Abstract
Background
Single nucleotide polymorphisms can create a genetic microenvironment in some tumors that affects the course of treatment, resistance, etc. Whether single nucleotide polymorphisms have an impact on gastrointestinal stromal tumor (GIST) development and disease progression is not yet accurately verified. KIT SNPM541L in exon 10 correlates with a worse prognosis of many cancers. The impact of KIT SNPM541L in GISTs is relatively unknown and, therefore, its analyses could have potential in patient therapy and could provide more detailed information on tumor character, clinical presentation, or tumor behavior in treatment.
Aim
The aim of the study was the analysis of the biological and clinical significance of the KIT SNPM541L polymorphism in exon 10.
Materials and methods
Paraffin sample tissues were obtained from the National GIST Register in Martin. Retrospective samples from 177 GIST patients were divided into several groups. Detection of SNPM541L was performed by Sanger sequencing. Statisitical analyses were performed to determine the prevalence of KIT SNPM541L in the Slovak GIST cohort, to search for correlation between c-KIT status and clinicopathological, molecular and biological data.
Results
Overall, 29 samples out of 177 showed KIT SNPM541L polymorphism.
Conclusion
Our results do not support the association between KIT SNPM541L and increased risk of relapse in localized primary GISTs. Additionally, we found a positive correlation between KIT SNPM541L occurrence and earlier onset of relapse in PDGFRa and WT subgroup of GISTs.
Keywords: Gastrointestinal stromal tumors, Single nucleotide polymorphism, c.1621 A > C, M541L, c-KIT
Introduction
Many diseases, especially cancer diseases, have a variety of pathological backgrounds responsible for abnormal cellular behavior exhibiting altered cell function or signal transduction. One possibility of these functional aberrations can be genetic change in the form of polymorphisms. Single nucleotide polymorphism is one of the most common and simplest forms of DNA variations across the human genome including introns, exons, promotors, etc. (Deng et al. 2017; Ghagane et al. 2016). Single nucleotide polymorphisms found in cell cycle regulation genes, immune response genes, respectively, in metabolism driving genes, and mRNA genes can affect promoter activity (Foster et al. 2008) that could trigger malignant cell behaviour (Brahmi et al. 2015; Laytragoon-Lewin et al. 2017). Therefore, studying, identifying and understanding their mechanism could uncover their potential as diagnostic and therapeutic targets of many cancers (Ghagane et al. 2016; Brahmi et al. 2015).
One of these polymorphisms is c.1621 A > C (M541L), with prevalence in the population of about 15–20% (Brahmi et al. 2015;) [4,6]. It is present in the transmembrane domain in exon 10 of KIT gene, which is one of the genes whose driver mutation causes GIST tumorigenesis (Foster et al. 2008). According to the catalog of tumor somatic mutations, M541L was also identified as a somatic change and considered a pathogenic variant (https://cancer.sanger.ac.uk).
It has been identified in several different tumor diseases, which may indicate that it is not tumor specific (Foster et al. 2008; Brahmi et al. 2015; Grabellus et al. 2011; Krasagakis et al. 2011; Dufresne et al. 2014; Hoade et al. 2018). In many diseases, it is associated with worse prognosis and aggressive behaviour (Brahmi et al. 2015).
GISTs, rare uncommon soft tissue sarcomas of gastroinstestinal tract, are characterized by their specificity, especially resistance to chemotherapy and radiotherapy (O’Brien et al. 2013). Their only possible biological treatment is with inhibitors of tyrosine kinase receptors (TKI). Driver mutations in KIT/PDGFRa oncogenes (present in more than 80% of GIST patients) encoding these receptors share a common origin in the interstitial cells of Cajal (ICC) (Niinuma et al. 2018). Based on the major occurrence of mutations in the mentioned genes, GISTs are divided into KIT/PDGFRa positive and KIT/PDGFRa negative, referred to as wild-type GISTs (WT GISTs), which are characterized by mutations in other genes: SDH-, NF1, BRAF, EGFR, PIK3CA, KRAS (Niinuma et al. 2018; Jasek et al. 2017; Lasota et al. 2016; Hurley et al. 2018).
A new identified mutation in KIT exon 8 has recently been described (Niinuma et al. 2018; Ito et al. 2014; Hus set al. 2013). Mutations in exons 9, 11, 13, 17 of KIT gene, exons 12, 14, 18 of PDGFRa gene as well as their prognostic and predictive significance and sensitivity for the treatment with tyrosine kinase receptor inhibitors are well described (Li et al. 2017; Hus set al. 2013). Only a small group of GISTs (10–15%) without KIT/PDGFRa mutations that form a heterogenous group of tumors with different molecular backgrounds, morphology and localization is usually less sensitive or does not respond to treatment with TKI (Nannini et al. 2014; Li et al. 2017).
In general, GIST genotype plays an important role in clinical management of diseases, so genetic testing should be part of standard diagnostics. The effect of SNP M541L on condition, disease behavior and overall GIST patient outcome is less known; therefore, their further analysis could provide more detailed information about tumor characteristics, tumor behavior in treatment or clinical presentation, and could be useful in patient therapy and personalized medicine based on the design of specific therapeutic agents.
Materials and methods
Patients and tumor characterization
The study was approved by the Ethical Committee of Jessenius Faculty of Medicine in Martin.
