ALL-kids

Recently identified genetic alterations in childhood ALL and the TARGET initiative (2011)

Update 2015: Moorman's article.

Update 2013: More chromosomal alterations in ALL are found: Charles Mullighan's article gives a good summary of the updated lists of alterations.

Also: for up-to-date information, visit the publications listing on the TARGET website:


(2011) In the last several years, several sub-types of childhood ALL exhibiting specific genetic alterations or gene expression profiles have been newly identified as "poor-prognosis" childhood ALL. These sub-types of ALL were discovered by biotechology assays called genomic profiling and gene expression profiling techniques. These techniques can examine a cells DNA and the expression of its genetic material in detail unheard of a few years ago. Parents of children with ALL will have new terms to learn, just as we learned about phenotypes, cytogenetics, and MRD.

My goal in this current article is to provide a little history, describe the techniques for finding new sub-types, name the discovered alterations, tie in targeted therapies, and let parents know about the TARGET initiative. The journal article that inspired me to study this topic is the review article by Stephen Hunger et. al. (epub February 2011) on improving outcomes for HR-ALL (high risk ALL). This article made me realize that the search for mutations in ALL that could be treated by targeted therapies is really stepping into gear. In the process, scientists are learning more about the biology of ALL cells and how they become cancer cells.

History

Targeted therapies are chemotherapy drugs that affect the cancer cells but not normal cells. The swing to the search for targeted treatments for cancer was given a boost in the late 1990s by the development of imatinib, or "Gleevec", for the treatment of chronic myelogenous leukemia (CML). CML is characterized by having the Philadelphia chromosome mutation (t(9,22) or BCR-ABL1), resulting in the expression of a form of the ABL1 protein (a tyrosine kinase) that is over-active. This in turn causes the cell to grow out of control: cancer. Researchers looked for a drug that would specifically inhibit BCR-ABL1 and leave cells with normal ABL1 alone.

The researchers screened a lot of compounds, and eventually developed the drug imatinib. It was tested in clinical trials, and found to successfully treat CML. This targeted therapy for cancer soon became standard treatment for CML, turning the cancer into a chronic condition rather than a death sentence. Brian Druker, Nicholas Lydon, and Charles Sawyers are credited for the discovery of imatinib; if you want to know more about this great story read:

CML is not the only cancer characterized by BCR-ABL1. Among others, a sub-type of childhood ALL called Ph+ ALL has the same mutation, and the same overactive tyrosine kinase. Clinical trials have shown that imatinib added to a combination chemotherapy strategy greatly improves the prognosis for children who have Ph+ ALL. The results of these studies are discussed in the Hunger article (also see my article on Targeted Therapies for Childhood ALL).

As an aside, chemotherapy drugs that affect only cancer cells include not only the new generation of "designed" drugs, but one drug that has been used in the treatment of childhood ALL since the 1980s. This drug is aspariginase - and it made a large difference in the success of childhood ALL treatment protocols.

Searching for sub-types of childhood ALL

Refresher: ALL mutations we know about and how they are found

Parents of children with ALL learn that ALL is a diverse disease, and that certain types have a better or worse prognosis. The first thing we usually learn about our child's ALL is whether it is early pre-B, pre-B, B-cell, or T-cell . This is the cells phenotype, and is determined by antigens that the cell displays on its surface. Phenotype is not a mutation, but it helped tell the doctors how to treat the leukemia.

