Application of HRM Analysis in Detection of PDGFRA Exon 10 Polymorphism in CML Patients with Imatinib Resistance

@#Introduction: Imatinib mesylate has been widely used as a standard treatment for chronic myeloid leukemia (CML). It acts as a selective competitive inhibitor of the BCR-ABL tyrosine kinase. Despite the excellent efficacy on CML treatment, some patients developed resistance to the treatment. Mutation in the PDGFRA may be one of the factors involved in the mechanism of resistance that affects the response to imatinib. The mutational status of PDGFRA is highly relevant for prognosis and treatment prediction in CML patients. Thus, this study is intended to establish and validate a High Resolution Melting (HRM) analysis for PDGFRA exon 10 c.1432 T>C polymorphism in CML patients. Methods: High resolution melting (HRM) analysis was used to identify the c.1432 T > C polymorphism in PDGFRA exon 10 (n =86; response = 43; resistance = 43). The results from HRM analysis were compared and validated with Sanger sequencing. The association between the polymorphism and treatment response was assessed by statistical analysis using binomial logistic regression analysis. Results: HRM analyses showed two different melt curves. One curve followed the shape of the reference, homozygous wild type (TT) and the other curve showed a different melting profile than the reference with the TC genotype (heterozygous variant). The results revealed that heterozygous variant (TC) genotype showed a high risk of acquiring resistance with an OR of 3.795; 95% CI: 1.502-9.591, with a statistically significant association, p = 0.005. HRM analysis also showed 100% sensitivity and specificity in the detection of PDGFRA exon 10. Conclusion: The HRM analysis of PDGFRA exon 10 c.1432 T>C was successfully established. The exon 10 c.1432 T>C polymorphism shows a higher risk for the development of resistance toward imatinib treatment.

Clinicopathological features and prognosis of gastrointestinal stromal tumors with KIT/PDGFRA gene "homozygous mutation": a multicenter retrospective cohort study / 中华胃肠外科杂志

