Insights on genetic origins of disease, treatment strategies

State-of-the-art genetic sequencing and analytics tools are being employed in medical research to gain an unprecedented understanding of disease mechanisms and treatments. In the search to improve understanding of disease, two studies describe the discovery of a novel genetic mutation that is commonly present in patients with myeloproliferative neoplasms, which may aid in diagnosis and treatment decisions. A third study uses similar technology to identify a genetic mutation that causes the very rare, inherited disease known as gray platelet syndrome. Turning the focus to the understanding of drugs, one study identifies the specific cellular mechanism of the commonly used drug lenalidomide and a second concludes that the therapeutic use of the standard-of-care treatment rituximab for pre-treated patients may be strengthened by combining it with a novel compound known as idelalisib. A final study explores the diagnostic criteria for pulmonary embolism and concludes that a simple age-adjusted algorithm may be beneficial in ruling out the dangerous clotting condition without the use of invasive imaging tests, a finding that may be particularly beneficial for elderly patients.

"The findings being presented during this year's Late-Breaking Abstracts session represent a number of particularly exciting areas of progress in hematology," said ASH President Janis L. Abkowitz, MD, of the University of Washington in Seattle. "From an improved understanding of the precise mechanisms in the body that drive disease or drug action, to the use of innovative technologies to better pinpoint disease activity, these data signal important advances that will help refine treatment strategies to create meaningful improvements for patients with a range of hematologic diseases."

Frequent Mutations in the Calreticulin Gene CALR in Myeloproliferative Neoplasms

Acquired mutations in the JAK2 and MPL genes have recently been associated with the development of myeloproliferative neoplasms (MPNs). The three classical MPN disease entities include polycythemia vera (PV), essential thrombocytopenia (ET), and primary myelofibrosis (PMF), a main feature of which is the overproduction of blood cells. While JAK2 and MPL mutations are valuable to accurately identify disease, they are only present in a segment of the population of patients with MPNs, leaving no explanation for the genetic origin of the disease among the remainder of the patient population.

In an effort to identify other genetic mutations associated with MPNs to improve diagnosis and inform treatment decisions, investigators performed advanced genetic sequencing of tumor tissue from six patients with MPNs. The team identified novel recurring mutations in the gene CALR encoding for calreticulin, a protein with a number of identified cellular functions. After identifying CALR mutations in the initial six patients, researchers screened more than 1,000 patients and found the CALR mutation in a majority of ET and PMF patients that did not have JAK2 or MPL mutations (67% and 88%, respectively). Notably, this mutation was not present in patients with certain leukemias and related blood disorders, indicating that it may be unique to MPNs. Patients with this genetic mutation generally had lower white blood cell counts and elevated platelet levels compared to patients with the JAK2 mutation. For both ET and PMF, patients with the CALR mutation had better overall survival than those with the JAK2 mutation. For ET patients specifically, those with CALR had a lower risk of thrombosis than those with the JAK2 mutation.

"Mutations in CALR are novel, specific molecular markers detected in the majority of MPN patients without JAK2 and MPL mutations. The identification of these mutations represents significant progress in our quest to fill the gap in knowledge that has remained about the genetic basis of these diseases," said study author Thorsten Klampfl, PhD, of the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences in Vienna. "This knowledge will not only help us to improve diagnosis in a greater portion of patients with MPNs, but it will also support future insights into the molecular basis of the disease as a whole. Furthermore, the mutations that we detected in CALR generate unique variants of the protein that might serve as targets for the development of disease-specific therapies."

The Genomic Landscape of Myeloproliferative Neoplasms: Somatic CALR Mutations in the Majority of JAK2-Wildtype Patients

In a separate study, investigators performed the first large-scale characterization the genetic mutations in myeloproliferative neoplasms by genetic sequencing and identified novel mutations in regulators of gene activity not previously associated with these diseases. Importantly, CALR mutations were identified as a novel biomarker in the vast majority of patients without JAK2 or MPL mutations. Of the patients screened, 97 percent showed a mutation in one of the three genes (JAK2, MPL, or CALR). The team screened for CALR mutations in a follow-up study of more than 3,000 patients with a range of hematologic malignancies or healthy controls. Overall, CALR mutations were found in 70-84 percent of patients with JAK2-unmutated myeloproliferative neoplasms, 8 percent of myelodysplasia patients, and occasional samples of other myeloid cancers but were not found in patients with lymphoid cancers, solid tumors, or in healthy controls. Compared with patients with the JAK2 mutation, patients with the CALR mutation had higher platelet counts and lower hemoglobin levels, suggesting differences in disease manifestation depending on the genetic mutation.

