ATM SEQUENCE VARIANTS ARE ...

ATM SEQUENCE VARIANTS ARE PREDICTIVE OF ADVERSE RADIOTHERAPY RESPONSE AMONG PATIENTS TREATED FOR PROSTATE CANCER, ...

[ Pobierz całość w formacie PDF ]
Int. J. Radiation Oncology Biol. Phys., Vol. 61, No. 1, pp. 196 –202, 2005
Copyright © 2005 Elsevier Inc.
Printed in the USA. All rights reserved
0360-3016/05/$–see front matter
doi:10.1016/j.ijrobp.2004.09.031
CLINICAL INVESTIGATION
Normal Tissues
ATM
SEQUENCE VARIANTS ARE PREDICTIVE OF ADVERSE
RADIOTHERAPY RESPONSE AMONG PATIENTS TREATED FOR
PROSTATE CANCER
J
AMIE
A. C
ESARETTI
, M.D.,* R
ICHARD
G. S
TOCK
, M.D.,* S
TEVEN
L
EHRER
, M.D.,*
†
D
AVID
A. A
TENCIO
,P
H
.D.,* J
ONINE
L. B
ERNSTEIN
,P
H
.D.,
‡
N
ELSON
N. S
TONE
, M.D.,
§
S
YLVAN
W
ALLENSTEIN
,P
H
.D.,
S
HERYL
G
REEN
, M.D.,* K
AREN
L
OEB
, M.D.,*
M
ARISA
K
OLLMEIER
, M.D.,* M
ICHAEL
S
MITH
, M.D.,*
AND
B
ARRY
S. R
OSENSTEIN
,P
H
.D.*
‡¶
*Departments of Radiation Oncology,
‡
Community and Preventive Medicine,
§
Urology, and
Biomathematical Sciences, Mount
Sinai School of Medicine, New York, NY;
¶
Department of Radiation Oncology, NYU School of Medicine, New York, NY;
†
Veterans Affairs Medical Center, Bronx, NY
Purpose:
To examine whether the presence of sequence variants in the
ATM
(mutated in ataxia-telangiectasia)
gene is predictive for the development of radiation-induced adverse responses resulting from
125
I prostate
brachytherapy for early-stage prostate cancer.
Materials and Methods:
Thirty-seven patients with a minimum of 1-year follow-up who underwent
125
I prostate
brachytherapy of early-stage prostate cancer were screened for DNA sequence variations in all 62 coding exons
of the
ATM
gene using denaturing high-performance liquid chromatography. The clinical course and postimplant
dosimetry for each genetically characterized patient were obtained from a database of 2,020 patients implanted
at Mount Sinai Hospital after 1990.
Results:
Twenty-one
ATM
sequence alterations located within exons, or in short intronic regions ﬂanking each
exon, were found in 16 of the 37 patients screened. For this group, 10 of 16 (63%) exhibited at least one form of
adverse response. In contrast, of the 21 patients who did not harbor an
ATM
sequence variation, only 3 of 21
(14%) manifested radiation-induced adverse responses (
p
0.004). Of the patients with missense variants, 5 of 9 (56%)
exhibited late rectal bleeding vs. 1 of 28 (4%) without such alterations (
p
0.002). Of those patients who were
at risk for developing erectile dysfunction, 5 of 8 (63%) patients with missense mutations developed prospectively
evaluated erectile dysfunction as opposed to 2 of 20 (10%) without these sequence alterations (
p
0.009).
Conclusions :
Possession of sequence variants in the
ATM
gene, particularly those that encode for an amino acid
substitution, is predictive for the development of adverse radiotherapy responses among patients treated with
125
I
prostate brachytherapy. © 2005 Elsevier Inc.
ATM gene, Radiation sensitivity, DHPLC, Prostate cancer, Brachytherapy.
INTRODUCTION
(
3
), including severe skin necrosis and organ dysfunction.
Understanding the function of the protein encoded by
ATM
advanced greatly after cloning of the
ATM
gene. Subsequent
elucidation of the activity of the ATM protein revealed a
central role orchestrating the cellular response to DNA
double-strand breaks (
4, 5
). ATM-dependent modiﬁcations
of the proteins encoded by the
p53
,
BRCA1
,
CHK2
,
NBS1
,
FANCD2
,
CDC25A
, and
RAD17
genes modulate cell cycle
progression and DNA repair in response to environmental
assaults and ionizing radiation (
6 –18
).
