Abstract
Somatostatin receptors (SSTRs) already act as important roles in gastroenteropancreatic neuroendocrine tumors (GEP-NETs) with high expression levels for prognosis predicting and octreotide LAR treatment purposes but less noticed in gastrointestinal stromal tumors (GISTs). Our study aims to fully evaluate the expression levels and prognostic values of SSTRs in GIST patients. For SSTRs expression detection, qPCR were used in 25 fresh GIST specimens, and then, 453 GIST samples (405 GISTs with operation only and 48 with imatinib adjuvant therapy after surgery) were collected for tissue microarrays (TMAs) construction and confirmed by immunohistochemistry (IHC). Clinicopathological data were confirmed by pathological diagnosis and clinical recorders, recurrence-free survivals (RFS) were evaluated in 453 GIST patients. With IHC performed, SSTR1 and SSTR2 present high positive proportion (81.9% and 87.6%) in 453 GISTs in our study, and positive expression rates of SSTR3, SSTR4 and SSTR5 are 56.1%, 8.8% and 47.2%, respectively. SSTR2 and SSTR5 negative expression are associated with decreased RFS when compared to positive cases by Kaplan-Meier survival analyses with log-rank test and univariate analysis in GISTs, furthermore, SSTR2 was an independent prognostic indicator for GISTs by multivariate analysis. In our study, detection of SSRT2 and SSTR5 expression helps to predict different prognosis in GIST patients. SSTR2 is a novel independent prognostic biomarker for GISTs. With high expression performance of SSTRs in GISTs, new therapeutic strategies such as octreotide or pasireotide LAR could be taken into consideration in selected advanced GIST patients.
Keywords: Gastrointestinal stromal tumor, somatostatin receptor, octreotide, pasireotide, prognosis
Introduction
Gastrointestinal stromal tumors (GISTs) is the most common types of gastrointestinal mesenchymal tumors with increased incidence in recent years [1-3]. Abnormal activation of tyrosine kinase proteins KIT or PDGFRa transcribed by oncogenic mutations were shown in most of GISTs [4]. NIH consensus criteria, Modified NIH criteria or AFIP criteria are common criteria wildly accepted as risk-stratification schemes for predicting prognosis in GISTs, with similar accuracy [5]. The mitosis count, tumor size and tumor site were important prognostic indicators in these schemes [5]. Following surgical resection, GIST patients often suffered disease recurrence and with no response to chemotherapy or radiation therapy. With wide application of imatinib mesylate (IM) in clinical practice for GISTs, the mortality rate of GIST patients has decreased significantly. Nevertheless, the recurrence or metastasis rates still remain high [6-8], and almost all advanced GIST patients eventually develop resistance to imatinib treatment [9]. Although sunitinib act as second-line treatment for advanced GISTs progressing on imatinib [10], and some other small molecule targeted therapies are in clinical trails, the reality is there are still few options we can choose in current GIST therapeutic strategies.
Somatostatin (SST) is a naturally growth hormone inhibitory neuropeptide with potent and broad antisecretory actions, and also performed negative regulation of cell proliferation in both normal and tumor cells [11-13]. Anti-proliferation effect of SST is cytostatic or cytotoxic by binding to seven trans-membrane G-protein coupled receptors (GPCRs), which were named as somatostatin receptors (SSTRs) with five subtypes SSTR 1-5 [14,15]. SST analogs such as octreotide or octreotide long-acting repeatable (octreotide LAR), similarly to somatostatin structure, was already developed and successfully used for control neuroendocrine symptoms and tumor progression in advanced or metastasis gastroenteropancreatic neuroendocrine tumors (GEP-NETs), which present high positive expression rates of SSTRs [16-18]. Detecting the expression of SSTRs helps to predict not only the efficacy of octreotide LAR treatment in GEP-NETs but also the prognosis in tumors [11,19]. However, the research about the roles of SSTRs in GISTs was very limited. Our study aims to fully evaluate the expression levels and prognostic values of SSTRs in 453 GIST patients.
Materials and methods
Ethics statement
This project was approved by ethics committee of Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine for the use of samples. Informed consents were obtained from all patients before study inclusion.