The study was carried out as a retrospective research on tissue samples from 177 GIST patients divided into two groups. Group 1 included primary untreated GISTs at diagnosis (primary GISTs). Group 2 consisted of patients with unresectable, locally advanced GIST or GISTs with metastases or tumor duality at the time of diagnosis. The study was conducted in collaboration with Biomedical Centre in Martin of JFM CU (Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava) and the Department of Pathological Anatomy JFM CU and University Hospital in Martin. All samples as well as the clinical and histopathological data were provided by the National Slovak GIST registry in Martin. Tissue samples were taken during biopsies or surgeries at the time of diagnosis. All patients signed informed consent and agreed to be included in the study. Prior the SNP analyses, all GISTs were analyzed for mutations in exons 9, 11, 13, and 17 of KIT and exons 12, 14, and 18 of PDGRa genes as part of national diagnostics and clinical studies (Jasek et al. 2017; Minarik et al. 2013; Plank et al. 2017). Tissue samples without mutations were referred to as WT GISTs.
DNA extraction
All molecular and statistical parts of the study were conducted in Biomedical Center. For SNP analysis we used DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue specimens using BlackPREP FFPE DNA Kit allowing simultaneous deparafinization and DNA isolation (Analytik Jena AG, Germany). DNA was isolated according to the manufacturer's instructions, using silica membrane columns. Sliced FFPE samples with added lysis solution and Proteinase K were incubated under continuous shaking for 1 h at 65 °C and 90 °C, respectively. Lysis was followed by washing steps with ethanol and wash buffer. After washing, DNA was eluted twice with 30 μl of elution buffer. Isolated DNA was quantitated using the Qubit Fluorometer (ThermoFisher Scientific, USA).
Sanger sequencing
SNP detection was carried out using the Sanger Sequencing provided by Applied Biosystems´genetic analyzer ABI Prism 3500 (ThermoFisher Scientific, USA). Before dideoxysequencing, we performed PCR to amplify KIT exon 10 with F: 5´TGG TAG AGA TCC CAT CCT GC 3′and R: 5′GCC ACT GGA GTT CCT TAA AGT 3′ (Péčová et al. 2018). Optimalized 25 µl reaction mixture contained: 0.1 mmol/L of each of four dNTPs (Applied Biosystems, USA), 2.5 mM MgCl2, 10 pmol/L of each primer (Sigma-Aldrich, USA) and 1 unit of FastStart Taq DNA Polymerase (Roche Diagnostics, Switzerland) with subsequent reaction conditions. The initial denaturation at 95 °C for 8 min was followed by 40 cycles of amplification steps: 95 °C for 20 s, 58 °C for 20 s (for both alleles), 72 °C for 1 min., and additional extension at 72 °C for 10 min. Sequencing primers were the same as those used in the PCR. Amplification products were separated by electrophoresis on 1.75% agarose gel stained with GelRed nucleic acid gel stain (Biotium, USA) and visualized on UV transilluminator. PCR products were purified by NucleoSpin® Gel and PCR Clean-up (Machery-Nagel, Germany).
Statistical analysis
Statistical analyses were performed using R statistical software (version 3.5.2). Pearson’s Chi-squared test with Yate’s continuity correction, Fisher’s exact test, Wilcoxon rank test, two-sample test for equality of proportions with continuity correction, and Welch two-sample t test were performed to (1) check KIT SNPM541L prevalence in GISTs; (2) study the distribution of known clinicopathological factors (tumor size, mitotic index, organ site) according to the presence of KIT SNPM541L; (3) evaluate the possible correlation between KIT SNPM541L and molecular and biological characteristics of GISTs (exons, mutations, relapse, risk category).
Results
Patients and clinicopathological findings
The study was performed on 177 specimens with 124 samples being the primary GISTs at diagnosis and 53 belonging to metastatic/locally advanced and unresectable GISTs or GIST patients with tumor duality at diagnosis. In Table 1 are listed clinical and histopathological data of 85 women and 92 men at mean age of 63.6 divided according to groups including size, localization, histological type, mitotic index, and proliferative activity. In general, the clinicopathological and genetic data from Group 1 (in percent) are not fundamentally different from the data of the other samples or from those described in literature. Within Group 1 (primary GISTs) tumor size was less than 2 cm in 17 samples (13.70%), while tumors of 2–5 cm and 5–10 cm showed up in 47 (37.90%) and 28 (22.58%) cases, respectively, and larger than 10 cm in 19 samples (15.32%). In 13 cases, we did not have the data. Most Group 1 samples we found more often in the stomach (62/50%), than in the small intestine (44/35.48%), colon (3/2.41%) or rectum (14/11.29%). Fourteen samples (11.29%) were not localized within the gastroinsteinal tract, referred to as extragastrointestinal GISTs (eGISTs). Spindle cell morphology has occurred in 80 samples (64.51%), epitheloid in 23 (18.54%) and mixed form in 20 (16.12%). We could not determine the morphology in 1 case (0.80%). Considering proliferation parameters, the number of mitoses was lower than 5/50HPF in 73 samples (58.87%), higher than 5/50 HPF in 42 (33.87%), and in 9 cases the data were not avalaible. Ki-67 index lower than ≤ 5 was found in 67 (54.03%), and higher than > 10 in 16 samples (12.90%). CD117 positive were 118 (95.16%) primary GISTs. No CD117 staining was noted in six cases (4.83%). CD34 staining was positive in 88 samples (70.96%). Other immunohistochemical data are shown in Table 1.
Table 1.
