ts and dpd mrna levels on formalin-fixed paraffin-embedded specimens as predictors for distant...
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Journal of Surgical Oncology 2012;105:529–534
TS and DPD mRNA Levels on Formalin-Fixed Paraffin-Embedded Specimens as
Predictors for Distant Recurrence of Rectal Cancer Treated With Preoperative
Chemoradiotherapy
KOJI TANAKA, MD, PhD,1* SUSUMU SAIGUSA, MD, PhD,1 YUJI TOIYAMA, MD, PhD,1 YUKI KOIKE, MD, PhD,1
YOSHINAGA OKUGAWA, MD, PhD,1 TAKESHI YOKOE, MD, PhD,1 YASUHIRO INOUE, MD, PhD,1
MINAKO KOBAYASHI, MD, PhD,2 CHIKAO MIKI, MD, PhD,1 ANDMASATO KUSUNOKI, MD, PhD1,2
1Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Mie, Japan2Department of Innovative Surgery, Mie University Graduate School of Medicine, Mie, Japan
Background and Objectives: Distant metastatic relapse occurs in approximately 20% of rectal cancer patients treated with 5-fluorouracil
(5-FU)-based chemoradiotherapy (CRT) followed by surgery. This study aimed to investigate mRNA level of 5-FU metabolizing enzymes in
post-treatment specimens and to evaluate their predictive value of distant recurrence after CRT.
Methods: Forty patients with rectal cancer underwent 5-FU-based CRT followed by surgery. After microdissection, total RNA of residual
cancer was isolated from formalin-fixed paraffin-embedded (FFPE) specimens. Thymidylate synthase (TS), dihydropyrimidine dehydrogenase
(DPD), thymidine phosphorylase (TP), and orotate phosphoribosyl transferase (OPRT) as 5-FU metabolizing enzyme were measured using
real-time reverse transcription polymerase chain reaction.
Results: Patients (n ¼ 6) who developed distant recurrence had a significantly higher TS (P ¼ 0.01), DPD (P ¼ 0.02), and TP (P ¼ 0.01)
levels, compared with those patients (n ¼ 34) without recurrence. High TS, DPD, and positive lymph node metastasis (pN) were significantly
poorer prognostic factors for DFS (TS: P < 0.01, DPD: P < 0.01, pN: P < 0.05).
Conclusions: High TS and DPD mRNA levels on FFPE specimens may predict distant recurrence of rectal cancer treated with 5-FU-based
preoperative CRT followed by surgery. Expression analysis of 5-FU metabolizing enzyme in residual cancer may be useful for treatment
stratification and clinical management in these patients.
J. Surg. Oncol. 2012;105:529–534. � 2011 Wiley Periodicals, Inc.
KEY WORDS: microdissection; chemoradiotherapy; rectal cancer; distant recurrence; thymidylate synthase;dihydropyrimidine dehydrogenase
INTRODUCTION
Preoperative chemoradiotherapy (CRT) followed by total meso-
rectal excision (TME) have improved sphincter preservation, local
pelvic control and survival [1–3], and has become the standard care
for rectal cancer in many institutions.
Despite significant improvements in the management of rectal
cancer patients, distant recurrence remains the major cause of
mortality in patients with preoperative CRT followed by TME [4–6].
Identification of predictive markers for distant recurrence should
improve clinical outcome and potential treatment strategies for such
patients. However, there is no clinical parameter or molecular marker
predicting distant recurrence of such patients in practice.
A number of studies have reported that clinicopathological varia-
bles or molecular biomarkers are associated with overall survival
(OS) or disease-free survival (DFS) of rectal cancer treated with pre-
operative CRT followed by TME [7–9]. Pathologic tumor response
or tumor regression grade may be of predictive value for survival
[10]. Post-CRT TNM stage also may be a prognostic indicator of
survival and recurrence [11].
Recently, the amount of residual cancer or pathologic response
has shown to predict the clinical outcome of oesophageal [12,13] or
gastric cancer [14,15] in patients after preoperative CRT followed by
surgery, rather than pre-CRT initial clinical stage. Because of clinical
significance of residual cancer, we focused on the gene expression of
residual cancer in formalin-fixed paraffin-embedded (FFPE) speci-
mens after preoperative CRT.
