Laboratory Assessment of Patients with Systemic Mastocytosis

系统性肥大细胞增多症患者的实验室评估

• 批准号: 7733668
• 负责人: irina maric
• 金额: $ 2.98万
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733668
• 关键词: 5q31; Agreement; Aspirate substance; Basophilia; Biological Assay; Bone Marrow; Bone marrow biopsy; C-KIT Gene; Chromosome abnormality; Chronic eosinophilic leukemia; Clinical; Collaborations; Confusion; Cytogenetic Analysis; Data; Detection; Diagnosis; Diagnostic; Disease; Disruption; Elements; Eosinophilia; Evaluation; Exons; Flow Cytometry; Fluorescent in Situ Hybridization; Gene Fusion; Hematopoietic; IL2RA gene; Imatinib; Immunophenotyping; Laboratories; Molecular; Molecular Analysis; Molecular Cytogenetics; Mutation; Myeloproliferative disease; National Institute of Allergy and Infectious Disease; Neoplastic Mast Cell; Nomenclature; PDGFRA gene; PDGFRB gene; PRKG2 gene; Pathogenesis; Patients; Peripheral; Peripheral Blood Eosinophilia; Phosphotransferases; Property; Protein Tyrosine Kinase; Proto-Oncogenes; Receptor Activation; Receptor Protein-Tyrosine Kinases; Reporting; Resolution; Risk Factors; Role; Score; Selection for Treatments; Somatic Mutation; Stem Cell Factor; Symptoms; Syndrome; System; Systemic Mastocytosis; Testing; Work; body system; eosinophil; fusion gene; human PRKG2 protein; inhibitor/antagonist; mast cell; mastocytosis; novel diagnostics; outcome forecast; prognostic; response

Laboratory evaluation of patients with suspected systemic mastocytosis (SM) includes: (1) morphologic review of bone marrow biopsies and aspirates, (2) molecular testing for the presence of the KIT mutation and (3) immunophenotypic analysis of mast cells and other hematopoietic elements by flow cytometry. Recently, we have developed RT-PCR/RFLP assay for detection of the most prevalent KIT mutation in mastocytosis patients (D816V).The KIT D816V mutation results in constitutive activation of the receptor tyrosine kinase and is believed to be involved in disease pathogenesis. We have also developed a flow cytometric assay for immunophenotypic analysis of mast cells, since it has been recently described that neoplastic mast cells display aberrant immunophenotype, most notably, aberrantly coexpress CD117 with CD25 and/or CD2. Aberrant expression of CD25 and CD2 is not detected in normal marrow mast cells. The identification of the KIT D816V mutation in patients with systemic mastocytosis has lately gained a major prognostic significance, largely because of the availability of tyrosine kinase receptor inhibitors such as imatinib. Imatinib was shown to be ineffective in patients carrying KIT D816V mutation, but effective in cases with some other mutations. Therefore, it is of paramount importance to correctly identify SM patients with KIT D816V mutation. Although most patients with SM do not have peripheral blood eosinophilia, both bone marrow and peripheral blood eosinophilia have been reported in D816V mutation-positive patients. In these cases, the broad and overlapping clinical manifestations between the patients with D816V KIT-associated systemic mastocytosis with eosinophilia and patients with chronic eosinophilic leukemia (CEL) associated with FIP1L1/PDGFRA fusion gene present diagnostic challenge. The increase in activated eosinophils and mast cells is seen in both disorders, and has led to confusion in the nomenclature. It is of paramount importance, however, to distinguish between these two groups of patients because of differences in clinical sequelae, prognoses and selection of treatment. This has profound consequences for patients with these disorders, since the FIP1L1/PDGFRA and D816V mutations respond dramatically differently to therapy. Most notably, the FIP1L1/PDGFRA fusion kinase, but not D816V KIT mutation, is inhibited by imatinib. We thus sought to identify clinical and laboratory features that could be used to distinguish these two diagnoses. We compared 12 patients with D816V-positive systemic mastocytosis with eosinophilia with 17 patients with FIP1L1/PDGFRA-positive CEL. Distinguishing features were used to create a risk factor scoring system. This system correctly classified 16/17 FIP1L1/PDGFRA-positive CEL patients, and all 12 systemic mastocytosis with eosinophilia patients. These results reinforce the hypothesis that the FIP1L1/PDGFRA gene fusion and D816V-KIT mutation cause distinct clinical syndromes. This novel diagnostic approach should prove helpful in clinical practice in the evaluation of patients with increased mast cells burden and peripheral eosinophilia. Detection of other molecular/cytogenetic abnormalities in SM patients is much less frequent. We identified an unusual case of a patient presenting with peripheral basophilia and systemic mastocytosis in whom cytogenetic analysis revealed a t(4;5)(q21.1;q31.3). Translocations involving region 5q31-32 (PDGFRB) have been reported in a variety of myeloproliferative diseases and are often associated with significant peripheral eosinophilia. We used molecular analysis to determine the role of PDGFRB in this case. Fluorescence in situ hybridization (FISH) documented a breakpoint in PDGFRB. In agreement with this, the patient responded very well to imatinib treatment with resolution of clinical symptoms, basophilia, and mast cell disease. Molecular analysis revealed that PDGFRB, encoding an imatinib-sensitive tyrosine kinase, was fused to PRKG2. The fusion gene incorporates the first two exons of PRKG2 fused to the truncated exon 12 of PDGFRB, resulting in the disruption of its juxtamembrane domain. Functional studies confirmed that the activity and transforming properties of PRKG2-PDGFRbeta were dependent on the disruption of the auto-inhibitory juxtamembrane domain. Our results identify a second case of the PRKG2-PDGFRB fusion and confirm the unusual PDGFRB breakpoint associated with this fusion. This work also illustrates the use of imatinib for the treatment of specific cases of systemic mastocytosis.

