We investigated the relationships between biomarkers related to endoplasmic reticulum stress proteins (glucose-regulated protein of molecular mass 78 [GRP78] and Cripto-1 [teratocarcinoma-derived growth factor 1 protein]), pathologic response, and prognosis in locally advanced rectal cancer.
All clinical stage II and III rectal cancer patients received 50.4 Gy over 5.5 weeks, plus 5-fluorouracil (400 mg/m2/day) and leucovorin (20 mg/m2/day) bolus on days 1 to 5 and 29 to 33, and surgery was performed at 7 to 10 weeks after completion of all therapies. Expression of GRP78 and Cripto-1 proteins was determined by immunohistochemistry and was assessed in 101 patients with rectal cancer treated with neoadjuvant chemoradiotherapy (CRT).
High expression of GRP78 and Cripto-1 proteins was observed in 86 patients (85.1%) and 49 patients (48.5%), respectively. Low expression of GRP78 protein was associated with a significantly high rate of down staging (80.0% vs. 52.3%, respectively; p=0.046) and a significantly low rate of recurrence (0% vs. 33.7%, respectively; p=0.008) compared with high expression of GRP78 protein. Mean recurrence-free survival according to GRP78 expression could not be estimated because the low expression group did not develop recurrence events but showed a significant correlation with time to recurrence, based on the log rank method (p=0.007). GRP78 also showed correlation with overall survival, based on the log rank method (p=0.045).
GRP78 expression is a predictive and prognostic factor for down staging, recurrence, and survival in rectal cancer patients treated with 5-fluorouracil and leucovorin neoadjuvant CRT.
Globally, colorectal cancer is one of the leading causes of cancer deaths and approximately one third of colorectal cancer arises from the rectum [
Tumor microenvironment is characterized by hypoxia, hypoglycemia, and acidosis, which results in chronic endoplasmic reticulum (ER) stress. Glucose-regulated protein of molecular mass 78 (GRP78), a major ER chaperone, is a member of the family of heat shock proteins. Under ER stress, GRP78 activates a pro-survival pathway using an autophagy mechanism, which in turn leads to the protection and survival of cancer cells, chemoresistance and radioresistance [
Many previous studies examining response indicators for rectal cancer used data from several institutes and various treatment strategies. This multi-institutional bias and variation of treatment strategy may affect data analysis. A single-institution experience with homogenous treatment and pathologic evaluation excludes some bias. We also limited the number of enrolled patients who underwent laparoscopic surgery in order to make a homogenous cohort group.
Therefore, the aim of the current study is to determine the clinical significance of GRP78 and Cripto-1 expression in patients with rectal cancer who underwent neoadjuvant CRT followed by laparoscopic surgery.
The study population consisted of 101 rectal cancer patients who underwent neoadjuvant CRT and laparoscopic surgery at St. Vincent’s Hospital between 2005 and 2008. The patients’ charts, radiographic findings, and postoperative pathologic slides were reviewed. Inclusion criteria were available archival paraffin-embedded tissue samples of initial colonoscopic biopsy, neoadjuvant CRT with 50.4 Gy (1.8 Gy/day in 28 fractions) over 5.5 weeks, plus 5-fluorouracil (400 mg/m2/day) and leucovorin (20 mg/m2/day) bolus on days 1 to 5 and 29 to 33, surgery performed at 7 to 10 weeks after completion of all therapies for all patients, and follow-up for at least 2 years for patients with clinical stage II-III rectal cancer. All patients had an initial work-up before CRT. Initial work-up included abdominal computed tomography (CT), chest CT, and positron emission tomography–CT, pelvis magnetic resonance imaging (MRI), and endorectal ultrasound. Initial clinical stage was decided comprehensively using these data. The patients also had a pre-surgical work-up including pelvis MRI and endorectal ultrasound. One surgical team, assisted by surgical residents in training, performed total mesorectal excision (TME) using laparoscopy. The quality of TME was classified as complete, near-complete, and incomplete. All patients had more than near-complete. Clinical stage and follow-up recurrence were performed in an independent review conducted by a radiologist, and pathologic stage, grade, and other pathological parameters were reviewed by two independent pathologists. Downstaging was defined as staging reduction from pretreatment stage (cStage) to pathologic staging (ypStage) (i.e., cIII to ypII, ypI or yp0; cII to ypI, or yp0). Pathologic response to CRT was reviewed and scored as follows: grade 0, no response; grade 1, necrosis or disappearance of tumor cells less than 2/3; grade 2, necrosis or disappearance of tumor cells more than 2/3; and grade 3, no viable cells (ypCR). We collected paraffin embedded tissue specimens from initial colonoscopic biopsies in these patients for immunohistochemistry staining.
