Role of Endothelin-1 in a Syndrome of Myelofibrosis and Osteosclerosis

Abstract

Context:

Primary myelofibrosis is one of the chronic myeloproliferative disorders characterized by bone marrow fibrosis associated with extramedullary hematopoiesis and osteosclerosis. Endothelin-1 (ET1) is a potent vasoconstrictor that is also a key mediator of osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation.

Case Description:

We report laboratory, radiographic, bone densitometry, and bone histology data of a patient presenting with newly diagnosed, biopsy-proven myelofibrosis and osteosclerosis. We were able to demonstrate abundant ET1 signaling in the bones of our patient.

Conclusions:

We believe that ET1 is responsible for the osteosclerosis that develops with advanced myelofibrosis and suggest that ET1 signaling may play a role in other osteosclerotic settings as well.

Primary myelofibrosis (PMF) is a clonal disorder of the hematopoietic stem cell characterized by marrow fibrosis, extramedullary hematopoiesis, marked splenomegaly, and constitutional symptoms (1). Half of the patients with PMF have an acquired Janus kinase 2 V617F mutation (2). Osteosclerosis can eventually develop in association with advanced marrow fibrosis (3). The pathogenesis of this skeletal syndrome in PMF remains poorly defined. Here, we provide new insights into the underlying mechanism responsible for the exuberant osteogenesis associated with PMF.

Case Report

A 60-year-old woman was seen in the hematology clinic because of leukocytosis and constitutional symptoms. She had been well until approximately 1 year before this evaluation, when she experienced gradual onset of pain in her legs accompanied by severe fatigue and unintentional weight loss. The pain did not decrease with physical therapy or a course of ibuprofen.

She had a history of atrial fibrillation, aortic stenosis (status post valve replacement), cardiac arrest (status post pacemaker), pulmonary hypertension, and depression. Medications included sotalol, sildenafil, Coumadin, oxycodone, duloxetine, and alprazolam. She had no known allergies. She was widowed and worked at a health care organization. She had a 40 pack-year smoking history and drank alcohol occasionally. Her father died at age 84 years with colon and bladder cancer, and her mother was older than age 90 with dementia; there was no family history of hematologic dyscrasias. On examination, the patient was alert, oriented, and in pain. There was tenderness on palpation of both thighs. Moderate splenomegaly was evident. The remainder of the examination was normal.

Radiographs of the thighs showed a periosteal reaction along the femoral shafts (Figure 1A). Radionuclide bone scans demonstrated increased technetium tracer uptake in the femora (Figure 1B). A recent bone densitometry showed a lowest recorded T-score of −2.3. Lumbar spine and total hip dual-energy x-ray absorptiometry were evaluated longitudinally over 12 years (Figure 1C.) The patient's axial skeletal demineralized substantially during the first 10 years; however, bone mineral density increased 16.3% at the total hip over the past 2 years.

Figure 1. A radiograph of the right femur reveals periosteal thickening in the femur (A, arrow).

A delayed-phase bone scintigraphy shows increased activity in femurs (B, arrows). Lumbar spine and total hip dual-energy x-ray absorptiometry obtained at the same institution in the same GE/Lunar imaging instrument (C). Following a steady decline, lumbar spine and total hip BMD measurements has increased substantially over the past 2 years, before PMF diagnosis. BMD, bone mineral density.

A white blood cell count was 30 × 10³/μL (reference range, 4.1–10.9), the hemoglobin was 11 g/dL (reference range, 12.9–17.3), and the platelet count was normal. Pathological examination of a bone marrow biopsy specimen revealed severe reticulin fibrosis (Figures 2A-B). Testing of peripheral blood leukocytes for the Janus kinase 2 V617F mutation was positive (Mayo Medical Laboratories). The diagnosis of PMF was confirmed.

Figure 2. Low- (A) and high- (B) powered light microscopy reveals thickened bony trabeculae with a lamellar bone pattern confirmed by polarization microscopy (C).

Immunohistochemistry demonstrating robust staining for ET1 in the tumor cells (D). Original magnification, ×400.

Tests of renal, liver, thyroid, and parathyroid function were normal, as were blood levels for calcium, phosphorus, vitamin D, creatine kinase, fluoride, and tissue transglutaminase. Hepatitis C serology was negative. Urinary collagen type 1 cross-linked N-telopeptide was 75 (reference range, 19–63) and the serum total alkaline phosphatase was 167 U/L (reference range, 40–125). Serum collagen type 1 cross-linked C-telopeptide, serum bone–specific alkaline phosphatase activity, γ-glutamyl transpeptidase level, and serum 5′ nucleotidase level were normal.