In the last decades of the twentieth century, cytogenetic testing detected translocations, deletions, diploidy, and trisomies in the chromosomes of a patient's leukemia cells. These are mutations in the cells DNA. The Ph+ mutation was one of the first studied. In Ph+ genomes, parts of two chromosomes, 9 and 22, swap places, as determined by FISH assays. Note that before Ph+ leukemia was known to have a defective tyrosine kinase ABL1 protein, this high-risk type of ALL was known for years only by its translocation, t(9,22). The same is becoming true of other sub-types of ALL identified first by their translocation, and now by a set of letters that designate more information about the mutation, as determined by newer techniques. The table below shows a few of the other translocations that parents have become familiar with. Some of these translocations are known to be good or poor prognostic factors, but many are simply observed and denote neither good nor poor prognosis.

parent-familiar name newer name
hyper- and hypodiploidy  
t(12;21) ETV6-RUNX1, used to be called "tel-AML-1 fusion"
trisomies 4 and 10  
t(1;19) E2A-PBX1
t(1;19)(q23;p13) TCF3-PBX1
t(11;19)(q23;p13) and t(4;11)(q21;q23) MLL
t(9,22) BRC-ABL1

Together, phenotype and cytogenetics, as well as biological and clinical factors such as age and white blood cell count at diagnosis and quick or slow response to therapy, shaped the COG ALL trials into "risk adjusted therapies". In the 2000s, very high risk ALL and relapsed ALL is treated by aggressive therapy at about the most toxic level possible without killing the patient. New treatments are needed and the sections below outline the directions that childhood ALL researchers are taking to find them.

New techniques for studying cancer cells

Today, new techniques for studying DNA are moving out of university research labs and into the commercial medical research and treatment facilities. A benchmark of this progress in the study of genomes is the sequencing of the entire human genome by the Human Genome Project (2000). As a result of these advances, researchers have a lot more tools to study a cells DNA than they used to.

Two types of assays that parents will become familiar with in the next decade are called genomic profiling and gene expression profiling. The techniques study genomes not only in great detail and but can be done on many samples in a short period of time. Use of these techniques has really taken hold in the last few years in the study of many types of adult cancers. But for once, childhood ALL is NOT being left behind. (Read on to the TARGET initiative, later in this article.)

Already, genomic profiling and gene expression profiling have identified several sub-types of childhood ALL that are high risk. First, a little about the assays.

Genomic Profiling

Genomic or genetic profiling detects small changes (mutations) in the sequence of DNA, as opposed to translocations of entire genes. The mutations are either deletions of a section of the DNA or changes in nucleotide sequence, less than 1 Mb in size, and are "sub-microscopic", unlike FISH assay results. The names of the genomic profiling methods discussed in the Hunger review article are:

While I do have an intellectual curiosity about these techniques, at this point in time I am not going to research and re-interpret these methods for lay readers. If you are interested, I suggest you Google the terms, or look them up on Wikipedia.

Gene Expression Profiles

Not all of the DNA in a cell is expressed, meaning, not all the DNA is translated into mRNA and hence to proteins. Gene expression profiling techniques determine which genes (DNA) are being expressed in a sample of cells. These tests can detect the expression of thousands of genes in one assay. The unique pattern of gene expression for a given sample of cells is referred to as its molecular signature.

Gene expression profiling is used in conjunction with genomic profiling (and other observations) to identify new subtypes of ALL. What this means is this. They indentify an alteration using genomic profiling, and then run gene expression profiles on the same ALL sample. Doing this, they found molecular signatures that are the same, even if though alterations are different. For instance, researchers identified an alteration that has the same molecular signature as Ph+ ALL, even though it has a different mutation. Moreover, they found that this type of ALL was at high risk of relapse. (More on this later.)

Recently identified mutations in childhood ALL

Using the new techniques, researchers recently identified over 50 recurring regions of genetic alteration in childhood ALL. (Ref.) The alterations are clustered in a few genes, as listed in the table below. The genetic alterations target genes and pathways that are involved in cell growth or tumorigenesis.

alterations in gene area: genes/pathways:
PAX5 lymphoid development
IKZF1 lymphoid development
EBF1 lymphoid development
ERG transcriptional regulator
ETV6 transcriptional regulator
TBL1XR1 transcriptional regulator
CDKN1A/B tumor suppressors, cell cycle regulators
RB1 tumor suppressors, cell cycle regulators
PTEN tumor suppressors, cell cycle regulators

