Regulators have moved quickly. After a positive opinion from the European Medicines Agency’s Committee for Medicinal Products for Human Use in June 2026, the European Commission approved pirtobrutinib in August 2026 for adults with CLL across all lines of therapy, and an equivalent decision from the US Food and Drug Administration is anticipated in the second half of 2026. An accompanying editorial by Davids argued that the two trials open the door for noncovalent BTK inhibition as initial therapy. On the surface, the case seems overwhelming: a drug that works at least as well as the incumbent, with dramatically better cardiovascular tolerability, seems destined to become the new default first choice.

But a correspondence published in Annals of Hematology by Boyi Chen and Shenxian Qian throws a careful, technically grounded brake on that enthusiasm. The authors highlight what they call a sequencing asymmetry, a structural imbalance in the evidence that neither of the new trials was designed to address. The order in which the two BTK inhibitor classes are given, they argue, matters enormously, and only one of the two possible orders is currently supported by randomized evidence. That order is the covalent-first sequence, and the argument for preserving it rests on the molecular biology of drug resistance as much as on clinical trial data.

The covalent BTK inhibitors, including ibrutinib, acalabrutinib and zanubrutinib, bind permanently to a cysteine residue at position 481 of the BTK kinase. Resistance to these drugs most often emerges through the C481S mutation, which replaces that cysteine with serine and blocks covalent attachment while leaving the binding pocket otherwise intact. Crucially, pirtobrutinib does not depend on covalent chemistry, so it retains activity against C481-mutant BTK. In the phase 1/2 BRUIN study, pirtobrutinib achieved a 73.3 percent response rate and a median progression-free survival of 19.6 months in patients whose disease had progressed on a covalent inhibitor, regardless of C481 status. In the phase III BRUIN CLL-321 trial, it outperformed idelalisib plus rituximab or bendamustine plus rituximab in this setting, with a hazard ratio of 0.54 and a median progression-free survival of 14.0 versus 8.7 months. A patient who begins treatment with a covalent inhibitor therefore keeps a rational, evidence-backed exit ramp if resistance develops.

The reverse sequence has no comparable data, and the resistance mechanisms give concrete reason for concern. Work by Wang and colleagues showed that acquired resistance to pirtobrutinib does not arise through C481 but through a distinct set of kinase-domain mutations: V416L, A428D, M437R, T474I and L528W. Of these, A428D and L528W conferred in vitro cross-resistance to ibrutinib and prevented multiple covalent BTK inhibitors from inhibiting the mutant enzyme. In other words, a patient who progresses on first-line pirtobrutinib may harbor leukemic clones that have compromised not just one drug but much of the covalent class, the very class that currently anchors relapse management. Notably, no patient in the Wang series acquired a new C481 mutation on pirtobrutinib, indicating that the two drug classes select largely distinct mutations, and that the mutations selected by the noncovalent agent can reduce sensitivity to covalent drugs. As Mato and colleagues observed in the original BRUIN publication, no information is currently available on the efficacy of covalent BTK inhibitors in patients who receive pirtobrutinib first and in whom resistance develops.

Davids offers a reasonable counterargument: many older patients with comorbidities may need only one or two lines of therapy over their remaining lifetime, making downstream resistance a less salient concern. For that subgroup, Chen and Qian concur that first-line pirtobrutinib is defensible, since a single effective line may be the realistic horizon. But CLL is not uniformly a disease of the very old. Younger patients, and those with unmutated IGHV genes or TP53 aberrations, can expect disease courses lasting ten to twenty years and multiple lines of treatment. Patients with del(17p) were even excluded from the CLL-313 trial. For these individuals, sequencing is not a footnote but the central strategic question, and spending the noncovalent agent first may forfeit options decades before they are needed.

Nor does toxicity, the argument that once favored abandoning covalent inhibitors altogether, justify skipping the class today. The cardiovascular case against ibrutinib has been substantially overtaken by the second-generation covalent agents. Zanubrutinib more than halved the rate of atrial fibrillation compared with ibrutinib in the ALPINE trial, 5.2 percent versus 13.3 percent, while improving progression-free survival, and acalabrutinib reduced atrial fibrillation in the ELEVATE-RR trial, 9.4 percent versus 16.0 percent, with non-inferior efficacy. The covalent-first strategy also preserves later options: beyond pirtobrutinib monotherapy after covalent inhibitor failure, the triplet of pirtobrutinib with venetoclax and rituximab has outperformed venetoclax plus rituximab in previously treated disease in BRUIN CLL-322, with a hazard ratio of 0.547. Frontline pirtobrutinib, by contrast, forgoes at present the only BTK-inhibitor class with phase III-validated salvage value.

