JAK2 V617F MUTATION SCANNING IN PATIENTS WITH ADRENAL INCIDENTALOMA

Abstract

Objective

Adrenal incidentaloma are lesions which are stated incidentally by imaging methods when there is no suspicion of any disease in adrenal gland. Inappropriate Jak2 signaling causes some solid and hematological malignancies. But the Jak2 mutation has not been previously evaluated with regard to adrenal tumors. In this study, we aimed to positivity of the Jak2 mutation in patients with non functioning adrenal incidentaloma (NFAI).

Methods

45 (38 female–7 male) patients, who were followed due to NFAI at Tepecik Training and Research Hospital, Department of Endocrinology and Internal Medicine between February 2014 and March 2015, and 45 (31 female–14 male) healthy controls were included in the study.

Results

The average age was 54.02±11.7 years and 38 patients were female, 7 were men. All patients underwent the following analyses for excluding a functioning adrenal mass, overnight dexamethasone suppression test, 24 hour urinary metanephrine and normetanephrine, plasma aldosterone/ renin activity ratio. Jak2 mutation of the patients who were diagnosed as NFAI was all negative.

Conclusion

We could not identify the JAK2 gene mutation positivity in any sample. Since other possible mechanisms may throw fresh light on the etiology of adrenal incidentaloma, further clinical studies are needed on this subject.

INTRODUCTION

Adrenal incidentalomas are located randomly by radiologic imaging such as computerized tomography (CT), abdominal ultrasound (USG) or magnetic resonance imaging (MRI). Development of abdominal imaging techniques and widespread use of imaging methods resulted by increased frequency of adrenal incidentalomas (1). The majority of adrenal incidentalomas are benign and clinically silent (2). The majority of surgically excised adrenal incidentalomas are non-functional cortical adrenal adenomas. The etiology of AI is not clear. It was stated that inappropriate JAK2 signaling is the cause of some solid and haematologic malignancies. There were previously reported three cases about Jak2 V617F mutation with nonfunctional adrenal adenoma in literature; one of them was published by us (3, 4). JAK 2 mutations have not been previously evaluated in adrenal incidentalomas. In this report we aimed to investigate this relationship. In the adrenal cortex, as in other tissues, oncology recapitulates ontogeny. Signaling pathways involved in embryonic/fetal development and in stem cell maintenance/activation are often deranged in adrenocortical tumors. Jak family (Jak1, Jak2, Jak3 and Tyk2) connects to the subunits of intracellular receptors, phosphorylated and establish connection areas for adaptor proteins which resulted by activation of their tyrosine kinase functions, following phosphorylation of signaling proteins such as STAT (5). Several cytokines are known to activate many different STAT proteins (6). STAT proteins stimulate the transcriptions of target genes by interaction of specific response series on DNA. To our knowledge luteinizing hormone (LH), insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (FGF) use JAK-STAT pathway and several studies show that they are related to adrenocortical tumorigenesis and proliferation. In this study we aimed to search the frequency of Jak2 V617F mutation positivity in patients with non-functional adrenal incidentalomas (NFAI).

MATERIALS AND METHODS

After getting the approval of the ethics committee, 45 patients who were admitted to internal medicine and endocrine outpatient clinics and diagnosed with AI (which is thought to be of benign nature and has proven to be non-functional with non-surgical excision indication, in patients with unrelated complaints) by USG, CT or MRI. Voluntary informed consent form has been obtained. All the patients’ age, gender, initial complaints, symptoms and smoking habits were recorded. All patients were examined about comorbidities such as diabetes mellitus (DM), hypertension (HT), coronary artery disease (CAH). The signs and symptoms of pheochromocytoma, Cushing syndrome, primary hyperaldosteronism were asked. Control group was designed with patients older than 18-years who did not have any malignancies, haematologic diseases or adrenal diseases. The study was designed in accordance with the Declaration of Helsinki; written informed consents were obtained from all subjects.

