Time now for tumor mutational burden?

The turnaround time for the assay at Dr. Nowak’s lab is typically two weeks and is limited by the NGS process itself rather than by any TMB-specific step(s). This includes a review for sample adequacy, DNA isolation, all the initial preparation steps for sequencing, the sequencing itself, pipeline analysis, and variant interpretation and report generation. “I don’t see any shortcuts that might tell you about TMB without actually sequencing the genes themselves,” he says.

Meanwhile, clinicians are filling out their own dance cards a little differently. At UC, Dr. Ritterhouse says her clinical colleagues devote very little time to discussing cutoffs. Or, as she puts it, “They don’t worry about it as much as I do.”

Some are more interested in TMB’s negative predictive value, Dr. Ritterhouse says. “I’ve gotten that from quite a few oncologists.” In a PD-L1-negative case, for example, a negative TMB might steer them away from using immunotherapy, particularly if the patient has comorbidities, is elderly, etc.

At Brigham and Women’s, TMB results are generated automatically. Dr. Nowak says his oncologist colleagues showed little excitement when he first spoke to them about offering TMB results. If they couldn’t act on the number, they didn’t want to see it. A year in, however, many more oncologists appreciate having the information, “and it’s something we routinely discuss in our tumor boards.” Even though it’s not quite actionable yet, “we’re clearly starting to at least informally distinguish between tumors that have a higher or a lower mutational burden on average than we might expect for that tumor type.”
The conversations sometimes take a funny turn, he continues. “We’ll go to tumor boards, and they’ll look at a report and say, ‘Oh, this tumor has a mutational burden of 12—it’s a little high.’ And then they’ll look at another tumor [of the same type] that has a mutational burden of 8, and they say, ‘Oh, it’s less than 10—it’s low.’”

How quickly the new becomes norm. The time to unwind these so-called standards is now, before they get caught in the brain like the Kars4Kids jingle. Says Dr. Nowak: “I always caution them that, based on the specifics of our assay, there is not a substantial difference between a tumor with a mutational burden of 8 versus 12.”

Dr. Nowak foresees a time when clinicians might want to apply TMB to situations apart from predicting response to therapy, including the occasional diagnosis. “We’ve seen this repeatedly now in carcinomas of unknown primary.” If such a tumor has an elevated mutational burden, it’s often possible to look at its pattern of mutations to discern an underlying mutagenic process. “So if the tumor has an elevated TMB and is also MMR deficient, you might think this is a tumor that could have come from the colon,” he says, whereas a tumor with an elevated TMB and a tobacco smoke signature could instead be suggestive of metastatic lung cancer.

“Occasionally, we have sort of the reverse example,” he continues, “perhaps a squamous cell carcinoma of the lung that everyone assumes to be a primary lung cancer. Rarely, we’ve seen that those tumors will harbor a high mutational burden with an ultraviolet light exposure signature. And so that’s actually strong evidence suggesting that the tumor has metastasized from a sun-exposed cutaneous site and is not a primary lung cancer at all.”

While this goes a bit beyond measuring TMB, it reinforces the idea that an elevated TMB might prompt pathologists to consider the underlying “why.” This might be helpful in only a small number of cases right now, Dr. Nowak says, but “when it is helpful, it is extremely helpful.”

NGS panels are capturing this granular information within TMB, whether it’s CTGA changes, dinucleotide changes, or frame shifts. Perhaps this can help determine therapy and provide hints to etiology, pathogenesis, and environmental exposures. It might even be more helpful, ultimately, to look at overall mutational spectrum and pattern, says Dr. Ritterhouse, rather than look only for a list of mutations, variants, or fusions.

Dr. Ritterhouse says the discussions with her colleagues have been lively if not definitive. The thoracic oncology department regularly requests TMB testing for research purposes. The director “would love for us to be reporting TMB,” she says. And in general, “I think oncologists want every bit of data they can have, regardless of whether there’s great evidence for it.”

At the same time, she knows some physicians remain skeptical of the data suggesting TMB’s utility. (More broadly, when talking to clinical colleagues from other institutions, she’s found they want to see more data, such as overall survival.) That skepticism may turn out to be useful, in fact. “It’s giving us a little extra time to figure this out.” She welcomes this bit of breathing room. No one is saying they’ll go elsewhere for testing if her lab isn’t reporting TMB within, say, the end of the year.