Clinicopathological features of GISTs cohortand divided according to analyzed series
| Clinicopathological data | All samples | Group 11 | Group 22 |
|---|---|---|---|
| Gender (F/M)/mean age | Gender (F/M)/age | Gender (F/M)/age | Gender (F/M)/age |
| Female/mean age/range | 85/64.06/17–89 | 62/64.14/17–89 | 23/64.40/39–86 |
| Male/mean age/range | 92/63.06/32–89 | 62/63.33/32–89 | 30/62.51/43–83 |
| ND* | 0/2 | 0/0 | 0/2 |
| n (%) | n (%) | n (%) | ||
|---|---|---|---|---|
| Tumor size (cm) | ||||
| ≤ 2 | 23 (12.99) | 17 (13.70) | 6 (11.32) | |
| 2–5 | 61 (34.44) | 47 (37.90) | 14 (26.41) | |
| 5–10 | 43 (24.29) | 28 (22.58) | 15 (28.30) | |
| > 10 | 24 (13.55) | 19 (15.32) | 5 (9.43) | |
| ND* | 26 (14.68) | 13 (10.48) | 13 (24.52) | |
| Morphologic features | ||||
| Spindle | 116 (65.53) | 80 (64.51) | 36 (67.92) | |
| Epithelioid | 27 (15.25) | 23 (18.54) | 4 (7.54) | |
| Mixed** | 31 (17.51) | 20 (16.12) | 11 (20.75) | |
| ND* | 3 (1.69) | 1 (0.80) | 2 (3.77) | |
| Location | ||||
| Stomach | 82 (46.32) | 62 (50.00) | 20 (37.73) | |
| Small intestine | 61 (34.46) | 44 (35.48) | 17 (32.07) | |
| Colon | 5 (2.82) | 3 (2.41) | 2 (3.77) | |
| e-GIST | 25 (14.12) | 14 (11.29) | 11 (20.75) | |
| Rectum | 2 (1.12) | 1 (0.08) | 1 (1.88) | |
| ND* | 2 (1.12) | 0 (0) | 2 (3.77) | |
| Mitoses/50 HPF | ||||
| ≤ 5 | 89 (50.28) | 73 (58.87) | 16 (30.18) | |
| ≥ 5 | 75 (42.37) | 42 (33.87) | 33 (62.26) | |
| ND* | 13 (7.34) | 9 (7.25) | 4 (7.54) | |
| Ki-67 index | ||||
| ≤ 5 | 89 (50.28) | 67 (54.03) | 22 (41.50) | |
| > 5– ≤ 10 | 42 (23.72) | 31 (25.00) | 11 (20.75) | |
| > 10 | 29 (16.38) | 16 (12.90) | 13 (24.52) | |
| ND* | 17 (9.60) | 10 (8.06) | 7 (13.20) | |
| Event | ||||
| Primary | 131 (85.71) | 95 (85.71) | 36 (85.71) | |
| Recurrence*** | 46 (2.86) | 29 (2.86) | 17 (2.86) | |
| ND* | 0 (0) | 0 (0) | 0 (0) | |
| CD117 | ||||
| Positive staining | 169 (95.48) | 118 (95.16) | 51 (76.22) | |
| No staining | 7 (3.95) | 6 (4.83) | 1 (1.88) | |
| ND* | 1 (0.56) | 0 (0) | 1 (1.88) | |
| CD34 | ||||
| Positive staining | 125 (70.62) | 88 (70.96) | 37 (69.81) | |
| Weak staining | 15 (8.47) | 10 (8.06) | 5 (9.43) | |
| No staining | 33 (18.64) | 23 (18.54) | 10 (18.86) | |
| ND* | 4 (2.25) | 3 (2.41) | 1 (1.88) | |
| DOG-1 | ||||
| Positive staining | 133 (75.14) | 99 (79.83) | 34 (64.15) | |
| Weak staining | 1 (0.56) | 1 (0.80) | 0 (0) | |
| No staining | 2 (1.12) | 0 (0) | 2 (3.77) | |
| ND* | 41 (23.16) | 24 (19.35) | 17 (32.07) | |
| S-100 | ||||
| Positive staining | 4 (2.25) | 2 (1.61) | 2 (3.77) | |
| Weak staining | 4 (2.25) | 2 (1.61) | 2 (3.77) | |
| No staining | 145 (81.92) | 106 (85.48) | 39 (73.58) | |
| ND* | 24 (13.55) | 14 (11.29) | 10 (18.86) | |
| DEZ | ||||
| Positive staining | 2 (1.12) | 0 (0) | 2 (3.77) | |
| Weak staining | 8 (4.51) | 8 (6.45) | 0 (0) | |
| No staining | 139 (78.53) | 99 (79.83) | 40 (75.47) | |
| ND* | 28 (15.81) | 17 (13.70) | 11 (20.75) | |
| VIM | ||||
| Positive staining | 100 (56.49) | 75 (60.40) | 25 (47.16) | |
| Weak staining | 1 (0.56) | 1 (0.80) | 0 (0) | |
| No staining | 3 (1.69) | 0 (0) | 3 (5.66) | |
| ND* | 73 (41.24) | 48 (38.7) | 25 (47.16) | |
| SMA | ||||
| Positive staining | 54 (30.50) | 38 (30.64) | 16 (30.18) | |
| Weak staining | 28 (15.81) | 20 (16.12) | 8 (15.09) | |
| No staining | 55 (31.07) | 41 (33.06) | 14 (26.41) | |
| ND* | 40 (22.59) | 25 (20.16) | 15 (28.30) | |
1Primary GISTs
2GISTs with metastases/unresectable/locally advanced/with tumor duality; ND* not determined; mixed** spindle-epitheloid; recurrence*** local/metastatic; mts ± metastases
Within Group 2, proliferation parameters were higher than in Group 1: higher mitotic index (≥ 5/50 HPF) 62.62% versus 33.87%, higher average mitotic index 14.22/50 HPF versus 8.75/50 HPF. Accordingly, there was also higher amount of samples with Ki-67 index above 10% (24.52% versus 12.9%). The average percentage of Ki-67 was 10.06% versus 7.39%. As expected, within Group 2 there was a relatively earlier average onset of relapse (35.8 versus 40.3 months) and higher percentage in the very high risk category according to Miettinen and Lasota’s risk assessment from 2006 (62.26% versus 39.52%) than in Group 1.