Patients and specimens
25 fresh GIST tissues obtained from patients during surgical resection between January 2013 to May 2014 were collected for detecting mRNA expression level of SSTRs by quantitative real-time PCR.
GIST patients inclusion criteria for immunohistochemistry and prognostic evaluation of SSTRs were as follows: 1) a confirmative pathologic diagnosis of GISTs; 2) underwent R0 resection, R0 resection in our study defined as margin-free resection and no metastasis detected before and during the surgery; 3) no radiotherapy, chemotherapy, nor other anti-cancer therapies prior to the surgery; and 4) complete clinicopathologic and follow-up data were available. The risk of potential malignancy in recurrence was calculated according to the modified NIH criteria [20], which classified GISTs into very low, low, intermediate, and high-risk categories. High-risk GIST patients with imatinib adjuvant therapy required at least 12 months uninterrupted drugs taking with 400 mg/day in our study. The parameters, including patient age, gender, tumor site, tumor size and number of mitoses/50 high-power fields (HPF) were recorded in the official pathology database.
453 paraffin-embedded tumor tissue samples from GIST patients (405 GISTs with operation only and 48 high-risk GISTs received imatinib adjuvant therapy after radical surgery), which met the inclusion criteria, were collected at Ren Ji Hospital, Shanghai Jiao Tong University, School of Medicine from June 2004 to May 2013 for tissue microarrays (TMAs) construction and immunohistochemistry staining. Complete follow-up data until May 2014 for GIST patients were available. Recurrence free survival (RFS) was calculated from the date of tumor resection until the detection of tumor recurrence or last observation. The median follow-up of 405 GISTs with operation only was 53 months (range, 8-113 months). In high-risk GISTs with imatinib adjuvant therapy, The median follow-up was 45 months (range 2274 months). Computed tomography (CT) and/or magnetic resonance imaging (MRI) were used to verify tumor recurrence in suspected cases.
Total RNA extraction and quantitative real-time PCR
Total RNA was extracted from 25 fresh GIST tissues using Trizol reagent (Takara) followed the manufacturer instructions. The reverse-transcription reactions were carried out with random primers and M-MLV Reverse Transcriptase (Takara). The 25 cases of cDNA were used for quantitative real-time PCR reaction in SYBR-Green method. The specific primer sequences of SSTR1-5 and 18 s were as follow: SSTR1 [forward: 5’-TATCTGCCTGTGCTACGTGC-3’; reverse: 5’-GATGACCGACAGCTGACTCA-3’], SSTR2 [forward: 5’-ATGCCAAGATGAAGACCATCAC-3’; reverse: 5’-TGAACTGATTGATGCCATCCA-3’], SSTR3 [forward: 5’-CTGGGTAACTCGCTTGGTCATCTA-3’; reverse: 5’-AGCGCCAGGTTGAGGATGTA-3’], SSTR4 [forward: 5’-GTGATCCTTCGCTACGCCAA-3’; reverse: 5’-CACGGTGAGACAGAAGACGC-3’], SSTR5 [forward: 5’-GTGACAACAGGACGCTGGT-3’; reverse: 5’-TGGTGACGGTCTTCATCTTG-3’] and 18 s [forward: 5’-TGCGAGTACTCAACACCAACA-3’; reverse: 5’-GCATATCTTCGGCCCACA-3’]. 18 s was used as an internal control. Relative SSTRs expression levels were quantified by the 2-ΔCt method.
Immunohistochemistry
Immunohistochemistry (IHC) was performed using a two-step protocol. After citrate buffer (pH 6.0) antigen retrieval, tissues were incubated with SSTR1 antibody (rabbit polyclonal antibody, Abcam), SSTR2 antibody (rabbit monoclonal antibody, Epitomics), SSTR3 antibody (rabbit polyclonal antibody, Abcam), SSTR4 antibody (rabbit polyclonal antibody, Pierce) or SSTR5 antibody (rabbit monoclonal antibody, Epitomics) overnight at 4°C. Next day, following incubated with goat anti-rabbit IgG-HRP (HUABIO) secondary antibody for one hour at room temperature, sections were developed in DAB solution under microscopic observation and counterstained with hematoxylin.