Ataxia-telangiectasia (A-T) is a rare autosomal recessive
genetic syndrome caused by genetic mutations in both cop-
ies of the
ATM
gene (
1
). Generally, these mutations result in
truncation of the encoded protein (
2
). A-T is characterized
clinically by cerebellar degeneration, ocular telangiectasias,
and immunodeﬁciency. Of particular interest has been the
observation that radiotherapy patients with A-T experience
devastating side effects after exposure to ionizing radiation
Reprint requests to: Jamie A. Cesaretti, M.D., Department of
Radiation Oncology, Mount Sinai School of Medicine, Box 1236,
New York, NY 10029. Tel: (212) 241-7502; Fax: (212) 410-7194;
E-mail: jamie.cesaretti@msnyuhealth.org
Acknowledgment
—We would like to thank both Patrick Concan-
non, Ph.D., and Juliet C. Park, M.D., for their thoughtful sugges-
tions during preparation of this article.
Received Apr 28, 2004, and in revised form Sep 15, 2004.
Accepted for publication Sep 16, 2004.
196
0.005). Nine of the patients with sequence alterations
speciﬁcally possessed missense mutations, which encode for amino acid substitutions and are therefore more
likely to possess functional importance. For this group, 7 of 9 (78%) exhibited at least one form of adverse
response. In contrast, of the 28 patients who did not have a missense alteration, only 6 of 28 (21%) manifested
any form of adverse response to the radiotherapy (
p
ATM
variants and adverse radiotherapy response
●
J. A. C
ESARETTI
et al
.
197
Table 1. Patient characteristics in addition to baseline urinary,
rectal, and erectile function
Table 2. Clinical tumor characteristics
Characteristic
Number of patients (%)
Characteristic
Number of patients (%)
PSA (ng/mL)
(range: 1.2–15, median: 6)
Median age
63 years (range: 48–78 years)
4
3 (8)
Coronary artery disease
12 (32)
4–10
31 (84)
Angioplasty
4 (11)
10–20
3 (8)
Hypertension
6 (16)
Gleason score
5
Coronary bypass surgery
3 (8)
5 (14)
Myocardial infarction
2 (5)
6
31 (84)
Not otherwise speciﬁed
1 (3)
7
1 (3)
Active smoker
4 (11)
Stage (AJCC 2002)
T1c
Reformed smoker
9 (24)
25 (68)
Diabetes
3 (8)
T2a
8 (22)
Pretreatment American Urologic
Association urinary
function score
Good (0–7)
T2b
4 (11)
28 (76)
One study, screening the
ATM
gene of 46 breast cancer
patients treated with radiotherapy, revealed that 3 of 4 patients
possessing an
ATM
missense mutation developed Grade 3– 4
skin ﬁbrosis. In contrast, none of the patients without a mis-
sense mutation developed this type of adverse radiotherapy
response (
26
). Another study with a more limited genetic
analysis of the
ATM
gene in which only 8 speciﬁc variants
were genotyped reported that 4 of 6 breast cancer patients
homozygous for the G3A transition polymorphism at nucle-
otide 5557, which transforms an aspartic acid into an aspara-
gine at position 1853 of the protein, exhibited clinically abnor-
mal radiosensitivity (
25
). In addition, it was reported that a
patient discovered to be heterozygous for insertion of a gua-
nine at position 3637, resulting in a frame-shift leading to a
stop codon (TAG) at nucleotide 3681, experienced severe skin
and subcutaneous tissue effects after conventional radiation
therapy in the adjuvant setting for breast cancer (
21
). Cells
from this patient displayed a radiosensitivity between the val-
ues for normal cells and those from patients with AT. Finally,
Hall
et al
. reported that 3 of 17 prostate cancer patients exhib-
iting radiation-related morbidity after radiotherapy possessed
ATM
mutations (
27
).