SNP detection by direct sequencing
Sequencing analyses were performed on genomic DNA samples (primary, locally advanced, metastatic, unresectable, tumor duality). The combination of primers flanking the KIT exon 10 amplified 107 bp long product. Importantly, 29 samples showed KIT SNPM541L polymorphism (Fig. 1a) while its incidence was statistically higher in Group 2 with metastases at diagnosis (or unresectable, locally advanced, with tumor duality) than in non-metastatic Group 1 (Table 2). Twelve samples out of 124 (9.67%) in Group 1 and 17 out of 53 (32.07%) in Group 2 (n = 17/53, 32.07% versus 12/124, 9.67%; Pearson’s Chi-squared test with Yates’continuity correction, p = 0.0005). This trend of statistically higher rate of KIT SNPM541L in Group 2 was also confirmed by the analysis of KIT SNPM541L prevalence in the non-relaptic and relaptic part of both groups. Pooling 29 relapsing patients from Group 1 with 17 relapsing patients from the Group 2 we got a group of 46 patients, where KIT SNPM541L significantly correlated with a higher rate of local or metastatic relapse in those from Group 2 (6/17, 35.29% versus 2/29, 6.89%; Fischer’s exact test, p = 0.038; two-sample t test, p = 0.04 and in non-relaptic part: n = 11/36, 30.55% versus 10/95, 10.52%; Fisher’s exact test, p = 0.0081).
Fig. 1.
a Representative chromatograms of KIT exon 10 sequence in GIST patients. The anlyses show the presence of the genomic variant c.1621 A > C (evidenced by arrows). b Representative chromatograms of KIT exon 10 sequence in GIST patients. The anlyses show the presence of the genomic variant c.1638 A > G (evidenced by arrows)
Table 2.
Molecular analyses of GISTs cohort and divided according to the analyzed series
| Mutation status | All samples | Group 11 | Group 22 |
|---|---|---|---|
| n (%) | n (%) | n (%) | |
| KIT gene | |||
| Exon 9 | 13 (7.34) | 11 (8.87) | 2 (3.77) |
| Exon 11 | 118 (66.66) | 81 (65.32) | 37 (69.81) |
| Exon 13 | 4 (2.25) | 3 (2.41) | 1 (1.88) |
| Exon 17 | 3 (1.69) | 3 (2.41) | 0 (0) |
| PDGFRa gene | |||
| Exon 12 | 6 (3.38) | 5 (4.03) | 1 (1.88) |
| Exon 14 | 2 (1.12) | 1 (0.80) | 1 (1.88) |
| Exon 18 | 20 (11.29) | 14 (11.29) | 6 (11.32) |
| D842V** | 13 (7.34) | 10 (8.06) | 3 (5.66) |
| D842V*** | (65.00) | (71.42) | (50.00) |
| KIT/PDGFRa negative* | |||
| WT GISTs | 17 (9.60) | 11 (8.87) | 6 (11.32) |
| KIT SNPM541L | |||
| Exon 10 | 29 (16.38) | 12 (9.67) | 17 (32.07) |
| KIT SNPK546K | |||
| Exon 10 | 24 (13.55) | 14 (11.29) | 10 (18.86) |
1Primary GISTs
2GISTs with metastases/unresectable/locally advanced/with tumor duality; KIT/PDGFRa negative* samples without any mutation in KIT/PDGFRa gene; D842V** mutation predicting resistance to tyrosine kinase receptor inhibitors is included in total number of mutations in exon 18, the percentage is calculated from the total number of samples; D842V*** is the proportion of total exon 18 mutations
Clinicopathological and molecular KIT SNPM541L-positive GIST data are summarized in Table 3. Average age of patients was 62.81 years. Thirteen patients were female and sixteen male with a mean age of 61.83 and 63.8 years, respectively. Average tumor size was 6.13 cm. Twenty-three tumors were of spindle-cell, two of epitheloid and three of mixed morphology. They were localized mostly in the small intestine (13 cases), stomach (nine cases), colon (three cases), and rectum (one case); three tumors were extragastrointestinal. In 15 cases, the tumors showed low mitotic activity (5 mitoses/50 HPF) and 12 tumors showed higher mitotic activity. Twenty-one samples belonged to the primary GISTs and eight cases were local or metastatic relapse with a majority of KIT exon 11 mutations (n = 17; 58.62%). Other immunohistochemical data are shown in Table 3.
Table 3.