The authors are careful to qualify their own argument. The cross-resistance evidence is preclinical, resting on in vitro potency and binding studies, and every patient in the Wang series who developed a non-C481 mutation had prior exposure to ibrutinib, so whether frontline pirtobrutinib selects the same mutational spectrum is unknown. Cross-resistance is also not strictly one-directional, since mutations such as L528W have been reported, less frequently, after covalent inhibitor therapy as well. These uncertainties, the authors stress, argue for caution and surveillance rather than regulatory delay. They also caution against treating BTK degraders, an emerging class of drugs that eliminate BTK rather than merely blocking it, as a ready safety net. Degraders such as NX-2127 can degrade kinase-impaired mutants including L528W, and early-phase studies of BGB-16673 and bexobrutideg report rapid, durable responses, but the evidence remains at the abstract level, pivotal trials are still accruing patients, and A428D can itself confer resistance to degrader therapy.

From this analysis flow four concrete recommendations. First, frontline trials of noncovalent BTK inhibitors should mandate BTK and PLCG2 mutation testing at progression, so the field learns which mutations first-line pirtobrutinib actually selects in previously untreated patients. Second, real-world registries should systematically track responses to covalent agents after pirtobrutinib failure, which the authors identify as the single most urgent evidence gap. Third, routine practice should incorporate BTK and PLCG2 testing whenever a patient progresses on any BTK inhibitor, since the mutational profile at relapse directly informs what can work next. Fourth, forthcoming guideline updates should explicitly address sequencing, and post-pirtobrutinib outcomes should be collected systematically while reverse-sequence data remain absent.

The BRUIN program has undeniably given CLL a highly effective drug, and its first-line efficacy is not in doubt. What remains unknown is what early use may cost at relapse. Until prospective sequencing data emerge, the authors conclude, the uncertainty argues for an individualized, sequencing-aware choice rather than a wholesale shift of the treatment default, particularly for the younger patients whose long disease courses give the order of therapy its greatest weight.

Subject of Research: Treatment sequencing and resistance mechanisms for first-line pirtobrutinib in chronic lymphocytic leukemia

Article Title: Sequencing asymmetry: the case for caution before first-line pirtobrutinib in chronic lymphocytic leukemia

Article References: Chen, B., & Qian, S. (2026). Sequencing asymmetry: the case for caution before first-line pirtobrutinib in chronic lymphocytic leukemia. Annals of Hematology, 105(9), Article 404. https://doi.org/10.1007/s00277-026-07264-x

Image Credits: AI Generated

DOI: 10.1007/s00277-026-07264-x

Keywords: chronic lymphocytic leukemia, pirtobrutinib, BTK inhibitor, drug resistance, treatment sequencing, BTK C481S, kinase-domain mutations, BRUIN trials, cross-resistance, BTK degraders, hematology, clinical trials

The research team, led by Yucheng Wang and Fei Yang of the University of Minnesota together with David L. Kerns of Texas A&M University, Graham P. Head of Bayer Crop Science, Dawson Kerns of Louisiana State University, and Bruce E. Tabashnik of the University of Arizona, set out to answer two questions with direct consequences for resistance management. First, does selection for resistance to Vip3Aa cause cross-resistance to Cry proteins, which would undermine the pyramided crops that produce both toxin classes? Second, do Cry proteins and Vip3Aa interact synergistically against insects that are already resistant, a property that could extend the useful life of multi-toxin Bt crops?

To address these questions, the researchers generated five strains of H. zea carrying more than 100-fold resistance to Vip3Aa in a susceptible genetic background. The strains originated from F2 screens of field populations collected in Louisiana, Mississippi, and Texas between 2019 and 2020, a technique that exposes hidden recessive resistance alleles carried by mating pairs of field-collected insects. Each strain was then crossed repeatedly with a laboratory-susceptible strain, BZ-SS, obtained from a commercial supplier, and reselected with Vip3Aa in the laboratory. After five to six rounds of backcrossing and selection, the resulting strains, designated LT70-Vip, AC4-Vip, M1-Vip, R2-Vip, and R15-Vip, showed such profound resistance that even the highest concentration of Vip3Aa tested, 100 micrograms per square centimeter of diet, killed no more than 15 percent of larvae, implying resistance ratios exceeding 500-fold.

The team then tested these Vip3Aa-resistant strains against five Cry proteins used in commercial Bt crops: Cry1Ab, Cry1Ac, Cry1A.105, Cry1Fa, and Cry2Ab. The verdict was strikingly inconsistent. Across 23 bioassay comparisons, seven LC50 values were significantly higher than those of the susceptible strain, seven were significantly lower, and nine did not differ significantly, a seven-to-seven split that matches exactly what would be expected if no consistent cross-resistance existed. The overall mean cross-resistance ratio was 1.2, statistically indistinguishable from the value of 1.0 expected if selection with Vip3Aa had no effect on susceptibility to Cry proteins. Only Cry1Ac showed a statistically significant mean positive cross-resistance, and only one strain, AC4-Vip, displayed significant overall positive cross-resistance across the Cry proteins tested.