The purpose of this study was to examine the hypothesis
that the presence of
ATM
sequence alterations is predictive
for the development of adverse radiotherapy responses
among prostate cancer patients. We have screened the ex-
pressed portions of
ATM
and short adjacent intronic regions
that may encompass putative splice sites for DNA sequence
variations (
28
). This work was accomplished using dena-
turing high-performance liquid chromatography (DHPLC)
with DNA samples derived from lymphocytes obtained
from an unselected group of 37 men treated with low-dose-
rate
125
I brachytherapy for prostate cancer. We explore any
potential association of acute and late erectile, rectal, and
urinary functional outcomes with
ATM
alterations using
standard morbidity measuring tools.
Moderate (8–19)
7 (19)
Severe (20–35)
2 (5)
History of transurethral prostate
resection before implant
1 (3)
Preimplant ultrasound prostate
volume
35 cm
3
8 (22)
36–50 cm
3
20 (54)
50
9 (24)
Erectile function
3 - Optimal
22 (60)
2 - Suboptimal but sufﬁcient
6 (16)
1 - Insufﬁcient
5 (14)
0 - None
4 (11)
Ulcerative colitis/Crohn disease
1 (3)
Hemorrhoids
7 (19)
Although the occurrence of alterations in both copies of
the
ATM
gene is rare, individuals who are heterozygous
carriers of a single
ATM
mutation may constitute more than
1% of the general population. It has been shown that cells
derived from heterozygous individuals exhibit an interme-
diate degree of radiosensitivity between those of wild-type
and homozygously mutated cells derived from people with
A-T (
19 –21
). Animal studies have found that heterozygous
ATM
/
mice are more susceptible to radiation-induced
cataracts compared with wild-type
ATM
/
counterparts
(
22
). These discoveries have led to the hypothesis that
possession of one altered copy of the
ATM
gene may pre-
dispose patients receiving radiotherapy to adverse reactions
associated with this treatment.
Several studies have screened the
ATM
gene in patients
who displayed clinically abnormal radiosensitivity. Initially,
the results of these studies were negative, primarily because
the samples were analyzed using a test for protein truncation
(
23, 24
). However, it is now recognized that the most
prevalent
ATM
sequence alterations detected speciﬁcally in
cancer patients are missense mutations causing amino acid
substitution in the encoded protein (
2
). In view of this
understanding, further studies were conducted using assays
designed to detect this class of genetic alterations, and
several positive ﬁndings correlating clinical radiosensitivity
and
ATM
mutations have since been reported (
21, 25, 26
).
METHODS AND MATERIALS
Patients
Peripheral blood lymphocytes were collected from a consecu-
tive series of 37 patients seen for periodic evaluation who under-
198
I. J. Radiation Oncology
●
Biology
●
Physics
Volume 61, Number 1, 2005
Table 3. The postimplant dosimetric parameters of all patients
Implant characteristics
Median (range)
Total activity (mCi)
42 (27.3–62.6)
Needle number
24 (16–29)
Seed number
103 (70–171)
Dose to 90% of the prostate (Gy)
196 (156–220)
Dose to 100% of the prostate (Gy)
111 (78–139)
Volume of prostate receiving
150% of prescription dose (%)
68 (36–84.3)
Dose to 30% of the urethra (Gy)
228 (23–265)
Amount of rectum receiving 100%
prescription dose (cm
3
)
0.7 (0.01–3.56)
went
125
I prostate brachytherapy for early-stage prostate cancer
between June 1997 and April 2002. All patients had biopsy-proven
adenocarcinoma with central pathology review performed on all
specimens. Patients were staged according to American Joint Can-
cer Commission standard (
29
). Patient and tumor characteristics
are outlined in
Tables 1
and
2
. Brachytherapy was administered via
the transperineal approach using a transrectal ultrasound probe to
direct the placement of each radioactive source within the prostate
(
30
). The implant characteristics are enumerated in
Table 3
. The
prescription dose for all implants was 160 Gy corrected for TG-43
recommendations (
31
). Patients returned at approximately 4 weeks
after the implant for detailed CT-based dosimetric analysis. In this
study, a comprehensive dose–volume histogram analysis was
available for the bladder, rectum, urethra, and prostate of each
patient. Patient follow-up included digital rectal examinations and
serial PSA measurements. Biochemical failure was deﬁned using
the American Society for Therapeutic Radiation and Oncology
consensus deﬁnition (
32
).