Judgment for immunoreactivity of SSTRs in GISTs was referred from Edris et al’s research [21]. TMAs were scored as follows: 0: absence of any staining; 1: weak staining whether diffusely or focally present in the tumor; 2: strong staining whether diffusely or focally present in the tumor. Score 0-1 was considered as negative (-) and a score of 2 was positive (+) for subsequent statistical analyses [21].
Statistical analysis
Statistical analyses were conducted by using SPSS (version 21.0) and MedCalc (version 11.4.2.0). RFS was calculated according to Kaplan-Meier method and log-rank test was used for comparing the survival distributions. Univariate and multivariate analyses were based on the cox proportional hazards regression model. All statistical tests were two-sided. P value less than 0.05 was considered statistically significant.
Results
SSTRs mRNA and protein expression in GISTs
For determining the differences in mRNA expressions of SSTRs in GISTs, mRNA transcript levels were analyzed by quantitative real-time PCR from 25 GIST samples. Scatter dot plot for mRNA relative expression levels (2-ΔCt) of SSTRs in GISTs were shown in Figure 1 and the clinicopathological characters for real-time PCR were shown in Table 1.
Figure 1.
Relative expression levels of SSTRs in GISTs by quantitative real-time PCR.
Table 1.
Clinicopathological characters of GISTs for real-time PCR
| Number | (%) | |
|---|---|---|
| Age (years) | ||
| ≤ 50 | 3 | 12.0 |
| > 50 | 22 | 88.0 |
| Gender | ||
| Male | 14 | 56.0 |
| Female | 11 | 44.0 |
| Tumor site | ||
| Stomach | 19 | 76.0 |
| Small bowel | 6 | 24.0 |
| Tumor size (cm) | ||
| 2.1-5.0 | 5 | 20.0 |
| 5.1-10.0 | 13 | 52.0 |
| > 10.0 | 7 | 28.0 |
| Mitoses per 50 HPFs | ||
| ≤ 5 | 16 | 64.0 |
| 6-10 | 4 | 16.0 |
| > 10 | 5 | 20.0 |
| Modified NIH criteria | ||
| Low risk | 6 | 24.0 |
| Intermediate risk | 6 | 24.0 |
| High risk | 13 | 52.0 |
| Total | 25 | 100.0 |
To confirm SSTRs expressions, we performed IHC study using TMAs that contained 453 GISTs (405 GISTs with operation only and 48 high-risk GISTs with imatinib adjuvant therapy). Representative stains of SSTR1-5 were shown in Figure 2. Positive expression proportion of SSTR1 and SSTR2 were 81.9% (371/453) and 87.6 % (397/405) in our study, which indicate high expression performance of SSTR1 and SSTR2 in GIST patients. Positive expression rates of SSTR3, SSTR4 and SSTR5 are 56.1%, 8.8% and 47.2% respectively in our study. These IHC results are similar to our real-time PCR findings. Clinicopathological characters of 453 GISTs could be referred from Tables 2 and 3. In 405 GISTs with operation only, sub-group study was designed based on modified NIH criteria. High-risk sub-group of GIST patients which suffered worst prognosis than very low, low and intermediate-risk was the most important population deserved attention in GISTs. Given the IHC reactivity of SSTRs observed, we focused remainder of our study on examining the prognostic value of SSTRs in GISTs.
Figure 2.
Representative immunohistochemical stains for SSTRs in GISTs.
Table 2.