实验室评估可疑的全身肥大症患者（SM）包括：（1）对骨髓活检和抽吸物的形态学综述，（2）通过流式细胞术对肥大细胞和其他造血元素对KIT突变的存在以及（3）分子检测。最近，我们开发了RT-PCR/RFLP测定法，用于检测肥大细胞增多症患者最普遍的试剂盒突变（D816V）。试剂盒D816V突变导致受体酪氨酸激酶的本构激活，并被认为与疾病病原体有关。我们还开发了一种用于对肥大细胞的免疫表型分析的流式细胞仪测定，因为最近已经描述了肿瘤肥大细胞显示出异常的免疫表型，最值得注意的是，与CD25和/或CD2异常共表达CD117。在正常骨髓肥大细胞中未检测到CD25和CD2的异常表达。 最近，全身性肥大症患者的试剂盒D816V突变的鉴定已获得了主要的预后意义，这主要是因为酪氨酸激酶受体抑制剂（如伊马替尼）的可用性。伊马替尼在携带KIT D816V突变的患者中无效，但在其他一些突变的情况下有效。因此，正确识别具有KIT D816V突变的SM患者至关重要。尽管大多数SM患者没有外周血嗜酸性粒细胞，但在D816V突变阳性患者中，骨髓和外周血嗜酸性粒细胞均已报道。在这些情况下，与嗜酸性粒细胞增多的D816V试剂盒相关的全身性肥大症与慢性嗜酸性白血病（CEL）患者与FIP1L1/PDGFRA融合基因相关的慢性嗜酸性白血病（CEL）患者之间的广泛而重叠的临床表现。在两种疾病中都可以看到活化的嗜酸性粒细胞和肥大细胞的增加，并导致命名法的混乱。然而，由于临床后遗症，预后和治疗选择的差异，区分这两组患者至关重要。由于FIP1L1/PDGFRA和D816V突变对治疗的反应很大，因此这对这些疾病患者产生了深远的影响。 最值得注意的是，伊马替尼抑制了FIP1L1/PDGFRA融合激酶，而不是D816V试剂盒突变。 因此，我们试图确定可用于区分这两个诊断的临床和实验室特征。我们与17例FIP1L1/PDGFRA阳性CEL的患者进行了比较了12例D816V阳性全身性肥大性肥大性肥大症患者。区分功能用于创建风险因素评分系统。该系统正确地将16/17 FIP1L1/PDGFRA阳性CEL患者和嗜酸性粒细胞增生的所有全身性肥大性分类。这些结果加强了以下假设：FIP1L1/PDGFRA基因融合和D816V-KIT突变会导致不同的临床综合征。这种新型的诊断方法应证明对临床实践有所帮助，以评估肥大细胞负担和周围嗜酸性粒细胞增长的患者。 SM患者中其他分子/细胞遗传学异常的检测要少得多。我们确定了一个异常情况，即患者出现外周嗜碱性嗜碱性嗜碱性粒细胞增多症和全身性肥大症，其中细胞遗传学分析显示T（4; 5）（Q21.1; Q31.3）。 涉及区域5q31-32区（PDGFRB）的易位已在多种骨髓增生性疾病中报道，并且通常与明显的周围嗜酸性粒细胞增多有关。我们使用分子分析来确定PDGFRB在这种情况下的作用。荧光原位杂交（FISH）记录了PDGFRB中的断点。与此一致，患者对伊马替尼治疗的反应很好，可以解决临床症状，嗜碱性嗜碱性疾病和肥大细胞疾病。分子分析表明，编码伊马替尼敏感酪氨酸激酶的PDGFRB与PRKG2融合。融合基因融合了PRKG2的前两个外显子与PDGFRB的截短外显子12融合，从而破坏了其叶膜域。功能研究证实，PRKG2-PDGFRBETA的活性和转化特性取决于自动抑制性近膜域的破坏。我们的结果确定了PRKG2-PDGFRB融合的第二种情况，并确认与此融合相关的异常PDGFRB断点。这项工作还说明了伊马替尼用于治疗系统性肥大病的特定病例。