PLCG2 mutation

P1, N=110, Recruiting, Carna Biosciences, Inc. | Phase classification: P1b --> P1

Re-emergent clones lacked BTK mutation and retained sensitivity to ibrutinib upon retreatment. NCT02251548.

Acquired mutations in BTK, PLCG2 and BCL2 are associated with resistance to continuous targeted agent therapy in chronic lymphocytic leukemia (CLL). Here, we discuss new evidence that limiting the duration of CLL therapy may prevent the evolution of such resistance mutations, potentially facilitating effective re-treatment strategies.

The results of Western blot showed that the protein expression levels of P65PI3K and p53 in the single drug group and the combination group were down-regulated in varying degrees, especially in the combination group（Figure 2). ConclusionIbrutinib combined with curcumin has synergistic killing effect on TP53- mutated CLL cells; Curcumin increases the killing effect of ibrutinib on TP53-mutated CLL cells by inhibiting PI3K and NFκB pathway, and degrading p53mt protein.

Of the 29 patients with PD, 19 have required retreatment (single-agent ibrutinib, n = 16, or ibrutinib plus venetoclax, n = 3); 17 achieved partial response or better, 1 achieved stable disease, and 1 is pending response assessment. First-line fixed-duration combination treatment with ibrutinib plus venetoclax may mitigate development of resistance mechanisms associated with continuous single-agent targeted therapies, allowing for effective retreatment.

Ibrutinib, Zanubrutinib, Orelabrutinib were administered in 55.3%,18.3% and 17.9% of patients respectively, in addition to 6 patients with Acalabrutinib and one with Loxo-305, among them 47.7% (125/262) received BTKi as first line treatment...Venetoclax-based regimen might be an effective salvage therapy which could showed a benefit trend in PFS as compared to other post BTKi therapy (10.1 months vs 3.1 months, p= 0.1818)...Acquired BTK/PLCG2 mutations remained to be key drivers of BTKis resistance and BTKC481S mutation was the dominant mutation, while BTKT474 mutation was only detected in Orelabrutinib-resistant patients. The prognosis was rather poor for relapsed patients especially for RTs, treatment strategy after disease progression remains to be optimized.

The majority of IR DP with BTK/PLCG2 mutations (6/8), were 100% homologous to germline IGHV unmutated, suggesting an inherent higher risk disease profile as a risk factor for acquiring BTKmt with prolonged BTKi therapy. BTK/PLCG2 mutations were enriched amongst very late progressors with 6/8 pts progressing shortly after stopping therapy at 72 mo., which is later than previously reported for relapsed/refractory CLL pts.

Blood samples from 28 patients from GELLC7 trial (NCT03280160, ibrutinib followed by ofatumumab consolidation) were collected before treatment (BT), and 1, 3, 6 and 12 months on treatment (at least two timepoints). Ibrutinib in T cells reduced the expression of exhaustion markers, Tregs and Tfh cells. In CLL cells, we observed a downregulation of markers related to adhesion, immunosuppression and migration, but an overexpression of TMBIM6 in CLL cells that retained migrative capacity towards CXCL12 under ibrutinib. Finally, we identified TMBIM6 expression as an independent poor prognostic factor and a potential novel target for CLL.

To permit a focused analysis of PF that would allow us to intercept this evolutionary loop, we characterized the single cell transcriptomes of PF from 4 U-CLL and 4 M-CLL patients immediately prior to ibrutinib treatment...To extend on these observations, we have documented that the unique surface markers expressed in these PF subfractions positively correlate with our transcriptomic results. Taken together, these results not only highlighted the molecular heterogeneity of CLL, but also lead to the identification of two PF subpopulations that may provide novel actionable therapeutic targets to significantly halt CLL clonal evolution.

ABBV-MALT1 has also shown activity in a range of preclinical lymphoma models as both monotherapy and in combination with venetoclax, including robust antitumor activity in malignant B-cell models that are resistant to BTK inhibitors. Pts are being enrolled in 25 sites across the USA, Australia, Belgium, France, Germany, Israel, Spain, and UK. As of August 1, 2023, 2 pts had been treated.