Objective: To investigate the clinicopathological features of gastrointestinal stromal tumor (GIST) with KIT/PDGFRA "homozygous mutation", the efficacy of targeted therapy and the prognosis. Methods: A retrospective cohort study and propensity score matching were used. "Homozygous mutation" was defined as the detection of KIT/PDGFRA gene status of GIST by Sanger sequencing, which showed that there was only mutant gene sequence in the sequencing map, lack of wild-type sequence or the peak height of mutant gene sequence was much higher than that of wild-type gene sequence (> 3 times). "Heterozygous mutation" was defined as the mutant gene sequences coexisted with wild type gene sequences, and the peak height was similar (3 times or less). The clinicopathological data and follow-up information of 92 GIST patients with KIT/PDGFRA "homozygous mutation" were collected from 4 hospitals in Shanghai from January 2008 to May 2021 (Renji Hospital, Shanghai Jiaotong University School of Medicine: 70 cases; Zhongshan Hospital, Fudan University: 14 cases; Changhai Hospital, Naval Military Medical University: 6 cases and Ruijin Hospital, Shanghai Jiaotong University School of Medicine: 2 cases). Patients with perioperative death, other malignancies, and incomplete clinicopathological information were excluded. The clinicopathological features of the patients and the efficacy of targeted drug therapy were observed and analyzed. The efficacy was evaluated using Choi criteria, which were divided into complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). In addition, a total of 230 patients with high-risk GIST with "heterozygous mutation" in exon 11 of KIT gene and 117 patients with recurrent or metastatic GIST with "heterozygous mutation" in exon 11 of KIT gene were included. The propensity score matching method was used to match GIST patients with "heterozygous" and "homozygous" mutations in exon 11 of KIT gene (1∶1) for survival analysis. The disease-free survival (DFS) between two groups of high-risk GIST patients who underwent complete surgical resection were compared. And progression-free survival (PFS) in patients with recurrent or metastatic GIST were compared. Results: Of the 92 GIST cases with KIT/PDGFRA "homozygous mutation", 58 were males and 34 were females, with a median onset age of 62 (31-91) years. Primary GIST 83 cases. Primary high-risk GIST (53 cases), metastatic GIST (21 cases) and recurrent GIST (9 cases) accounted for 90.2% (83/92). There were 90 cases of KIT gene"homozygous mutation" (exon 11 for 88 cases, exon 13 for 1 case, exon 17 for 1 case), and 2 cases of PDGFRA gene "homozygous mutation" (exon 12 for 1 case, exon 18 for 1 case). The median follow-up time was 49 (8-181) months. Among the 61 cases of primary localized GIST undergoing complete surgical resection, 2 cases were intermediate-risk GIST, 5 cases were low-risk GIST, and 1 case was very low-risk GIST, of whom 1 case of intermediate-risk GIST received 1-year adjuvant imatinib mesylate (IM) therapy after operation, and no tumor recurrence developed during the follow-up period. The remaining 53 cases were high-risk GIST, and follow-up data were obtained from 50 cases, of whom 22 developed tumor recurrence during follow-up. Of 9 patients directly receiving neoadjuvant targeted therapy (IM or avapritinib), 5 had complete imaging follow-up data, and the evaluation of efficacy achieved PR. Of all the 92 GIST cases with KIT/PDGFRA "homozygous mutation", 50 (54.4%) had tumor metastasis or tumor recurrence or progression during follow-up, and 12 (13.0%) died of the tumor. Survival analysis combined with propensity score showed that in 100 cases of high-risk GISTs with complete resection, GISTs with "homozygous mutation" in exon 11 of KIT gene had shorter disease-free survival (DFS) than GISTs with "heterozygous mutation" in exon 11 of KIT gene (median DFS: 72 months vs. 148 months, P=0.015). In 60 cases of recurrent or metastatic GISTs with KIT gene exon 11 mutation, IM was used as the first-line treatment, and the progression-free survival (PFS) of GISTs with "homozygous mutation" was shorter compared to GISTs with "heterozygous mutation" (median PFS: 38 months vs. 69 months, P=0.044). The differences were statistically significant. Conclusions: "Homozygous mutation" in KIT/PDGFRA gene is associated with the progression of GIST. The corresponding targeted therapeutic drugs are still effective for GIST with KIT/PDGFRA gene "homozygous mutation". Compared with GIST patients with "heterozygous mutation" in KIT exon 11, GIST patients with "homozygous mutation" in KIT exon 11 are more likely to relapse after surgery and to develop resistance to IM. Therefore, it is still necessary to seek more effective treatment methods for this subset of cases.

Loeffler endocarditis in chronic eosinophilic leukemia with FIP1L1/PDGFRA rearrangement: full recovery with low dose imatinib

No abstract available.

Recent advances in the classification and management of hypereosinophilia

Numerous disorders and etiologies may underlie increased eosinophil counts. Hypereosinophilia (HE) is defined as a peripheral blood eosinophil count greater than 1,500/mm3 and may be potentially harmful because of tissue damage. Hypereosinophilic syndrome (HES) also represents a heterogeneous disorder characterized by persistent HE with the evidence of organ dysfunction, clinical symptoms, or both caused by eosinophilia. The refining criteria and subclassification of HE and HES are currently being revised on cellular and molecular based diagnostic methods. Initial approaches focus on evaluating various underlying causes, including helminthic infections, adverse drug reactions, allergic diseases, and neoplastic diseases. When secondary causes of HE are excluded, the workup should proceed to the evaluation of primary/clonal bone marrow disease, including fip 1-like 1-platelet driven growth factor receptor alpha (FIP1L1-PDGFRA) mutation. Concurrently, if the patient has symptoms and signs, organ damage or dysfunction must be evaluated. Although, corticosteroids are the mainstay of therapy in confirmed HES, imatinib is considered a definitive treatment for FIP1L1-PDGFRA, platelet driven growth factor receptor beta rearranged HE and HES. In this article, we discuss recent advances in the classification of and practical approaches to HE and HES. In addition, we introduce several promising therapies for HE and HES.