Biochemical Examination

All patients were administered 1 mg of dexamethasone at 23:00 for screening hypercortisolemia and blood samples were taken at 8:00 am. Subclinic Cushing syndrome (SCS) was excluded if cortisol level was < 1.8 μg/dL (50 nmol/L). 24 hours of urine metanephrine, nor-metanephrine levels were measured by HPLC method. To prevent false positive tests, tricyclic antidepressants, anxiolytics, paracetamol, non selective alpha blockers, phenoxybenzamine, β-blockers, monoamine oxidase (MAO) inhibitors, sympathomimetics were withheld 2 weeks before. Alpha-1 blockers were given during 2 weeks in hypertensive patients. At the end of 2 weeks, patients collected 24h urine in a container with 20 mL 6N HCl. To exclude primary hyperaldosteronism, blood samples were examined for aldosterone (ALD) and plasma renin activity (PRA). To prevent false positive results, diuretics were stopped 6 weeks before and beta blockers, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARBs), dihydropyridines were withheld 2 weeks before sampling. Before taking blood sample, serum potassium levels were examined. After obtaining normal results, the test was performed. Plasma ALD ve PRA was measured by RIA (radioimmune assay). ALD/PRA ratio was used for diagnosis of primary hyperaldosteronism. Patients with ALD/PRA ratio >30 and PRA level below reference rate, after correction were diagnosed as primary hyperaldosteronism. Blood samples were collected from patients with non-functional adenoma.

Genetic Analysis

Genomic DNA is extracted from peripheral whole blood using MagPurix Blood DNA Extraction Kit by MagPurix 12 (Zinexts Life Science Corp., Taiwan). The genotyping procedure consisted of polymerase chain reaction (PCR) amplification and SNP detection of the V617F mutation (rs77375493) of JAK2 gene using the following pair of primers:

Forward 5’-TGCTGAAAGTAGGAGAAA GTGCAT-3’ and Reverse 5’-TCCTACAGTGTTTT CAGTTTCAA-3’ by direct sequencing.

PCR products are purified as follows: 5 µL PCR products are treated with 2 µL of ExoSAP-IT enzyme (USB Affymetrix, USA) at 37°C for 30 min and at 85°C for 15 min.

Products of sequence PCR are purified (second purification) by spin colon (ZR DNA Sequencing Clean-up Kit™, Zymo Research, USA).

Sanger sequencing is performed by capillary electrophoresis after 5 min denaturation (3500 Genetic Analyzer, Lifetechnologies, USA). The obtained sequences are analyzed by using SeqScape® Software v3.0.

Radiologic Assessment

Images of the patients were re-assessed by experienced radiologists. Diagnostic criteria for adrenal adenoma were that the mass was bigger than 1 cm, smooth, homogenously hypodense lesions.

Statistical Analysis

SPSS 18.0 program was used for statistical analyses. For continuous variables, the median (minimum-maximum) values, for categorical variables n and percentage were given. Mann-Whitney U test was planned to compare the two independent groups.

RESULTS

Thirty-eight of the patients (84.4%) were women, 7 were (15.6%) men. Mean age was 54.02 in the patient group. 22 patients (48.9%) had HT, 8 patients (17.8%) had DM, one patient had CAD history. There was no evidence to suggest hyperaldosteronism in any patient with hypertension. History of malignancy is excluded from the study. The age, sex, comorbidity, smoking and diabetes rates of the patients and control group are shown in Table 1.

Table 1.

The age, sex, comorbidity, smoking and insulin resistance rates of the patients and control group

	Patient N (%)	Control group N (%
Sex
Male	7 (15.6)	14 (31.1)
Female	38 (84.4)	31 (68.9)
Comorbidity
Hypertension	22 (48.9)	0
Diabetes mellitus	8 (17.8)	0
Coronary artery disease	1(2.2)	0
Malignancy	0	0
Smoking	12 (26.7)	21 (46.7)