And where would they go, exactly? Enter the Romulus and Remus of laboratory testing: standardization and validation. These two pillars, always important, have yet to be settled in TMB. In fact, the two key TMB studies relied on different tests and different cutoffs.

Every step in the TMB dance—and there are many—becomes more intricate across institutions. Ideally studies will have been done on the same sequencing platform, analyzed by the same pipeline, with the same reference genome and transcript settings, and analyzed the same tumor content. But this is a purity of line generally reserved for the Rockettes. In laboratories, “This is almost never the case,” Dr. Nowak says.

Some algorithms look at essentially the entire DNA sequence of every gene that’s coded. Other groups say it’s too expensive and too inefficient to analyze such vast territory. It’s possible, they say, to look at a subset of genes, albeit still millions of bases, and come up with a number that reasonably approximates the number obtained by looking at all the coding sequences.

But that raises the question of “how much genomic real estate you need to sequence before you have a number that fairly matches what you would see from whole exome sequencing,” says Dr. Nowak. “Is it enough to sequence 100 genes? 200? 500? 1,000?” Moreover, for most targeted sequencing, the gene panels are not chosen at random; generally, specific genes are selected because they’re important in cancer. “So you’re sequencing a biased subset of all the genes you would be analyzing by whole exome sequencing.” Could that introduce a bias into the TMB calculation? “I think that’s a real possibility.”

For those interested in even greater detail, says Dr. Borczuk, there’s another consideration. “If you have to produce a protein that’s mutated in order to get an immune response, then only the mutations that result in a protein sequence change should matter,” he says. Again, the algorithms have varied from one paper to the next. “Some have been more inclusive, some less inclusive.”

The challenge of converting acceptable research test methods to a uniform test plagued PD-L1 testing, Dr. Borczuk says. Labs have to work hard to figure out if they are re-creating what was done in various studies, with their different antibodies and different platforms. He fears TMB will be messy as well. In a sense, it’s like rescuing lost choreography. You think this is what Martha Graham meant, but one is never quite sure if this is how the steps should look.

How does this problem get solved? “I’m not sure of the best answer,” Dr. Borczuk replies.

Since the drugs and the tests are proprietary, he says, “until the full FDA approval occurs, sharing of that is difficult. It’s limited by whatever rules have been put in place between the participants [pharmaceutical and diagnostics companies] in the clinical trials.” By the time pathologists outside the clinical trials become involved in the process—“We become much more engaged once the approval has come through,” he says—“we’re potentially six months to a year behind. Sometimes more.”

Timing is crucial, Dr. Ritterhouse agrees. She notes that several groups, including Friends of Cancer Research, are trying to standardize TMB. But she remains worried. By the time useful guidance emerges, “it may be too long after everyone has needed to report it.”

Dr. Borczuk’s biggest concern is that “we don’t have the biologically relevant samples to test. What we end up doing is test validation within the laboratory, but we can never do a full validation in a true cohort of responders and nonresponders. That’s a huge limitation.”

Fortunately, he says, matters have improved since the PD-L1 process. The major pharmaceutical companies “are asking what we think about things now,” he says. “And that’s a great help, because when it was being filtered only through their oncology contacts in medical centers, they were getting a skewed perspective of the scope of the problem.”

Nonetheless, for oncologists now bringing these drugs into clinical practice, “their instinct is to go with a commercial laboratory that has the most upfront brand for the test.” That means FoundationOne, the test used for CheckMate 227, becomes the de facto standard, even “if that’s not intentional.”

Looming larger is the issue that clinicians simply “want a number to work off of, and they want to justify why it’s valid,” Dr. Borczuk says. For labs, he continues, it’s often difficult after the fact to be able to say that “the number that you produce in your own laboratory is equally valid as the one that was used in the study trial.” At this point, he says, if FDA approval were given for nivolumab and ipilimumab for stage IV lung cancer, in the near term, he’d feel compelled to use Foundation’s test. “The same thing happened with PD-L1,” he recalls. “The day that was made public, we had to choose a reference lab” from among the small number doing the FDA-approved test.

He’s worried that laboratories will continue to fall down this rabbit hole again and again. “This seems to be the way it’s just going to happen every single time.”

