The practical application of genetic testing in the clinical management of gastrointestinal mesenchymal tumors was investigated by performing genetic testing in 13 patients with immunohistochemical diagnosis of gastrointestinal mesenchymal tumors (GIST). METHODS: Fifteen genetic tests were performed on 13 patients with GIST by PCR gene amplification, and their results and clinical data were retrospectively analyzed. RESULTS: Ten mutations in exon 11 of the C-kit gene were found in the 15 genetic tests, among which the mutation types were deletion mutation in 5 cases, heterozygous deletion in 2 cases, point mutation in 2 cases and insertion mutation in 1 case, and one mutation in exon 9 of the C-kit gene, which was a duplication mutation. There were 4 negative results of genetic testing. CONCLUSION: With the progress of science and technology, gene testing is important for the diagnosis of gastrointestinal mesenchymal tumors and the selection of targeted therapy, and should strive to become a routine test for gastrointestinal mesenchymal tumors.
[Keywords] Genetic testing; gastrointestinal mesenchymal tumor
With the establishment of GIST diagnostic criteria, the incidence of gastrointestinal mesenchymal tumor in clinical practice has increased year by year, and with the increase of cases, clinical problems have also gradually increased. As early as 2007, targeted therapy for advanced and recurrent gastrointestinal mesenchymal tumors has been carried out in large numbers in China, and postoperative targeted adjuvant therapy for gastrointestinal mesenchymal tumors is now also widely carried out in major hospitals. the technology of genetic testing for GIST has become increasingly mature, and the price of its testing has also decreased compared with previous years. In this context, we retrospectively analyzed 13 cases of gastrointestinal mesenchymal tumors managed in our department, and discussed their practical application in clinical practice, in order to play a role in the standardized treatment of gastrointestinal mesenchymal tumors.
1. Data and methods
1.1 General information
All the cases in the group were clinical immunohistochemistry patients who were clearly considered to have gastrointestinal mesenchymal tumor (GIST) and were intended to be treated with targeted drugs. Among them, there were 7 males and 6 females, aged 36-86 years, with a mean age of 57.8. There were 8 cases of gastric mesenchymal tumor, 2 cases of small intestinal mesenchymal tumor (including one case of duodenal mesenchymal tumor), 1 case of mesenchymal tumor of the esophagogastric junction, 1 case of mesenchymal tumor of the transverse colon and 1 case of mesenchymal tumor of the rectum. 6 of the 13 patients with GIST were treated with postoperative adjuvant therapy with imatinib (Gleevec), and 6 cases were surviving with tumor. Among them, 4 were treated with Gleevec-targeted therapy and 2 with sunitinib (Sotan). One case abandoned post-operative adjuvant therapy due to economic reasons.
1.2 Genetic testing methods
In this group of cases, due to the limited clinical conditions, the specimens were sent for examination by paraffin-embedded white slices, and the detection method was PCR gene amplification. The detection period was from 2009 to 2012. Nine of the cases were tested at Guangzhou Jinwei Medical Laboratory Center, two cases at the Affiliated Hospital of Fudan University, two cases at the Pathology Center of Zhejiang Second Hospital, one case at Zhejiang First Hospital, and one case at the Pathology Center of Peking University.
2.Results
A total of 15 genetic tests were performed in 13 patients with gastrointestinal mesenchymal tumors. There were 2 cases with 2 tests. In one case, the primary tumor gene test of esophagogastric combined mesenchymal tumor suggested a deletion mutation in exon 11 of the C-kit gene, and because of the early onset in 2007, no adjuvant targeted therapy was performed after surgery. The other case was a small intestinal mesenchymal tumor, and the genetic test in the pathology department of Zhejiang Second Hospital suggested wild-type changes, while the test in the pathology department of Peking University suggested exon 11 point mutation of C-kit gene. In summary, a total of 10 mutations in exon 11 of the C-kit gene were found in 15 genetic tests, of which the mutation types were deletion mutations in 5 cases, heterozygous deletions in 2 cases, point mutations in 2 cases, insertion mutations in 1 case, and duplication mutations in exon 9 of the C-kit gene in 1 case.
There were 4 negative genetic tests. The results of the genetic tests performed in this group suggest that mutations in the C-kit gene are common in patients with GIST, and that exon 11 mutations are predominant, which is consistent with previous literature. However, due to the small number of cases in this group, no platelet-derived growth factor receptor (PDGFR-α) gene mutations were detected. The genetic test in this group showed wild-type manifestation as many as 4 times, which is not consistent with the literature, considering that the specimens in this group were sent by paraffin-embedded white slices, and the specimens were prone to false negatives.
