Research in acute leukemia reveals genetic markers that alone and in combination can help determine prognosis and drive treatment decisions in patients with cytogenetically normal AML.
By Darrell E. Ward, Photograph by Roman Sapecki
The cancer cells in about half of patients with acute myeloid leukemia (AML) show characteristic chromosome damage that includes inversions, deletions and translocations—bits of chromosomes are reversed, lost or shifted from one chromosome to another.
|Clara D. Bloomfield, MD|
Distinguished university professor and the William G. Pace III Professor in Cancer Research at the Ohio State University
At first, these changes were assumed to be caused by the cancer and of no clinical importance. Then research showed just the opposite—when part of one chromosome broke free and stuck to another, it could fuse two genes together in ways that disrupted normal cell proliferation, differentiation and death. Other changes caused the loss or silencing of genes that protect against cancer.
Further research, including seminal work by Clara D. Bloomfield, MD, an internationally renowned leukemia specialist at the Ohio State University Comprehensive Cancer Center (OSUCCC), revealed that the type of chromosome damage present often predicted a patient’s response to therapy. This work showed that cytogenetic abnormalities could identify patients who have a low risk of relapse and are likely to be cured by standard therapy, versus those who have a high risk of relapse and require aggressive or experimental therapy.
But 40 to 45 percent of AML cases lack these cytogenetic abnormalities. Nonetheless, some patients with cytogenetically normal AML (CN-AML) respond well to therapy, while others quickly relapse and die.
“We could cure 40 percent of these patients, but until recently we’ve been unable to distinguish those individuals from the 60 percent we cannot cure,” Bloomfield says.
Clearly, although the chromosomes look intact in this AML subtype, they are far from normal.
In the early 1990s, Bloomfield and a group of researchers, many of whom are now at the OSUCCC, found the first molecular alteration in CN-AML. Since then, many studies by Bloomfield and her colleagues, and by others in the field, have revealed that CN-AML harbors multiple gene mutations, gene-expression differences and microRNA expression changes that may have prognostic and therapeutic importance.
|The Flt3 ITD can add three to 33 or more amino acids to the FLT3 protein, causing the constant activation of this tyrosine kinase receptor. A less common point mutation in this gene has a similar effect.|
“This work, along with future findings, will likely have a major impact on the clinical management of cytogenetically normal AML and on personalizing the care of acute leukemia patients,” Bloomfield says.
She notes that it is already becoming possible to separate CN-AML patients into prognostic subgroups, and that this is leading to improved therapy and the development of targeted agents. For example, both the 2008 World Health Organization AML classification and the National Comprehensive Cancer Network Clinical Practice Guidelines for AML now incorporate molecular markers.
Discovery of the first mutation in CN-AML grew from the investigation of a potent anomaly seen in cytogenetically abnormal AML. In the early 1990s, Bloomfield and Carlo M. Croce, MD, both internationally known cancer gene researchers at the OSUCCC, teamed up with Michael A. Caligiuri, MD, now director of the OSUCCC, and others to study the role of chromosome 11 in AML development.
About 3 to 4 percent of all AML cases have translocations that involve chromosome 11. The break almost always occurs on the long arm at band 23 (i.e., 11q23), and the wayward fragment can combine with other chromosomes in over 80 different ways. In rare AML cases, trisomy 11 (an extra copy of the chromosome) is the only abnormality present, and it signals a poor prognosis.
Of the genes spanning the 11q23 region, the researchers focused on the mixed lineage leukemia gene, or MLL (also known as ALL1). Within that gene, they discovered a novel mutation, an internal partial tandem duplication, or PTD, in which a piece of the gene is repeated, like a stutter in the DNA.
Susan Whitman, PhD
They found this odd mutation in three of four AML patients with trisomy 11, but even more exciting, they found it in two of 19 CN-AML patients included in the study.
Additional research by the investigators over the next four years suggested that MLL PTD, found in about 8 percent of CN-AML patients, could promote leukemia development even without a chromosomal translocation, and that it could help predict patient outcome.
For that study, Bloomfield and her colleagues screened 98 diagnostic bone-marrow samples from CN-AML patients and found the mutation in 11 cases. These patients relapsed in seven months, on average, while those with a normal MLL relapsed in 23 months.
“These findings suggested for the first time that molecular changes in AML with normal cytogenetics could have prognostic significance,” Bloomfield says.
The MLL PTD mutation seemed to define a subset of CN-AML patients who did more poorly following standard chemotherapy, and who might benefit from more intense therapy, she says.
