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48uep6bbph|2000F98CTab_Articles|Fulltext

Myeloproliferative disorders (MPD) like polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis(PMF) are responsible for 50% cases of Budd- Chiari syndrome (BCS) manifesting as hepatic venous thrombosis (HVT) and/or inferior vena cava thrombosis (IVCT); and 35% cases of portal venous thrombosis (PVT) in western series.[1,2,3] Diagnosis of MPD pose a challenge given their latency of clinical and hematological abnormalities. Latent forms of MPD lack the characteristic blood picture and may be classified as idiopathic thrombotic disorder. Recently the discovery of a point mutation at Val617Phe of Janus kinase 2 tyrosine kinase gene (JAK2V617F mutation) lead to drastic change in diagnostic protocol for MPD. JAK2V617F mutations have been noted to occurs in a high proportion of MPD patients.[4]

The JAK2 mutation under study is extremely rare in healthy individuals and is located on chromosome 9p. This mutation causes an amino acid substitution of valine with phenylalanine in the antiregulatory JH2 pseudokinase domain of the JAK2 gene. This gain of function mutation leads to growth factor hypersensitivity and dependence resulting in upregulated constitutive kinase activity causing enhanced hematopoiesis.[5] It is a constitutively active tyrosine kinase and has transforming properties both in vitro and in vivo. Amplified JAK-STAT signaling seems to be central to the pathogenesis of MPD. Further studies on JAK2V617F negative cases of MPD have identified mutations in JAK2 axon 12 (in PV) and MPL (in ET and PMF), which also results in constitutive activation of JAK-STAT signaling leading to MPD.[4]

Previous diagnostic criteria for MPD were inadequate and latent/ atypical forms of the disorder lacking typical blood picture were missed frequently. JAK2V617F mutation analysis is now a major diagnostic criterion for all forms of MPD as per updated WHO criteria.[6]

BCS refers to hepatic venous outflow tract obstruction (HVOTO) starting from the level of small hepatic veins (HV) through large HV and inferior vena cava (IVC) to the junction of IVC and right atrium.[1,7,8] This includes both hepatic vein thrombosis (HVT) and inferior vena caval obstruction (IVCT) or obliterative hepatocavopathy. In contrast to the western world, there is a striking predominance of IVCT (mainly from membrane or web) or combined IVCT-HVT rather than HVT alone, in Indian patients.

Furthermore, idiopathic cases are among the most common presentation of BCS.[9] Prevalence of JAK2V617F mutation has been reported in 30 to 75% patients with BCS and 18 to 40% patients with PVT.[10] BCS is often associated with thrombophilic abnormalities and/or hematopoietic disorders like MPD. Defining these abnormalities is important as anticoagulation is the answer to the former disorder and cytoreductive therapy for the latter.[10] As there is very limited data available from India on MPD as a cause of intra-abdominal venous thrombosis (IAVT), this cross sectional study was planned to determine the prevalence of JAK2 mutations in IAVT.

Methods

In this prospective cross sectional study, a total of 170 subjects were tested for presence of JAK2V617F mutations, from December 2006 to January 2009. These 170 subjects included 58 consecutive patients of IAVT without cirrhosis, cancer, pancreatitis or trauma, (comprising of 20 patients with BCS, 35 patients with PVT and 3 patients with combined BCS and PVT); 58 consecutive patients of MPD without thrombosis; 31 consecutive patients with non-MPD hematological disorders and 23 healthy volunteers attending our health check-up clinics, who served as controls.

MPD was diagnosed and classified as per the WHO 2008 diagnostic criteria. Diagnosis of BCS was based on imaging (ultrasonography, CT scan, CT angiography, and/or MR angiography) or angiographic evidence of HVOTO (i.e. obstruction of IVC and/or HV) and/or histological evidence of BCS (sinusoidal dilation with centrilobular congestion with variable amount of pericentral hepatocyte necrosis and pericentral fibrosis).

All the patients with BCS were tested for hypercoagulable state before starting any treatment. Tests for protein C, protein S and antithrombin III levels (corrections for liver dysfunction were done);10 serum homocysteine levels; factor V Leiden, prothrombin gene 20210 and MTHFR gene mutations; lupus anticoagulant; anticardiolipin and antiphospholipid antibodies; tests for paroxysmal nocturnal hemoglobinuria (PNH) (sucrose lysis and Ham’s tests); complete blood counts for MPD and bone marrow histopathology (BMH)/ cytogenetic studies (whenever feasible for suspected MPD) were performed.

Imaging (ultrasonography, CT and/or MR imaging of abdomen), serological markers (including tumor markers and tests for amoebiasis or hydatid cyst) and/or histology were done to identify underlying etiology of BCS as and when appropriate. All female patients were subjected to urine test for pregnancy and were questioned regarding use of oral contraceptive pills. All the patients with diagnosis of BCS were checked for presence of JAK2V617F mutations. Those tested positive for the mutation were subjected to BMH and cytogenetic analysis to diagnose latent MPD.