Clinicopathological characters of 405 GISTs with operation only and 48 high-risks with imatinib adjuvant therapy for IHC
| Operation only | Imatinib adjuvant therapy | |||
|---|---|---|---|---|
|
|
|
|||
| Number | (%) | Number | (%) | |
| Age (years) | ||||
| ≤ 50 | 81 | 20.0 | 7 | 14.6 |
| > 50 | 324 | 80.0 | 41 | 85.4 |
| Gender | ||||
| Male | 215 | 53.1 | 28 | 58.3 |
| Female | 190 | 46.9 | 20 | 41.7 |
| Tumor site | ||||
| Stomach | 238 | 58.8 | 18 | 37.5 |
| Small bowel | 129 | 31.9 | 23 | 47.9 |
| Colorectum | 19 | 4.7 | 3 | 6.3 |
| Others | 19 | 4.7 | 4 | 8.3 |
| Tumor size (cm) | ||||
| ≤ 2.0 | 36 | 8.9 | 1 | 2.1 |
| 2.1-5.0 | 194 | 47.9 | 3 | 6.3 |
| 5.1-10.0 | 115 | 28.4 | 24 | 50.0 |
| > 10.0 | 60 | 14.8 | 20 | 41.7 |
| Mitoses per 50 HPFs | ||||
| ≤ 5 | 327 | 80.7 | 17 | 35.4 |
| 6-10 | 43 | 10.6 | 16 | 33.3 |
| > 10 | 35 | 8.6 | 15 | 31.3 |
| Modified NIH criteria | ||||
| Very low risk | 32 | 7.9 | 0 | 0 |
| Low risk | 187 | 46.2 | 0 | 0 |
| Intermediate risk | 62 | 15.3 | 0 | 0 |
| High risk | 124 | 30.6 | 48 | 100.0 |
| Total | 405 | 100.0 | 48 | 100.0 |
Table 3.
IHC expression for SSTRs in 453 GIST patients (405 with operation only and 48 high-risks with imatinib adjuvant therapy)
| Total | SSTR1 | SSTR2 | SSTR3 | SSTR4 | SSTR5 | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
|
|
|
|
|||||||||||||||||
| — | (%) | + | (%) | — | (%) | + | (%) | — | (%) | + | (%) | — | (%) | + | (%) | — | (%) | + | (%) | ||
| Age (years) | |||||||||||||||||||||
| ≤ 50 | 88 | 13 | 2.9 | 75 | 16.6 | 7 | 1.5 | 81 | 17.9 | 37 | 8.2 | 51 | 11.3 | 80 | 17.7 | 8 | 1.8 | 39 | 8.6 | 49 | 10.8 |
| > 50 | 365 | 69 | 15.2 | 296 | 65.3 | 49 | 10.8 | 316 | 69.8 | 162 | 35.8 | 203 | 44.8 | 333 | 73.5 | 32 | 7.1 | 200 | 44.2 | 165 | 36.4 |
| Gender | |||||||||||||||||||||
| Male | 243 | 42 | 9.3 | 201 | 44.4 | 27 | 6.0 | 216 | 47.7 | 112 | 24.7 | 131 | 28.9 | 221 | 48.8 | 22 | 4.9 | 125 | 27.6 | 118 | 26.0 |
| Female | 210 | 40 | 8.8 | 170 | 37.5 | 29 | 6.4 | 181 | 40.0 | 87 | 19.2 | 123 | 27.2 | 192 | 42.4 | 18 | 4.0 | 114 | 25.2 | 96 | 21.2 |
| Tumor site | |||||||||||||||||||||
| Stomach | 256 | 44 | 9.7 | 212 | 46.8 | 33 | 7.3 | 223 | 49.2 | 131 | 28.9 | 125 | 27.6 | 234 | 51.7 | 22 | 4.9 | 138 | 30.5 | 118 | 26.0 |
| Small bowel | 152 | 29 | 6.4 | 123 | 27.2 | 18 | 4.0 | 134 | 29.6 | 47 | 10.4 | 105 | 23.2 | 136 | 30.0 | 16 | 3.5 | 72 | 15.9 | 80 | 17.7 |
| Colorectum | 22 | 7 | 1.5 | 15 | 3.3 | 2 | 0.4 | 20 | 4.4 | 12 | 2.6 | 10 | 2.2 | 21 | 4.6 | 1 | 0.2 | 16 | 3.5 | 6 | 1.3 |