3. Discussion
3.1 Molecular biological features and pathogenesis of gastrointestinal mesenchymal tumors
To understand the clinical application of genetic testing in gastrointestinal mesenchymal tumors, it is important to understand the molecular biological features and mechanisms of their pathogenesis. The pathogenesis of gastrointestinal mesenchymal tumors at the genetic level is relatively well studied compared to most multigene mutated tumors. Gastrointestinal mesenchymal tumors are mainly characterized by mutations in the C-kit gene and PDGFR-α gene. Both genes encode C-kit protein and platelet-derived growth factor receptor (PDGFR-α), which belong to the type III receptor tyrosine kinase family. The abnormal proteins generated by mutations have the ability to generate dimers without relying on ligands, leading to tyrosine kinase autophosphorylation, activation of downstream signaling pathways such as mitogen-activated protein kinases and signal transduction and activation proteins, and sustained activation of tyrosine kinases, which affects the proliferation-apoptosis process under normal circumstances, leading to uncontrolled cell proliferation and tumor formation. Genetically, about 80-85% of GISTs have mutations in the kit gene and 5-10% have mutations in the PDGFR-α gene, with mutations in both showing mutually exclusive characteristics. Another approximately 10% of GIST patients are wild-type, with no detectable mutations.
The most common type of mutation is a deletion/deletion-insertion mutation, accounting for 66% of the cases, resulting in the loss of one or several amino acids and occasionally the insertion of one or two amino acids, concentrated in the 5, end of exon 11, involving the 1669-1704 (Lys550-Glu561) region of the codon. The deletion of the shear acceptor site in exon 11 generates a new in-exon pre-mRNA 3, shear acceptor site, resulting in an in-frame Lys550-Lys558 deletion at the protein level. the second common type of mutation in exon 11 is a missense mutation, accounting for about 21.5% of the mutations, also concentrated in the 5, end of exon 11, mainly involving codons 557, 559 and 560 codons.
C-kit mutations are about 5%-15% of the mutations in exon 9, which mainly occur in small intestinal mesenchymal tumors and have a high biological aggressiveness, which is one of the reasons why small intestinal mesenchymal tumors are more malignant and have a poorer prognosis than gastric mesenchymal tumors. Essentially all mutations in exon 9 are six-nucleotide repeats encoding Ala502-Tyr503dup (1525-1530dupGCCTAT). Additional partial C-kit gene mutations, occurring in exon 13, exon 14, and exon 17, the latter of which is thought to be associated with resistance to Gleevec. The rate of primary mutations in these latter three exons is low, less than 2%.
In patients with GIST without mutations in the C-kit gene, PDGFR-α mutations are detected in approximately one-third of patients, mainly in exons 12, 14 and 18. PDGFR-α mutations occur mainly in the stomach and the microscopic morphology of mesenchymal tumors often shows a predominantly epithelial pattern, with mutation types including point mutations, deletions, deletions-insertions and insertions, with the most common 1821C>A In addition, C-kit gene and PDGFR-α gene mutations are not detectable in about 10% of GIST patients and are referred to as wild-type GIST; interestingly, most of these mesenchymal tumor patients may still have detectable C-kit protein (CD117 positive), and the pathogenesis of wild-type patients at the genetic level is not fully The pathogenesis of wild-type patients at the genetic level is not fully understood.
3.2 Genetic testing and targeted therapy
Gene testing plays an important role in the targeted therapy of gastrointestinal mesenchymal tumors. First, genetic testing has an irreplaceable role in the diagnosis of mesenchymal tumors. Some clinicians may question this, believing that the combination of immunohistochemistry and microscopic morphological judgment leaves no doubt about the diagnosis of GIST. Indeed, most of the GIST tumor cells are predominantly spindle-shaped (50%-70%) or epithelial-like cells (20%-40%) and mixed type (10%) under microscopy, and immunohistochemistry is positive for CD117 and CD34, so that the diagnosis is clear. But clinically, we can also encounter the aforementioned patients with negative CD117 expression, at this time, the genetic testing has irreplaceable significance in the diagnosis.
In my department, there was a case of postoperative pathological diagnosis of gastrointestinal mesenchymal tumor with smooth muscle differentiation or smooth muscle sarcoma, but immunohistochemistry in two hospitals was negative for CD117, so if the diagnosis of mesenchymal tumor could not be made clearly or mesenchymal tumor could not be ruled out clearly, it was difficult to decide whether to perform postoperative adjuvant targeted therapy for this patient. The genetic test performed on paraffin sections of the specimen indicated that there were no mutations in the C-kit gene and PDGFR-α gene, and combined with the genetic test results, the patient was first considered to be a smooth muscle sarcoma. The clinical management was to abandon postoperative adjuvant therapy with Gleevec, and the patient has been followed up for more than 1 year with no recurrence of metastasis.
In this group, there is a patient with mesenchymal tumor of the esophagogastric junction, who developed local recurrence 2 years after resection of the primary lesion. The morphology of the recurrent lesion was considered to be postoperative recurrence of gastrointestinal mesenchymal tumor, but immunohistochemistry indicated that CD117 and CD34 were negative, and then genetic testing indicated exon 11 mutation of C-kit gene, which clarified the diagnosis. The treatment was switched to Sotan for one year after surgery, and no recurrence of metastasis has been indicated at the follow-up for more than 3 years. In summary, genetic testing is of diagnostic importance for patients with clinically suspected mesenchymal tumors that cannot be confirmed by immunohistochemistry. For this group of patients, clinical consideration of targeted therapy or not must rely on genetic testing to make a judgment.
