Two cases with hypereosinophilic syndrome shown with real-time PCR and responding well to imatinib treatment
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- Selvi, N., Kaymaz, B.T., Şahin, H.H. et al. Mol Biol Rep (2013) 40: 1591. doi:10.1007/s11033-012-2207-6
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The aim of this work was to report two cases of hypereosinophilic syndrome (HES). FIP1L1-PDGFRA fusion was assessed with two protocols at RNA level. The fusion transcript was found positive at the RNA level with both PCR methods in two cases. In this study, the efficiency of imatinib treatment and a dramatic response in two HES cases with multisystemic involvement showing the characteristics of a chronic myeloproliferative disease were presented. Both cases showed complete responses confirming that imatinib mesylate treatment could be successful even in patients with advanced HES having myeloproliferative disease.
KeywordsHesImatinibReal-time PCRFIP1L1-PDGFRA fusion
Eosinophiles might cause organ infiltration and therefore damage in relation to non-hematological (reactive-secondary) and hematological (primary-clonal) diseases. Hypereosinophilic syndrome (HES) is a heterogeneous disease characterized by chronic eosinophilia in peripheral blood (>1500 μL for at least 6 months) and dysfunction associated with eosinophilia in various organs . A diagnosis is made by the morphological evaluation of peripheral blood and bone marrow and by showing the histopathological or clonal increase with cytogenetics and FISH techniques. No efficient treatment of the disease has been defined until recently. It has recently been reported that imatinib mesylate that inhibits BCR-ABL, c-kit and FIP1L1-PDGRFα tyrosine kinases in some of the patients with HES provides clinical and hematological responses [2–4]. A subgroup of HES that displays the characteristics of a chronic myeloproliferative disease characterized by high serum tryptase levels with FIP1L1-PDGRFα fusion gene resulting from interstitial deletion in chromosome 4q12 leading to a tyrosine kinase activity, tissue fibrosis, a poor prognosis and a response to imatinib has been defined . Imatinib mesylate has a potential inhibitory effect on c-abl, bcr-abl, c-kit, platelet-derived growth factor receptor. Imatinib is effectively used in Philadelphia chromosome positive CML . In this study, the efficiency of imatinib treatment and a dramatic response in two HES cases with multisystemic involvement showing the characteristics of a chronic myeloproliferative disease were presented.
52-year-old, a male patient, complaints about pruritus and jaundice. The patient was hospitalized for the regulation of Type 2 diabetes in March 2010 by the Endocrinology Department. In his physical examination, it was detected that his general state was good, that his ECOG performance status was 1 and that the liver was 4 cm palpable and the spleen was 2 cm palpable, while the results of laboratory examination were found as follows: leukocyte: 10,200 mm3 (5 % eosinophile); Hb: 13 g/dL; Hct: 37 %; thrombocyte: 56,000 mm3; ALT: 82; AST: 45; ALP: 1,060; GGT: 580; T. bilirubin: 3.72; D. bilirubin: 3.7; urea: 40 mg/dL; creatinine: 0.7 mg/dL; and albumin: 3.4 g/dL. The autoantibodies and hepatitis markers (-), abdominal USG, MRCP and ERCP performed as a result of the gastroenterology consultation were found normal. The patient was followed with ursodeoxycholic acid without being hospitalized. The results of the physical examination were detected as follows: fever: 38.5; ECOG performance status: 3; and general state: moderate-bad. In his laboratory examination, the following were detected: leukocyte: 22,800 mm3 (16 % eosinophile); Hb: 11 g/dL; Hct: 33 %; thrombocyte: 38,000 mm3; ALT: 98; AST: 56; ALP: 1,100; GGT: 590; T. bilirubin: 4.5; D. bilirubin: 4; albumin: 3 g/dL; urea: 150 mg/dL; and creatinine: 5.8 mg/dL. The patient, who was consulted to us, was hospitalized at our clinic. It was detected that his infection screening was (-), that the ECHO was normal and that a 12 % increase in eosinophilia was found in the peripheral smear. In the evaluation of the bone marrow, an increase in megakaryocytes accompanied by 20 % eosinophile and degree 3 myelofibrosis were detected, along with a positive FIP1L1-PDGRFα from the bone marrow. In September 2010, 200 mg/day of imatinib mesylate was started as treatment. As of the 2nd week from the initiation of the treatment, a dramatic improvement of the clinical findings began in the patient, whose all clinical and hematological disorders except for renal functions completely normalized in the 2nd month. A clear improvement was achieved in the renal functions of the patient that had started as an acute renal failure and become chronic. In his last control in October 2011, the results of the physical examination were detected as follows: general state: good; ECOG performance status: 1; and liver and spleen: nonpalpable. The results of laboratory examination were as follows: leukocyte: 7,200 mm3 (68 % neutrophile, 2 % eosinophile); Hb: 12 g/dL; Hct: 48 %; thrombocyte: 248,000 mm3; albumin: 4.3 g/dL; ALT: 32; AST: 25; ALP: 179; GGT: 42; T. bilirubin: 1.5; D. bilirubin: 0.5; urea: 60 mg/dL; and creatinine: 2.1 mg/dL. During his follow-ups, it was determined that the eosinophilic series in the bone marrow was normal and that FIP1L1-PDGRFα became negative. The patient is still under his treatment with 200 mg/day of imatinib mesylate, and although his renal functions have not completely normalized, they have been stable for a year.