| Others | 23 | 2 | 0.4 | 21 | 4.6 | 3 | 0.7 | 20 | 4.4 | 9 | 2.0 | 14 | 3.1 | 22 | 4.9 | 1 | 0.2 | 13 | 2.9 | 10 | 2.2 |
| Tumor size (cm) | |||||||||||||||||||||
| ≤ 2.0 | 37 | 10 | 2.2 | 27 | 6.0 | 3 | 0.7 | 34 | 7.5 | 23 | 5.1 | 14 | 3.1 | 37 | 8.2 | 0 | 0.0 | 18 | 4.0 | 19 | 4.2 |
| 2.1-5.0 | 197 | 27 | 6.0 | 170 | 37.5 | 16 | 3.5 | 181 | 40.0 | 89 | 19.6 | 108 | 23.8 | 178 | 39.3 | 19 | 4.2 | 90 | 19.9 | 107 | 23.6 |
| 5.1-10.0 | 139 | 25 | 5.5 | 114 | 25.2 | 16 | 3.5 | 123 | 27.2 | 54 | 11.9 | 85 | 18.8 | 130 | 28.7 | 9 | 2.0 | 73 | 16.1 | 66 | 14.6 |
| > 10.0 | 80 | 20 | 4.4 | 60 | 13.2 | 21 | 4.6 | 59 | 13.0 | 33 | 7.3 | 47 | 10.4 | 68 | 15.0 | 12 | 2.6 | 58 | 12.8 | 22 | 4.9 |
| Mitoses per 50 HPFs | |||||||||||||||||||||
| ≤ 5 | 344 | 55 | 12.1 | 289 | 63.8 | 26 | 5.7 | 318 | 70.2 | 157 | 34.7 | 187 | 41.3 | 315 | 69.5 | 29 | 6.4 | 158 | 34.9 | 186 | 41.1 |
| 6-10 | 59 | 15 | 3.3 | 44 | 9.7 | 13 | 2.9 | 46 | 10.2 | 18 | 4.0 | 41 | 9.1 | 50 | 11.0 | 9 | 2.0 | 41 | 9.1 | 18 | 4.0 |
| > 10 | 50 | 12 | 2.6 | 38 | 8.4 | 17 | 3.8 | 33 | 7.3 | 24 | 5.3 | 26 | 5.7 | 48 | 10.6 | 2 | 0.4 | 40 | 8.8 | 10 | 2.2 |
| Modified NIH criteria | |||||||||||||||||||||
| Very low risk | 32 | 7 | 1.5 | 25 | 5.5 | 2 | 0.4 | 30 | 6.6 | 20 | 4.4 | 12 | 2.6 | 32 | 7.1 | 0 | 0.0 | 16 | 3.5 | 16 | 3.5 |
| Low risk | 187 | 28 | 6.2 | 159 | 35.1 | 15 | 3.3 | 172 | 38.0 | 88 | 19.4 | 99 | 21.9 | 168 | 37.1 | 19 | 4.2 | 80 | 17.7 | 107 | 23.6 |
| Intermediate risk | 62 | 8 | 1.8 | 54 | 11.9 | 1 | 0.2 | 61 | 13.5 | 28 | 6.2 | 34 | 7.5 | 58 | 12.8 | 4 | 0.9 | 28 | 6.2 | 34 | 7.5 |
| High risk | 172 | 39 | 8.6 | 133 | 29.4 | 38 | 8.4 | 134 | 29.6 | 63 | 13.9 | 109 | 24.1 | 155 | 34.2 | 17 | 3.8 | 115 | 25.4 | 57 | 12.6 |
| Total | 453 | 82 | 18.1 | 371 | 81.9 | 56 | 12.4 | 397 | 87.6 | 199 | 43.9 | 254 | 56.1 | 413 | 91.2 | 40 | 8.8 | 239 | 52.8 | 214 | 47.2 |
SSTR2 acts as an independent prognostic indicator for GISTs
In GISTs with operation only, we found SSTR2 negative expression significantly associated with disease recurrence by univariate and multivariate cox proportional hazards model analyses (P < 0.001 and P = 0.002, Table 4). Further sub-group univariate and multivariate studies also showed SSTR2 was an independent prognostic indicator for high-risk GISTs (P < 0.001 and P = 0.004, Table 4). Kaplan-Meier survival analyses with log-rank test for RFS showed sharply decreased curves in SSTR2 negative expression tumors compared with positive cases in GISTs (Figure 3A) and high-risk sub-group (Figure 3B).
Table 4.
