at least one sample with
both 9p LOH and 17p LOH as well as a separate sample with LOH on only
one of the arms, and the losses must have the same STR patterns. 17p
LOH was rarely detected before 9p LOH in one (5%) of 21 patients, but
9p LOH was detected before 17p LOH in 11 (52%) of 21. The two
abnormalities were always detected together in nine (43%) of 21
patients (i.e., all samples had lost the same allele and loci on each
chromosome arm). In an additional four patients, 9p LOH and 17p LOH
were detected in mutually exclusive clones, none of which had both 9p
LOH and 17p LOH in the same sample. In 19 other patients, we detected
either 9p LOH or 17p LOH but not both. We detected 9p LOH without 17p
LOH in 10 patients and 17p LOH without 9p LOH in nine patients,
supporting the concept that either abnormality can occur in the absence
of the other.
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We investigated clonal diversity in premalignant tissue by assessing
9p LOH and 17p LOH systematically throughout the Barrett's segment in
all patients. The ploidy, lost allele (i.e., smaller or larger allele
in base- pair size), and patterns of loss along each chromosome as well
as between the two chromosome arms (9p and 17p) were used to define
clonal populations and to determine the number of distinct clones that
were present in the evaluated segment (Table 4).
Forty-four patients had LOH at one or more STRs. In 14 (32%) of the 44
patients, we found evidence for only a single clone (e.g., Fig.
2,
A; case No. 252). In these 14 cases, all abnormal
samples had the same ploidy and LOH pattern for both chromosomes.
However, for the majority of patients, we found evidence that the
Barrett's segment contained a mosaic of clones and subclones with
different patterns of LOH and ploidies. For example, 13 (30%) of the
44 patients had the same LOH pattern in both the diploid and the
aneuploid populations (e.g., Fig. 2,
B; case No. 916). In these
patients, LOH could have developed in diploid progenitor cells before
the evolution of aneuploid progeny. Four (9%) of the 44 patients had
mutually exclusive clones, with 9p LOH in one subset of samples and 17p
LOH in others but never both 9p LOH and 17p LOH in the same sample
(e.g., Fig. 2,
D and E; case Nos. 368 and 790, respectively). These
clones arose either independently of each other or from a common
progenitor that had one or more abnormalities not being assessed in
this study. Seven (16%) of the 44 patients had alternate alleles lost
in different samples (e.g., Fig. 2,
F; case No. 306). In these cases,
one or more samples had LOH of the larger allele, whereas other samples
from the same patient had LOH of the smaller allele. We also found
evidence for clonal evolution of LOH in 13 (30%) of 44 patients, as
evidenced by the accumulation of loss on different chromosomes in
different samples or the expansion of the region of loss along the
length of the chromosome. For example, in Fig. 2,
C (case No. 994), the
1.8N population had both 9p LOH and 17p LOH, whereas other samples had
only 9p LOH, suggesting that a diploid clone with 9p LOH was the
progenitor for the 1.8N aneuploid progeny with both 9p LOH and 17p LOH.
Expansion of loss along the length of the chromosome was identified in
three patients (e.g., Fig. 3
). In this patient, the
sorted G1 fraction at the 34-cm level had 9p LOH involving
STR polymorphic markers GATA62F03, D9S935, D9S925, and D9S932 with
retention of heterozygosity at D9S1121 and D9S1118, consistent with a
terminal deletion of 9p, but the sorted G1 fraction at the
36-cm level had 9p LOH involving all STRs evaluated, indicating the
evolution of additional losses.
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The extent to which a clone with a specific somatic genetic
abnormality can expand within Barrett's mucosa may influence the risk
of progression by increasing the size of the population in which
subsequent genetic events can occur. Furthermore, the extent of clonal
expansion may also determine whether a given biopsy protocol can detect
the abnormality and, therefore, its utility as an intermediate marker
of progression. We assessed the extent to which different clones
expanded in the Barrett's segment (Fig. 4.) Some
clones showed little expansion, being detected at only a single 2-cm
level of the Barrett's segment, whereas others expanded to involve the
entire evaluated Barrett's segment length. Many clones showed
incomplete expansion and were detected at multiple 2-cm intervals of
the Barrett's segment, even though they did not involve the entire
segment. Because the length of the Barrett's segment differs among
patients, the number of levels evaluated for each patient varied,
ranging from 2 to 20 cm per patient (average, 6 cm per patient). Some
diploid clones with LOH can undergo complete expansion to involve the
entire evaluated Barrett's segment. Clones expanding to multiple 2-cm
intervals in the Barrett's segment were detected in 17 (77%) of 22
diploid clones with 9p LOH only, in four (36%) of 11 diploid clones
with 17p LOH only (P = .05), and in six (50%) of 12 diploid
clones with both 9p LOH and 17p LOH (P = .13). Aneuploid and
increased 4N populations with 9p LOH, 17p LOH, or both could also
expand over variable Barrett's segment lengths from 2 to 20 cm.
Aneuploid or increased 4N clones showed expansion in 29%, 40%, and
39% of the clones with 9p LOH, 17p LOH, or both, respectively. Diploid
clones with 9p LOH only were typically detected over a greater extent
of Barrett's epithelium than aneuploid and increased 4N populations
(P = .02) and diploid populations with either 17p LOH only or
both 9p LOH and 17p LOH (P = .05).