With the widespread development of targeted therapy for gastrointestinal mesenchymal tumors, there is now a general consensus on the relationship between genetic mutations and targeted therapy in GIST patients, and the relationship between the type of genetic mutations in GIST patients and the most commonly used first-line targeted therapy drug, Gleevec, is the aspect that has been most intensively studied. It is now generally accepted that for the common C-kit gene exon 11 mutation, targeted therapy with Gleevec has the greatest sensitivity in patients with such mutations. It has been reported to achieve 6% CR, 61% PR, 25% SD, and 3% PD [1]. In patients with exon 9 mutations in the C-kit gene, good sensitivity was obtained due to the observation that second-line drug sunitinib treatment was administered after the development of gleevec resistance, and significantly greater results were achieved with sunitinib treatment compared to the group with increased gleevec dose [2].
Some experts now believe that early conversion to sunitinib therapy can be considered for patients with genetic test results for exon 9 mutations in the C-kit gene who develop drug resistance. Even more radical view is that once the C-kit gene exon 9 mutation is detected, first-line targeted therapy with sunitinib can be considered directly. There is a patient with exon 9 mutation in our group, who developed local recurrence and liver metastasis 8 years after gastric mesenchymal tumor surgery. For wild-type GIST patients, the effect of targeted therapy is relatively not as good as that of those with clear genetic mutations.
In this group of patients with genetic testing, there was a case of transverse mesenteric mesenchymal tumor, which was clinically judged to be moderate to high risk and needed postoperative adjuvant target therapy, but its genetic test was wild type, although wild type patients with tumor survival also had 23% PR and 50% SD with targeted therapy, but also had 19% PD. We decided not to use post-operative adjuvant targeted therapy for the time being and followed up the patient closely for 9 months. For patients with PDGFR-α mutation, the relationship with Gleevec targeted therapy is not completely clear due to its low prevalence, but it is clear that exon 12 and 14 mutations of PDGFR-α gene are sensitive to Gleevec therapy in in vitro test, but exon 18 mutation, especially D842V mutation form is resistant to Gleevec therapy [3].
3.3 Genetic testing and prognosis of patients with gastrointestinal mesenchymal tumors
Genetic testing is closely related to the prognosis of gastrointestinal mesenchymal tumors. First, as mentioned earlier, patients with different types of mesenchymal stromal tumor mutations have different sensitivities to Gleevec-targeted therapy, and currently, the only first-line drug available for the standardized treatment of gastrointestinal mesenchymal tumors is Gleevec; therefore, how sensitive to Gleevec-targeted therapy is directly related to patient prognosis, especially for those patients with local recurrence, unresolved tumors, or liver metastases. It is now believed that the prognosis of patients with gastrointestinal mesenchymal tumors correlates with the site of tumorigenesis, or that the malignancy of gastrointestinal mesenchymal tumors is not only related to the size of the tumor and the number of nuclear divisions under high magnification, but also closely related to the site of tumorigenesis. In general, the prognosis of mesenchymal tumors occurring in the stomach is better than that in the small intestine.
At the genetic level, gastric mesenchymal tumors have more C-kit gene exon 11 mutations, while small intestinal mesenchymal tumors have relatively more C-kit gene exon 9 mutations. As mentioned earlier, the former are more sensitive to targeted therapy with Gleevec. In addition, the type of mutation correlates with patient prognosis. It has been shown that the occurrence of mutations in the C-kit gene is an independent prognostic indicator, with those with mutations having a higher postoperative recurrence rate and a lower 5-year survival rate than those without mutations [4]. In general, the prognosis of those with deletion mutations in the C-kit gene is worse. It has been noted that gastric mesenchymal tumors with deletion mutations in exon 11 of the C-kit gene are more aggressive than those with replacement mutations, but there is no significant difference between those with deletion mutations in exon 11 of the C-kit gene and those with point mutations in small intestinal mesenchymal tumors. Due to the small number of cases in this group, a subgroup comparison analysis of the relationship between mutation type and prognosis was not performed. The direct correlation between mutations in exon 9 of the C-kit gene and clinical prognosis is not clear at present. mutations in PDGFR-α gene are mostly seen in gastric mesenchymal tumors and suggest a better prognosis overall.
Overall, genetic testing of patients with gastrointestinal mesenchymal tumors is important both from the perspective of further clarifying the diagnosis, determining prognosis, further understanding sensitivity to targeted therapies, or judging the timing of switching to sotan treatment in the event of Gleevec resistance. Currently, with the continuous progress of clinical testing technology, the price of genetic testing for patients with gastrointestinal mesenchymal tumors has decreased significantly compared to the previous one. With the current price of targeted therapies still very expensive, genetic testing is an important guideline. It is recommended that genetic testing be routinely performed if the patient’s financial condition permits. Genetic testing is especially recommended for patients who are to undergo targeted therapy or for patients with mesenchymal tumors that are clinically relapsed or surviving with tumors, and is particularly important for our clinical management and treatment. However, there are still problems in genetic testing such as inconsistent standards, inconsistent results when tested by multiple units, and specimens are still sent mainly in paraffin sections, which are prone to false negatives, etc. It is expected that with further development of genetic testing technology, improvements will be made.