 

All of the gene alterations in the table (above) have not been correlated with low- or high-risk ALL (yet). However, a new type of ALL that researchers are calling "BCR-ABL1-like" ALL has been identified. This type of ALL is associated with IKZF1 deletions. IKZF1 encodes the early lymphoid transcription factor IKAROS, and is a a hallmark of high-risk ALL. IKZF1 deletions are present in 80% of the cases of Ph+ ALL. Gene expression profiles of cases of ALL that do not have BCR-ABL (and thus are called Ph-negative) but do have IKZF1 deletions are similar to gene expression profiles of Ph+ ALL. They call this "BCR-ABL1-like" ALL, and it is a high-risk type of ALL, with a history of poor treatment outcomes.

One-third of BCR-ABL1-like ALL cases also have rearrangements in CRLF2 and/or JAK1 and JAK2. CRLF2 encodes "cytokine receptor like factor 2" and JAK refers to a family of kinases. ALL cases with mutations in CRLF2 and JAK that are not DS-ALL (Downs syndrome ALL) are high-risk. Following is a more detailed list of recurring gene alterations in BRC-ABL-like ALL:

These genetic alterations are connected with the reason why the cells are continually growing out of control, because they are in areas of the genome with known cell pathway controllers, e.g., kinase signaling (among others). Some sub-types are more likely to have multiple mutations than other types. All of this information helps researchers understand the leukemogenesis of ALL. Leukemogenesis is defined as "the onset, development, or progression of leukemia" or more technically as "a process in which successive transformational events enhance the ability of hematopoietic progenitor cells to proliferate, differentiate, and survive". (The Free Dictionary.)

How recent findings might translate into better treatments for childhood ALL and the TARGET initiative

The first sentence in the summary section of the Hunger article reads: "To improve outcome in ALL we must develop mechanisms to identify patients at HR of early BM relapse at initial diagnosis and develop new molecularly targeted therapies for these patients."

This is what it is all about: (1) identify newly-diagnosed patients who have high risk ALL and (2) treat these patients with targeted therapies, just as Ph+ patients are now treated with imatinib. As parents, our next question might be: "Is this happening?" And happily, the answer is "Yes!" And this is because of the TARGET project.

TARGET is the acronym for Therapeutically Applicable Research to Generate Effective Treatments. TARGET is under the Office of Cancer Genomics (OCG), which is in turn under the National Cancer Institute (NCI). OCG was initiated in 1996, but the comprehensive effort announcement and the first entry on the website is 2005 (read the press release of the OCG launch). TARGET, launched March 2008, studies ALL, AML, neuroblastoma, osteosarcoma, and high-risk Wilms. The initiative includes COG, St Jude, UNM, and NIH researchers. It is an exciting collaboration that gathers samples of childhood cancers for analysis.

According to the TARGET website [2011], TARGET employs modern genomics technologies to identify therapeutic targets in childhood cancers. They use "high-throughput genomics and nucleotide sequencing to identify genes and pathways that are consistently altered in HR-ALL."

The TARGET website lists both newsroom lay articles and journal publications that come from the researchers contributing to the project. For instance, the IKZF1, CRLF2, and JAK mutation discoveries are all listed on the website (2011). Keep checking the following links for the latest research reports:

Members of the COG ALL committee are working on assessing the frequency of the IKZF1, CRLF2, and JAK mutations in real time in patients enrolled on COG (and other) clinical trials, in order to identify patients who might benefit from targeted therapies. (Hunger article, conclusion, p. 989) The current "Classification" COG ALL trial is AALLO8B1 (activated 8/2010), and this protocol addresses the collection of samples for testing in collaboration with TARGET. TARGET is a coordinated effort of almost all childhood cancer research cooperatives to gather data from a large number of patients and follow them to correlate the findings with treatment outcomes.