The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Twenty-three (76.7%) of the patients were on ibrutinib, while 7 (23.3%) were on acalabrutinib...Six patients (20.0%) continued on the same BTKi with addition of venetoclax, 10 (33.3%) patients were switched to venetoclax and immunotherapy (either obinutuzumab or rituximab), and 3 (10.0%) patients were started on single agent venetoclax...Three (10.0%) patients were taken to chimeric T cell receptor (CAR-T) cell therapy of which one patient had no response and was started on salvage treatment with duvelisib... Our results reveal that durable responses can be achieved by switching to venetoclax based regimens in patients with BTKi. Though the early results of the use of noncovalent BTKi in this setting aree encouraging, the durability of response is limited as new BTK mutations are selected and future therapeutic alternatives are needed for these subset of patients.

Understanding of acquired resistance to cBTKi therapy has largely come from data on patients (pts) treated with the first-in-class cBTKi ibrutinib...In contrast, much less is known about genetic mechanisms of drug resistance in pts treated with next generation cBTKi acalabrutinib and zanubrutinib... After a median follow-up of 6.5 years (IQR 2.9-6.3), 23/48 (48%) pts developed PD (20 CLL, 3 RT). PD occurred in 17/32 (53%) R/R pts and 6/16 (38%) TN pts. Median progression-free survival was reached at 6.0 years.

To gain further insight into the genetic mechanisms of cBTKi resistance in a randomized population of pts with CLL, we performed next-generation sequencing (NGS) on samples from pts who had progression on zanubrutinib (zanu) or ibrutinib (ibr) in the phase 3 ALPINE study (NCT03734016; Brown et al. Of the 52 pts, most (82.6%) did not have acquired BTK or PLCG2 mutations. Among the zanu pts, 3/24 (12.5%) developed non-C481 BTK mutations. This rate was lower than that reported by Woyach et al (ICML 2023); shorter follow-up and fewer prior therapies in the ALPINE study may explain this discrepancy.

BTK Cysteine 481 substitution is known to contribute to cBTKi acquired resistance to ibrutinib, acalabrutinib, and zanubrutinib. Despite this cohort representing the first relapsing CLL patients from BRUIN and presenting with frequent baseline BTK mutations, response to pirtobrutinib was high, with an ORR of 83%, and substantial clearance of BTK C481 clones. At progression, the majority of pts (56%) either acquired non-BTK mutations or did not acquire any resistance mutations in this targeted panel, suggesting alternative resistance mechanisms. A smaller group of patients (44%) displayed emergence of non-C481 clones, particularly gatekeeper T474 and kinase-impaired L528W mutations.

In summary, our study highlighted the clinical implications of CSF-ctDNA in CNSi detection of ND DLBCL pts (Figure 1M): (1) CSF-ctDNA exhibits higher sensitivity than CM in detecting CNSi in ND DLBCL. (2) Different genomic and clinical landscapes were observed between SCNSL and PCNSL. (3) The CNSi-IPI is a robust, highly reproducible tool that can be used to estimate the risk of CNSi in ND DLBCL.

We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib...In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S, PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment including known resistance mutations p.C481R, p.T474I and p.C481S...For example, BTK p.L528W in patient R001 increased in frequency more than 1000-fold (from CCF 0.02% to 35%), and p.T474I in patient R002 increased in CCF from 0.03% to 4.2% (more than 100-fold). Our data illuminates the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.

P1/2, N=209, Active, not recruiting, Juno Therapeutics, a Subsidiary of Celgene | Trial completion date: Jul 2027 --> Jul 2026 | Trial primary completion date: Jul 2027 --> Jul 2026

This systematic investigation describes development of mutations over time in patients without PD and informs the potential clinical opportunity to optimize ongoing benefits for such patients.

(1) Ibrutinib combined with curcumin has synergistic killing effect on TP53- mutated CLL cells; (2) Curcumin increases the killing effect of ibrutinib on TP53-mutated CLL cells by inhibiting PI3K and NFκB pathway, and degrading p53mt protein.

Novel, non-C481 BTK mutations have been described, including BTK L528W, which has been reported in patients treated with ibrutinib, zanubrutinib, and pirtobrutinib (Maddocks, JAMA Oncol. This is the largest dataset characterizing the incidence and patterns of BTK and PLCG2 mutations in patients with CLL treated with ibrutinib. BTK C481 mutations are the most frequently occurring mutations. PLCG2 mutations occur across the gene at a low incidence per locus (<5%).