Hypereosinophilic Syndrome: A rare differential diagnosis for profound leucocytosis

@#A broad range of disorders are present with eosinophilia and these include infectious, allergic, rheumatic, neoplastic, endocrine and idiopathic disorders which range from benign to life-threatening illnesses. All these conditions create a heterogeneous list of clinical presentation that patients may display, thus creating a diagnostic challenge for clinicians. We present a patient with a broad range of clinical features and hematological counts that fit into the diagnosis of Hypereosinophilic Syndrome (HES). He was investigated reasonably to rule the various possible differentials and subsequently started on Prednisone therapy.

Características genético-moleculares de los tumores estromales gastrointestinales (GIST) / Molecular and genetic features of gastrointestinal stromal tumors (GIST)

Los tumores estromales gastrointestinales (GIST) son neoplasias mesenquimales que típicamente surgen a nivel del estómago, intestino delgado, colon, y otros sitios en la cavidad abdominal y su identificación se ha incrementado por mejoras en los criterios de detección. La mayor parte de los tumores GIST son causados por mutaciones activadoras en los genes de receptores transmembranares tirosina quinasa c-KIT y receptor alpha del factor de crecimiento derivado de plaquetas (PDGFRA). Las mutaciones causales de GIST se restringen solo a ciertas regiones del gen que corresponden a importantes zonas funcionales de c-KIT o PDGFRA. Se reporta que hasta 70% de casos de GIST se debe a mutaciones en el exón 11 del gen c-Kit que corresponde a la región yuxtamembrana del receptor. La región y el tipo de mutación determinan diferencialmente cómo se desarrolla la neoplasia, el pronóstico y su respuesta a inhibidores de las tirosina quinasas como el Imanatib. Por tal motivo, el genotipado de KIT y PDGFRA es importante para el diagnóstico y establecimiento de la sensibilidad a los inhibidores tirosina quinasa.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms typically arising in the stomach, small intestine, colon, and other sites in the abdominal cavity and its identification has improved dramatically mainly due to better criteria in its detection. Most GISTs tumors harbor activating mutations in the tyrosine kinase receptor c-KIT or platelet derived growth factor receptor-alpha (PDGFRA). Those mutations are restricted to a few regions corresponding to important functional domains of c-KIT or PDGFRA. Upto 70% of cases are due to mutations in exon 11 of c-KIT corresponding to its juxtamembrane region of the receptor. The location and type of mutation will differentially determine the development of the disease, its prognosis and the response to inhibitors of tyrosine kinases as Imanatib. For this reason, genotyping c-KIT and PDGFRA is important for GIST diagnosis and assessment of sensitivity to tyrosine kinase inhibitors.

An Approach to Diagnosing Gastrointestinal Stromal Tumors Using Immunohistochemistry of c-kit and PDGFRA with Molecular Analysis

BACKGROUND: Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors in the gastrointestinal tract. Recently, many methods for the diagnosis of GIST have been developed including molecular diagnosis. METHODS: We selected 90 cases of GIST that had presented at Kyungpook National University Hospital between 1998 and 2007. Tissue microarrays were made using core areas of tumor tissues. Immunohistochemical staining for c-kit, protein kinase C-theta, and platelet-derived growth factor receptor alpha (PDGFRA) was done. Direct sequencing of hot spot exonal areas for c-kit and PDGFRA were done using extracted DNAs of all 90 paraffin block tissues. RESULTS: Among the 90 cases, 83.3% (75/90) were c-kit positive, 16.6% (15/90) were c-kit negative, 93.3% (84/90) were PDGFRA positive, and 6.6% (6/90) cases were PDGFRA negative. Fifteen cases of c-kit negative GIST included 1 case of PDGFRA negative and 5 cases of PDGFRA negative GIST were ckit positive. The one case in which both c-kit and PDGFRA were negative, showed a c-kit mutation in exon 11. CONCLUSIONS: Combined immunohistochemical staining of c-kit, discovered on GIST 1 (DOG1) and PDGFRA is helpful for the diagnosis of GIST. When all staining tests are negative for immunoreactivity, c-kit mutation analysis for exon 11, 9 should be done. Genotyping of kit and PDGFRA do not need to be examined initially, if it is only for the diagnosis of GIST.