Fig. 1. An example of a wild-type and mutant chromatogram and
resultant base pattern alteration.
better quantify late erectile dysfunction (ED) (
37
). A score of 0 –2
was judged as an adverse response. The last completed form was
used for this study, because the relatively recent development of
the IIEF-5 did not allow for a prospective evaluation in most
patients.
The goals of the project were discussed with each patient as
outlined by the guidelines approved in the institutional review
board protocol, and written informed consent was obtained.
ATM
exon characterization
DNA isolation from lymphocytes was accomplished using Fi-
coll separation as described previously (
38
). Polymerase chain
reaction (PCR) was used to amplify each of the 62 exons, and short
intronic regions ﬂanking each exon, that comprise the coding
region of the
ATM
gene using primers previously described (
39
).
DHPLC analysis was performed on a WAVE Nucleic Acid Frag-
ment Analysis System (Transgenomic, Omaha, NE) using buffer
gradient and temperature conditions calculated using WAVE-
maker software (version 3.3; Transgenomic) designed for this
purpose. An example of a wild-type and mutant chromatogram and
resultant base pattern alteration is seen in
Fig. 1
. Exons with an
aberrant DHPLC chromatogram underwent DNA forward and
reverse sequencing using an ABI PRISM 377 DNA Sequencer
(Foster City, CA).
Deﬁnition of adverse response
Patient clinical data were available from the departmental pros-
tate cancer tissue repository database, which prospectively col-
lected data for the 2,020 patients who underwent prostate brachy-
therapy at Mount Sinai between June 1990 and February 2004. All
patients underwent a detailed history and physical examination
before implantation followed by a directed history and physical
examination at 6-month-interval follow-up evaluations. Acute and
late rectal toxicities were graded using the Radiation Therapy
Oncology Group (RTOG) morbidity criteria (
33
). Patients who
developed either RTOG grade level 1 or 2 rectal effects were
classiﬁed as having an adverse response. Urinary tract morbidity
was prospectively measured using the American Urologic Asso-
ciation Symptom Score (AUASS) sheet that was administered
before the implant and at each follow-up evaluation (
34
). The
urinary quality of life score from the AUASS was used for analysis
with a score of 6 or “terrible” long-term urinary quality of life
classiﬁed as an adverse response. Erectile function was assessed
using the following scoring system: 0, complete inability to have
erections; 1, able to have erections but insufﬁcient for intercourse;
2, can have erections sufﬁcient for intercourse but considered
suboptimal; and 3, normal erectile function. The derivation and
relevance of this scoring system have been previously described
(
35, 36
). For this analysis, a decline by 2 points was considered a
signiﬁcant prospective decline in erection function, and these
patients were classiﬁed as having an adverse response. In addition,
beginning in June 2000, the validated International Index of Erec-
tile Function (IIEF-5) was used as a complementary method to
Statistical analysis
Analyses were performed using the Statistical Package for So-
cial Sciences (SPSS, Chicago, IL) software. Differences in pro-
portions were derived using the Fisher’s exact
t-
test. A two-sided
p
value of
0.05 was considered to indicate statistical signiﬁ-
cance.
RESULTS
A total of 21
ATM
sequence variants, representing 17
different alterations, were detected in expressed portions of
the gene, or within 10 nucleotides of each exon encompass-
ing potential splice sites, in 16 of the 37 patients screened
(
Table 4
). It should be noted that most of the sequence
variants detected in this group of patients represent genetic
ATM
variants and adverse radiotherapy response
●
J. A. C
ESARETTI
et al
.
