Clinical Cancer Research Vol. 6, 1410-1414, April 2000
© 2000 American Association for Cancer Research
Molecular Oncology, Markers, Clinical Correlates |
Allelic Loss and Microsatellite Alterations of Chromosome 3p14.2 Are More Frequent in Recurrent Cervical Dysplasias1
W. Michael Lin,
Eugenia A. Michalopulos,
Nina Dhurander,
Pui C. Cheng,
William Robinson,
Raheela Ashfaq,
Robert L. Coleman and
Carolyn Y. Muller2
Departments of Obstetrics and Gynecology [W. M. L., R. L. C., C. Y. M.] and Pathology [R. A.] and the Hamon Center for Therapeutic Oncology Research [W. M. L., E. A. M., C. Y. M.], University of Texas Southwestern Medical Center, Dallas, Texas 75235, and Departments of Obstetrics and Gynecology [P. C. C., W. R.] and Pathology [N. D.], Tulane University Hospital, New Orleans, Louisiana 70112
 |
ABSTRACT
|
|---|
Epidemiological
studies have documented the unpredictable clinical progression or
recurrence of cervical dysplasia. Recent studies have shown several
molecular changes in cervical cancers and their associated dysplasia.
We conducted molecular analyses on a retrospectively ascertained cohort
of recurrent and nonrecurrent cervical dysplasia cases in an attempt to
define molecular biomarkers to predict progressive or recurrent
disease. Cases were chosen if long-term follow-up (3–5 years after
conization) and biopsy confirmation were available. Paraffin-embedded,
postconization cervical tissues from 19 recurrent and 18 nonrecurrent
dysplasias were analyzed. Human papillomavirus (HPV) was identified by
PCR for general and type-specific (HPV-16 and HPV-18) primers.
Allelotyping analysis was performed by multiplex PCR using a panel of
16 microsatellite markers targeting putative tumor suppressor gene
regions on chromosomes 3p, 5p, 6p, 9p, 11q, and 17p. The overall rate
of HPV infection was similar in both groups. In the allelotyping
analysis, loss of heterozygosity at the fragile histidine triad region
in 3p14.2 was significantly higher in the recurrent group than in the
nonrecurrent group (P = 0.005). Furthermore,
microsatellite alterations (MAs) were more frequent in the recurrent
group (mean MA index, 0.254) as compared with the nonrecurrent group
(mean MA index, 0.085; P = 0.0025). These findings
suggest that HPV status alone does not predict recurrence and that loss
of heterozygosity at the fragile histidine triad region may represent a
potential biomarker in predicting recurrence. Frequent MAs in the
recurrent group may represent an underlying genomic instability that
creates susceptibility for allelic loss, thus increasing the risk for
recurrence or progression.
|
INTRODUCTION
|
|---|
Whereas evidence has been mounting in recent years establishing
HPV3
as the
etiological agent in cervical cancer, it is clear that other factors
are involved because the majority of patients with HPV infections do
not develop invasive lesions. As is well known, the natural history of
cervical dysplasia is characterized by regression in the majority of
mild and moderate dysplasias and progression only in a minority of
mild, moderate, and even severe dysplasias (1)
.
It is widely accepted that there are multistep molecular changes
leading to malignant transformation from preneoplastic lesions to
invasive tumors. However, the sequence of molecular events responsible
for cervical carcinogenesis has not yet been elucidated. To date, there
are no reliable clinical or molecular biomarkers to predict an
individual’s cervical dysplasia progression risk. The current
management strategies are based on outcome statistics and patient
compliance issues. This current system often leads to expensive and
uncomfortable evaluations and overtreatment for millions of women.
Numerous studies in recent years report a high LOH rate in different
chromosomal regions, indicating hot spots for potential TSGs in
cervical carcinogenesis (2, 3, 4, 5)
. Some of these losses were
also demonstrated in the dysplastic lesions associated with the
invasive tumors, suggesting that the LOH in these critical regions may
be an early event (2
, 3
, 6
, 7)
. Our group and others have
demonstrated that the FHIT gene, a putative TSG that
overlaps FRA3B in chromosome 3p14.2, is frequently altered in cervical
cancer, whereas LOH, homozygous deletions, and aberrant transcripts are
frequently detected in these invasive cancerous lesions
(8)
. Despite these interesting molecular findings, the
lack of protein-altering mutations and tumor heterogeneity brings into
question the exact role of the FHIT gene as a TSG in
cervical carcinogenesis.