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DISCUSSION |
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High-grade dysplasia is a risk factor for progression to cancer in Barrett's esophagus, but prospective studies (19,20) have shown that many patients do not progress to cancer. We have found striking somatic genetic heterogeneity in the Barrett's mucosa of patients with high-grade dysplasia. For example, 23% of the evaluable patients had no detectable LOH, 16% had only 9p LOH, 16% had only 17p LOH, and 45% had both. Although prospective validation will be essential, it may be that somatic genetic assessment of 9p LOH and 17p LOH can identify low- and high-risk patients. For example, the fraction of patients with both LOH events is comparable to the proportion of patients previously diagnosed with high-grade dysplasia in the Seattle Barrett's Esophagus Study whose disease progressed (Reid BJ: unpublished results).
Our results extend our understanding of the biologic behavior of clones with 9p LOH, 17p LOH, or both in human neoplastic progression in vivo. A previous study (41) in patients whose disease had already progressed to cancer suggested that there was no obligate order of 9p LOH and 17p LOH during neoplastic progression in Barrett's esophagus. Our results clearly indicate that 9p LOH or 17p LOH can each arise without the other in premalignant Barrett's epithelium. 9p LOH, however, was more frequently detected before 17p LOH in patients in whom an order could be determined. The observation that 9p LOH can be detected before other genetic abnormalities during progression is consistent with those in other cancers in which it has been suggested that 9p LOH is an early event (42,58-60). Clones with 17p LOH were more likely to be associated with increased 4N fractions or aneuploid cell populations, probably as a consequence of loss of cell cycle checkpoint functions of p53 with subsequent genetic instability (28,61,62). In contrast, diploid clones with 9p LOH were rarely associated with aneuploidy or increased 4N fractions in the absence of 17p LOH and tended to spread to large regions of esophageal mucosa, possibly as a consequence of inactivation of p16's G1/S-phase- or senescence-related functions (63,64). Our observations might result from clonal expansion of diploid cells with 9p LOH, creating a field in which 17p LOH could develop as a later event. An alternative hypothesis is that inactivation of p53 alone is not as permissive for clonal expansion, thereby limiting detection of clones with only 17p LOH.
We have shown previously that, in patients whose disease had already progressed to cancer,
the
Barrett's epithelium surrounding the malignancy contained multiple aneuploid cell
populations and
neoplastic cell lineages with multiple molecular abnormalities (31,41,51,56,65).
Our present results extend these observations by demonstrating that the metaplastic epithelium of
patients who have not yet developed cancer can consist of a mosaic of clones and subclones.
Some
mosaics appear to develop from a common progenitor that evolves progeny with additional LOH
events or changes in ploidy (Fig. 2, C). However, a minority of mosaics
may
also develop from
neoplastic cell lineages that arise independently (Fig. 2,
D and E). In these
cases, some clones have 9p
LOH, whereas others have 17p LOH, none have both, or patients have neoplastic cell lineages
with
loss of alternate 9p or 17p alleles (Fig. 2,
F). It is possible in these cases
that
an unknown common
progenitor evolved bifurcating neoplastic cell lineages leading to apparently independent
pathways.
These apparently independent clones are consistent with other studies, suggesting possible
multifocal
clonal evolution of human neoplasms (59,66-68).
Although LOH is a common lesion found in the vast majority of human cancers and precancerous lesions, it has been difficult to use LOH as a biomarker in large-scale clinical or population-based studies. We recently described a method for high-throughput analysis of LOH in small, clinical samples (44). Here, we demonstrate that this method can be applied to a human premalignant condition to detect the presence, extent, and biologic characteristics of clones with 9p LOH, 17p LOH, or both. Our results suggest a model of clonal evolution during neoplastic progression in Barrett's esophagus. Inactivation of one allele of p16 and p53, as assessed by 9p and 17p LOH, is a common genetic alteration in premalignant Barrett's epithelium and can develop before flow-cytometric biomarkers of progression, such as increased 4N fractions and aneuploidy. Diploid populations with 9p LOH spread to involve variable regions of esophageal mucosa, sometimes involving the entire Barrett's segment. Although either 9p LOH or 17p LOH can occur without the other, 9p LOH tends to be detected before 17p LOH when the two abnormalities can be ordered relative to each other in single patients. Progenitor clones can either arise independently or evolve bifurcations that lead to divergent progeny and extensive clonal heterogeneity within premalignant tissue. Most patients with aneuploidy and increased 4N fractions have both 9p LOH and 17p LOH, and it seems likely that the acquisition of 9p LOH and 17p LOH predisposes to the evolution of aneuploid cell populations and other genetic abnormalities that culminate in the development of cancer. Prospective follow-up will be required to determine which, if any, of the abnormal clones with 9p LOH and/or 17p LOH will undergo further progression. Our findings indicate that, while human neoplastic progression is complex, the techniques and protocols used in this study can allow future studies that will assess the usefulness of LOH as a biomarker of progression.
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NOTES |
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We thank Michael Barrett, Thomas Paulson, and David Wong (Fred Hutchinson Cancer Research Center, Seattle, WA) for their critical review of this manuscript.
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Manuscript received April 17, 1999; revised September 14, 1999; accepted October 5, 1999.
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