Fresh incentive to look for Ph-like ALL

They began by taking samples from children who failed treatment and looking at the molecular characterization of about 1,000 cases, starting with gene expression profiling and ultimately moving to transcriptomic RNA seq or whole exome sequencing.

“The interesting thing about the children who failed therapy was that their genetic lesions weren’t known in the majority of cases,” she says. “So we thought it would be a particularly fruitful group to study.” The project was picked up by the NCI and evolved into the first TARGET program—the first pediatric cancer project under The Cancer Genome Atlas—with researchers from UNM, St. Jude, COG, and Children’s Hospital of Philadelphia.

Some 15 to 20 percent of the high-risk kids, it turned out, had a gene expression signature barely reminiscent of BCR-ABL1. As their work progressed, they also found, through directed gene sequencing, that several of the children had a mutation in the JAK2 tyrosine kinase that is often mutated in myelodysplastic syndrome—but this one appeared in a different region of the gene.

But the real bombshell was the transcriptomic sequencing results. “We were completely blown away. It was almost like every child was different,” says Dr. Willman, with their own, specific tyrosine kinase fusion or point mutation. The number of variants currently sits at about 200. “It’s dramatic,” she says. Thirteen different kinase mutations have been described, with 48 (emphasis hers) different fusion gene partners. “It’s just sort of shocking.” And none of them recur frequently. “In other words, it’s not like 50 percent of the kids have one of these. They’re spread out,” not unlike people in Chicago who vote Republican.

Subsequent sequencing work had led to a gene expression profile that has been developed into a clinical diagnostic assay used for screening. “We’ve done a lot of testing now of more than 8,000 ALL cases of this type, from children, adolescents, and adults,” she says.

The test is a gene expression signature for either an eight- or 15-gene signature (which incorporates the eight genes). It’s FDA approved in the context of clinical trials. UNM is in the process of licensing and commercializing the screen.

Interest is growing, Dr. Willman reports. “We continue to get calls from pathologists and clinicians as they become more aware of Ph-like ALL.” This often comes up when a patient is not attaining remission on standard induction therapy, “which is pretty rare these days in the treatment of leukemia,” she says. They may also have a high white count, or (as noted) ethnicity might offer a clue. (She notes that the GATA3 risk allele variant frequency is three- to fourfold higher in Hispanics and Indigenous Americans than whites.) Another clue: absence of the classic recurrent cytogenetic abnormalities pathologists typically see in other leukemia subtypes, such as a t(12;21) or a t(9;22). “People are beginning to think of this disease now and want to do a screen to make sure they get that child on a trial. We spend a lot of time on the phone with pathology groups,” Dr. Willman says.

Dr. Hunger fields plenty of calls himself. Ideally, he says, patients will undergo real-time screening using the new assay, “which we think is pretty accurate. And we have the ability then to enroll patients in clinical trials.” Here he interjects a note of caution. “At least from my perspective, the targeted therapies have not yet been proven to be effective. There’s a reasonable hope they will be, but clinical trials are needed to prove that.”

“I think the more common challenge,” he continues, “is the patient’s not enrolled in a clinical trial, and one of these abnormalities is identified by one of a variety of methodologies.” Next steps depend on how a patient is responding to standard therapy. “If they’ve responded very well, often physicians will keep this knowledge in their back pocket to use potentially if the patient were to relapse.”

The newer subtypes of ALL have an increased risk of relapse, he says. “A higher percentage of them will relapse than other, if you will, garden-variety ALL cases.” But not all will relapse, either. In particular, children with low-risk clinical features do quite well with conventional chemotherapy, even if they have a high-risk genetic abnormality.

Dr. Willman seconds the importance of accruing patients to one of the national clinical trials. Not only are molecular tests being developed in the context of these trials, but given the likelihood of eventual relapse, “they’re going to need another round of testing to determine what to do next.”

Treatments are initially dramatic, says Dr. Willman—typically two to three years remission, “and then you begin to get clonal evolution and relapse.” Clearly the targeted therapies are essential, but so too is monitoring.

One of the most powerful tools available in treating ALL is the measurement of minimal residual disease, or, as it’s increasingly known, measurable residual disease, says Dr. Mullighan. Typically results are mutation agnostic, he says, using leukemia immunophenotype and flow cytometry, or detecting antigen receptor rearrangements quantitatively, to track leukemia burden. “That information is one of the strongest predictors of outcome,” he says, “and it’s used to risk-adapt therapy—if patients respond poorly, for example, [it’s] intensified.