The objective of this study was to conduct molecular analyses on a
cohort of recurrent and nonrecurrent cervical dysplasia patients in an
attempt to define molecular predictors. Specifically, we performed HPV
and allelotyping analyses within these two cohorts, using 16
polymorphic markers covering previously defined regions of loss on
chromosomes 3p, 5p, 6p, 9p, 11q, and 17p. Our goal was to improve our
understanding of dysplasia recurrence with the hope of defining
molecular biomarkers to predict progression risk.
|
MATERIALS AND METHODS
|
|---|
Tissue Samples.
Paraffin-embedded histology slides cut from the cervical cone biopsy
specimens of 19 subsequent biopsy-proven recurrent cervical dysplasia
cases and 18 appropriately followed nonrecurrent cases were retrieved
from the Departments of Pathology of Tulane University Hospital and the
Parkland Health and Hospital System from 1993–1995. All patients were
followed regularly with Pap smears and colposcopy, when indicated, for
3.5–5 years from the time of their initial cone biopsy. Cases were
reviewed by two skilled gynecological pathologists (N. D. and R. A.),
and most severe areas of dysplastic epithelium were identified for
precision microdissection.
Microdissection and DNA Isolation.
Precision microdissection was performed on the targeted dysplastic
regions above the basement membrane to obtain a pure population of
dysplastic cells without normal stromal or infiltrating lymphocyte
contamination. Approximately 1000 cells were microdissected for each
case. Matched normal stroma was dissected and used as constitutional
normal DNA for each case. DNA was extracted using proteinase K
digestion as described previously (9)
.
Detection of HPV Sequences.
The presence of HPV DNA in the dysplasia specimens was determined by
PCR analysis using general and type-specific primers for HPV. The
general primer pairs (GP5+/GP6+) target the HPV L1 open reading frame,
and they detect a broad range of genital HPVs (10)
. The
type-specific primer pairs (TS-16 and TS-18) were used to identify the
oncogenic HPV-16 and HPV-18, respectively (11)
. HPV
analysis of the microdissected specimens was performed using the
modified two-round PCR from the method described previously
(2)
, in which the product of first-round PCR was used as
template and amplified using the same primers. DNA made from near
confluent cultures of the human cervical carcinoma cell lines CaSki
(HPV-16) and HeLa (HPV-18; American Type Culture Collection, Manassas,
VA) was used as positive controls.
LOH and MA Analyses.
To evaluate LOH and MAs within both cohorts of cervical dysplasia, a
panel of 16 microsatellite markers was used covering the following
chromosomal regions: (a) 3p21.2–24.2 (D3S1351,
ITIH, and D3S1478); (b) 3p14.2
(D3S1234 and D3S1600); (c)
5p15.1–15.2 (D5S406, D5S208, and D5S807);
(d) 6p21.2–24 (D6S277, D6S105, and
D6S291); (e) 9p21 (IFNA and
D9S1748); (f) 11q23.3 (D11S490 and
CD3D); and (g) 17p13 (p53CA).
These primers were selected for their reported high LOH rate
(2, 3, 4, 5)
. The sequences were obtained from the Genome
Database4
or the
respective reference and synthesized by Life Technologies, Inc.
(Gaithersburg, MD).