AI was detected by USG 4 (8.9%), MRG 23 (51.1%) and CT in 18 patients (40%), respectively. Mass was on the left and right side in 19 (42.2%) and 26 (57.8%) patients respectively. Median size of the mass was 20.20 mm. Patients with adrenal mases larger than 4 cm were excluded due to increased risk of cancer. Overnight 1 mg dexamethasone suppression test was normal (cortisol response in the morning was below 1.8 ug/dL) in all patients. 24 hours of urine metanephrine- normetanephrine levels were normal in all patients. Plasma aldosterone/renin activities (PRA) were normal in all patients. Control group consisted of 45 individuals (31 of them (68.9%) were women and 14 of them (31.1%) were men. Mean age was 34.8. In the control group, there was no DM, HT, CAD or malignancy. 21 people (46.7%) in the control group were smokers. Jak2 mutation was not detected in both AI patients and control group.

DISCUSSION

Adrenal incidentaloma is a popular topic in the field of endocrinology due to increasing prevalence. However there is no absolute etiology detected. Adrenocortical tumors are often benign and sporadic. On the other hand, it can be coexistent with hereditary carcinogenic syndromes such as Beckwith-Wiedemann syndrome, Li-Fraumeni syndrome and multiple endocrine neoplasia type 1.

It was reported that mean age peak was 5-7^th decades, with mean diagnosis age of 55, no difference between sexes (7). In our research, mean age of diagnosis was 54 years which is similar to the literature. The ratio of female/male was reported 1.2 to 1.3. This ratio was reported as 2.5 by Bednarczuk et al. This ratio was (8) found higher in our study compared with previous reports (9). It was reported that the incidentalomas were on the right, left side and bilateral in 50-60%, 30-40% and 10% of the patients, respectively. Incidentalomas were on the right and left side in 42.2% (19 patients) and 57.8% (26 patients), respectively in our study. Patient group had no patient with bilateral masses due to our study design.

IGF-I and IGF-II show mitogenic activity in adrenocortical tissue and overexpressing IGF-II causes adrenocortical hyperplasia in animal models. Studies have also shown that IGF II overexpression in the ACA is mainly responsible for the last step toward tumorigenesis to carcinogenesis in ACC patients. TGF and FGF also act as adrenocortical mitogens and are over-expressed in ACC compared with ACAs like IGF.

Activation of G-protein coupled receptors shows pleiotropic effects on cell growth, differentiation, and steroid production in adrenocortical tissue which is mediated through the cAMP-protein kinase A signaling pathway. Multinodular hyperplasia and ACA have been linked to ectopic expression of certain transmembrane G-protein coupled receptors, including receptors for LH. Some of these mechanisms could be involved in the oncogenesis of adrenal tumors with uncertain malignant potential (10).

LH, IGF, TGF and FGF use JAK-STAT pathway and Jak-2 V617F mutation may cause over signaling and increase the effectiveness of this substrate to promote adrenocortical adenoma development. Chromogranin A can be used as a neuroendocrine tumor marker for distinction between medulla and cortical adrenal tumors (11).

The fact that the mutation analysis was negative in all of our patients and we could not do mutation analysis at the tissue level, so this view cannot beyond the hypothesis and constitutes a weak feature and a limit of our study.

On the other hand about the previous case reports in the literature ‘Jak-2 mutation and adrenal incidentaloma’ all of the three patients had thrombocytosis, none in our study population. So it could be more logical to design the study in AI with thrombocytosis. Although we mentioned it could be a relationship between AI and Jak-2 V617F mutation. We think that in the previous case reports this relationship was a chance assocation.

There are no previous studies in the literature that examine the frequency of adrenal incidentaloma in myeloproliferative diseases and the incidence of adrenal imcidentaloma in Jak-2 V617F mutated patients and this requires further investigation.

In conclusion, identification of signaling pathways may refer to new approaches for specific medications for many diseases. JAK 2 V617F mutation analysis was negative in all of our patients and we could not do mutation analysis at the tissue level. This constitutes a weak direction of our study. We believe that this work is done more efficiently with obtained histopathological tissue and a larger number of patients.

Conflict of interest

The authors declare that they have no conflict of interest concerning this article.