“Where things are changing now,” Dr. Mullighan continues, “is how to use the new genomic information to influence the MRD testing.” Some fusions, for example, are being used as MRD assays (though not in Ph-like ALL). “You can already think that there might be a way you could track the specific rearrangement again—that would be flow cytometry to CRLF2 because if that’s part of the leukemia phenotype, you conveniently track that clone by flow cytometry over time.”

Given the diversity of mutations, no single, standard assay will likely emerge for testing MRD, says Dr. Willman. “In a lot of our clinical trials, [MRD] is being monitored using flow cytometric testing, which makes sense—when a new clone emerges, you can pick it up” and then submit it for another round of detailed molecular testing.

The other complexity, Dr. Mullighan says, is that kinase lesions can be subclonal events and can wax and wane over time. “We often make a decision to intervene, with a TKI or a more intensive approach, at completion of induction therapy. The question then arises, if you’re detecting positive MRD after a month of therapy, are the genetics in that sample going to be the same as what we found in the original diagnosis? Or will they have started to evolve? Could that impact the decision to use a TKI?” Dr. Mullighan and his colleagues are addressing this prospectively in a clinical trial. “That’s by no means standard of care anywhere, to my knowledge,” he says. “But it’s a very important point—how do you build mutation-specific MRD tests?”

Meanwhile, the hunt for new fusions continues apace. For known kinases or cytokine receptors that are rearranged, “We continue to find more fusion paths,” says Dr. Mullighan, including, recently, an FLT3 fusion that hadn’t been previously described in any cancer.

He and his colleagues have also taken a subset of cases, performing gene expression profiling to determine the group of cases that have Ph-like ALL, then applied other levels of sequencing. “We still can’t find the driver lesion. There are some cases that look, for all intents and purposes, extremely, strongly, Ph-like—yet we haven’t found a lesion. We are continuing to use whole genome sequencing and other approaches to try to find those drivers.”

“There have been a few surprises as this work has unfolded,” Dr. Hunger agrees. “One is just the diverse variety of genetic abnormalities that seem to get you to the same endpoint.”
“We continue to describe—this amazes me—new variants every month,” says Dr. Willman, sounding a bit like a highly caffeinated Adam, naming the inhabitants of the newly formed Earth far into the book of Genesis.

She finds this especially satisfying given her entree into the field. “When I submitted my first director’s challenge grant to do genomic characterization and sequencing in the acute leukemias, one of the reviewers said, ‘Oh, that’s stupid. Why would we fund leukemia? We know all those translocations already. This is a waste of money.’”

Though she didn’t share that misdirected certainty, she does concede surprise of her own. “I don’t think I expected that it would sort of expand or open up to such a broad discovery of so many genetic lesions.”

She’s also intrigued as she watches other researchers do transcriptomic sequencing in solid tumors. “They’re also finding these fusion genes. We’re discovering a much more complex spectrum of gene fusions present in these cancers than anyone ever thought. I think we were kind of one of those first windows into how complex the genomic lesions are in any one cancer. You don’t just have one mutation or one amplification or one fusion gene. There are actually several.”

The questions continue to emerge alongside each new discovery. “Is this the same clone that has all these mutations, or do we have multiple subclones that have slightly different constellations and mutations?” Dr. Willman asks. “I think the deeper sequencing studies have shown the latter to be the case. That most human cancers, even these leukemias I’m talking about, are highly clonally heterogeneous. As you begin to apply a therapy, and cause genetic evolution, you’re actually doing that with the therapy, probably clonal selection and evolution—you’re perturbing that heterogeneous mix.”

The only unpromising note in this story, she suggests, might be the name of the disease that is transforming ALL diagnostics. Philadelphia chromosome-like acute lymphoblastic leukemia hardly rolls off the tongue. And unlike the arts, where a clunky working title can eventually give way to one that shines—isn’t “The Sound of Music” more pleasing than “The Story of the Trapp Family Singers”?—medical nomenclature might be a little less flexible.

“We called it a terrible name,” she says. “None of us like it, but it sort of stuck. Unfortunately.”

Karen Titus is CAP TODAY contributing editor and co-managing editor.