Clinicopathological and molecular features of KIT SNPM541L-positive GIST cohort and divided according to analyzed series
| Clinicopathological data of KIT SNPM541L-positive GISTs | All samples | Group 11 | Group 22 |
|---|---|---|---|
| Gender (F/M)/mean age | Gender (F/M)/age | Gender (F/M)/age | Gender (F/M)/age |
| Female/mean age/range | 13/61.83/51–71 | 4/58.75/52–64 | 9/63.37/51–71 |
| Male/mean age/range | 16/63.80/49–84 | 8/63.25/49–84 | 8/64.42/56–75 |
| ND* | 0/2 | 0/0 | 0/2 |
| n (%) | n (%) | n (%) | |
|---|---|---|---|
| Tumor size (cm) | |||
| ≤ 2 | 3 (10.34) | 2 (16.66) | 1(5.88) |
| 2–5 | 9 (31.03) | 5 (41.66) | 4 (23.52) |
| 5–10 | 9 (31.03) | 3 (25.00) | 6(35.29) |
| > 10 | 3 (10.34) | 1 (8.33) | 2 (11.76) |
| ND* | 5 (17.24) | 1 (8.33) | 4(23.52) |
| Morphologic features | |||
| Spindle | 23 (79.31) | 9 (75.00) | 14 (82.35) |
| Epithelioid | 2 (6.89) | 1 (8.33) | 1 (5.88) |
| Mixed** | 3 (10.34) | 1 (8.33) | 2 (11.76) |
| ND* | 1 (3.44) | 1 (8.33) | 0(0) |
| Location | |||
| Stomach | 9 (31.03) | 5 (41.66) | 4 (23.52) |
| Small intestine | 13 (44.82) | 6 (50.00) | 7 (41.17) |
| Colon | 3 (10.34) | 1 (8.33) | 2 (11.76) |
| e-GIST | 3 (10.34) | 0 (0) | 3 (17.64) |
| Rectum | 1 (3.44) | 0 (0) | 1 (5.88) |
| ND* | 0 (0) | 0 (0) | 0 (0) |
| Mitoses/50 HPF | |||
| ≤ 5 | 15 (51.72) | 8 (66.66) | 7 (41.17) |
| ≥ 5 | 12 (41.37) | 3 (25.00) | 9 (52.94) |
| ND* | 2 (6.89) | 1 (8.33) | 1 (5.88) |
| Ki-67 index | |||
| ≤ 5 | 16 (55.17) | 7 (58.33) | 9 (52.94) |
| > 5- ≤ 10 | 7 (24.13) | 3 (25.00) | 4 (23.52) |
| > 10 | 3 (10.34) | 1 (8.33) | 2 (11.76) |
| ND* | 3 (10.34) | 1 (8.33) | 2 (11.76) |
| Event | |||
| Primary | 21 (72.41) | 10 (83.33) | 11 (64.70) |
| Recurrence*** | 8 (27.58) | 2 (16.66) | 6 (35.29) |
| Relapse+ | 30 | 48 | 24 |
| ND* | 0 (0) | 0 (0) | 0 (0) |
| CD117 | |||
| Positive staining | 27 (93.10) | 11 (91.66) | 16 (94.11) |
| No staining | 2 (6.89) | 1 (8.33) | 1 (5.88) |
| ND* | 0 (0) | 0 (0) | 0 (0) |
| CD34 | |||
| Positive staining | 18 (62.06) | 7 (58.33) | 11(64.70) |
| No staining | 1 (37.93) | 5 (41.66) | 6 (35.29) |
| ND* | 0 (0) | 0 (0) | 0 (0) |
| DOG-1 | |||
| Positive staining | 20 (68.96) | 10 (83.33) | 10 (58.82) |
| No staining | 2 (6.89) | 0 (0) | 2 (11.76) |
| ND* | 7 (24.13) | 2 (16.66) | 5 (29.41) |
| S-100 | |||
| Positive staining | 1 (3.44) | 0 (0) | 1 (5.88) |
| No staining | 24 (82.75) | 11 (91.66) | 13 (76.47) |
| ND* | 4 (13.79) | 1 (8.33) | 3 (17.64) |
| DEZ | |||
| Positive staining | 1 (3.44) | 1 (8.33) | 0 (0) |
| No staining | 22 (75.86) | 9 (75.00) | 13 (76.47) |
| ND* | 6 (20.68) | 2 (16.66) | 4 (23.52) |
| VIM | |||
| Positive staining | 12 (41.37) | 5 (41.66) | 7 (41.17) |
| No staining | 3 (10.34) | 0 (0) | 3 (17.64) |
| ND* | 14 (48.27) | 7 (58.33) | 7 (41.17) |
| SMA | |||
| Positive staining | 10 (34.48) | 5 (41.66) | 5 (29.41) |
| No staining | 10 (34.48) | 4 (33.33) | 6 (35.29) |
| ND* | 9 (31.03) | 3 (25.00) | 6 (35.29) |
| Mutation status | |||
| KIT mutations | 17 (58.62) | 8 (66.66) | 9 (52.94) |
| PDGFRa mutations | 6 (20.69) | 2 (16.67) | 4 (23.53) |
| KIT/PDGFRa negative+++ | 6 (20.69) | 2 (16.67) | 4 (23.53) |
1Primary GISTs
2GISTs with metastases/unresectable/locally advanced/ with tumor duality; ND* not determined; mixed** spindle-epitheloid; recurrence*** local/metastatic; mts ± metastases; relapse+ the average time of relapse in months; KIT/PDGFRa negative+++samples without any mutation in KIT/PDGFRa gene
The results from molecular analyses, the KIT/PDGFRa mutations and SNP polymorphisms, are summarized in Table 2. In addition, we identified c.1638 A > G (K456K) polymorphism in exon 10 of KIT gene. But since it does not change the amino acid and it is a silent variant, we do not discuss it further (Fig. 1b).