With this background, this study sought to investigate mRNA
level of 5-fluorouracil (5-FU) metabolizing enzymes in residual
cancer cells on FFPE specimens and to evaluate their predictive
value of distant recurrence after 5-FU-based CRT.
MATERIALS AND METHODS
Patients
A total of 40 patients with locally advanced rectal cancer were
included in the current analyses. All patients were treated with pre-
operative CRT followed by surgery at the Department of Gastrointes-
tinal and Paediatric Surgery in the Mie University Graduate School
of Medicine. Selection criteria were the availability of the quality of
Abbreviations: TS, thymidylate synthase; DPD, dihydropyrimidine dehy-drogenase; OPRT, orotate phosphoribosyl transferase; TP, thymidinephosphorylase; RT-PCR, reverse transcription-polymerase chain reaction.
Conflict of interest: None declared.
*Correspondence to: Koji Tanaka, MD, PhD, Department of Gastrointes-tinal and Paediatric Surgery, Mie University Graduate School of Medicine,2-174 Edobashi, Tsu, Mie 514-8507, Japan. Fax: 81-59-232-6968E-mail: [email protected]
Received 29 January 2011; Accepted 21 September 2011
DOI 10.1002/jso.22123
Published online 17 October 2011 in Wiley Online Library(wileyonlinelibrary.com).
� 2011 Wiley Periodicals, Inc.
isolated RNA for real-time polymerase chain reaction (RT-PCR)
with complete clinical data.
Treatment
The treatment included pelvic radiotherapy using a four-field box
technique and concomitant chemotherapy consisting of 5-FU given
as 600 mg/m2 administered intravenously for 24 hr and tegafur and
uracil (UFT) given as 400 mg/body weight administered orally for
5 days over a week.
In our institution, preoperative radiotherapy was delivered to the
whole pelvis at a dose of 20 Gy in four fractions (5 Gy per fraction)
within a week [16]. This short-course radiotherapy is based on the
irradiation schedule reported by the Swedish Rectal Cancer Group
[17]. All patients underwent computed tomography (CT) simulation
for three-dimensional radiotherapy planning and were treated with a
10 MV X-rays from a linear accelerator. The radiation field encom-
passed a volume that included the primary tumor, mesorectum, pre-
sacral space, the whole of the sacral hollow, and regional lymph
nodes. The superior border was placed at L5/S1 and the inferior bor-
der at 3 cm or more caudal to the primary tumor.
Rectal resection with TME was performed within 2 weeks after
the end of CRT.
Pathologic Evaluation and Treatment Response
Pathologic evaluation in resection specimens was performed
according to TNM classification [18]. Tumor regression of the pri-
mary tumor was semiquantitatively determined by the amount of via-
ble tumor versus the amount of fibrosis, ranging from no evidence of
any treatment effect to a complete response with no viable tumor
identified, as described by Dworak et al. [19]. Tumor regression
grade 0 was defined as no regression; TRG1, minor regression
(dominant tumor with fibrosis in 225% of the tumor mass); TRG2,
moderate regression (dominant tumor with fibrosis in 26–50% of the
tumor mass); TRG3, good regression (more than 50% tumor regres-
sion); and TRG4, total regression (no viable tumor cells, only fibrotic
mass).
Microdissection in FFPE Specimens
Tumor specimens were fixed in 10% formaldehyde solution v/v
and embedded in paraffin. Ten-micrometer-thick sections of FFPE
specimens were stained with nuclear fast red and subsequently
manually microdissected under microscope magnification (from �5
to �10). Residual cancer cells were scraped off using a sterile blade
and carefully collected with reference to hematoxylin and eosin
sections, containing more than 70% of cancer cells. Fibrotic tissue
areas, necrotic cells, and non-neoplastic cells were separated. If
necessary, they were collected as stromal cells, subjected to RNA
isolation.
RNA Extraction From FFPE Specimens
Microdissected samples were digested with proteinase K in lysis
buffer containing Tris–HCl, EDTA, and sodium dodecyl sulfate as
previously reported with minor modification [20]. RNA was purified
by phenol and chloroform extraction. Isolated RNA was purified by
ethanol precipitation. The concentration and quality of RNA were
measured by the UV absorbance at 260 and 280 nm (A260/280 ratio).
cDNA Synthesis
To reversely transcribe the fragmented mRNA from FFPE tissue
materials, we used random hexamer priming, instead of oligo (dT)-
based priming. cDNA was synthesized with random hexamer and
Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions.