Diagnosis of splanchnic vein thrombosis (portal vein, splenic vein and splanchnic vein) in the absence of cirrhosis, cancer, pancreatitis and trauma was established by Doppler ultrasound, CTA, and/or MRA, as deemed appropriate for each case under investigation. Patients with splanchnic vein thrombosis were tested for thrombophilia and grouped with BCS patients accordingly. All patients with splanchnic vein thrombosis underwent upper GI endoscopy for presence of varices and if detected they were managed with standard endoscopic and pharmacotherapy. All the patients were subjected to anticoagulation  therapy, while cytoreductive therapy was given to those who had evidence of MPD. All consecutive patients with BCS and splanchnic venous thrombosis both acute and chronic, were included in this study. The JAK2 mutation was tested on whole blood samples by the following method. Genomic DNA was extracted from 200 µl of whole blood (EDTA) using spin procedure (QIAamp DNA Blood Mini Kit) which gave a yield of 3-12 µg of DNA per sample. The JAK2V617F mutation was detected by allele specific polymerase chain reaction (PCR) (Ipsogen JAK2 MutaScreen Kit and applied Biosystems 7500 Real Time PCR system). It included a specific fluorescent dye labeled probe to differentiate the amplification of each allele (V617F FAMTM probe for the mutant allele and VICR probe for the wild type allele).

This study was approved by the Bombay Hospital  institutional review board. Statistical analysis was done using Chi square test and Student t test. A p-value of less than 0.05 was considered statistically significant.

Results

This study included 58 patients with IVAT (including 20 patients with BCS, 35 with PVT and 3 with combined BCS and PVT). Demographics and prevalence data of JAK2V617F mutation in the control group, in non-MPD hematological patients, in MPD patients with thrombosis and in MPD patients without thrombosis is tabulated in Table 1 and Table 2.

Of the various MPD sub-sets examined, JAK2V617F mutation was present in 42/49 (86%) patients of PV, 5/12 (42%) patients of ET, 9/13 (69%) patients of PMF and 3/4 (75%) patients of unclassified MPD.The distribution of JAK2V617F mutation in IAVT is tabulated in Table2.

Comparison of MPD patients with or without thrombosis and comparison of base line characteristics of patients with BCS with or without MPD is shown in Tables 3 & 4. Our cohort had 35 patients with PVT, of which 10 had acute illness while 25 had chronic PVT. Only 14% of these patients had JAK2 mutations. Three patients had both BCS and PVT and all of them presented with acute illness. A total of 23 persons attending our routine health check-up clinic consented to participate in the study and were enrolled as controls, but were not stringently matched with the patient groups.









Prevalence of JAK2 mutation was significantly higher in patients with BCS when compared with patients with PVT (40% vs. 15% p <0.001). Other coagulation abnormalities seen in patients with BCS were protein C deficiency in two patients, protein S deficiency in one, hyperhomocysteinemia in two, antithrombin III deficiency in 3, factor V leiden mutation in 5, anticardiolipin antibodies in 2, lupus anti-coagulant in 1,  PNH in 1 and multiple abnormalities in 4 patients. On the other hand other coagulation abnormalities seen in patients with PVT were protein C deficiency in six patients, protein S deficiency in four patients, hyperhomocysteinemia in one, factor V leiden mutation in 2, anticardiolipin antibodies in 2, lupus anticoagulant in 1, and multiple abnormalities in 7 patients.

Discussion

In our study JAK2V617F mutation was present in a significantly high proportion of MPD patients with or without thrombosis,when compared to controls and non-MPD hematological disorders. These results suggest relative specificity of JAK2V617F mutation for diagnosis of MPD. As a corollary all patients with JAK2V617F mutation should be monitored for presence or development of MPD. The JAK2V617F mutation was present in highest proportion in patients of PV as compared to patients of ET, PMF or unclassified MPD. JAK2V617F mutations have been reported in 23-69% of ET, 14- 58% of PMF, 54% of unclassified MPD and 65-97% of PV patients.[5,12,13,14,15,16,17] Our results of JAK2V617F mutation analysis corroborated with the results of these other studies. The JAK2V617F mutation has been reported to have very low prevalence in other hematological neoplasm like chronic myeloid leukemia (CML) (3-20%), myelodysplastic syndrome (5-20%), hypereosinophilic syndrome (0-2%) and acute myeloblastic leukemia (2.7%).[14] This mutation is reported to be present in 0.9% of general hospital population.[17] To date, JAK2V617F mutation has not been described in patients with reactive myeloproliferation, lymphoid disorders or solid tumors. The JAK2V617F mutation is not the gold standard for diagnosis of MPD, given its absence in ~10% of PV and ~50% of ET and PMF patients; yet it remains a very useful adjunct diagnostic test, especially in presence of thrombotic signs and symptoms.[18,19]

In our study, MPD with or without thrombosis had similar prevalence of this mutation. Previous reports on MPD with JAK2V617F mutations have noted a higher incidence of venous thrombosis than cases without JAK2V617F mutation. It is speculated that the natural history of JAK2V617F positive occult MPD might be different from that of typical MPD.[20,21,22]

Thrombotic events like IAVT take place in 5-10% of PV and ET patients.[17] In cases of IAVT the diagnosis of MPD can be resolved in 28-49% of BCS and 14-35% of PVT using conventional tests (78% of BCS and 48% of PVT using endogenous erythroid colony formation (EEC); and 53% of BCS and 28% of PVT using BMH criteria).[17,23,24,25] In contrast to performance of conventional tests, various series from different countries have reported the presence of JAK2V617F mutation in 40-58.5% of HVT, 17.2-42.8% of PVT and 17-29.4% of all IAVT cases.[17,19,22,23,26,27,28] In our study, JAK2V617F mutation when employed to define etiology, was found in around one third of patients with BCS, PVT or both.