Impact of KIT SNPM541L polymorphism on GISTs
The distribution of clinicopathological data for all patients between KIT SNPM541L-positive and KIT SNPM541L-negative samples is shown in Table 4. Although, there were no statistically significant differences between KIT SNPM541L-positive and KIT SNPM541L-negative samples, in the characteristics, size, morphology, localization, mitotic index, Ki-67 index and event, within the KIT SNPM541L-positive group, there was a trend of higher values associated with higher risk (Fletcher et al. 2002; Miettinen and Lasota 2006) and a higher percentage of tumors above 5 cm in size was observed (n = 12/29; 41.38/ versus n = 55/148; 37.16%). A higher percentage of KIT SNPM541L-positive GISTs were localized in small intestine (n = 13/29; 44.83% versus n = 48/148; 32.43%), colon (n = 3/29; 10.34% versus n = 2/148; 1.35%), and rectum (n = 1/29; 3.44% versus n = 1/148; 0.67%). The statistical difference in the incidence of the KIT SNPM541L women in Group 2 is an exception.
Table 4.
The comparison of clinicopathological and molecular features of two analyzed GIST series according to the presence of KIT SNPM541L polymorphism
| Clinicopathological data | KIT SNPM541L-positive GISTs | KIT SNPM541L-negative GISTs |
|---|---|---|
| Gender (F/M)/mean age | Gender (F/M)/age | Gender (F/M)/age |
| Female/mean age/range | 13/ 61.83/51–71 | 72/ 64.56/17–89 |
| Male/mean age/range | 16/63.80/49–84 | 76/62.91/32–89 |
| ND* | 0/2 | 0/0 |
| n (%) | n (%) | |
|---|---|---|
| Tumor size (cm) | ||
| ≤ 2 | 3 (10.34) | 20 (13.51) |
| 2–5 | 9 (31.03) | 52 (35.13) |
| > 5 | 12 (41.38) | 55 (37.16) |
| ND* | 5 (17.24) | 21 (14.18) |
| Morphologic features | ||
| Spindle | 23 (71.31) | 93 (62.83) |
| Epithelioid | 2 (6.89) | 25 (16.89) |
| Mixed** | 3 (10.34) | 28 (18.91) |
| ND* | 1 (3.44) | 2 (1.35) |
| Location | ||
| Stomach | 9 (31.03) | 73 (49.32) |
| Small intestine | 13 (44.82) | 48 (32.43) |
| Colon | 3 (10.34) | 2 (1.35) |
| e-GIST | 3 (10.34) | 22 (14.86) |
| Rectum | 1 (3.44) | 1 (0.67) |
| ND* | 0 (0) | 2 (1.35) |
| Mitoses/50 HPF | ||
| ≤ 5 | 15 (51.72) | 74 (50.00) |
| ≥ 5 | 12 (41.37) | 63 (42.56) |
| ND* | 2 (6.89) | 11 (7.43) |
| Ki-67 index | ||
| ≤ 5 | 16 (55.17) | 73 (49.32) |
| > 5– ≤ 10 | 7 (24.13) | 35 (23.64) |
| > 10 | 3 (10.34) | 26 (17.56) |
| ND* | 3 (10.34) | 14 (9.45) |
| Event | ||
| Primary | 21 (72.41) | 110 (74.32) |
| Recurrence*** | 8 (27.58) | 38 (25.67) |
| Relapse+ | 30 | 42,7 |
| ND* | 0 (0) | 0 (0) |
| Mutation status | ||
| KIT mutations | 17 (58.62) | 117 (79.05) |
| PDGFRa mutations | 6 (20.69) | 19 (12.84) |
| KIT/PDGFRa negative+++ | 6 (20.69) | 12 (8.11) |
| Type of series of GIST cohort | ||
| Primary GISTs (localized) | 12 (41.37) | 112 (75.67) |
| MTS/unresectable/locally advanced/tumor duality | 17 (58.62) | 36 (24.32) |
ND* not determined; mixed** spindle-epitheloid; recurrence*** local/metastatic; mts ± metastases; relapse+ the average time of relapse in months; KIT/PDGFRa negative+++ samples without any mutation in KIT/PDGFRa gene
In the assessment of relapse, depending on the presence of KIT SNPM541L, neither in both categories it was statistically different (Wilcoxon rank test, Group 1: p = 0.57 versus Group 2: p = 0.334), altough the trend of early onset of relapse in KIT SNPM541L-positive patients was observable (30 months in KIT SNPM541L-positive GISTs versus 42.7 months in KIT SNPM541L-negative GISTs) (Table 4).
Each sample was also assigned a Fletcher risk group from 2002 (Fletcher et al. 2002) and Miettinen and Lasota’s risk category from 2006 (Miettinen and Lasota 2006) as part of clinical and immunohistochemical diagnostics. Category 0/1 represented no to low risk, 2 moderate risk and 3 high risk of malignant behavior. We also did not find a positive correlation of the KIT SNPM541L with the high risk group 3 as originally expected, but it is interesting that the rising trend of higher KITSNPM541L prevalence is visible in group 2 (moderate risk), even according to both assessment criteria (Fig. 2a, b). The percentage of KIT SNPM541L of the risk groups in both evaluation criteria was as follows: 0/1–11.46%, 2–25%, 3–17.05% according to Fletcher et al.; 0/1–12.33%, 2–46.67%, 3–18.84% according to Miettinen and Lasota.