Real-Time Quantitative RT-PCR
Real-time quantitative RT-PCR analysis was performed by a fluo-
rescence-based real-time detection method (TaqMan) using an ABI
PRISM 7700 Sequence Detection System (Applied Biosystems, Inc.,
Foster City, CA). In terms of specificity, we used TaqMan-based
detection, preferable to SYBR-Green-based detection because of less
false positivity.
Primers were selected or designed to be intron spanning to avoid
amplification from contaminated genomic DNA. Probes were also
selected or designed placing over exon–exon junction to reduce
amplification of contaminating genomic DNA. Target sequences
were kept as small as possible (<100 bp) to ensure the detection
of fragmented and partially degraded RNA. To confirm primer speci-
ficity, a single band of expected amplicon size for each target gene
was verified by analyzing electrophoretically on 2% agarose gels and
visualizing by ethidium bromide staining.
According to the above-mentioned criteria, primers and probes
for beta actin, thymidine phosphorylase (TP), and orotate phosphor-
ibosyl transferase (OPRT) were designed with primer3 software
(Biology Workbench Version 3.2, San Diego Supercomputer Center,
at the University of California, San Diego, CA). Primers and probes
for thymidylate synthase (TS) and dihydropyrimidine dehydrogenase
(DPD) were synthesized according to previously published sequences
[21]. Sequence and the size of expected PCR product are as follows;
TS (sense, 50-GCCTCGGTGTGCCTTTCA-30; antisense, 50-CCCG-TGATGTGCGCAAT-30; probe, 50-TCGCCAGCTACGCCCTGCT-CA-30; 67 bp), DPD (sense, 50-AGGACGCAAGGAGGGTTTG-30;antisense, 50-GTCCGCCGAGTCCTTACTGA-30; probe, 50-CAGTG-CCTACAGTCTCGAGTCTGCCAGTG-30; 84 bp), TP (sense, 50-GCTGGAGTCTATTCCTGGATTC-30; antisense, 50-TCTGACCCA-CGATACAGCAG-30; probe, 50-TCCAGAGCCCAGAGCAGATG-CA-30; 92 bp), OPRT (sense, 50-CCAGGAGTTCAGTTGGAAGC-30;antisense, 50-GGAACCTCGTTTGCCAATAA-30; probe, 50-TTGT-ACTGTTGGCCAAGATTATCTCCTCCT-30; 85 bp),and beta-actin
(sense, 50-ACAGAGCCTCGCCTTTGC-30; antisense, 50-GCGGC-GATATCATCATCC-30; probe, 50-CCGCCGCCAGCTCACCAT-30;75 bp).
PCR was carried out in a final volume of 25 ml with a Taqman
Universal PCR Master Mix (Applied Biosystems) using 0.5 mlcDNA, 900 nM of each primer and 200 nM of probe for the respec-
tive genes. Cycling conditions were 508C for 2 min and 958C for
10 min followed by 40 cycles at 958C for 15 s and 608C for 1 min.
Relative mRNA Levels of Target Genes
The parameter Ct (threshold cycle) is defined as the fractional
cycle number at which the fluorescence generated by cleavage of the
probe passes a fixed threshold above baseline. The Ct is inversely
proportional to the amount of cDNA, that is, a higher Ct value means
that more PCR cycles are required to reach a certain level of
detection.
Relative mRNA levels were determined by the standard curve
method. The standard curves and line equations were generated
using fivefold serially diluted solutions of cDNA from colon cancer
cell line, Lovo. All standard curves were linear in the analyzed range
with an acceptable correlation coefficient (R2). The amount of target
gene expression was calculated from the standard curve.
Quantitative normalization of cDNA in each sample was
performed using the expression of the beta actin gene as an internal
control. Finally, mRNA levels of the target gene were presented
as ratios between the genes of interest and internal reference gene
530 Tanaka et al.
Journal of Surgical Oncology
(beta actin). Real-time PCR assays were performed twice for each
sample and mean values were used for calculations of the mRNA
levels.
Statistical Analysis
All statistical analyses were completed using JMP version 5 (SAS
Institute Inc. Cary, NC). Values of each target gene are expressed as
a median value (inter-quartile range).
Associations between gene expression levels (continuous varia-
bles) and clinicopathological variables (categorical variables) were
evaluated using Mann–Whitney U-test for two groups or Kruskal–
Wallis test for multiple groups.