The other key study used Memorial Sloan Kettering’s IMPACT panel. But those researchers did not address the combination immunotherapy scenario used in CheckMate 227. That could lead to a situation where combination immunotherapy would use the Foundation cutoff of 10, but a monotherapy with a laboratory-developed test would require a different cutoff. Dr. Borczuk sees potential for matters “spiraling out of control,” with every indication potentially having its own test and its own cutoff. “So this is a problem,” he says. “There’s no question.”

The impact of using different platforms, different bioinformatics, and different variant calling is real. Dr. Ritterhouse says she and her UC colleagues used an outside laboratory to run the whole exome sequencing. “They did the variant calling and gave us the TMB number, and we did it separately.” The results gave them pause. “It was amazing—the exact same data, the same samples, the same sequencer—how vastly different the numbers were. And it’s just that their whole exome variant calling pipeline was nascent, shall we say, and not heavily tested. It was, in fact, wildly different. Wildly inaccurate.”

It was a useful exercise in demonstrating how all the so-called simple steps, such as variant calling, can make a big impact, she continues. Unfortunately, the way matters stand now, “it’s like every lab is trying to reinvent the wheel themselves.”

UC is part of a larger consortium, called GOAL, consisting of 17 academic institutions, which is trying to develop a consensus gene list that could be shared for cross-site, cross-testing methodology concordance studies. Demonstrating concordance between labs would be a solid step toward standardization, she says.

Beyond laboratory standardization, it will likely be important to harmonize reporting strategies as well. Dr. Nowak is grappling with “Do we need to dichotomize into low and high? Or low, intermediate, and high? Or do we need a more granular, quantitative breakdown?”

Given all the testing complications, the biology of TMB seems almost like a quaint afterthought. Nevertheless, says Dr. Nowak, “It’s probably important to think about this.”

How “active,” so to speak, is TMB? Does it change over the tumor’s lifespan? Dr. Borczuk says he suspects it does. But, he notes, current discussions revolve around late-stage tumors. Early-stage tumors might indeed have different burdens, but that’s not currently clinically relevant.

On a related note, however, pathologists should be testing the most recent sample—the one that documents stage IV disease—rather than an earlier one, perhaps from a resection, Dr. Borczuk suggests. Possibly down the road, if it does change over time, it might be a future marker for tumors that are becoming more aggressive, he posits.

Some research suggests that the real question isn’t necessarily if the TMB itself changes, but rather if a change in biology—possibly a mutation in the antigen processing and presentation pathway—enables the tumor to acquire a mechanism to essentially hide its neoantigens from the immune system. “You might still have a cell with a high mutational burden and many predicted neoantigens,” explains Dr. Nowak, “but maybe they never actually make it to the surface of the cell to be recognized.” At least for patients undergoing immunotherapy, he says, there could be “a strong selective pressure to somehow block your neoantigens from being expressed and recognized” on the cell surface.

TMB may not be equally useful in all tumors. Lung cancer, melanoma, and cutaneous squamous cell carcinoma, for example, all tend to have very high mutational burdens. Many pediatric tumors, on the other hand, which may be driven by translocations, often have very low TMB. But Dr. Nowak has also seen wide variation—over at least one order of magnitude, but sometimes several—within a single tumor type.

He suggests that the broader group of tumors that are MMR deficient will likely incur additional benefit from some type of TMB measurement. The option to give pembrolizumab is currently a binary decision. Is the tumor MMR deficient or proficient? But a number of sequencing studies have shown that not all MMR deficiency is equal, so to speak, in terms of mutational burden. Some MMR-deficient tumors have a TMB that’s very slightly above MMR-proficient or microsatellite-stable tumors. Others have 10 times as many mutations, or more. It seems reasonable, Dr. Nowak says, to expect that those tumors might respond differentially to immunotherapy and to have different prognoses. Studies looking at this are underway, which might show potential use of TMB as a marker to stratify within MMR-deficient or even MMR-proficient tumors.

In the meantime, TMB is, if nothing else, quietly making a run at becoming the Miss Congeniality of biomarkers. For any lab doing some type of genomic sequencing over sufficient genomic real estate, TMB values can be calculated fairly easily across all tumor types, says Dr. Nowak. “And almost every tumor has some number—it’s not going to be zero.” That simplicity is appealing. “We don’t have too many markers that we can calculate and compare at a pan-tumor level.”

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