A two-round multiplex PCR method was used to amplify the microsatellite
markers from microdissected cells as described previously
(12)
. Multiplex (two to six primer sets in each reaction)
PCR was done during the first amplification, followed by uniplex PCR
incorporating [32P]dCTP using the optimal
annealing temperature for each individual marker. These results were
confirmed in duplicate. The final products were separated on a 6%
denaturing polyacrylamide gel and subjected to autoradiography. Areas
of stroma that contained the highest density of infiltrating
lymphocytes were used as constitutional normal DNA for each case. The
sizes and relative intensities of alleles from the normal stroma and
invasive tumors were compared directly. Markers that identified two
distinguishable alleles of different sizes but similar intensity in the
lane having constitutional normal DNA were termed "informative"
(heterozygous). Markers that gave a single major band in the normal DNA
were termed "noninformative" (homozygous). LOH was scored by visual
detection of the complete absence of the upper or lower allele. Markers
that gave shifted bands or expanded bands as compared with the normal
control DNA were scored as having MAs.
Statistical Analyses.
FRL (FRL = the number of patients with LOH in that region/the
number of patients informative in that region) was used to evaluate
LOH. If any marker for a region was informative, the region was
regarded as informative. If one or more markers showed LOH, we regarded
the region as demonstrating loss. We used the RMA (RMA = the
number of patients with MA in that region/the total number of patients)
to evaluate the regional instability, and we used the MA index (number
of markers with MA/total number of markers evaluated per patient) to
evaluate the overall genomic instability. The differences in the FRL
rate and the RMA rate between the recurrent and nonrecurrent groups
were analyzed by a two-sided 
2 test or by
Fisher’s exact test. The difference in the MA index between the
recurrent and nonrecurrent dysplasias was analyzed by Wilcoxon’s
rank-sum test. P < 0.05 was considered significant.
|
RESULTS
|
|---|
Case Demographics.
The mean age of the patients was similar in both groups (Table 1)
. African Americans were predominantly
represented in each group (n = 15), leaving an equal
racial distribution. High-grade intraepithelial lesions (CIN II, CIN
III, or carcinoma in situ) accounted for 18 cases in each
group, with only one case of CIN I seen in the recurrent group. Of
interest, there were four cases of recurrent dysplasias in women known
to be infected with HIV, but none in the nonrecurrent group. However,
the presence of HIV sequence was not determined molecularly in these
specimens. The positive surgical margin in the conization specimens was
also distributed equally between the two groups (Table 1)
.
Detection of HPV Sequences.
Overall, HPV was detected in 18 of 19 cases (95%) in the recurrent
group versus 15 of 18 (83%) cases in the nonrecurrent group
(P = 0.264; Table 1
). Seven of 19 (37%) recurrent
cases were positive for HPV-16 and/or HPV-18, whereas 7 of 18 (39%)
cases in the nonrecurrent group were positive for HPV-16 and/or HPV-18
(P = 0.90). Three cases in the nonrecurrent group
demonstrated coinfection with both oncogenic HPVs as compared with only
one case in the recurrent group. Cases positive for HPV general primers
but negative for HPV-16- or HPV-18-specific primers were not further
classified. Fig. 1
demonstrates the
PCR-based HPV subtyping results.
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1. HPV analysis for eight dysplasia cases.
Top, PCR amplification using general primers (GP5+/GP6+)
that identify the presence of any HPV DNA. Middle, PCR
amplification specific for HPV-16. Bottom, PCR
amplification specific for HPV-18. Lane 1, negative (no
DNA) control. CaSki and HeLa are the positive controls for HPV-16 and
HPV-18, respectively. Lanes 4–11, cases 1–8 of the
dysplasia cases evaluated. Lane 2, cervical cancer
cell line Caski; Lane 3, cervical cancer cell line HeLa.
|
|
LOH Analysis.
Of the seven chromosomal regions evaluated in all of the dysplasia
cases, 5p15.1 demonstrated the highest regional loss rate of 42% (14
of 33 informative cases). With a trend toward significance, LOH at any
5p15.1 marker was greater than twice that in the nonrecurrent group
(P = 0.062; Table 2
).
When comparing the regional loss rate between the recurrent and the
nonrecurrent groups, only the FHIT region at 3p14.2 demonstrated a
significantly higher loss rate in the the recurrent dysplasia group
(P = 0.0048). Fig. 2
shows examples of the allelotyping analysis with LOH and MAs.