Fig. 2.
a Risk categories according to Fletcher et al. (2002); 0–1: low or no risk, 2: medium risk, 3: high risk; SNP negative: samples without KIT SNPM541L polymorphism; SNP positive: samples bearing KIT SNPM541L polymorphism. b Risk categories according to Miettinen and Lasota (2006); 0–1: low or no risk, 2: medium risk, 3: high risk; SNP negative: samples without KIT SNPM541L polymorphism; SNP positive: samples bearing KIT SNPM541L polymorphism
We divided the samples into two subgroups: (1) KIT positive and (2) WT and PDGFRa-mutated GISTs. KIT SNPM541L was significantly associated with the subgroup 2 (n = 17/134; 12.67% versus 12/43; 27.91%; Pearson’s Chi-squared test with Yates’ continuity correction, p = 0.0349). The average time of relapse was significantly shorter in KIT SNPM541L-positive patients in subgroup 2 (24 versus 37.88 months; Welch two-sample t test, p = 0.02762) (Fig. 3).
Fig. 3.
The average time to relaps in months in WT and PDGFRa subgroup of GISTs according to the presence of KIT SNPM541L polymorphism
Discussion
The need for the detection of genetic variants as tumor genetic microenvironment, and their impact on the patient is becoming increasingly important. Diagnosis of any disease is incomplete, unless we can predict whether the patient will relapse and which treatment is suitable.
Although, there are only a few SNP GIST studies, the associations between GISTs and other SNPs that play a role in clinical patients outcome, clinical features, tumorigenesis, and relapses have been observed (Brahmi et al. 2015; Minarik et al. 2013; Angelini et al. 2015; O'Brien et al. 2013). For example, the SNP in CYP1B1 gene strongly associates with the deletion of 557–558 codons in KIT exon 11. Other, potentially hazardous, SNPs have been found in the ERCC2, GSTM1, RAD23B genes (O'Brien et al. 2013).
In our study, we found the presence of KIT SNPM541L in 29 out of 177 samples, with 12 samples being in Group 1 of localized GISTs and 17 in the Group 2 of metastatic GISTs (or locally advanced, with tumor duality, unresectable). We have not been able to demonstrate the nature (somatic, germinal) of these substitutions, as neither blood nor healthy patient tissues were available. KIT SNPM541L was originally identified only in neoplastic cells (Krasagakis et al. 2011; Iurlo et al. 2014). This variant is thought to be associated with an increased risk of some cancer, e.g. aggressive fibromatosis (Dufresne et al. 2014). It has also been identified in other types of tumors (chronic myelogenous leukemia, chronic eosinophilic leukemia, GISTs, ring cell colorectal cancer). Thus, it does not seem to be tumor specific or GIST predictive and should be classified not as a mutation but as a single nucleotide polymorphism. This is also supported by the fact that larger studies have shown that the frequency of KIT SNPM541L in some tumors does not differ from that in the general population (Brahmi et al. 2015; Dufresne et al. 2014; Inokuchi et al. 2002; Rocha et al. 2010; Krüger et al. 2006). According to Brahmi et al. (2015), there is a prevalence of KIT SNPM541L in the population of about 20%, which is not distinctly different from the prevalence of KIT SNPM541L in GISTs (Brahmi et al. 2015), which is in accordance with our GIST study (29/177; 16.4%) and occurrence in ring cell colorectal cancer (25%) (Alvi et al. 2017). However, Inokuchi was the first to observe an increased frequency of KIT SNPM541L in patients compared with healthy controls (Inokuchi et al. 2002).
The KIT receptor as the transmembrane tyrosine kinase protein, coded by a gene located on chromosome four, is composed of several domains. Its transmembrane domain is encoded by exon 10 (Li et al. 2017; Krüger et al. 2006). The KIT gene is expressed in multiple cells: interstitial cells of Cajal (ICC), mast cells, melanocytes, germ cells, and hematopoietic progenitor cells (Jasek et al. 2017; Iurlo et al. 2014). Intracellular activity of the KIT protein is activated by binding of stem-cell factor protein (SCF) to the extracellular domain of the KIT receptor, triggering a series of signaling events and downstream pathways (Ke et al. 2016; Quek and George 2010). Most KIT mutated GIST patients benefit from treatment with tyrosine kinase inhibitor-imatinib (Noujaim et al. 2016) but it should be noted that KIT mutations have antagonistic activity. They increase cell proliferation (O'Brien et al. 2013), but at the same time improve sensitivity to imatinib in some types of tumors (Li et al. 2017; O'Brien et al. 2013; Masago et al. 2015). The efficacy of targeted imatinib therapy appears to depend not only on the type of mutation but also on variations and polymorphisms (Chevrier et al. 2014). In several in vitro studies, the interaction of KIT SNPM541L with SCF (Iurlo et al. 2014) has been shown to alter receptor activity. It is suggested that in patients with hypereosinophilia without PDGFRα/b rearrangements, KIT SNPM541L is strongly associated with response to imatinib (Iurlo et al. 2014). These claims are supported by the fact that KIT SNPM541L has been reported to increase the sensitivity of the KIT receptor to SCF (Krüger et al. 2006). But they are in contradiction with the fact that in the ClinVar database, KIT SNPM541L is classified as benign/probably benign (https://www.ncbi.nlm.nih.gov/clinvar), which is in contradiction with the claim that KIT SNPM541L is pathogenic with a score of 0.74 (https://cancer.sanger.ac.uk).