Developing/incorporating new targeted therapies

Another quote from the Hunger article: "Specific challenges for pediatric cancer include finding potentially relevant drugs that are being developed for more common diseases in adults; and to find industry partners who are willing to test new agents in children." He goes on to say that one example of a target for which inhibitors have been developed is the JAK family of kinases; as discussed above, JAK mutations are found in newly identified types of HR-ALL. COG (partnered with Incyte/Novartis) is bringing INCB18424, which targets JAK, into a single-agent phase I clinical trial for several childhood cancers, including ALL.

Other targeted therapeutics that have been brought to clinical trial by COG for relapsed ALL are lestaurtinib for MLL infant ALL, dasatinib for Ph+ ALL, and bortezomib for relapsed ALL. These trials are a combination of the targeted therapeutic and conventional chemotherapy. I discuss these in my article on Targeted Therapies for Childhood ALL. While I was sort of disappointed that targeted therapes are not eliminating cytotoxic chemoterapy in my Targeted Therapies article, I now realize that these new therapies have the potential to reduce the amount of cytotoxic chemotherapy, and this very important.

There are other issues over and above identifying targets and suitable targeted drugs. Decisions about appropriate doses for children must be made, for instance. Studies of drug metabolism in children are necessary. The companies that produce the drugs need to supply the drug in liquid suspension or at least make sure it can be crushed to be put in food or drink for children. The companies are not always used to dealing with specific issues of children, from dosing to ethics to practical matters.

I'll end with a direct quote of the final sentence of the Hunger review article:

"Ultimately, the goal should be to move new agents safely into frontline therapies in order to prevent relapse and perhaps even to spare some toxicities of our currently used cytotoxic drugs. In this way, we will truly realize the promise of the exciting recent discoveries underlying the pathogenesis of childhood leukemia."

References

2011: Improving outcomes for high-risk ALL: Translating new discoveries into clinical care. Stephen P. Hunger et. al., Pediatric Blood & Cancer, first published online: 15 FEB 2011. Abstract.

Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Mullighan CG, Goorha S, Radtke I, et al., Nature 2007;446:758–764.

High resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression. Kuiper RP, Schoenmakers EF, van Reijmersdal SV, et al., Leukemia 2007; 21:1258–1266.

Molecular allelokaryotyping of pediatric acute lymphoblastic leukemias by high-resolution single nucleotide polymorphism oligonucleotide genomic microarray. Kawamata N, Ogawa S, Zimmermann M, et al., Blood 2008;111:776–784.

Mullighan CG, Zhang J, Harvey RC, et al: JAK mutations in high-risk childhood acute lymphoblastic leukemia. Proc Natl Acad Sci U S A. 2009 Jun 9 106(23):9414-8. Epub May 22, 2009

Genome-wide profiling of genetic alterations in acute lymphoblastic leukemia: Recent insights and future directions. Mullighan CG, Downing JR, Leukemia 2009;23:1209–1218. (Review article.)

JAK mutations in high-risk childhood acute lymphoblastic leukemia. Mullighan CG et al., Proc Natl Acad Sci U S A. 2009. PubMed Abstract.

"Here, we report activating mutations in the Janus kinases JAK1 (n = 3), JAK2 (n = 16), and JAK3 (n = 1) in 20 (10.7%) of 187 BCR-ABL1-negative, high-risk pediatric ALL cases. The JAK1 and JAK2 mutations involved highly conserved residues in the kinase and pseudokinase domains and resulted in constitutive JAK-STAT activation and growth factor independence of Ba/F3-EpoR cells. The presence of JAK mutations was significantly associated with alteration of IKZF1 (70% of all JAK-mutated cases and 87.5% of cases with JAK2 mutations; P = 0.001) and deletion of CDKN2A/B (70% of all JAK-mutated cases and 68.9% of JAK2-mutated cases). The JAK-mutated cases had a gene expression signature similar to BCR-ABL1 pediatric ALL, and they had a poor outcome. These results suggest that inhibition of JAK signaling is a logical target for therapeutic intervention in JAK mutated ALL."

