Analyzing the Significance of Genetic Variants in Acute Lymphoblastic Leukemia

Charles G. Mullighan, MBBS, MSc, MD

Charles G. Mullighan, MBBS, MSc, MD

Although most research in terms of genetic predisposition to acute lymphoblastic leukemia (ALL) has been done in the pediatric setting, some differences have been noted in patients with ALL across the age spectrum, according to a presentation by Charles G. Mullighan, MBBS, MSc, MD.

For example, highly deleterious germline variants, such as those in the P53mutation, have been determined as age-dependent disease factors. The P53mutation, in particular, has been associated with Li-Fraumeni syndrome, which can increase the risk of developing numerous cancer types and denotes the need to test family members for cancer risk as well. This mutation is found in pediatric patients with hyperdiploid ALL as germline events, whereas in adult patients the P53mutation appears as a sporadic event.

Overall, the genetic features of ALL vary between pediatric and adult patients, but more research is still necessary, according to Mullighan. An interesting area under investigation now, however, is in how the germline variants may affect treatment decision. Gene variations in a gene such as IKAROS (IKZF1) have been associated with a direct effect on responsiveness to chemotherapy in patients with ALL.

“I think we have a lot to learn about addition genes that predispose [patients] to leukemia,” said Mullighan, the deputy director of the Comprehensive Cancer Center, co-leader of the Hematological Malignancies Program, medical director of the St. Jude Biorepository, and the William E. Evans Endowed Chair at St. Jude Children’s Research Hospital, “and each of these will require a similarly detailed look at the functional effects of these variants.”

In an interview withTargeted Oncology,Mullighan discussed the role of genetic variants in understanding a patient’s predisposition to ALL. He highlighted findings in research that may help in utilizing the current understanding of these genetic variants to make treatment decisions for patients with ALL.

TARGETED ONCOLOGY:Can you provide us with an overview of your talk?

Mullighan:My presentation reviewed the current state of the art of understanding the nature of germline predisposition to ALL. It touched on several themes; the first was the type of genetic variants, whether they are silent and noncoding or whether they are non-silent variants that actually affect genes, whether they are present in families that are known to have a history of cancer or ALL, or as we increasingly understand, [whether] we can find similar variants in the germline of cases that are not known to have any evidence of familial history of cancer.

Another theme that was covered was the notion that there can be a very strong relationship between an individual gene or even mutations or variants within that gene and the features of the leukemia. Not all variants will generally cause an increasing risk for developing leukemia; some of the variants that have been identified predispose [patients] to developing a specific type of leukemia. They can be found in different combinations with tumor acquired or somatic events, and more recently, we have some evidence that particular genes when they are harboring alterations in the germline can influence drug response and outcome as well. This is indicating a new paradigm where variants need to be detected not only because they inform how we counsel our patients and their families, but also because they have implications on how leukemias might be treated or how well they will respond.

TARGETED ONCOLOGY:Can you expand more on the significance of genetic variants in ALL?

Mullighan:If we consider the first targeted genetic variants the cause associated with developing leukemia, these are the ones that have been recognized for the longest period of time, so a decade or more, and these are single nucleotide type polymorphism alleles, often intergenic or noncoding variants that have been identified typically from microarray-based genome-wide association studies. These studies have found 6 or more variants now that rather subtly increase the risk of developing leukemia, so they may be associated with a ratio, if you will, of less than 2. In isolation, there’s a relatively low risk of increasing susceptibility to leukemia.

Again, these variants are interesting because they often involve or are adjacent to genes that we know are very important in lymphoid biology or are also the targets of somatic genetic change, genes such as IKAROS,CDKN2A, another transcription factor called ERK. They are sometimes associated with particular subtypes of leukemia. A nice example is an association betweenGATA3and a high-risk leukemia like Ph-like (Philadelphia chromosome—like) ALL that’s associated with poor treatment response and treatment failure.

What is less well understood is how these genes are influencing the risk of leukemia whether they have some role, for example, in expanding the pool of cells that are susceptible to somatic mutations that drive leukemogenesis, or whether they directly influence the function of the gene they are most closely related to, for example the level of expression of a transcription factor. For some of these associations, there is some evidence that that’s the case and that they can directly affect the level of expression or function of the associated gene.

TARGETED ONCOLOGY:Are some of these disease characteristics more common in pediatric versus adult patients with ALL?

Mullighan:The answer here is somewhat limited in what I can tell you because most work in this area has been derived from studies of childhood leukemia. It’s the most common setting where leukemia arises and so far, there has been much less comprehensive genomic analyses of adult ALL, although that’s starting to change, in particular in work that we and others are doing now looking at many hundreds if not thousands of cases of leukemia across the age spectrum.

However, we do know there are some differences; firstly, there are very clear differences in the subtype of leukemia according to age. Those subtypes typically are defined by their somatic genetic changes, so the different rearrangements or types of aneuploidy are associated with different frequencies of leukemia across the age spectrum. Some of the highly deleterious germline variants, for example mutations in P53,are age-dependent. P53 germline mutations are the cause of many patients with Li-Fraumeni syndrome, familial syndrome of multiple different types of cancer. It is now recognized that P53 mutations are found in children as germline events with hyperdiploid ALL, that’s leukemia with losses of whole chromosomes. This form of leukemia is also seen in adults at quite a high frequency—approximately 15% of adult cases have the same form of leukemia. They also have P53 mutations but as sporadic events; they rarely, if ever, have those mutations as germline events.