Immunohistochemistry was performed using formalin fixed, paraffin-embedded tissue samples from initial colonoscopic biopsies to examine the expression of GRP78 and Cripto-1 proteins. The antibodies used in this study were GRP78 (Novus Biologicals, Littleton, CO) and Cripto-1 (Novus Biologicals). The tissue samples were deparaffinized in xylene and rehydrated in graded alcohols and water. Endogenous peroxidase was blocked by soaking in 3% H2O2 at 45°C for 4 minutes. The slides were microwaved in citrate buffer at 120°C for 15 minutes to unmask the antigen and treated with a protein-blocking reagent before incubation at 4°C overnight with primary antibodies at a 1:50 dilution, as recommended by the supplier. After extensive washing, the sections were incubated at room temperature for 10 minutes with Immpress anti-Rabbit immunoglobulin antibodies (Vector Laboratories Inc., Burlingame, CA) at a 1:20 dilution and subsequently with streptavidin-biotin peroxidase complexes at a 1:25 dilution. Reaction products were visualized by immersing the slides in 3,3′-diaminobenzidine tetrahydrochloride. Counterstaining was performed with hematoxylin. All series included positive and negative controls. The negative controls were prepared by omitting the primary antibodies, and known positive controls were included in each run. The results of immunohistochemical staining were evaluated independently by two pathologists in a blinded fashion. For the discordant samples, slides were reviewed jointly and a consensus was reached for the results. The results were scored according to staining intensity and staining percentage. Immunostaining intensity (a) was classified as lack of staining (0), weak staining (1), or moderate to strong staining (2); the percentage of positive tumor cells (b) was semiquantitatively divided according to three grades: no positive staining or positive staining in < 10% of tumor cells (0), 10%-50% staining (1), and > 50% staining (2). The score for each section was measured as (a)×(b), the result was defined as 0 (score 0), 1+ (score 1), 2+ (score 2), or 3+ (score 4). For statistical analysis, the expression was grouped according to low (0, 1+) or high (2+, 3+) level expression.
IBM SPSS ver. 21.0 (IBM Co., Armonk, NY) was used for statistical analysis. To evaluate the association between GRP78 and Cripto-1 expression and clinicopathologic factors including recurrence, correlation analysis was performed using Pearson chi-square or Fisher exact test as appropriate. Recurrence-free survival (RFS) was defined as the time from the date of operation to relapse or death for patients who died without relapse. Overall survival (OS) was defined as the time between the date of operation and death. RFS and OS were calculated using the Kaplan-Meier method. Simple variable analysis was performed using the log rank method and multivariate analysis using the Cox regression model. Survival rates and odds ratios are presented with their 95% confidence intervals and p-value of < 0.05 was considered statistically significant.
This study included 69 male and 32 female patients with a median age of 62 years (range, 53 to 69 years). Clinical stages II and III accounted for 51.5% and 48.5% in each group. The down-staging rate of this neoadjuvant CRT was 56.4% (57 patients) and ypCR was 19.8% (20 patients). Down-staging after neoadjuvant CRT presented 52 cII patients to 17 ypI and 9 yp0, and 49 cIII patients to 15 ypII, 5 ypI and 11 yp0. At a median follow-up period of 51 months (range, 38 to 64 months), 81 patients (80.2%) were alive, and two cases were non-cancer-related deaths. Overall recurrence rate was 31.7% (32 patients). Eight patients (7.9%) had loco-regional recurrence, and the rest of patients with recurrence had distant recurrence only. The RFS and OS did not reach the median. Three-year RFS was 66.3% and 3-year OS was 78.2%. Patients’ characteristics and their response to neoadjuvant CRT are shown in
High expression of GRP78 and Cripto-1 proteins was observed in 86 patients (85.1%) and 49 patients (48.5%), respectively. Expression of GRP78 and Cripto-1 proteins is shown in
Associations between expression of these biomarkers and clinical parameters are shown in
The current study attempted to evaluate the clinical significance of GRP78 and Cripto-1 expression in patients with LARC who underwent neoadjuvant CRT followed by laparoscopic surgery. Our results suggested the predictive and prognostic role of GRP78. In patients with low expression of GRP78, higher response rate (down staging) and prolonged survival were demonstrated and no recurrence was observed.
Emerging data have shown the relationship of ER stress and human cancer, in terms of tumorigenesis, aggressiveness, and resistance to anti-tumor treatment including chemotherapy and radiotherapy [
Cripto-1, a cell surface protein, plays an important role during embryogenesis and disappears in adult tissues, except in stem cells. Cripto-1 reappears in tumorous conditions, which promotes cell proliferation, epithelial to mesenchymal transition, migration, invasion, and angiogenesis. Cripto-1 interacts with an extensive range of signaling molecules, including TGF, Wnt/β-catenin, erbB4, Notch, and GRP78, which accounts for its diverse biological effects [
This current study has limitations. Preoperative biopsied tissue was used in our study and there is some risk in regarding biopsied tissue as a representative sample for the whole tumor. Median RFS has not yet been reached, thus, further follow up and a large number of study subjects is required in order to properly assess the prognostic roles of GRP78 and Cripto-1.