View larger version (42K):
[in this window]
[in a new window]
|
Fig. 2. Allelotype analysis of cervical dysplasia. LOH
is demonstrated in the left and center
panels at chromosome loci D5S208 in a case of
moderate cervical dysplasia (CIN2) and at chromosome
loci D11S144 in severe cervical dysplasia
(CIN3), respectively. Complete loss of the upper allele
is seen in both cases as compared with the matched normal stroma
(STR) used as constitutional normal DNA. Right
panel, a case of MA at the D3S1478 locus in the
same CINIII lesion. The entire allele is shifted upward (expanded) as
compared with the normal stroma.
|
|
MA Analysis.
Of the seven chromosomal regions evaluated, 5p15.1 demonstrated the
highest RMA rate of 43% (16 of 37). When comparing the two groups, the
FHIT region at 3p14.2 and the p53 region at 17p13
demonstrated a significantly higher RMA rate in the recurrent group
(P = 0.009). When we examined the MA index of each
individual case, overall, there is more MA in the recurrent group than
in the nonrecurrent group (P < 0.0025; Fig. 3
; Table 3
).
View larger version (74K):
[in this window]
[in a new window]
|
Fig. 3. MA index is depicted for each case of recurrent
and nonrecurrent dysplasia. The MA index for individual cases
was defined as the number of microsatellite markers with MA/total
number of markers evaluated per patient. The mean index was calculated
for each group and is shown in the figure.
|
|
|
DISCUSSION
|
|---|
Although it is a causative agent in most invasive cervical cancers
and high-grade dysplastic lesions, HPV is only one of multiple somatic
cell errors that leads to cervical cancer. The elucidation of the
molecular pathogenesis can eventually lead to the development of
clinically useful risk assessment biomarkers, which can help select
those patients at risk for neoplastic progression and may allow
individualized follow-up and treatment for those identified as being at
risk. In addition, intermediate molecular markers will allow rapid
advances in the field of chemoprevention for those at risk for cervical
dysplasia.
Many studies have documented that allelic losses are common in various
chromosomes in cervical cancer. Some chromosomal arms consistently have
higher loss rates, indicating hot spots for potential TSGs (2, 3, 4, 5
, 7
, 13)
. Of all of the candidate loci, only 3p14.2 has been
studied extensively. The FHIT gene, a putative TSG that
overlaps the common fragile site FRA3B in chromosome 3p14.2, is
frequently altered in cervical cancer. This is also the location where
LOH, homozygous deletions, and aberrant transcripts are frequently
detected in cancerous lesions. FRA3B, the most common of the
constitutive aphidicolin-inducible fragile sites, is a specific
chromosomal region prone to forming gaps or breaks and is associated
with recurring abnormalities in tumors (8)
.
This study examined the HPV status and the allelic status of a cohort
of patients with recurrent and nonrecurrent cervical dysplasia with an
average of 3–5 years of follow-up. It is not surprising that the HPV
rates are similar in both groups because HPV appears to be the earliest
event in the pathway to cervical carcinogenesis. Numerous studies have
shown that HPV infection is probably the initiating event in the
development of cervical dysplasia and that other factors are
responsible for persistence, recurrence, or progression. Although there
is the possibility of a lack of statistical power in this cohort study
to observe the difference in HPV rate between the two groups, the HPV
findings here are consistent with the high rates of HPV infection in
high-grade dysplasias reported previously.
It is interesting that in the LOH analysis, only the 3p14.2
FHIT region showed a significantly higher loss rate in the
recurrent group than in the nonrecurrent group. It is not known whether
this reflects the importance of the FHIT gene as a TSG in
dysplasia destined to recur or whether it merely reflects a higher
level of instability within the FRA3B fragile site. Chromosomal region
5p15.1 demonstrated the highest overall loss rate in both groups but
was not statistically different between these small cohorts. The
importance of the 5p15.1 locus is not ruled out by the lack of
statistical significance that may reflect the small sample size or the
relatively short follow-up period of 3–5 years. Of interest, LOH at
5p15.1 was more common than that seen within the 3p14.2 region, which
is lower than we reported previously (2)
. It is possible
that the lower rate of LOH seen within 3p14.2 could be attributed to
the choice of markers used in this study (markers widely flanking
FHIT versus the intragenic markers
D3S1300 and D3S4103) or the geographic and racial
differences in the cohorts. It is also possible that epigenetic or
environmental differences in certain cohorts can elicit different
molecular mechanisms that result in the same phenotype of cervical
dysplasia.