"This discovery of CALR mutations has major implications for how MPN patients can now be diagnosed, as we will be able to quickly and accurately diagnose these patients through a blood test in the clinic in a similar manner to how JAK2 testing has had a great impact on diagnosis worldwide," said study author Jyoti Nangalia, MB Bchir, FRCPath, of the University of Cambridge, Wellcome Trust Sanger Institute, United Kingdom. "Furthermore, by identifying a mutation in a biological pathway that has not previously been associated with mutations, we have provided the research community with a new target for the development of therapies to improve treatment of MPNs over time."

A Dominant-Negative GFI1B Mutation in Gray Platelet Syndrome

Patients with gray platelet syndrome (GPS) have reduced numbers of platelets that are atypically large and have a gray appearance, caused by a lack of alpha-granules essential to normal platelet activity. This rare, hereditary bleeding disorder can be accompanied by myelofibrosis and spontaneous bleedings.

To identify the genetic mutation that causes this disease, investigators analyzed samples of genetic material from members of a large family from the Netherlands with autosomal-dominant GPS. Following whole-genome analyses, the research team focused their investigation on mutations of one specific gene (growth factor independence 1B, or GFI1B) because of its role in regulating the genes responsible for the development of the cells that produce platelets (megakaryocytes) and therefore affect bleeding. Sequencing analyses uncovered a GFI1B mutation that appeared to be linked to the GPS disease that inhibited the function of the normal GFI1B gene. Researchers next evaluated the megakaryocytes of a mouse model expressing the GFI1B mutation and observed that they were developing abnormally (either too large or small) and were dividing too quickly, indicating a developmental defect caused by the inhibition of the normal gene. Furthermore, they observed that the mutation caused dysplastic features in megakaryocytes similar to what would be observed in human GPS cells.

"In addition to identifying a highly valuable new target for therapeutic development in GPS, this research confirms the central role of the GFI1B gene as a master regulator of megakaryocyte and platelet production -- an insight that could be useful in research on a wide range of platelet disorders." said Bert Van der Reijden, PhD, of the Radboud University Medical Center in Nijmegen, Netherlands.

Age-Adjusted D-Dimer Cut-Off Levels to Rule out Pulmonary Embolism: A Prospective Outcome Study

A simple blood test evaluating levels of D-dimer -- a small protein fragment present in the blood after a clot is dissolved -- is instrumental to the diagnosis of blood clots and can rule out pulmonary embolism (PE, a dangerous condition that occurs when a clot detaches and travels to the lungs, blocking blood flow) without the need for imaging tests. While the test is very useful in outpatients younger than 60, it is less useful for more elderly patients, as it has lower specificity in this sub-group of patients. This limitation of the test has prompted researchers to develop an age-adjusted D-dimer level -- determined by multiplying the patient's age by 10 in patients older than 50. This new cut-off may improve the proportion of elderly patients in whom PE may be excluded on the basis of D-Dimer measurement.

To evaluate the new age-adjusted estimate, investigators evaluated more than 3,000 patients who visited emergency rooms in several countries for suspected PE (average age of 62) over a three-year period. Those who were found to have a D-Dimer level between the usual cut-off of 500 micrograms/L (µg/L) and the age-adjusted value were neither imaged nor treated with anticoagulants and were followed for three months. In total, PE was confirmed in 19 percent of the patients, and a large segment of patients were considered to have a low or moderate clinical probability of PE (2,898 patients). Of the 331 patients in whom D-Dimer measurement was between the usual cut-off of 500 µg/L and the age-adjusted value, seven patients died of a cause other than PE and seven underwent further testing during the follow-up period for suspected venous thromboembolism (VTE). Of these suspected VTE cases, only one was considered positive, giving the test a failure rate of 0.3 percent (1/331). Among the 766 patients aged 75 years or older, of whom 673 had a non-high clinical probability, use of the age-adjusted cut-off (instead of the 500 µg/L cut-off) increased the proportion of patients in whom PE could be excluded on the basis of D-Dimer from 43/673 (6.4%) to 200/673 (29.7%) without any additional false-negative test.

"Our data suggest that this age-adjusted cut-off has significant potential in clinical practice to rule out suspected PE with a similar safety than the usual cut-off set at 500 µg/L," said study author Marc Righini, MD, of the Division of Angiology and Hemostasis at the Geneva University Hospital in Switzerland. "When combined with clinical probability, the age-adjusted cut-off could increase the proportion of elderly patients in whom PE can be excluded without the use of more invasive imaging tests."