There will likely be some subtlety to this; it’s not all children or not all adults. There is an age spectrum, and we know that the genetic features of ALL, the somatic features, shift quite appreciatively between young children, older children, adolescents, and older adults. My expectation will be that we will see differences in the relative role of germline variants as well according to age, but that’s still an ongoing subject of investigation.

TARGETED ONCOLOGY:Can you expand on how some germline variants may affect treatment responses or resistance, and thereby treatment decisions as well, in patient with ALL?

Mullighan:This is a very interesting area. I mentioned that some of the germline variants we know are associated with a subtype of leukemia. Some of those subtypes of leukemia are known to have a greater or lesser risk of treatment failure and relapse. Hyperdiploid ALL is another great example; typically, this is a form of leukemia that has a high rate of treatment failure and relapse.

What is not clear is that specific genetic variations can directly influence treatment responsiveness and outcome. One of the interesting studies that we were involved with that was published last year was describing variations in a gene called IKAROS (IKZF1), which encodes a lymphoid transcription factor. This gene is known to be mutated as a somatic event in particular subtypes of ALL, particularly those subtypes of leukemia that have kinase-driving alterations, like BCR-ABL. We and others have identified families that had IKAROS alterations in the germline, and then sequencing of large numbers of cases of sporadic leukemia, examining germline DNA, found that 1% to 2% of cases had non-silent variations in the IKAROS gene.

In a series of studies looking at the effects of these mutations, it was shown that many of these germline variations were firstly deleterious and directly affected the function of the transcription factor; they changed cellular behavior, specifically in that they caused the cells to become much more sticky or inherent to each other and to the bone marrow microenvironment; and finally, they directly affected the responsiveness of those leukemic cells to chemotherapy in a very mutation-specific way. Many of the mutations were more or less deleterious, and a few of them were not deleterious at all.

This, to us, is a very striking example. It’s 1 of the first where mutations have a direct effect on drug responsiveness. How broadly applicable this will be remains to be seen. I think we have a lot more to learn about additional genes that predispose [patients] to leukemia, and each of these will require a similarly detailed look at the functional effects of these variants.

TARGETED ONCOLOGY:Where do you see the research in this field headed?

Mullighan:Research is heading in several directions. Specifically related to germline predisposition, there are very intensive efforts from many groups looking at an order of magnitude greater cases in the genome-wide analyses approach because many of the variants that have been found have been very modest in their effect on leukemic predisposition; it’s very clear that to identify all variants you have to have not just hundreds but tens of thousands of cases. These efforts to understand the genetic basis of germline predisposition are firstly expanding in scale.

A second area of investigation is looking beyond the coding genome. When we consider non-silent variants, typically, those studies have been based on sequencing gene panels just looking at the coding region or exome sequencing again, just looking at the coding region of the genome, but we now know from work from my group and from others that some families have highly deleterious variants that are directly predisposing them to the leukemia; they are structural events, and they may not be found by just focusing efforts on the coding genome. The careful analysis of non-sequence variants at a structural variance is needed to identify these variants.

A third area of investigation is even more broad scale, and that is now integrating germline variants and somatic variants, both to understand the constellations of these variants that are needed to drive leukemia, but also the notion of which variants are most important in determining disease response. This is a large-scale undertaking that many are involved with. My group is part of a collaboration with colleagues at St. Jude’s Children Oncology Group and other groups as well, but many are engaged in this both in using more detailed sequencing, whole-genome sequencing, noncoding analysis of large cohorts, and cohorts that are clinically well annotated that are at some level relatively uniformly treated so we can make associations between genetic and clinical features and outcomes. Also, we have the scale and power to be able to integrate germline variants, somatic variants, and some of these features such as response and outcome.

St. Jude’s has really supported all of these studies, and they have been extremely collaborative between many institutions and colleagues.

TARGETED ONCOLOGY:What is your main take home message from this?

Mullighan:For those that are seeing patients in the clinic, the message is a very clear one; we are now truly in the genomic era for understanding our patients and how they should be diagnosed and treated. There is still some work to do to implement genomic testing from the clinical perspective, but this is becoming increasingly widespread. Even if we just think about somatic genetic alterations, these are extremely powerful determinants of initial subtyping risk stratification and also responsiveness to treatment. The previous, if you like, somewhat old-fashion methods, of genetic testing that were not able to resolve all of these genetic alterations are now not sufficient, and we need to move toward genomic-sequencing approaches whether it is just RNA sequencing or even a more comprehensive approach.

The second implication is that we must think of ALL as being a disease of 2 genomes, particularly in children and perhaps to some extent in adults that patients have alterations in their germline genome as well as the somatic events that interact with each other and are key determinants of developing leukemic response. There are some red flags, and there are some subtypes of leukemia, for example hyperdiploid leukemia as well as some others, where if you see that form of leukemia, there should be a high index of suspicion that patients should have a germline variant and that patients should be offered appropriate genetic counseling and testing where needed.