Our study also examined the significance of MAs in these cohorts. The
exact functional significance of MA has not been determined; however,
there is evidence that HIV-positive patients have a higher rate of MA
and greater genomic instability that may play a critical role in the
development of HIV-associated cancer (12)
. The mean MA
index in our study is significantly higher in the recurrent group than
in the nonrecurrent group, reflecting the underlying genomic
instability. Our recurrent group did include four HIV + patients, and
this group also had a higher mean MA index of 0.218 as compared to the
nonrecurrent group. One of four HIV + patients had both MA and LOH in
chromosomal regions 3p14.2 and 5p15.1. It is possible that the
HIV-related genomic instability contributed to the higher loss rate in
the recurrent group; however, because the number of HIV+ cases is
small, we did not attempt to stratify our data according to HIV status.
In the MA analysis, the FHIT locus at 3p14.2 again
demonstrated a significantly higher MA rate in the recurrent group.
Similar to the LOH rate, chromosomal region 5p15.1 demonstrated the
highest overall rate of MA; however, the difference was not
statistically significant. Of interest, the other significant regions
of difference regarding MA in the recurrent cohort involved the markers
within the p16 and p53 TSG regions. Combining the LOH and MA findings,
we speculate that the two processes may be related and may act
synergistically to inactivate critical TSGs. Conversely, regional
instability reflected by the difference in MA may lead to allelic loss
and subsequent development of neoplasia.
In summary, we have demonstrated that HPV status alone does not predict
recurrence. A higher LOH rate in the FHIT region of the
recurrent cases and a significant rate of MA at the same locus suggest
an important role for the FHIT gene in cervical
carcinogenesis. Furthermore, we demonstrated that the recurrent
dysplasia group had higher RMAs and overall MAs, reflecting their
underlying genomic instability, which contributes to allelic loss, thus
increasing the risk for recurrence or progression. These findings
appear to provide evidence for genomic characteristics and chromosomal
locations implicated in HPV-associated cervical cancer and in
particular to the dysplasias that are destined to recur.
|
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by the Reproductive Scientist
Development Program through NIH Grant K12HD00849, the American
Association of Obstetrician Gynecologist Foundation (AAOGF), the
Cancer Research Foundation of North Texas, and the Texas Division of
the American Cancer Society. C. Y. M. is an AAOGF-National Institute
of Child Health and Development Fellow of the Reproductive
Scientist Development Program, and W. M. L. is supported by NIH T32
Training Grant in Surgical Oncology, CA66187-03. This study was
presented at the 28th Annual Meeting of the Western
Association of Gynecologic Oncologists, Victoria, British Columbia,
June 2–5, 1999. 
2 To whom requests for reprints should be
addressed, at Hamon Center for Therapeutic Oncology Research,
University of Texas Southwestern Medical Center, 5323 Harry Hines
Boulevard, Dallas, TX 75235-9032. Phone: (214) 648-3026; Fax: (214)
648-8404; E-mail: cmulle{at}mednet.swmed.edu
3 The abbreviations used are: HPV, human
papillomavirus; MA, microsatellite alteration; LOH, loss of
heterozygosity; TSG, tumor suppressor gene; FHIT, fragile histidine
triad; RMA, regional MA; FRL, fractional regional loss; CIN, cervical
intraepithelial neoplasia.
4 www.gdb.org.