199
Table 4. Each patient with toxicity, genetic, comorbid, and follow-up data
Patient
(#)
ATM
alteration
Prospective
erectile
decline
Last
follow-up
IIEF-5
Rectal
bleeding
Urinary
quality
of life
D
90
‡
(Gy)
Comorbidities
Follow-up
(months)
1
4473C
T, 149.1F
F
No
24
No
1
184
CAD
21
2
No
18
No
4
192
36
3
4578C
T, 1526P
P;
5557G
A, 1853D
N
Yes
2
RTOG 1
6
180
67
4
No
20
No
3
208
Tob
37
5
No
16
No
2
205
Tob
29
6
No
24
No
1
165
36
7
*
10
No
0
191
70
8
No
†
No
2
220
49
9
1810C
T, 604P
S
Yes
16
No
6
208
19
10
378T
A, 126D
E; IVS7-8insT;
1176C
G, 392G
G
Yes
1
No
2
197
DM
12
11
2685A
G, 895L
L; 2614C
T,
872P
S
Yes
1
RTOG 1
1
205
40
12
IVS38-8T
C
No
24
No
1
159
60
13
*
23
No
2
174
DM, CAD
31
14
No
1
No
3
210
CAD
20
15
IVS38-8T
C
No
19
No
4
164
Tob
39
16
No
14
No
0
183
59
17
*
5
No
0
169
44
18
No
22
No
2
220
40
19
No
12
No
2
206
26
20
*
21
No
2
199
Tob
37
21
*
2
No
2
174
DM, CAD
25
22
198A
C, 66K
K
*
1
No
1
217
40
23
No
23
No
1
160
25
24
Yes
9
No
2
184
39
25
*
6
No
4
218
32
26
4388T
G, 1463F
C;
1810C
T, 604P
S
*
2
RTOG 2
2
209
CAD
13
27
No
15
No
4
205
32
28
5071A
C, 1691S
R
Yes
1
RTOG 2
2
192
45
29
3161C
G, 1054P
R
No
19
No
2
197
27
30
IVS62
8A
C
No
19
RTOG 1
0
217
CAD
47
31
4578C
T, 1526P
P
Yes
8
No
0
193
26
32
2038T
C, 680F
L
No
19
RTOG 1
0
219
31
33
No
24
No
2
162
71
34
*
3
No
0
168
CAD
69
35
5557G
A, 1853D
N
No
20
No
0
186
58
36
No
18
No
1
197
43
37
IVS22-6T
G
No
22
No
3
210
29
Abbreviations:
CAD
coronary artery disease; DM
diabetes mellitus; RTOG
Radiation Therapy Oncology Group; Tob
active
smoker.
* Patient had a suboptimal erectile function before implant.
†
Patient did not ﬁll out IIEF-5.
‡
Dose to 90% of the prostate gland via brachytherapy.
alterations that have been previously reported as polymor-
phisms in
ATM
(
40–42
). For this group, 10 of 16 (63%)
exhibited at least one form of adverse radiotherapy re-
sponse. In contrast, of the 21 patients who did not harbor an
ATM
sequence variation, only 3 of 21 (14%) manifested any
form of adverse response (
p
0.004). More-
over, 5 of 9 (56%) patients with missense mutations exhib-
ited an adverse response in two or three of the three organ
systems evaluated (Patients 3, 9, 11, 26, and 28), whereas
none of the remaining 28 patients without such sequence
changes exhibited morbidity in more than one evaluated
organ system (
p
0.005). There were 9 patients
found carrying missense mutations encoding for amino acid
substitutions in the
ATM
protein. Missense mutations rep-
resent sequence alterations that are more likely to impact
functional integrity. Of the 9 patients with missense muta-
tions, 7 (78%) exhibited at least one form of adverse re-
0.003).
RTOG Grade 1 or 2 rectal bleeding was seen in 5 of 9
(56%) patients with missense mutations vs. 1 of 28 (4%) of
sponse. In contrast, of the 28 patients who did not have a
missense mutation, only 6 of 28 (21%) manifested any form
of adverse response to the radiotherapy (
p
200
I. J. Radiation Oncology
●
Biology
●
Physics
Volume 61, Number 1, 2005
Table 5. Univariate analysis of variables that may predict for urinary, erectile, and rectal morbidity. All
p
values derived from 2-sided
Fisher’s exact
t
-test
Variable
Two radiation
morbidities
SHIM erectile
decline
Prospective
erectile decline
Rectal Bleeding
RTOG 1,2
Urinary quality
of life “terrible”
Dose
210 Gy
1
0.34
0.29
0.14
1
Diabetes
1
0.12
0.25
1
1
Smoking
1
0.56
0.55
1
1
Coronary artery
disease
1
0.17
0.55
0.32
1
ATM
alteration
0.0003
0.01
0.009
0.002
0.05
Abbreviations:
RTOG
Radiation Therapy Oncology Group; SHIM
Sexual Health Inventory for Men.