In fact, a follow-up study by Susan P. Whitman, PhD, an OSUCCC research scientist, along with Bloomfield and a group of colleagues showed that more intense therapy at least neutralized the negative effect of the mutation.
This 2007 study evaluated 238 adults with CN-AML under age 60. All the patients had participated in Cancer and Leukemia Group B (CALGB) clinical cooperative group trials and had received aggressive induction therapy. Twenty-four (10 percent) of the patients carried the MLL PTD mutation, and 22 of these achieved complete remission. These patients then received aggressive consolidation therapy, usually an autologous stem-cell transplant.
In less than a year and a half, 13 of those patients relapsed, but nine (41 percent) remained in remission when the study ended, with disease-free periods ranging from two to almost eight years.
“Patients with MLL PTD who received intensive consolidation therapy during first remission did better,” says Whitman, the study’s first author. “It didn’t result in a high cure rate, but it did remove the adverse impact of the mutation. Eight to 10 years ago, nearly 100 percent of these patients relapsed and died within two years.”
The MLL PTD is a duplication of the beginning region of the MLL gene. The duplicated portion is inserted into the MLL gene, giving rise to an elongated protein that has altered function. The duplication occurs as a result of aberrant DNA replication that recombines repetitive pieces of the MLL gene.
The OSU investigators had discovered this first abnormality in CN-AML patients in 1994, and by 2007, they had shown that autologous transplantation could eliminate its adverse prognostic significance.
Japanese researchers discovered the second prognostic marker in CN-AML, a mutation of FLT3, a gene involved in the survival and differentiation of multipotent hematopoietic stem-cells. Mutated FLT3, or FLT3 ITD, is also a gene rearrangement, an internal tandem duplication that occurs in about a third of CN-AML patients, according to Guido Marcucci, MD, an AML specialist and translational researcher at Ohio State.
Initial reports linked the mutation to a poor prognostic outcome. In 2001, Whitman, Bloomfield and Caligiuri led a CALGB study that confirmed this and yielded an unexpected finding. The investigators examined blood samples from 82 CN-AML patients treated with dose-intensive regimen through a CALGB trial. Twenty-three of the cases had a mutated FLT3; the remaining 59 had normal copies of the gene. Comparing the outcomes of the two groups showed that those with the mutation relapsed sooner, but there was no difference in overall survival.
To try to understand why, Whitman and her colleagues divided the study patients into three genetic groups: those having two normal FLT3 genes (59 cases), those having one mutated and one normal FLT3 (15 cases), and those with a mutated FLT3 and no normal copy of the gene (eight cases).
This study presented a new picture of overall survival. After one year, 74 percent of the patients with two normal FLT3 genes were alive, as were 65 percent of patients with one normal and one mutated FLT3. But of the patients with no normal copy of the gene, only 13 percent—one patient of the eight—was alive after one year.
“Patients having only the mutated copy of FLT3 also had significantly poorer disease-free survival,” Whitman says. “We hypothesized that these AML cells have a growth advantage, resulting in a more aggressive form of the disease.”
An international phase III trial, CALGB 10603, began in April to learn if the FLT3 inhibitor, midostaurin, added to standard chemotherapy, will increase survival in these patients.
The Ohio State University is the trial’s designated screening center for determining the FLT3 mutational status for all North American cases. Marcucci directs the laboratory and is the trial’s principal investigator at Ohio State.
A Marker of Expression
In addition to gene mutations, changes in expression of certain genes may also be prognostically important. In 2002, cytogeneticist Krzysztof Mrózek, MD, PhD, Bloomfield, and two OSUCCC colleagues made a discovery that enabled identification of a gene whose level of expression has prognostic significance in CN-AML.
Krzysztof MrÓzek, MD, PhD, Cytogeneticist and AML researcher
Using spectral karyotyping (SKY), the researchers examined samples of 29 adults with AML and abnormal, complex karyotypes—those with three or more chromosome abnormalities. These patients generally have a very poor outcome. SKY labels all the pairs of chromosomes so that each chromosome pair is depicted in a different color (see illustration on page 20).
Among their findings, the researchers discovered an amplified region on the long arm of chromosome 21 in eight of eight patients who appeared to have only a single copy of the intact chromosome 21. In all these cases, SKY analysis showed that material from the seemingly missing chromosome 21 was hidden in chromosomes with abnormal structure, such as ring or marker chromosomes.
Further investigations of the amplified region by Albert de la Chapelle, MD, PhD, and colleagues led to the ETS-related gene, ERG, which encodes a protein critical for regulating proliferation, differentiation and apoptosis. High ERG expression was seen not only in complex karyotype AML but also, surprisingly, in some patients with CN-AML. The question arose—did the level of ERG expression have consequences for these patients?