Bone marrow biopsy specimens showed thickened bone trabeculae occupying >50% of the bone marrow space (Figure 2A-B). Polarized microscopy demonstrated normal lamellar architecture (Figure 2C). Staining for endothelin-1 (ET1) was clearly positive (Figure 2D).

Discussion

PMF is one of the chronic myeloproliferative disorders that are collectively characterized by clonal proliferation of myeloid cells with variable morphologic maturity and hematopoietic efficiency (1). Most patients with myelofibrosis are initially diagnosed in the overt fibrotic stage. In this stage, the bone marrow biopsy demonstrates clear-cut reticulin or collagen fibrosis (1).

Osteosclerosis refers to trabecular bone thickening, leading to increased skeletal mass. Osteosclerosis has been reported to occur with several metabolic disorders such as fluorosis, renal osteodystrophy, hypothyroidism, and hyperparathyroidism. It may also occur secondary to hepatitis C infection, Paget's bone disease, mastocytosis, lymphomas, and sarcoidosis. We excluded all of these disorders through appropriate investigations.

Osteosclerosis is often an accompanying feature of PMF. The radiographic findings of osteosclerosis were detected in 30–70% of patients with PMF (3). However, the densitometric measurements were rarely reported (4). In our study, we found a substantial increase in axial bone mass (+16.3% at the total hip) over the 2 years before the PMF diagnosis.

Histologically, the 2008 World Health Organization classification of hematolymphoid tumors describes the relationship between advanced myelofibrosis and bony changes as follows, “Osteoid seams or appositional new bone formation in bud-like endophytic plaques may be observed. In this osteosclerotic phase, the bone may form broad, irregular trabeculae that can occupy >50% of the bone marrow space” (2). Similar bone changes were identified in our case.

ET1 is a potent vasoactive peptide that signals through a pair of G protein–coupled receptors, termed the A (EDNRA) and B (EDNRB) type receptors (5). The endothelin system is essential in development, with mouse knockouts of both receptors and ET1 having a lethal phenotype (5). ET1's main site of synthesis is endothelium, where it is secreted in response to shear stress. It is secreted as a biologically inert precursor, big ET1, which is proteolytically cleaved by endothelin converting enzymes 1 and 2 or any of several other extracellular proteases to yield active ET1 (5). ET1, acting primarily via both EDNRA and EDNRB, in smooth muscle leads to vasoconstriction; however, binding to EDNRB in endothelial cells causes vasodilation (6).

Guise and colleagues (7) proposed a model to explain the interactions between a tumor cell and bone that are responsible for osteoblastic bone metastases formation. The basic outline of this model has been validated in metastatic prostate cancer and osteosclerotic breast cancer (8–10). Tumor cells housed in bone produce factors, such as ET1, which stimulate new bone formation via EDNRA on osteoblasts. This results in abundant and disorganized new bone matrix that is characteristic of osteoblastic metastases. Growth factors produced by the osteoblast have the potential to stimulate tumor growth as well as further increase tumor production of ET1.

Canonical WNT signaling is a critical pathway in osteoblastogenesis; Dickkopf homolog 1 (DKK1) is a secreted inhibitor of this pathway. New evidence supports a mechanism whereby the anabolic action of ET1 occurs through WNT signaling pathway activation consequent to decreased inhibitory DKK1 (11). We recently demonstrated that osteoblasts undergo autocrine ET1 signaling, with inhibition of the pathway resulting in decreased mineralization in an in vitro differentiation setting (12). We found that ET1 signaling reduced expression of both DKK1 and SOST, inhibitors of canonical WNT signaling. We also found that ET1 stimulated secretion of insulin growth factor 1, which may contribute to the survival and proliferation of nearby tumor cells.

Conversely, inhibition of the WNT signaling pathway leading to functional defect of osteoblasts is important in lytic bone disease that occurs in tumors such as breast and lung cancer and multiple myeloma. Tian et al (13) found an overexpression of DKK1 in myeloma microenvironment of patients with extensive osteolytic lesions. Moreover, it has been shown that DKK1 serum concentration levels correlated significantly with myeloma stage and the number of bone lesions.

Few papers in the literature have studied the underlying pathomechanism responsible for the osteogenesis associated with PMF (14). We were able to demonstrate abundant ET1 signaling in the bone of our patient. We believe, therefore, that ET1 is responsible for the osteosclerosis that developed with advanced myelofibrosis. Furthermore, despite the histological appearance of trabecular bone thickening, we observed normal lamellar architecture of the patient's bone on polarized microscopy that represented organized regulated bone growth. There are no prior reports of increased ET1 signaling in the setting of myelofibrosis, and we suspect that ET1 signaling may play a role in other osteosclerotic settings as well.