0.002). The
median amount of rectal tissue exposed to the prescription
dose of 160 Gy among the individuals with rectal bleeding
was 0.87 cm
3
(range, 0.04 –1.24), which is below previously
published rectal dosing parameters for prostate brachyther-
apy and predicts a low probability of late radiation-induced
proctitis based upon dose alone (
43
).
Severe ED as quantiﬁed by IIEF-5 occurred in 5 of 9
(56%) patients with missense mutations compared with 3
of 27 (12%) of patients without these sequence abnor-
malities (
p
predictive for the development of adverse responses result-
ing from radiotherapy.
Radiation-induced permanent sexual dysfunction has a
substantial negative impact on the quality of life of men
treated for prostate cancer. Brachytherapy series have
reported a widely variable incidence of reduced sexual
potency after implantation (
35, 36, 44 – 48
), ranging from
14% to 50%. In this unselected series, 30% (11 of 37) of
patients overall had erectile dysfunction, a ﬁgure that is
consistent with previous reports. Of even greater signif-
icance, however, is that 63% of patients in this study with
good preirradiation erectile function developed prospec-
tively evaluated ED if they possessed an
ATM
missense
mutation vs. 10% of men without such an alteration. The
correlation of ED with
ATM
missense mutations was also
apparent when men were evaluated only at last follow-up
with the validated IIEF-5. Using this evaluation tool, it
was found that 56% of patients with missense mutations,
vs. 12% without these genetic changes, developed severe
ED. These ﬁndings attest to the predictive power of
ATM
mutational status for ED and warrant validation of this
striking correlation in a larger group of individuals.
A second signiﬁcant correlation observed in this study
is that of postradiation rectal bleeding with
ATM
se-
quence alterations. All of the patients who experienced
late rectal bleeding had
ATM
sequence alterations. The 2
patients who manifested comparatively severe rectal
bleeding, RTOG Grade 2, had DNA missense mutations.
In particular, the patient with the most serious rectal
bleeding was a carrier of two nonconservative missense
mutations and displayed this morbidity at only 5 months
after radioactive seed implantation, rather than the more
typical 1.5 to 2 years. This patient underwent colonos-
copy and biopsy, which identiﬁed distal proctitis and an
absence of the classic telangiectasias. Patients who un-
dergo brachytherapy receive relatively low rectal doses
compared with the use of external beam irradiation in-
volving a larger pelvic ﬁeld. Most radiation-related rectal
bleeding secondary to prostate cancer radiotherapy is
self-limited and innocuous, but there are patients who are
inordinately affected and develop rectourethral ﬁstulas
(
49, 50
). In these instances, it could prove even more
0.01). When considering only patients with
sufﬁcient erectile function before radiotherapy prospec-
tively, a signiﬁcant correlation was also noted between
the development of erectile dysfunction in men with
missense mutations, 5 of 8 (63%), as opposed to 2 of 20
(10%) in men without these types of variants (
p
0.009).
In addition, both patients who reported a “terrible” uri-
nary quality of life had
ATM
missense alterations (2 of 9,
22%) vs. 0 of 28 patients without missense alterations (
p
0.05).
The effects of total dose, diabetes, coronary artery dis-
ease, and active tobacco use were analyzed separately in
relation to each of the adverse responses deﬁned. No inde-
pendent variable achieved statistical signiﬁcance (
Table 5
),
other than the presence of an
ATM
sequence alteration. In
addition, none of the patients experienced a palpable local
or biochemical disease recurrence.
DISCUSSION
Sixty-three percent (10 of 16) of prostate cancer patients
treated with
125
I brachytherapy who were found to be car-
riers of sequence variants either within the exons or in short
intronic regions ﬂanking exons of the
ATM
gene developed
at least one form of urinary, sexual, or rectal adverse re-
sponse. In contrast, only 14% (3 of 21) of patients without
ATM
sequence variations displayed some form of adverse
response. Furthermore, when only those patients speciﬁ-
cally harboring missense mutations are considered, 78% of
these patients developed adverse responses compared with
21% who did not possess these types of sequence abnor-
malities. The results of this study are supportive of the
hypothesis that genetic alterations in the
ATM
gene are
those without these genetic alterations (
p
[ Pobierz całość w formacie PDF ]

zanotowane.pl

doc.pisz.pl

pdf.pisz.pl

dodatni.htw.pl

Podobne