49-year-old, a male patient. In November 2009, he applied to the emergency service with complaints about dyspnea, which had started a year earlier and gradually increased, weakness, exertional dyspnea (Class III), chronic cough and swelling in the lower extremities. The patient was hospitalized at our clinic with the following results in the physical and laboratory examinations: general state: moderate; bedridden; ECOG performance status: 3; liver was 4 cm palpable and spleen was 2 cm palpable; pretibial 2+ edema; leukocyte: 49,500 mm3 (82 % eosinophile); Hb: 12 g/dL; Hct: 37 %; thrombocyte: 33,400 mm3; ALT: 245; AST: 102; urea: 40 mg/dL; creatinine: 0.7 mg/dL; and albumin: 2.4 g/dL. Giant thrombocytes, hypogranular neutrophiles and a clear increase in eosinophilia were detected in the peripheral smear. A common coarse reticulonodular pattern change and thickening of the peribronchovascular wall were detected in thorax HRCT, while severe enlargement of the right cardiac cavities, compression on the left cardiac cavities, 125 mmHg of pulmonary artery pressure, severe tricuspid regurgitation and 55 % left ventricular ejection fraction were detected in echocardiography. Inflammation developing with an increase of a large number of eosinophiles was detected in the lamina propria in the biopsy that was taken upon the detection of polyps in the colonoscopy performed due to constipation. After the other diseases developing with eosinophilia had been excluded, the bone marrow was evaluated. An increase in megakaryocytes accompanied by an increase in the eosinophilic series (82 %) and degree I myelofibrosis were detected in the bone marrow. FIP1L1-PDGRFα from the bone marrow was found positive. In April 2010, 200 mg/day of imatinib mesylate was started as treatment. As of the 2nd week from the initiation of the treatment, an improvement of the clinical findings began in the patient, whose all clinical and hematological disorders completely normalized in the 2nd month. In his last control in October 2011, the results of physical examination were as follows: general state: good; ECOG performance status: 1; exertional dyspnea: class I; no edema; liver was 1 cm palpable and spleen was nonpalpable; leukocyte: 9,610 mm3 (68 % neutrophile, 2 % eosinophile); Hb: 15.4 g/dL; Hct: 48 %; thrombocyte: 248,000 mm3; and albumin: 4.3 g/dL. It was also determined that the pulmonary artery pressure decreased to 80 mmHg and the SVEF rose to 60 in echocardiography and that during his follow-ups, the eosinophilic series was normal and FIP1L1-PDGRFα became negative in the bone marrow. The patient is still under his treatment with 200 mg/day of imatinib mesylate.
Both cases show that complete responses can be received with the imatinib mesylate treatment even in patients with advanced HES having the myeloproliferative disease characteristics, developing with fibrotic findings and having multisystemic involvement and that it should be used in such patients as well.
Material and method
Genomic RNA isolation from the bone marrow sample of the cases was performed by using the High Pure DNA Isolation Kit (Roche Applied Science). The obtained RNAs were measured with the Nanodrop spectrophotometer device and evaluated as 68 and 58 ng/μL respectively.
An evaluation of FIP1L1-PDGFRA fusion with the real-time PCR
In the sample taken from the bone marrow, FIP1L1-PDGFRA fusion was examined with two different protocols at the RNA level. The reaction protocols were performed with the primers and probes that were designed and synthesized specifically to the transcript and without probes based on the manufacturer’s instructions and were adapted to the real-time PCR device LightCycler (LC) 2.0.
In conclusion, the fusion transcript was found positive at the RNA level with both PCR methods in two cases followed in our center.