In conclusion, this current study indicates that GRP78 and Cripto-1 were highly expressed in the majority of LARC patients and high expression of GRP78 was related to poor response and poor prognosis; this suggests correlation of GRP78 with chemoresistance and radioresistance. So far in rectal cancer, little is known about GRP78 and Cripto-1 and predictive molecular markers in CRT remain elusive. Prediction of tumor response is important in selection of promising candidates for preoperative CRT and candidates who need additional adjuvant therapy after surgery. GRP78 is an important prognostic marker due to correlation with OS. Although further studies are needed, including further follow-up, standardized validation, and
GRP78 expression is a predictive and prognostic factor for down staging, recurrence, and OS in rectal cancer patients treated with 5-flurouracil and leucovorin neoadjuvant CRT.
Conflict of interest relevant to this article was not reported.
The authors wish to acknowledge the financial support of the Catholic Medical Center Research Foundation made in the program year of 2013.
These are representative tissue sections used in immunohistochemical analyses. (A) Low expression of glucose-regulated protein of molecular mass 78 (GRP78, ×400). (B) High cytoplasmic expression of GRP78 (×400). (C) Low expression of Cripto-1 (×400). (D) High cytoplasmic expression of Cripto-1 (×400).
(A) Down staging (DS) rate according to expression of glucose-regulated protein of molecular mass 78 (GRP78) protein (low expression of GRP78 [80.0%] vs. high expression of GRP78 [52.3%], respectively; p=0.046). (B) Recurrence rate (RR) according to expression of GRP78 protein (low expression of GRP78 [0%] vs. high expression of GRP78 [37.2%], respectively; p=0.004).
Kaplan-Meier survival curves are calculated using expression patterns of glucose-regulated protein of molecular mass 78 (GRP78) for recurrence-free survival.
Patients' characteristics
Characteristic | No. of patients (n=101) |
---|---|
Age (yr) | 62 (53-69) |
Gender (malefemale) | 69:32 |
ECOG PS (1:0) | 45:56 |
Differentiation | |
Well | 18 |
Moderate | 76 |
Poor | 7 |
CEA (ng/mL) | 3.48 (1.83-6.87) |
LDH (IU/L) | 285.0 (235.0-321.5) |
Values are presented as median (range) or number. ECOGPS, Eastern Cooperative Oncology Group performance status; CEA, carcinoembryonic antigen; LDH, lactate dehydrogenase.
Treatment response of neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer
Variable | Clinical stage | Pathologic stage | |
---|---|---|---|
T stage | |||
0 | 0 | 20 (19.8) | |
1 | 0 | 5 (5.0) | |
2 | 2 (2.0) | 23 (22.8) | |
3 | 96 (95.0) | 50 (49.5) | |
4 | 3 (3.0) | 3 (3.0) | |
N stage | |||
0 | 52 (51.5) | 71 (70.3) | |
1 | 32 (31.7) | 24 (23.8) | |
2 | 17 (16.8) | 6 (5.9) | |
TNM stage | |||
0 | 0 | 20 (19.8) | |
I | 0 | 22 (21.8) | |
II | 52 (51.5) | 29 (28.7) | |
III | 49 (48.5) | 30 (29.7) | |
Pathologic response | |||
Grade 0 | 19 (18.8) | ||
Grade 1 | 33 (32.7) | ||
Grade 2 | 29 (28.7) | ||
Grade 3 | 20 (19.8) |
Values are presented as number (%).
Immunohistochemistry results of endoplasmic reticulum stress-related proteins
Variable | Intensity (a) | Portion (b) | Score (axb) |
---|---|---|---|
GRP78 | |||
0 | 0 | 0 | - |
1 | 15 (14.9) | 16 (15.8) | 15 (14.9) |
2 | 86 (85.1) | 85 (84.2) | 1 (1.0) |
4 | 85 (84.2) | ||
Cripto-1 | |||
0 | 32 (31.7) | 32 (31.7) | 32 (31.7) |
1 | 31 (30.7) | 30 (29.7) | 20 (19.8) |
2 | 38 (37.6) | 39 (38.6) | 21 (20.8) |
4 | - | - | 28 (27.7) |
Values are presented as number (%). GRP78, glucose-regulated protein of molecular mass 78.