DFS was calculated from the date of surgery to the date of dis-
ease recurrence. OS was calculated from the date of surgery to the
date of death due to rectal cancer or last follow-up. Survival was
evaluated using the Kaplan–Meier method. The log-rank test was
used to compare the cumulative survival durations in the patient
groups.
A non-parametric receiver operating characteristic (ROC) analysis
was performed to calculate the best cut-off values predictive of
distant recurrence using Medcalc 7.2 for Windows (Mariakerke,
Belgium).
P-values <0.05 were considered statistically significant.
RESULTS
Patient Characteristics and Survival
Forty patients were included in this study. The median age was
65 years (range, 37–78 years); the male to female ratio was 3.4:1.
The post-CRT clinicopathological variables were shown in Table I.
Medians of OS and DFS were 38.1 months (range; 6.8–86.3) and
41.3 months (range; 2.5–86.3), respectively. As shown in Figure 1,
the presence of distant recurrence and pathological stage were
significantly associated with OS (P ¼ 0.0018 and P ¼ 0.0174,
respectively). Having an age over 65 years and pathological positive
lymph nodes were significantly associated with DFS (P ¼ 0.0486
and P ¼ 0.0469, respectively). Other clinical parameters were not
associated with OS or DFS.
Pathologic Response to Chemoradiotherapy
The results of TRG were also shown in Table I. No TRG 0 was
observed, indicating that some degree of CRT response was found
in all patients. There was no TRG 4 in this study because of no
availability of residual cancer cells. Tumor or node downstaging was
demonstrated in 23 patients (57.5%).
RNA Isolation From Microdissected
Residual Cancer on FFPE Specimens
The A260/280 ratio of total RNA from FFPE tissue materials
were 1.66 � 0.07 (mean � SD), ranging from 1.43 to 1.83. Their
concentration (ng/ml) were 789.1 � 5.13.3, ranging from 196 to
2,492. The average Ct value for beta-actin was 27.7 � 3.7.
Associations Between 5-FU Metabolizing Gene Levels
and Clinicopathological Variables
Median value of post-CRT tumoral TS, DPD, TP, and OPRT
mRNA levels on FFPE specimens were 2.32 (inter-quartile range
1.02–4.51), 5.99 (3.64–14.81), 3.99 (2.39–9.06), and 0.10 (0.05–
0.15), respectively. Associations between TS, DPD, TP, and OPRT
mRNA levels and clinicopathological variables were summarized
in Table I. TS and TP were significantly higher in female patients
TABLE I. Clinicopathological Variables and mRNA Levels of 5-FU Metabolizing Enzymes
Variable Number TS P-value DPD P-value TP P-value OPRT P-value
Gender
Male 31 1.89 5.8 3.84 0.12
Female 9 3.53 0.02 15.88 0.07 10.15 0.03 0.05 0.53
Age (median: 65 years)
265 21 2.06 5.8 3.84 0.09
>65 19 2.53 0.28 6.64 0.34 4.01 0.42 0.12 0.95
Pathologic T category
T1 4 1.81 5.24 3.43 0.16
T2 10 1.97 6.34 4.25 0.09
T3 25 2.71 6.29 4.41 0.08
T4 1 1.03 0.58 2.91 0.59 1.97 0.58 0.23 0.14
Pathologic N category
Present 13 2.05 6.7 4.01 0.09
Absent 27 2.51 0.83 5.93 0.99 3.88 0.9 0.12 0.38
Histology
Well/moderate 34 2.1 5.9 3.93 0.11
Poor/muc 6 3.33 0.34 15.29 0.29 9.31 0.29 0.07 0.52
Lymphatic invasion
Present 31 2.05 5.8 3.88 0.09
Absent 9 3.53 0.22 8.52 0.15 6.53 0.16 0.12 0.29
Vascular invasion
Present 26 2.6 6.17 4.21 0.08
Absent 14 1.58 0.38 5.9 0.73 3.71 0.57 0.13 0.01
Tumor Regression Grading
TRG 1 7 3.06 8.52 5.93 0.12
TRG 2 17 1.89 5.65 3.86 0.12
TRG 3 16 2.06 0.64 6.17 0.56 4.51 0.66 0.06 0.22
T and N categories indicate extent of the primary tumor and presence of lymph node metastasis. Values of each target gene are expressed as median value. Bold
type indicates a significant value.