HES is a rare disease (1:50,000) and more frequently seen in the male gender . The clinical onset might be silent and the increase in eosinophilia might be randomly detected or the onset might be acute and develop with life-threatening complications . The WHO accepts all myeloproliferative variants of the HES as a neoplastic (clonal) disease . HES is a myeloid type of disease that develops with an increase in eosinophiles in blood and bone marrow and sometimes with an increase in blasts, that might cause multiple organ involvement and that has a poor prognosis [7, 8]. The FIP1L1-PDGRFα fusion protein resulting from 4q12 deletion is an oncogene with a tyrosine kinase activity. In cases with HES, it is positive in eight of every 15 patients and the tyrosine kinase inhibitor responds well to the imatinib treatment . As the FIP1L1-PDGRFα fusion protein plays a significant role in the etiology of HES, it has become the main target in treatment and the importance of imatinib treatment has increased . HES is a potential fetal disease and might develop with multiple organ involvement. Renal involvement and membranous glomerulopathy are rare findings and should be treated quickly [11, 12]. Three mechanisms were considered in the renal involvement of HES. The harmful granules of eosinophiles were put forward in the first mechanism. In the second mechanism, it was suggested that the immune complexes caused by eosinophiles accumulated in the kidney, whereas in another mechanism, it was suggested that the immuno-allergic mechanism triggered by eosinophiles played a role in pathogenesis . In another study by Garella and Marra  arteriolar glomerulonephritis and vasculitis were considered. Richardson et al. took a renal biopsy upon the presence of a rapidly developing acute renal failure in a patient who was being treated for polyneuropathy, allergic asthma, acute myocardial infarction and diagnosed with HES; they detected crescentic glomerulonephritis caused by eosinophile infiltration; and the findings improved quickly upon the initiation of steroid treatment . In a study reported by Hirsszel et al.  acute tubular necrosis and tubular absorption disorder developed in a patient in blastic crisis that occurred together with autoimmune hemolytic anemia due to the considerable amount of Charcot-Leyden crystals in the urine. When compared with renal involvement, cardiac involvement and the complications connected with it are more frequently seen in HES. Cardiac anomalies were detected at the rate of 60 % in a study by Wojciechowska et al. and at the rate of 50–60 % in a study by Ten et al. In both studies, it was reported that cardiac involvement was frequent and the most important cause of mortality in HES [17, 18]. It was shown that cardiac infiltration and organ damage developed in three steps in HES. The first phase is the necrotic phase which is caused by the granule content of the eosinophiles formed due to the infiltration of myocard by eosinophiles. This phase is generally asymptomatic and it is difficult to make a diagnosis in this phase. The second phase is the thrombotic phase. Due to the thrombosis occurring particularly along the wall in the left ventricle in endocardium, the endocard tissue in time leaves its place to the granulation tissue. The third and last phase is the fibrotic phase. It is the phase in which the granulation tissue, where the eosinophilic infiltrate is less, leaves its place to hyalin fibrosis and leads to the formation of restrictive cardiomyopathy and the complications connected with it . Because cardiac involvement is frequent in HES, sample cases were reported in many studies. Ten et al.  identified acute ischemic infarction developing with ST elevation in V1-2 and reciprocal ST depression in V2 in ECG and left atrial dilatation in ECHO in a 52-year-old female patient. Chen et al.  detected Hypereosinophilia-dependent ST segment variations and mural thrombus in a 17-year-old patient who had applied with fever, palpitation and chest pain and diagnosed with acute infarction. Cincin et al. made a diagnosis of HES in a patient with resistance to medical treatment and with heart failure and apical thrombus. They detected a clear improvement in the cardiac functions after the early steroid treatment . Patients with HES respond well to the glucocorticoid and imatinib treatments . Since the FIP1L1-PDGRFα fusion protein plays a significant role in the etiology of HES, it has become the main target in treatment and the importance of imatinib treatment has increased . Mortinelli et al. obtained a quick response in 3 weeks with the early imatinib treatment in a HES patient with a positive FIP1L1-PDGRFα fusion protein. The treatment was carried on for 17 months; with the treatment, it was ensured that the patient remained in remission; and a maintenance treatment was recommended, but no time was defined . In a multicentric study conducted by Jovanovic et al.  a good response to a low dose of imatinib treatment was received in HES cases with a positive FIP1L1-PDGRFα fusion protein. Baccarani et al. started imatinib in 63 of 72 HES patients with a positive FIP1L1-PDGRFα fusion protein; complete remission was achieved in 27 patients who persevered in treatment; and in six patients who abandoned the treatment and whose FIP1L1-PDGRFα fusion protein became positive again, complete remission was again achieved upon restarting of the imatinib treatment . Vigna et al.  achieved an improvement in clinical findings (diplopia) on the 7th day of the imatinib treatment in a HES patient with a retroorbital mass and a positive FIP1L1-PDGRFα fusion protein. Butterfield et al. prepared a review about treatment of HES and defended the idea of newer medications included pegylated forms of IFN-aα and IFN-α2b, first- and second-generation tyrosine kinase inhibitors (imatinib mesylate, nilotinib), and monoclonal antibodies to interleukin (IL)-5 and CD52 .
Our both cases are acute-onset myeloid-type HES cases developing with multiple organ involvement including the kidney. Symptoms of kidney that is more rarely involved in HES are outstanding in the first case, whereas symptoms of heart that is quite frequently involved and associated more closely with mortality are outstanding in the second case. Quickly-progressing multiple organ involvement was present at the time of diagnosis in both patients. A dramatic recovery was detected in the patients under imatinib treatment. In the first case, a partial response was received in kidney and a complete response in other organs. In the other case, however, the cardiac response was partial and the response in the other organs was complete. Both cases showed complete responses confirming that imatinib mesylate treatment could be successful even in patients with advanced HES having myeloproliferative disease characteristics, developing with fibrotic findings and having multisystemic involvement and that it should be used in such patients as well.