Marcucci, Bloomfield and their colleagues discovered the answer in a series of studies published in the Journal of Clinical Oncology. The work involved leukemic cells from patients under age 60 who had participated in CALGB trials.
A 2005 study of 84 CN-AML patients showed that high ERG levels signal a poor outcome when considered alone and in combination with other markers. The researchers stratified the cases into four groups, ranging from high to low ERG expression levels, and looked at relapse rates: 81 percent of the high-ERG expressers relapsed after five years versus 33 percent of patients in the three other groups. In combination with MLL PTD, high-ERG expressers were four times more likely to relapse than the patients with low ERG.
An example of hidden high-level amplification of chromosome 21 in a complex karyotype detected using spectral karyotyping (SKY) in a patient with de novo AML.
A: Metaphase cell with G-banded chromosomes, in which an arrow denotes a large ring chromosome shown by SKY to be composed of at least six segments from 21q [r(21)], and an arrowhead identifies one normal copy of chromosome 21.
B: The same metaphase cell with chromosomes shown in SKY classification colors.
C: Same metaphase cell shown as a SKY karyotype in classification colors.
This showed that high-ERG expressers lived about 1.2 years following treatment, while the other three groups had not reached their average survival by the study’s end. When other markers were also considered, patients with low ERG expression and low BAALC expression did best, with 70 percent surviving after five years.
The researchers then investigated the clinical implications of ERG expression in a study of 148 CN-AML patients. It showed that high-ERG expressers had poorer disease-free survival than low expressers, with estimated rates of 42 percent and 72 percent, respectively, after two years. This remained unchanged by favorable gene markers such as normal FLT3 and a mutated NPM1. High ERG expressers also had fewer complete remissions and worse event-free survival.
“High ERG activity seems to cause an aggressive form of AML that requires intense therapy, such as an allogeneic stem-cell transplant,” Marcucci says.
Marcucci noted that ERG also plays an important role in prostate-cancer development, although through a different mechanism. “This makes ERG interesting,” Marcucci says. “It is an example of how studies like ours in AML may also improve the understanding of other types of cancer.”
Changes in microRNA, short stretches of RNA 19 to 25 nucleotides long that regulate gene expression, may also one day provide important prognostic markers and therapeutic targets in AML.
Marcucci and Bloomfield led a 2008 CALGB study published in the New England Journal of Medicine investigating microRNA levels and clinical outcome in CN-AML patients with high-risk molecular features. This subgroup—which had mutated FLT3, unmutated NPM1, or both—encompasses about two-thirds of CN-AML patients and one-third of all AML patients under age 60.
The researchers identified a set of 12 microRNAs that distinguished between two groups of high-risk patients, those with an estimated five-year event-free survival rate of 11 percent and another with a rate of 36 percent. The study also suggested that low expression of the microRNA-181 family contributes to an aggressive leukemia subtype.
“These findings suggest that microRNA profiling might be prognostically significant and independent of the influence of gene mutations,” Marcucci says.
OSUCCC translational, clinical and basic investigators have been at the forefront of discovery of many of gene alterations in CN-AML or in determining their prognostic or therapeutic relevance (see sidebar).
“Just as chromosomal alterations divide AML patients into high- and low-risk groups, we are now using molecular alterations to define risk groups for AML patients with normal cytogenetics,” Bloomfield says. “This research is benefiting our patients now, and it should lead to targeted therapies that will improve and personalize their care in the future.”
Forever In New Genes
Albert de la Chapelle, MD, PhD, led research that discovered the brain and acute leukemia, cytoplasmic (BAALC) gene, the first prognostic factor for CN-AML based on expression change. Later OSUCCC research showed that this gene’s over-expression in AML indicates a poor prognosis, suggesting that the gene plays a key role in leukemia.
GUIDO MARCUCCI, MD
Hematologist and oncologist and associate professor of Internal Medicine
Clara Bloomfield, MD, and Guido Marcucci, MD, led research showing that mutations in the Wilms tumor 1 (WT1) gene in CN-AML may forecast a short remission (nine months on average) and recurrent disease that resists therapy.
Bloomfield, Marcucci and colleagues recently evaluated the prognostic significance of CEBPA mutations. They showed that pretreatment testing for CEBPA mutations in CN-AML predicts better disease-free and overall survival independently of other molecular and clinical prognosticators. The findings were published Oct. 20 in the Journal of Clinical Oncology.