Received 8/18/99;
revised 1/11/00;
accepted 1/17/00.
|
REFERENCES
|
|---|
-
Holowaty P., Miller A. B., Rohan T., To T. Natural history of dysplasia of the uterine cervix. J. Natl. Cancer Inst., 91: 252-258, 1999.[Abstract/Free Full Text]
-
Wistuba I. I., Montellano F. D., Milchgrub S., Virmani A. K., Behrens C., Chen H. L., Ahmadian M., Nowak J. A., Muller C., Minna J. D., Gazdar A. F. Deletions of chromosome 3p are frequent and early events in the pathogenesis of uterine cervical carcinoma. Cancer Res., 57: 3154-3158, 1997.[Abstract/Free Full Text]
-
Mitra A. B., Murty V. V., Singh V., Li R. G., Pratap M., Sodhani P., Luthra U. K., Chaganti R. S. Genetic alterations at 5p15: a potential marker for progression of precancerous lesions of the uterine cervix. J. Natl. Cancer Inst., 87: 742-745, 1995.[Abstract/Free Full Text]
-
Rader J. S., Kamarasova T., Huettner P. C., Li L., Li Y., Gerhard D. S. Allelotyping of all chromosomal arms in invasive cervical cancer. Oncogene, 13: 2737-2741, 1996.[Medline]
-
Hampton G. M., Penny L. A., Baergen R. N., Larson A., Brewer C., Liao S., Busby-Earle R. M., Williams A. W., Steel C. M., Bird C. C., Stanbridge E. J., Evans G. A. Loss of heterozygosity in cervical carcinoma: subchromosomal localization of a putative tumor-suppressor gene to chromosome 11q22–q24. Proc. Natl. Acad. Sci. USA, 91: 6953-6957, 1994.[Abstract/Free Full Text]
-
Larson A. A., Liao S. Y., Stanbridge E. J., Cavenee W. K., Hampton G. M. Genetic alterations accumulate during cervical tumorigenesis and indicate a common origin for multifocal lesions. Cancer Res., 57: 4171-4176, 1997.[Abstract/Free Full Text]
-
Evans M. F., Koreth J., Bakkenist C. J., Herrington C. S., McGee J. O. Allelic deletion at 11q23.3–q25 is an early event in cervical neoplasia. Oncogene, 16: 2557-2564, 1998.[CrossRef][Medline]
-
Muller C. Y., O’ Boyle J., Fong K. M., Wistuba I. I., Biesterveld E., Ahmadian M., Miller D. S., Gazdar A. F., Minna J. D. Abnormalities of fragile histidine triad genomic and complementary DNAs in human cervical cancer with human papilloma virus type. J. Natl. Cancer Inst., 90: 433-439, 1998.[Abstract/Free Full Text]
-
Hung J., Kishimoto Y., Sugio K., Virmani A., McIntire D. D., Minna J. D., Gazdar A. F. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. J. Am. Med. Assoc., 273: 558-563, 1995.[Abstract]
-
de Roda Husman A. M., Walboomers J. M., van den Brule A. J., Meijer C. J., Snijders P. J. The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J. Gen. Virol., 76: 1057-1062, 1995.[Abstract/Free Full Text]
-
Baay M. F., Quint W. G., Koudstaal J., Hollema H., Duk J. M., Burger M. P., Stolz E., Herbrink P. Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J. Clin. Microbiol., 34: 745-747, 1996.[Abstract]
-
Wistuba I. I., Behrens C., Milchgrub S., Virmani A. K., Jagirdar J., Thomas B., Ioachim H. L., Litzky L. A., Brambilla E. M., Minna J. D., Gazdar A. F. Comparison of molecular changes in lung cancers in HIV-positive and HIV- indeterminate subjects. J. Am. Med. Assoc., 279: 1554-1559, 1998.[Abstract/Free Full Text]
-
Larson A. A., Kern S., Curtiss S., Gordon R., Cavenee W. K., Hampton G. M. High resolution analysis of chromosome 3p alterations in cervical carcinoma. Cancer Res., 57: 4082-4090, 1997.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
D. C. Connolly, D. L. Greenspan, R. Wu, X. Ren, R. L. Dunn, K. V. Shah, R. W. Jones, F. X. Bosch, N. Muñoz, and K. R. Cho
Loss of Fhit Expression in Invasive Cervical Carcinomas and Intraepithelial Lesions Associated with Invasive Disease
Clin. Cancer Res.,
September 1, 2000;
6(9):
3505 - 3510.
[Abstract]
[Full Text]
|
|
|
Top
ABSTRACT
INTRODUCTION