Univariate & multivariate cox proportional hazards model to predict factors associated with RFS in GISTs with operation only
| Variable | Univariate | Multivariate | ||
|---|---|---|---|---|
|
|
|
|||
| Hazard Ratio (95% Cl) | P value | Hazard Ratio (95% Cl) | P value | |
| Total | ||||
| Age | 1.414 (0.692-2.887) | 0.342 | 0.883 (0.406-1.918) | 0.753 |
| Gender | 0.413 (0.231-0.738) | 0.003** | 0.485 (0.267-0.881) | 0.017* |
| Tumor site | 1.728 (1.309-2.281) | < 0.001** | 1.353 (1.004-1.823) | 0.047* |
| Tumor size | 4.280 (2.990-6.127) | < 0.001** | 2.887 (1.939-4.299) | < 0.001** |
| Mitosis count | 4.251 (3.180-5.681) | < 0.001** | 2.351 (1.594-3.468) | < 0.001** |
| SSTR1 | 0.707 (0.373-1.339) | 0.287 | 1.282 (0.567-2.896) | 0.551 |
| SSTR2 | 0.176 (0.100-0.310) | < 0.001** | 0.333 (0.166-0.669) | 0.002** |
| SSTR3 | 1.297 (0.754-2.230) | 0.347 | 1.833 (0.955-3.519) | 0.069 |
| SSTR4 | 0.776 (0.281-2.147) | 0.819 | 0.713 (0.250-2.036) | 0.528 |
| SSTR5 | 0.446 (0.255-0.782) | 0.005** | 0.892 (0.449-1.770) | 0.743 |
| High-risk sub-group | ||||
| Age | 1.120 (0.524-2.393) | 0.770 | 0.836 (0.367-1.906) | 0.671 |
| Gender | 0.584 (0.315-1.081) | 0.087 | 0.537 (0.287-1.007) | 0.053 |
| Tumor site | 1.062 (0.770-1.466) | 0.714 | 1.179 (0.850-1.636) | 0.324 |
| Tumor size | 1.582 (1.001-2.500) | 0.049* | 1.752 (1.070-2.869) | 0.026* |
| Mitosis count | 2.060 (1.480-2.868) | < 0.001** | 1.819 (1.201-2.756) | 0.005** |
| SSTR1 | 0.878 (0.450-1.712) | 0.702 | 1.253 (0.546-2.874) | 0.595 |
| SSTR2 | 0.236 (0.130-0.430) | < 0.001** | 0.361 (0.181-0.721) | 0.004** |
| SSTR3 | 1.108 (0.573-1.810) | 0.951 | 1.759 (0.885-3.496) | 0.107 |
| SSTR4 | 0.635 (0.229-1.762) | 0.383 | 0.782 (0.272-2.246) | 0.648 |
| SSTR5 | 0.508 (0.274-0.941) | 0.031* | 0.743 (0.360-1.530) | 0.420 |
P < 0.05;
P < 0.01.
Figure 3.
SSTR2 negative expression predicts poor prognosis in GISTs (A) and high-risk sub-group (B), and SSTR5 negative expression also predicts poor prognosis in GISTs (C) and high-risk sub-group (D).
SSTR5 negative expression can predict poor prognosis of GISTs
Kaplan-Meier survival analysis with log-rank test and univariate analysis show GIST tumors with SSTR5 negative expression were associated with decreased RFS when compared to cases expressed SSTR5 positively in GISTs and high-risk sub-group (Figure 3C & 3D, Table 4), but there is no statistic significance between SSTR5 and RFS by multivariate analysis (Table 4).
Expressions of SSTRs show no relationship with efficacy of imatinib adjuvant therapy
Although negative expression of SSTR2 and SSTR5 can predict adverse prognosis in GISTs with operation only, no relationship was found between SSTRs and RFS in high-risk GIST patients with imatinib adjuvant therapy by univariate analysis (Table 5).
Table 5.