My lay interpretation: Almost 200 cases of high risk ALL were studied. These cases were not Ph+, and so are designated "BCR-ABL1-negative". Genomic profiling showed that 10% of these cases have mutations in one of the Janus kinase genes: shorthand notation is JAK1, 2, or 3. Of these cases that have JAK mutations, 70% also showed alterations in the IKZF1 gene. Furthermore, gene expression profiles determined that the JAK-mutated cases have a molecular signature similar to BCR-ABL1-positive (Ph+ ALL).

Bottom line: Of HR-ALL (as determined by, probably, relapse rate or MRD, you could pull full text to figure this out) cases, ten percent can be identified as having JAK mutations by genomic profiling and by gene expression profiles. Since they are JAK-mutated, JAK tyrosine kinase inhibitors that are currently used for other cancers and/or are in development might work to increase successful treatment.

Identification of novel cluster groups in pediatric high-risk B-precursor acute lymphoblastic leukemia with gene expression profiling: correlation with genome-wide DNA copy number alterations, clinical characteristics, and outcome. Harvey RC, et al., Blood. 2010 Dec 2;116(23):4874-84. PubMed abstract.

To resolve the genetic heterogeneity within pediatric high-risk B-precursor acute lymphoblastic leukemia (ALL), a clinically defined poor-risk group with few known recurring cytogenetic abnormalities, we performed gene expression profiling in a cohort of 207 uniformly treated children with high-risk ALL. Expression profiles were correlated with genome-wide DNA copy number abnormalities and clinical and outcome features. Unsupervised clustering of gene expression profiling data revealed 8 unique cluster groups within these high-risk ALL patients, 2 of which were associated with known chromosomal translocations (t(1;19)(TCF3-PBX1) or MLL), and 6 of which lacked any previously known cytogenetic lesion. One unique cluster was characterized by high expression of distinct outlier genes AGAP1, CCNJ, CHST2/7, CLEC12A/B, and PTPRM; ERG DNA deletions; and 4-year relapse-free survival of 94.7% ± 5.1%, compared with 63.5% ± 3.7% for the cohort (P = .01). A second cluster, characterized by high expression of BMPR1B, CRLF2, GPR110, and MUC4; frequent deletion of EBF1, IKZF1, RAG1-2, and IL3RA-CSF2RA; JAK mutations and CRLF2 rearrangements (P < .0001); and Hispanic ethnicity (P < .001) had a very poor 4-year relapse-free survival (21.0% ± 9.5%; P < .001). These studies reveal striking clinical and genetic heterogeneity in high-risk ALL and point to novel genes that may serve as new targets for diagnosis, risk classification, and therapy.

My lay interpretation: Again, about 200 cases were studied. The test is gene expression profiling and was correlated with DNA copy number abnormalities and clinical outcome. Found: 8 unique cluster groups, 2 of which are similar to MLL, 6 lack any previously known cytogenetic lesions. One of the 6 clusters is associated with particularly poor survival; this cluster is characterized by genomic profiling as having IKZF1 deletions, JAK mutations, and CRLF2 rearrangements.

Also see: New Treatments for ALL

Cost-effective multiplexing before capture allows screening of 25 000 clinically relevant SNPs in childhood acute lymphoblastic leukemia. A Wesolowska et al., Leukemia (2011) 25, 1001–1006. Abstract.


Footnote

*CBS News had a story on a "Cancer Breakthrough" how a man with "incurable" lung cancer was tested and found to have a rare mutation found in only 4% of lung cancer patients, and they offered him a treatment that targeted this specific mutation and is now doing well (June 2011). This didn't surprise me, as I've been studying targeted therapies for awhile, but it did make me happy. This is the way cancer treatments are starting to go, and hopefully, thanks to research and the TARGET initiative (later on this), this will be a happy story in the treatment of childhood cancers too.