Cancer Diagnostics

Detecting molecular genetic abnormalities guide outcomes in patients with acute myeloid leukemia

An overabundance of white blood cells and a diminished supply of red blood cells and platelets can signal acute myeloid leukemia (AML). Another indicator of this cancer of the blood and bone marrow, associated with variable growth but inevitable bone marrow failure, is the presence of blast cells, or immature white blood cells, normally found in bone marrow but rarely seen in the peripheral blood.

Leukemia can be detected with a blood test, however, a definitive diagnosis usually requires a bone marrow aspirate and biopsy in which a needle is used to remove bone marrow samples from the posterior iliac crest.

In order to differentiate AML from other types of leukemia and to classify the specific disease subtype, the blood or marrow sample is examined via light microscopy flow cytometry and immunohistochemical stains. In recent years, genetic approaches have emerged as a central component in the diagnosis and classification of AML.

“Molecular tools complement the cytogenetic and flow cytometric methods which have long been used to diagnose and characterize AML,” explained Jonathan E. Kolitz, MD, associate chief, hematologic oncology, North Shore-LIJ Cancer Institute, and professor of medicine at Hofstra North Shore-LIJ School of Medicine. “The principal method used for risk assessment has been cytogenetics.”

In addition to the standard karyotype, molecular diagnostic tests have emerged as important tools to detect risk, monitor disease and guide the treatment strategy to ensure best outcomes. According to Kolitz, about half of patients diagnosed with AML have normal karyotypes yet these patients have disparate outcomes. Among patients with normal karyotypes, the two most common mutations are found in the FMS-like tyrosine kinase 3 (flt-3) and Nucleophosmin (NPM-1) genes, with almost half of patients having one or both mutations, he told ADVANCE.

“Depending on the presence or absence of molecular genetic abnormalities, such as mutations in the flt-3 or NPM-1genes, relapse-free and overall survival rates are markedly affected,” Kolitz said. “Mutations in genes such as flt-3, for example, confer an unfavorable prognosis while NPM-1 mutations are associated with more favorable outcomes.”

According to Kolitz, the favorable impact of NPM-1 mutations is countered by the negative effect of flt-3 mutations, which can occur as an internal tandem duplication of the gene or as a mutation in its tyrosine kinase domain.

Molecular Diagnostic Tests/Assays

For patients with AML and normal cytogenetics, the presence of mutations in FLT3, NPM1, and CCAAT/enhancer-binding protein alpha (CEBPA) genes can hold prognostic significance. AML diagnostics are used to define a patient’s genetic profile in order to guide and optimize therapeutic approaches.

Nucleotide sequencing is widely considered the gold standard for CEBPA heterogeneous mutational screening and Polymerase chain reaction (PCR)-based fragment length analysis is relied on for its speed and sensitivity in detecting FLT3 and NPM1 mutations.

According to Kolitz, experts are paying increasing attention to detecting abnormalities in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) genes. “Multiple gene panels have been proposed to help refine prognostic analysis; the minimum number of genes needed to provide the most accurate and clinically useful predictive model remains to be defined,” he observed.

Once a cytogenetic and molecular profile is defined, Kolitz explained, the findings can be used to inform in some instances choice of therapeutics, in particular decisions regarding the need for allogeneic hematopoietic stem cell transplantation. Novel therapeutic agents are in development to block the negative effects of gene products such as mutated flt-3.

New Research Studies and Tests

The most important change in the management of patients with AML is the stratification of risk profiles based on cytogenetic and molecular genetic findings, Kolitz explained. By complementing cytogenetics and molecular genetics, DNA sequencing technologies may contribute to refining prognostic models of AML and bring the industry closer to adopting a personalized medicine model.

“For now, we have our hands full ensuring that we recognize the most important known genes involved in leukemogenesis and develop treatment strategies appropriate for each patient,” Kolitz said. “Even as we can foresee the likelihood that yet more individualized treatment models will make inroads into the management of patients with AML.”

At present, assignment of risk helps determine which patients are best served by continuing intensive chemotherapy or proceeding with a search for a suitable donor for allogeneic transplantation. “There are constellations of prognostic findings that are so adverse as to justify using investigational strategies whenever possible,” Kolitz said. “In general, all patients with AML should be encouraged to participate in well-designed investigational protocols.”

Kolitz believes the field needs to expand and refine the prognostic model so that a suitable panel of clinically useful genetic findings can be used to assign risk to patients in a timely manner. Additionally, it’s important to provide clinicians with potentially “targetable” findings, including available and emerging therapeutics that could be incorporated into treatment plans.

According to Kolitz, techniques exist-and are in development-to introduce genetic material in various packages into patients, leading to the synthesis in vivo of normal gene products able to reverse the effects of loss of function mutations. “Ultimately, one can foresee the possibility of more extensive genetic profiling and even sequencing providing yet more powerful diagnostic and therapeutic paradigms,” he stated.

Orally available inhibitors of flt-3 as well as another mutated enzyme seen in AML, IDH2, have demonstrated unequivocal and potent anti-leukemic effects in the clinic, Kolitz shared. The flt-3 inhibitors include midostaurin, quizartinib and AG-221, an inhibitor of mutated IDH-2 that was recently granted fast-track designation for approval in IDH2-mutated AML by the U.S. Food and Drug Administration.

Gene mutations can also be used as markers for residual disease following therapy. “PCR-based assays exist and are being developed to aid in defining which patients have minimal residual disease and who consequently are at risk of relapse,” Kolitz said. “The early identification of such patients can lead to preemptive treatment approaches.”