In a similar study from India, JAK2 mutations have been reported in 3% patients with BCS, 8.8% PVT and none of the healthy controls.[29] Prevalence of JAK2 mutations in patients of chronic liver disease with or without PVT was reported to be 24% and 7%, respectively; while the prevalence in healthy controls was 2% in another study from India.[30]

The above data calls attention to the importance of defining MPD as an etiological factor in IAVT and to the use of JAK2V617F mutation analysis as a diagnostic tool for all cases of IAVT. All persons with JAK2V617F mutation should be monitored for the presence or development of IAVT. As shown previously JAK2V617F mutation is a better diagnostic tool for MPD in IAVT than traditional hematological investigations.[23,26,31] Portal hypertension in patients with IAVT masks the features of MPD. Spleen size may be affected by the presence of portal hypertension.[18] Gastrointestinal blood loss (occult or variceal), hypersplenism and hemodilution due to splenomegaly, and portal hypertension itself makes the peripheral blood counts unreliable.18Among other traditional diagnostic tests, erythropoietin levels may be low in the presence of IAVT; and EEC is diagnostic only in less than 50% IAVT cases. The diagnostic utility of EEC is limited because it’s neither widely accepted, nor widely available and is fraught with technical difficulties and lack of lab to lab standardization. BMH on the other hand remains the only diagnostic tool which offers >90% sensitivity, but suffers from the disadvantages of being an invasive test which is difficult to employ in patients on anticoagulant therapy, and has difficult test interpretation introducing lab to lab variation.[17,19,26,32]In contrast to the conventional tests, JAK2V617F mutation analysis is a simple, reliable test with a high positive predictive value to enable a gastroenterologist to objectively suspect MPD in IAVT cases. As evident from our data, JAK2V617F mutations helped diagnose nearly two-thirds of latent MPD cases in our patients of IAVT.

It is important to identify cases of MPD because the management of IAVT with MPD differs from IAVT without MPD. The former requires aspirin and cytoreductive therapy in addition to the usual management and if not managed appropriately, these patients are prone to recurrent thrombotic events.18Erstwhile only a few studies have examined the prognostic significance of detecting JAK2V617F mutations in IAVT patients.[26,33] In a study by Kildjian26 BCS patients with MPD had severe liver dysfunction at the time of diagnosis and required early interventional therapy, but the management offered demonstrated no impact on their 5-year survival rate. In another study by Smalberg et al,[33] the 10 year survival for BCS patients with MPD was 92%, which was no different from the survival rates in BCS patients without MPD.

Application of JAK2V617F mutation analysis has few limitations in clinical practice. Variable frequency of JAK2V617F mutations in different studies may be a consequence of subclassification of MPD cases, the type and sensitivity of the molecular assays employed.[15] Current information on predicting disease prognosis based on JAK2 mutations is inconclusive. Further studies are required to define the precise role of JAK2V617F mutations in clonal transformation and expansion of bone marrow progenitors; as well as to define the efficacy of anti-JAK2 kinase inhibitors in risk reduction of thrombosis and in treatment protocols for MPD. Our study data presents a strong case for the use of this test in routine clinical practice.We suggest that JAK2V617F mutation should be included in work up for IAVT, bone marrow fibrosis and unexplained polycythemia, thrombocytosis or leukocytosis, and as a routine test to detect occult, latent or atypical MPD.

A recent meta-analysis assessed the utility of JAK2 mutations in BCS and PVT, examined cases across 23 studies involving 555 patients with BCS, 858 patients with PVT and 284 unclassified SVP. These studies were sub classified into: 1) patients with IAVT regardless of underlying etiological factor; 2) few studies which had excluded pre-existing MPD and 3) two studies of patients with cirrhosis with IAVT. The meta-analysis concluded a significant variation in prevalence of JAK2 mutations in IAVT. The authors also noted that JAK2 mutations are frequently associated with this condition and project a specific association with these conditions. Further, it was noted that JAK2 mutations are strongly associated with
the future development of MPD and this meta-analysis recommends routine screening of JAK2 mutations in clinical practice.[34]

In conclusion, JAK2V617F mutations occur at a high frequency in patients with MPD (particularly PV) and in patients with idiopathic IAVT. All cases of idiopathic IAVT must be screened for JAK2V617F mutation, to detect latent or occult cases of MPD, as MPD is major underlying cause of IAVT. Detection of latent or occult MPD by JAK2V617F mutation in IAVT may thus change the treatment strategy and patient outcome.

Acknowledgements

The authors are extremely thankful to Birla Smarak Kosh for financial support to carry out this study.

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