Gene mutation and targeted therapy development of gastrointestinal stromal tumors / 国际外科学杂志

Gastrointestinal stromal tumors are the common mesenchymal tumors of the gastrointestinal tract. The constitutive activation of KIT is one of the earlies ttransforming events in GISTs and occurs mainly through activating mutations in the kit gene. PDGFRa is associated with the pathogenesis of GISTs too. The main mutative site of KIT gene are exon11, exon9, exon13 and exon17 and those of PDGFRA are exon18, exon12 and exon14.The clinical parameter and the response to imatinib of GISTs are associated with the site and type of gene mutation. Imatinib mesylate is a small-molecule inhibitor that selectively inhibit the tyrosine kinase and the clinical use of it is one milestone of the targeted therapy of GISTs. We review the gene mutation and targeted therapy development of gastrointestinal stromal tumors in this article.

c-kit and PDGFRA gene mutation in gastrointestinal stromal tumors and its significance / 中国癌症杂志

Background and purpose:Gastrointestinal stromal tumor(GIST) is the most common mesenchymal tumor of the digestive tract. The mutation status of c-kit and PDGFRA gene in GIST appear to be in correlation with gleveec therapy. However, the prognostic significance of c-kit and PDGFRA mutations in GIST is still controversial. We investigated the status and significance of c-kit and PDGFRA gene mutation in GIST. Methods: Mutation of c-kit (exon 9,11,13,17) and PDGFRA gene (exon 12,18) in 50 cases of GIST were examined by PCR amplification and direct sequencing. Results:Mutations of c-kit exon 11 were identified in 54% (27/50) of patients and were heterogeneous, including 16 cases of in-frame deletion,10 cases of point mutation and 1 case of the two type mutation. These mutations mostly were clustered in 550-560 "hot spots" code at the 5′ end of the exon 11.1 case with mutation was detected in 2 cases of CD117-negative GIST. There was significant relationship between mutations of c-kit exon 11 and the categories of cell type (P0.05), respectively. Conclusion:c-kit mutation was common in GIST and had relatively fixed tendency and mostly was detected in spindle-cell GIST.The presence of c-kit mutation is not a significant prognostic factor in GIST.

Gastrointestinal Stromal Tumors associated with Neurofibromatosis Type I: A Report of Two Cases

Gastrointestinal stromal tumor (GIST) is the most common non-epithelial neoplasm arising in the gastrointestinal tract, but this tumor is rarely seen in association with type 1 neurofibromatosis (NF-1). We report here on two cases of multiple GISTs of the small intestine that occurred in NF-1 patients. We also analyzed the mutations of c-kit exons 9, 11, 13 and 17 and the plateletderived growth factor receptor-alpha (PDGFRA) exons 12 and 18 in two GIST patients. Histologically, the NF-1-associated GISTs were similar to those of non-the NF-1 GISTs, but they characteristically revealed hyperplastic interstitial cells of Cajal around the GISTs. Immunohistochemically, these tumors showed strong co-expressions of CD117 and CD34. The molecular genetic analysis of the GISTs showed that all of the c-kit and PDGFRA exons that were analyzed in the GISTs of the two patients were the wild-type, suggesting a limited role for the c-kit and PDGFRA mutations in the tumorigenesis of NF-1-associated GISTs.