73 patients had only one BTKi (ibrutinib (IBR), 64; acalabrutinib (ACA), 9). 12 pts had multiple BTKis, 8 with two drugs with IBR first followed by ACA (Nf3, 37.5%), vecabrutinib (Nf1, 12.5%), and PIR (Nf4, 50.0%); and 4 with three or more drugs... Our retrospective report summarizes mutations detected during BTKi treatment and shows that BTK L528W can occur during both covalent and non-covalent BTK inhibitor therapy. Four of six patients who progressed on PIR had T474 mutations. In addition, our results may suggest that activating mutations in RAS/RAF/MAPK pathway are related to BTKi resistance.

In CLL cells, we observed a downregulation of markers related to adhesion, immunosuppression, and migration, and an overexpression of TMBIM6 in CLL cells that retained migrative capacity towards CXCL12 under ibrutinib. Furthermore, we identified TMBIM6 high expression as an independent poor prognostic variable and demonstrated that BIA induces apoptosis of CLL cells ex vivo revealing TMBIM6 as a potential novel target for CLL.

Introduction: The drug-resistant mechanisms of the first-generation Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib, has been extensively explored in Chronic Lymphocytic Leukemia (CLL) patients. Integrated multi-omics were performed in our zanubrutinib-resistant CLL patients’ cohort. BTK Cys481 and Leu528 were two main BTK resistant mutations in zanubrutinib resistant CLL patients. Due to spatial heterogeneity and clonal evolution among patients, deep targeted-gene NGS and ddPCR should be used comprehensively to evaluate the emergence of resistant clones.

If prior CIT, I no longer recommend re-treatment with CIT, regardless of duration of previous response, given the consistent evidence for superiority of targeted agents over CIT (discussed below), unless low intensity therapy such as oral chlorambucil or cyclophosphamide is used for purely palliative symptom control...The resulting score was clustered as low (0–1), intermediate (2–3) or high (4) and stratified probability of 24-month overall survival as 88%, 64%, and 44%, respectively in an external validation cohort.41 Among the choices of targeted agent drug classes, my least preferred are the PI3K inhibitors such as Idelalisib (±rituximab) as the efficacy is inferior and the toxicity profile,35, 42 especially infection risk and solid organ autoimmune complications (colitis/hepatitis/pneumonitis) are less favorable than the alternatives, as directly established in comparison against acalabrutinib in the ASCEND study.36 The major data from the most impactful randomized studies in R/R CLL are summarized in Table 2...The ELEVATE R/R study38 with acalabrutinib showed non-inferior PFS to ibrutinib (consistent in all biological subsets) but a clearly superior toxicity profile with lower rates of atrial arrhythmias, hypertension and cumulatively a lower cumulative toxicity burden.48 The ALPINE study compared zanubrutinib to ibrutinib and also displayed superior tolerability, fewer discontinuations and a lower rate of arrhythmias, but no difference in rates of hypertension (all grade; 21.9 vs. 19.8% and grade 3; 14.8 vs. 11.1%).39 A potential major advantage for zanubrutinib is that it also achieved statistically significantly superior PFS over ibrutinib (24 month rates of 79.5% vs. 67.3%; HR 0.65, p = 0.0024), but the PFS difference may in part be attributable to a relative “under performance” of ibrutinib in ALPINE relative to RESONATE and ELEVATE R/R.49 I personally prefer acalabrutinib or zanubrutinib over ibrtuinib noting that both have the slight inconvenience of twice daily dosing...2 TIME-LIMITED VENETOCLAX The early clinical evaluation and first regulatory approvals of venetoclax used a continuous single-agent treatment schedule,52 the largest clinical experience in the del(17p) subset used that approach,53, 54 and the most robust evaluation of prognostic factors for outcomes with venetoclax therapy was performed in a cohort predominantly so treated,55 identifying the following factors predictive of less durable disease control: bulky adenopathy (≥5 cm), disease refractoriness to either fludarabine or prior B-cell receptor inhibitors, TP53 aberrancy, and NOTCH1 mutation.55 While less durable than in BTKi-naïve disease, if BTKi refractory or intolerant, venetoclax is the preferred treatment with 70% response rate and median PFS of ∼24 months23. Combination therapy with an anti-CD20 antibody achieves a higher rate of uMRD (above 60% in R/R cohorts),56 enabling time-limited therapy for deep responders without evidence of impairing PFS relative to continuous therapy,57 and maintaining the opportunity for re-treatment with prolongation of duration of venetoclax benefit.57, 58 That approach with a fixed 24-month treatment duration was used in the MURANO study and compared to Bendamustine-rituximab.24, 30, 59, 60 The logistic complexities and resource burdens of the venetoclax dose-ramp up and tumor-lysis syndrome mitigation and monitoring, inconvenience of the parenteral anti-CD20 antibody administration are draw-backs from this approach, perhaps counterbalanced by the excellent tolerance and low adverse event rate beyond the first 6 months combination treatment period, and the attraction of time-limited therapy...4 RICHTER SYNDROME The development of Richter transformation as a clonally related evolution of underlying CLL is a profoundly adverse event and is usually fatal (median survival ∼6 months),1 unless able to receive a cellular immunotherapy (allogeneic transplant or CAR-T).65 The minority of patients where the DLBCL is clonally unrelated to their CLL, definitively recognized by comparative mutational analyses, but correlated with low expression of PD-1 by immunohistochemistry,66 is important to recognize, as their prognosis is far more favorable and may approach that of de novo DLBCL. Standard anthracycline- or ara-C/platinum-based regimens commonly used for DLBCL achieve response rates of 40%–65% but median PFS is consistently substantially less than 12 months (reviewed in ref #1) and clinical trials are therefore strongly preferred but have not shown major promise to date except for the addition of Venetoclax to DA-EPOCH-R,67 noting profound myelosuppression commonly encountered, and perhaps best utilized where subsequent allogeneic transplant can restore hematopoiesis,64 or inexplicably pirtobrutinib,68 despite low activity of other BTKi.

Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients. CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression.

There were 29 men, 16 women, median age 65.5 years (range 47-86), 43 patients received ibrutinib and 2 acalabrutinib. Our study shows that BTK and/or PLCG2 mutations were found in 64.4% of patients with progression of CLL during BTKi therapy, and in 35.6% of patients the cause of resistance has not yet been identified. Most mutations in our sample were detected in the C481 codon of BTK gene after 2 years of treatment, suggesting that regular screening with simple PCR tests starting from the second year of treatment is a reasonable approach. NGS may expand data in cases with undetectable mutations.

Background: The drug-resistant mechanisms of the first-generation Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, has been extensively explored in Chronic Lymphocytic Leukemia (CLL) patients. NGS and ddPCR should be used comprehensively to evaluate the emergence of resistant clones. BTK Cys481 and Leu528 were two main BTK mutations leading to zanubrutinib resistance. The shift of BCL2 anti-apoptotic family gene expression indicates that higher MCL-1 expression of RT might lead to insensitivity to venetoclax treatment following zanubrutinib resistance.

Gene mutation is common in pediatric acute B lymphoblastic leukemia, especially the RAS signal pathway relatedgene mutation. Gene mutations involve multiple signaling pathways. Prognostic analysis finds that multiple gene mutations are associated with prognosis.

CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression.

P1/2, N=209, Active, not recruiting, Juno Therapeutics, a Subsidiary of Celgene | Trial completion date: Feb 2027 --> Jul 2027 | Trial primary completion date: Feb 2027 --> Jul 2027

Detection of acquired BTK and PLCG2 mutations in ctDNA while on therapy is feasible and may herald clinical disease progression. This trial was registered at https://anzctr.org.au/ as ACTRN12619000024145.

P1/2, N=252, Active, not recruiting, Juno Therapeutics, a Subsidiary of Celgene | N=188 --> 252

Finally, no difference in TP53 mutation burden was observed between BTKmut versus BTKwt relapsing cases, and ibrutinib treatment did not appear to favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance.

P1/2, N=188, Active, not recruiting, Juno Therapeutics, a Subsidiary of Celgene | Trial completion date: Sep 2026 --> Dec 2026 | Trial primary completion date: Sep 2026 --> Dec 2026

Introduction Progressive disease (PD) is driven by clonal evolution in patients with chronic lymphocytic leukemia (CLL) treated with the Bruton tyrosine kinase inhibitor (BTKi) ibrutinib. Acquired mutations in NOTCH1, ZNF292, and BIRC3 were detected in PD patients without BTK or PLCG2 mutations. Longitudinal mutation profiling could deepen understanding of acalabrutinib resistance mechanisms in CLL.

However, some patients (pts) have experienced toxicities to BTK inhibitors ibrutinib (ibr) and acalabrutinib (acala), which lead to dose reduction or treatment discontinuation. Exploratory analysis results confirmed that cell cycle, DNA damage, and NOTCH1 pathway genes were frequently mutated in pts with B-cell malignancies on study BGB-3111-215 (pts intolerant to ibr and/or acala). Pts with mutations associated with poor prognosis at baseline were more likely to develop PD.