Brahmi et al. (2015) noticed that KIT SNPM541L has similar activation and maturation as WT KIT, but also a transduction pathway similar to KIT-mutated signaling. It was demonstrated that cells bearing KIT SNPM541L show an expanded proliferative response and cell survival only in the case of reduced SCF concentration (Foster et al. 2008; Dufresne et al. 2014; Inokuchi et al. 2002). This may alter the receptor’s ability to trigger PI3K/Akt and MAPK signaling pathways as compared to WT GISTs (Brahmi et al. 2015; Ke et al. 2016; Pedersen et al. 2009).
Inokuchi et al. presented the role of KIT SNPM541L in chronic myeloid leukemia (Inokuchi et al. 2002). In in vitro studies, transfected Ba/F3 cells showed increased activation of the KIT receptor in the presence of low doses of SCF compared to WT KIT cells (Inokuchi et al. 2002). In chronic eosinophilic leukemia, KITSNPM541L carriers showed a good response to low doses of imatinib while during treatment there was a remission of the disease in all cases (Iurlo et al. 2014). Whithin the GISTs, KIT SNPM541L was not considered to be a predictive biomarker for efficacy of Imatinib (Brahmi et al. 2015), as well as in aggressive fibromatosis, where a correlation between KIT SNPM541L and imatinib activity was not demonstrated (Dufresne et al. 2014). And although, in this study we did not have the necessary data to evaluate the efficacy of imatinib in KIT SNPM541L-positive patients, we believe that KIT SNPM541L deserves further studies as a potential therapeutic target as it can help identify a subset of cases that may benefit from low dose imatinib treatment despite contradictory studies so far.
Assessing the impact of KIT SNPM541L on GISTs revealed an increased risk of progression and dissemination of tumors. We demonstrated a statistically increased KIT SNPM541L prevalence in the higher-risk Group 2 with metastases at diagnosis (or tumor duality, unresectable, locally advanced) compared with Group 1 of localized GISTs. Pooling the nonrelaptic/relaptic (metastatic or local relapse) samples from both groups, a statistically significant proportion of KIT SNPM541L was also found in Group 2. Brahmi et al. (2015) confirmed a significant correlation of KIT SNPM541L with relapse in the first group of localized GISTs (Fisher’s exact test, p = 0.008), resulting in reduced relapse-free survival (RFS), p = 0.001. It is also important to note that the reduced RFS is associated with clinical parameters in multivariate analyses: size, p = 0.03; mitotic index, p = 0.006 (Brahmi et al. 2015). In our study, the average time to relapse was shorter by 12 months in KITSNPM541L-positive samples, although this was not statistically significant. However, by dividing the samples according to the mutated oncogene, a statistically shorter time of relapse in the PDGFRa and WT subgroup was demonstrated. And KIT SNPM541L frequency was also statistically higher in the PDGFRa and WT subgroup. Compared to the only other study about KIT SNPM541L in GISTs, we failed to demonstrate association of KIT SNPM541L with an increased risk of relapse or with lower RFS in Group 1. However, it could be concluded that this variant may be a prognostic factor of progression, dissemination and relapse of tumors.
We evaluated the prevalence and significance of KIT SNPM541L by direct sequencing of KIT exon 10, which was performed on genomic DNA obtained from formalin-fixed paraffin-embedded tissue. Inconsistency, controversy as well as ambiguous results may be due to the used method. In heterogeneous GISTs, especially in relapses, this method with a sensitivity of 10–20% (Jasek et al. 2017; Iurlo et al. 2014; Barbano et al. 2015) may not be sufficiently sensitive. Using a more sensitive method (castPCR) with a sensitivity below 1%, KIT SNPM541L alleles would be more detected in multiple-relapse samples.
In groups divided according to Fletcher and Miettinen and Lasota evaluation criteria, we had expected an increased KIT SNPM541L prevalence in the group with highest risk. Surprisingly, the prevalence of KIT SNPM541L was higher in the medium-risk group, which deserves further analysis as to whether KIT SNPM541L could represent the predictive factor for the medium risk group.
However, despite the inconsistent results and missing statistical correlations in many parameters in GISTs, in many cases we observed a higher trend of KIT SNPM541L prevalence within GIST risk parameters: size, localization in small intestine, colon, rectum (Miettinen and Lasota 2006). We believe that KIT SNPM541L certainly belongs to the new types of predictive markers. However, the decision whether to include KIT SNPM541L in diagnostic markers requires further extensive studies and investigation.
The detection of KIT SNPM541L could be useful in clinical management of GIST patients as it could help identify a subgroup of cases that may benefit from treatment with low dose imatinib.
Conlusion
In conclusion, we did not find KIT SNPM541L to be associated with the risk of relapse in localized GISTs. Given the positive correlations of prevalence and average time to relapse in the PDGFRα and WT subgroup, further studies in this subgroup would be appropriate, including monitoring imatinib’s efficacy (despite no evidence of greater efficacy of imatinib across all GIST specimens), thus giving KIT SNPM541L-positive patients the potential advantage in the treatment.
Funding
This publication is the result of the project implementation: “CENTER OF EXCELLENCE FOR RESEARCH IN PERSONALIZED THERAPY (CEVYPET)”, ITMS: 26220120053 supported by the Operational Programme Research and Innovation funded by the ERDF. This study was also supported by the project of Slovak Research and Development Agency no. APVV-16-0066 as well as by the Grant of The Ministry of Education, Science, Research and Sport of the Slovak Republic no.VEGA-1/0380/18.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Informed consent
Informed consent was obtained from all individual participants included in the study.
Footnotes
Publisher's Note
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Contributor Information
Peter Kruzliak, Email: peter.kruzliak@savba.sk.
Zora Lasabova, Email: zora.lasabova@uniba.sk.
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