What astonished the researchers was not the absence of a trend but the sheer magnitude of the variation around it. The 23 cross-resistance ratios spanned an extraordinary 1100-fold range, from 0.06, indicating 17-fold negative cross-resistance to Cry1Fa in strain LT70-Vip, to 65-fold positive cross-resistance to Cry1A.105 in strain AC4-Vip. That range is roughly 49 to 70 times wider than those reported in previous reviews of cross-resistance between Vip3 and Cry proteins in other lepidopteran species. The authors suggest that the genetic diversity of the field-derived resistance alleles, which complementation tests place at three distinct loci among the five strains, likely contributes to this variability, as does the unknown mechanism of resistance in most of the strains. Only LT70-Vip has a biochemically characterized mechanism, reduced binding of Vip3Aa to midgut brush border membrane vesicles, and as expected under the receptor model of Bt toxicity, that strain showed no significant cross-resistance to any Cry protein.

The second half of the study tackled synergy, an interaction in which two toxins combined kill more insects than would be predicted from the independent action of each toxin alone. Previous surveys suggested synergy between Cry and Vip proteins is relatively rare, appearing in only about 19 percent of bioassay evaluations reviewed in 2020, though more recent studies on fall armyworm and peach fruit moth reported far higher frequencies. Critically, nearly all prior synergy work used susceptible insects. To test synergy against resistant insects, the team created a triple-resistant strain, TRE-RR, by crossing the Vip3Aa-resistant LT70-Vip strain with a Cry-resistant strain derived from Texas field corn, then selecting successive generations with all three toxins. The resulting strain exhibited resistance ratios of 265 for Cry1Ac, 495 for Cry2Ab, and greater than 96 for Vip3Aa.

Against this formidable laboratory adversary, synergy emerged clearly. In diet bioassays conducted at two concentrations, combinations of Vip3Aa with Cry1Ac produced significantly lower observed survival than expected under independent action at both concentrations tested, and Cry2Ab combined with Vip3Aa was synergistic at the higher concentration. Most remarkably, the three-toxin combination of Cry1Ac, Cry2Ab, and Vip3Aa was synergistic at both concentrations, meaning that even larvae equipped with resistance to every toxin individually died more often when the toxins were delivered together than their separate toxicities would predict. The finding is consistent with the authors’ earlier analysis of field data from Bt corn showing synergy between Cry1Ab and Vip3Aa against susceptible H. zea, but it is, to the authors’ knowledge, the first demonstration of Cry-Vip synergy against a strain resistant to both toxin classes.

The practical implications are substantial. If synergy between Cry proteins and Vip3Aa operates in the field against resistant H. zea, as it appears to against susceptible populations, it would kill some insects that survive either toxin alone, reducing the intensity of selection for Vip3Aa resistance and potentially slowing its evolution. That would be welcome news for an industry and a grower community watching early warning signs of Vip3Aa resistance accumulate in the United States, where practical resistance to Vip3Aa has already been documented in Brazil. The synergy finding also carries a cautionary note for resistance monitoring. Current surveillance relies partly on sentinel plots of corn producing Cry1Ab plus Vip3Aa, from which a phenotypic frequency of resistance is calculated by comparing larval abundance in Bt ears with that in non-Bt ears. If synergy kills some larvae that are resistant to either toxin alone, this approach could underestimate the true frequency of Vip3Aa resistance, a discrepancy the authors recommend resolving by comparing estimates from F2 screens with those from sentinel plots.

The study also refines the scientific understanding of how Bt resistance evolves and spreads. Because Vip3Aa and Cry proteins bind to different midgut receptors, strong cross-resistance between them was not expected, and the new results largely vindicate that expectation while revealing how messy the underlying biology can be. Mechanisms other than altered receptor binding, such as changes in toxin processing within the midgut, can confer broad cross-resistance, and the authors note that determining the resistance mechanisms in the remaining strains could explain the puzzling differences between R2-Vip and R15-Vip, which share a resistance locus yet differed 13-fold in their mean cross-resistance to Cry proteins. For now, the message for resistance management is cautiously optimistic: pyramiding Vip3Aa with Cry proteins remains a sound strategy, the lack of consistent cross-resistance means Cry resistance alleles should not compromise Vip3Aa efficacy, and synergy may quietly buy precious time for one of agriculture’s most valuable pest control technologies as its chief adversary continues to adapt.

Subject of Research: Cross-resistance and synergy between the Bt toxins Vip3Aa and Cry proteins in Vip3Aa-resistant strains of the corn earworm Helicoverpa zea

Article Title: Evaluating cross-resistance and synergy between Vip3Aa and Cry proteins from Bt in six strains of Helicoverpa zea derived via F2 screens

Article References: Evaluating cross-resistance and synergy between Vip3Aa and Cry proteins from Bt in six strains of Helicoverpa zea derived via F2 screens. (n.d.). https://doi.org/10.1007/s44297-026-00073-8

Image Credits: AI Generated

DOI: 10.1007/s44297-026-00073-8

Keywords: Helicoverpa zea, corn earworm, Bt crops, Vip3Aa, Cry proteins, cross-resistance, synergy, insect resistance, transgenic crops, resistance management, Bacillus thuringiensis, pest control