Univariate cox proportional hazard model to predict factors associated with RFS in high-risk GISTs with imatinib adjuvant therapy
| Variable | Hazard Ratio (95% Cl) | P value |
|---|---|---|
| Age | 0.684 (0.188-2.498) | 0.566 |
| Gender | 0.993 (0.332-2.974) | 0.990 |
| Tumor site | 1.340 (0.701-2.561) | 0.375 |
| Tumor size | 1.811 (0.704-4.659) | 0.218 |
| Mitosis count | 1.514 (0.749-3.059) | 0.248 |
| SSTR1 | 1.435 (0.440-4.682) | 0.550 |
| SSTR2 | 0.466 (0.153-1.417) | 0.179 |
| SSTR3 | 0.616 (0.205-1.846) | 0.387 |
| SSTR4 | 1.688 (0.210-13.566) | 0.622 |
| SSTR5 | 1.170 (0.321-4.269) | 0.812 |
Discussion
SSTRs act as important roles in GEP-NETs with high expression levels for prognosis predicting and octreotide LAR treatment purposes [19,22]. However, the studies about relationship between SSTRs and GISTs were very limited. Arne et al’s research detected SSTR1-5 expression levels in 34 GISTs which presented 100% positive expression rates of SSTR1, 2 and relative lower expression rates of SSTR3, 4, 5 in GISTs [23]. Here, we present the first large-scale characterization of SSTRs expression in GIST patients. Initial detection of GISTs mRNA expression were conducted by quantitative real-time PCR, and then 453 GIST cases (405 cases with operation only and 48 high-risk cases received imatinib adjuvant therapy after radical surgery) were confirmed by IHC on TMAs. SSTR1 and SSTR2 also present high positive proportion (81.9% and 87.6%) in 453 GISTs in our study, and positive expression rates of SSTR3, SSTR4 and SSTR5 are 56.1%, 8.8% and 47.2%, respectively.
The next we focused on examining the prognostic value of SSTRs in GISTs. Negative expression of SSTR2 and SSTR5 showed adverse prognosis of GIST patients by univariate cox model analysis and Log-rank test, furthermore, SSTR2 acted as an independent prognostic indicator in GISTs in our study by multivariate analysis. This result was similar in GEP-NETs, which indicated SSTR2 negative expression also as an independent adverse prognostic indicator although high positive expression rate presented [19,22]. There were no significant associations for prognosis were found between SSTR1, SSTR3 or SSTR4 and GISTs, and no relationship between SSTRs and imatinib adjuvant therapy.
With high positive expression performance of SSTR1 (81.9%) and SSTR2 (87.6%), and approximately half of the GISTs expression SSTR3 (56.1%) and SSTR5 (47.2%) in our study, relative therapeutic strategies are reasonably considerable. Agitating SSTR1, 2, and 3 can transduce their antiproliferative functions by stimulating one or more PTPs, which have inhibitory effects on mitogenic MAPK and survival PI3K pathways [24]. Octreotide LAR, mainly agitate SSTR2 (IC50, 0.75 nM) and SSTR5 (IC50, 7 nM) [25], was demonstrated its antiproliferative effect in the randomized, double-blind, placebo-controlled PROMID study in patients with advanced intestinal NETs [18]. By application of pasireotide (SOM230), which is new generation of SST analog with high-efficiency of agitating abilities in SSTR1 (IC50, 9.3 nM), SSTR2 (IC50, 1 nM), SSTR3 (IC50, 0.5 nM) and SSTR5 (IC50, 0.16 nM) [25], 56.5% (13/23) patients with advanced NETs refractory or resistant to octreotide LAR showed stable disease status in a phase II study [26]. Like GEP-NETs, GISTs is also a relatively rare neoplastic disorder with limited therapeutic options. Such high positive expression performance of SSTR1 and SSTR2, and approximate half of the GISTs with SSTR3 and SSTR5 expression, may provide new possible choices by applications of octreotide or pasireotide LAR in selected advanced GISTs alone or accompany with current imatinib/sunitinib treatment procedure.
In summary, detection of SSTR2 and SSTR5 help to predict outcomes of GIST patients. Negative expression of SSTR2 is an independent adverse prognostic indicator in GISTs. High expression performance of SSTRs in GISTs may provide new therapeutic strategies in selected advanced GISTs.
Acknowledgements
National Natural Science Foundation of China (No. 81272743); Shanghai Committee of Science and Technology, China (No. 13XD1402500 and 13411950902).
Disclosure of conflict of interest
None.
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