Correlation between expressions of endoplasmic reticulum stress-related proteins and clinicopathologic factors
Variable | No. | GRP78 |
Cripto-1 |
||||
---|---|---|---|---|---|---|---|
Low | High | p-value | Low | High | p-value | ||
Age (yr) | 0.431 | 0.292 | |||||
≤ 65 | 65 | 11 | 54 | 36 | 29 | ||
> 65 | 36 | 4 | 32 | 16 | 20 | ||
Histologic differentiation | 0.623 | 0.317 | |||||
Well | 18 | 4 | 14 | 11 | 7 | ||
Moderate | 76 | 10 | 66 | 36 | 40 | ||
Poor | 7 | 1 | 6 | 5 | 2 | ||
Clinical T stage | 0.289 | 0.193 | |||||
2 | 2 | 1 | 1 | 1 | 1 | ||
3 | 96 | 14 | 82 | 51 | 45 | ||
4 | 3 | 0 | 3 | 0 | 3 | ||
Clinical N stage | 0.905 | 0.375 | |||||
0 | 52 | 7 | 45 | 27 | 25 | ||
1 | 32 | 5 | 27 | 14 | 18 | ||
2 | 17 | 3 | 14 | 11 | 6 | ||
cTNM stage | 0.686 | 0.928 | |||||
II | 52 | 7 | 45 | 27 | 25 | ||
III | 49 | 8 | 41 | 25 | 24 |
GRP78, glucose-regulated protein of molecular mass 78; cTNM stage, clinical tumor, node, metastasis stage.
Responses and recurrence after neoadjuvant chemoradiotherapy according to expressions of endoplasmic reticulum stress-related proteins
Variable | GRP78 expression |
Cripto-1 expression |
||||
---|---|---|---|---|---|---|
Low (n=15) | High (n=86) | p-value | Low (n=52) High (n=49) | p-value | ||
Pathologic response | 0.406 | 0.809 | ||||
Grade 0 | 2 | 17 | 11 | 8 | ||
Grade 1 | 3 | 30 | 18 | 15 | ||
Grade 2 | 5 | 24 | 13 | 16 | ||
Grade 3 | 5 | 15 | 10 | 10 | ||
Down staging (%) | 80 | 52.3 | 0.046 | 53.8 | 59.2 | 0.589 |
ypCR rate (%) | 33.3 | 17.4 | 0.154 | 19.2 | 20.4 | 0.882 |
Recurrence rate (%) | 0 | 37.2 | 0.004 | 32.7 | 30.6 | 0.882 |
GRP78, glucose-regulated protein of molecular mass 78; ypCR, pathologic complete response.
Univariate analysis for several factors related to recurrence and survival
Variable | No. | Relapse free survival (mo) |
Overall survival (mo) |
|||
---|---|---|---|---|---|---|
Relapse free survival (mo) | p-value | Mean (95% CI) | p-value | |||
Age (yr) | ≤ 65 | 65 | 56.1 (49.4-62.7) | 0.613 | 75.1 (69.7-80.5) | 0.16 |
> 65 | 36 | 53.5 (43.9-63.1) | 64.1 (56.3-71.9) | |||
Clinical stage | II | 52 | 60.5 (53.8-67.3) | 0.046 | 78.0 (72.6-83.5) | 0.023 |
III | 49 | 48.1 (39.9-56.3) | 67.0 (59.8-74.1) | |||
Down staging | Yes | 57 | 64.4 (58.5-70.2) | 0 | 75.7 (70.6-80.8) | 0.072 |
No | 44 | 43.1 (34.2-52.0) | 68.3 (60.4-76.2) | |||
Pathologic complete response | Yes | 20 | 68.4 (60.9-75.9) | 0.031 | 77.5 (70.2-84.8) | 0.261 |
No | 81 | 51.8 (45.5-58.2) | 71.5 (66.1-77.0) | |||
GRP78 | Low | 15 | Not estimate | 0.007 | Not estimate | 0.045 |
High | 86 | Not estimate | Not estimate | |||
Cripto-1 | Low | 52 | 51.7 (44.1-59.2) | 0.737 | 73.0 (66.2-79.9) | 0.860 |
High | 49 | 56.3 (48.7-64.0) | 67.5 (62.2-72.8) | |||
Carcinoembryonic antigen (ng/mL) | ≤ 5.0 | 67 | 53.0 (46.5-59.5) | 0.978 | 71.6 (65.6-77.6) | 0.472 |
> 5.0 | 34 | 55.1 (45.8-64.4) | 74.5 (67.5-81.4) | |||
Lactate dehydrogenase (IU/L) | ≤ 300 | 61 | 53.8 (46.5-61.1) | 0.663 | 70.0 (63.4-76.7) | 0.166 |
> 300 | 40 | 56.8 (48.6-65.1) | 71.7 (66.4-76.9) |
CI, confidence interval; GRP78, glucose-regulated protein of molecular mass 78.