Distant Recurrence of Rectal Cancer 531
Journal of Surgical Oncology
compared with male patients (TS; P ¼ 0.02, DPD; P ¼ 0.03). OPRT
was significantly higher in patients without vascular invasion than
those with it (P ¼ 0.01). No significant association between TRG
and expression of 5-FU metabolizing gene was seen.
Association Between 5-FU Metabolizing Gene Levelsand Occurrence of Distant Recurrence
No patient had local recurrence. Patterns of distant recurrence
were liver and lung metastases in two patients, lung metastases alone
in three patients and peritoneal metastasis in one patient.
As shown in Table II, six patients who developed distant recur-
rence had a significantly higher TS (P ¼ 0.01), DPD (P ¼ 0.02),
and TP (P ¼ 0.01) compared with those patients without recurrence.
No significant association between distant recurrence and OPRT was
observed.
Predictive Value of TS, DPD, and TP for
Distant Recurrence
On the basis of these results, ROC analysis was used to identify
each cut-off value of TS, DPD, and TP predictive of distant
recurrence. A non-parametric ROC analysis showed that the best
cut-off values of TS, DPD, and TP were 10.26, 41.00, and 3.85,
respectively.
As shown in Figure 2, patients with TS or DPD above the cut-off
value showed significantly worse DFS (TS, P < 0.0001, DPD,
P ¼ 0.0007). There was no significant difference of DFS between
patients with TP above the cut-off value and those with TP below
cut-off value (P ¼ 0.24).
DISCUSSION
Gene Expression in Residual Cancer
Post-CRT TRG has been significantly associated with DFS in
rectal cancer patients who were treated with 5-FU-based CRT
followed by TME [10]. Although a variety of TRG has been pro-
posed, Dworak’s TRG is originally based on the percentage of
residual tumor cells to stroma of entire tumor beds after CRT [17].
The amount of residual cancer has also been reported to predict the
clinical outcome of oesophageal [12,13] or gastric cancer [14,15] in
patients after preoperative CRT followed by surgery. In addition to
the clinical importance regarding the quantity of residual cancer,
gene expression profiles in residual cancer may have a significant
role in the treatment of patients who undergo preoperative CRT
followed by surgery. These lines of evidence prompted us to focus
on the gene expression of residual cancer in FFPE specimens.
TS and DPD Levels After Chemoradiotherapy
Expressions of 5-FU metabolizing enzymes have been analyzed
with respect to local recurrence and distant metastases of colorectal
cancer after postoperative 5-FU-based chemotherapy. High expressions
Fig. 1. Overall survival and disease-free survivals according to clinicopathological variables.
532 Tanaka et al.
Journal of Surgical Oncology
of TS or DPD or TP are associated with worse clinical outcome in
colorectal cancer [22,23]. In this study, we showed that patients who
developed distant recurrence had a significantly higher TS, DPD, and
TP, compared with those patients without recurrence. Furthermore,
high TS and DPD mRNA levels on FFPE specimens and pathologic
lymph node metastasis were significant predictive factors for worse
DFS of rectal cancer patients who were treated with 5-FU-based
CRT followed by TME.
Jakob et al. [24] also showed that post-therapeutic TS-gene
expression on surgical specimens was significantly higher in rectal
cancer patients with cancer recurrence after 5-FU-based CRT.
Recently, local recurrence rate have been shown to be decreasing at
<10% [1–3]. However, distant recurrence will develop in as high as
20–30% of patients with CRT followed by TME [4–6]. To improve
clinical outcome of patients with distant recurrence, predictive
markers for distant recurrence are needed to assist in the manage-
ment of rectal cancer. TS and DPD mRNA levels on FFPE speci-
mens may be a useful predictor for distant recurrence.
However, data should be interpreted with caution. The major limi-
tation of this study was the relatively small number of patients
(n ¼ 40) especially patients with those (n ¼ 6). Large prospective
trials will be needed to confirm the validity of the predictive value of
TS and DPD levels for distant recurrence.
In conclusion, high TS and DPD mRNA levels on FFPE speci-
mens may predict distant recurrence of rectal cancer treated with 5-
FU-based preoperative CRT followed by TME. Expression analysis
of 5-FU metabolizing enzyme in residual cancer may be useful for
treatment stratification and clinical management in these patients.
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