Citations

Background Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. Methods We developed transgenic mice carrying D oxycycline (Dox)- i nducible SB mutag e nesis s y stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8 + T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 + T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively. Results Under chronic stimulation, SB -mutagenized CD8 + T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. Conclusions Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

PURPOSE Leptomeningeal metastatic disease (LMD) is associated with poor prognosis and imposes severe symptomatic limitations on functional status and quality of life. In recent years, the range of diagnostic and therapeutic options available to patients with LMD has expanded, but the impact of these innovations on clinical characteristics and outcomes remains unclear. We conducted a retrospective study to define the LMD patient population, symptoms experienced, and the frequency of accessing specialty palliative care (PC) services, at a large tertiary referral center. METHODS We conducted a retrospective electronic medical record review on the basis of a convenience sample of 104 patients diagnosed and treated with LMD with dates of diagnosis between January 1, 2016, and October 1, 2022. Variables were generated on the basis of patient demographics, reported symptoms, and clinical course. Data were analyzed using Stata 16. RESULTS Of 104 patients in the analysis, the most common symptoms reported were headache and extremity weakness (52% of patients reported each). The mean number of weeks between LMD diagnosis and date of death was 41.4, with a median of 27.9 weeks, and a range of 1.4-143.4 weeks. Forty-five percent of patients had at least one documented visit with an inpatient or outpatient PC provider. CONCLUSION Patients with an LMD diagnosis face a high symptom burden. Our findings highlight that prognosis may be longer than what is typically quoted in the literature, but further study would be needed to understand this discrepancy. Fewer than half of the patients in our sample had a documented visit with a PC specialist, suggesting a missed opportunity in this population to connect with providers specialized in managing symptom burden and doing nuanced advance care planning.

ABSTRACT Background and hypothesis Podocytopathy associated with likely pathogenic/pathogenic variants of Transient receptor potential cation channel subfamily C member 6 (TRPC6) (TRPC6-AP) has been recognized for about 20 years. As a result of its rarity however, the spectrum of clinical phenotypes and genotype–phenotype correlation of TRPC6-AP remains poorly understood. Here, we characterized clinical, histological and genetic correlates of familial and sporadic patients with TRPC6-AP. Methods In this multicentre observational study, an online questionnaire followed by a systematic literature review was performed to create a cohort with comprehensive data on genetic and clinical outcomes [age of onset, clinical presentation, treatment response, kidney biopsy findings and progression to kidney failure (KF)]. Logistic regression, Cox proportional hazards model and Kaplan–Meier analyses investigated the associations between genetic variants and disease progression. Results Among 87 families (96 familial and 45 sporadic cases), 31 distinct missense TRPC6 variants (including 2 novel) were identified, with c.2683C>T p.(Arg895Cys) and c.523C>T p.(Arg175Trp) the commonest variants. Proteinuric kidney disease/nephrotic syndrome was the most common clinical presentation (83.7%), while focal segmental glomerulosclerosis was the most common histological finding (89.4%). By 33 (interquartile range 17–40) years, 48.9% (69/141) of patients had progressed to KF. Sporadic TRPC6-AP demonstrated an earlier progression to KF than familial cases (P = .001) and were more likely to present with nephrotic syndrome [odds ratio 4.34 (1.85–10.15); P = .001]. Gain-of-function TRPC6 variants were more frequent in familial than sporadic TRPC6-AP (70.8% vs 44.4%; P = .004). Compared with patients with other TRPC6 variants, patients with TRPC6 p.R175W and p.R895C variants progressed to KF earlier [median kidney survival of 21 years, hazard ratio 2.985 (95% confidence interval 1.40–5.79); and 38 years, hazard ratio 1.65 (95% confidence interval 1.01–2.81), respectively, log-rank P = .005]. Conclusion Our study shows unique clinical and genetic correlations of TRPC6-AP, which may enable personalized care and promising novel therapies.

Importance There is a lack of long-term efficacy and safety data on hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) and on RNA interference (RNAi) therapeutics in general. This study presents the longest-term data to date on patisiran for hATTR-PN. Objective To present the long-term efficacy and safety of patisiran in adults with hATTR-PN. Design, Setting, and Participants This global open-label extension (OLE) of the APOLLO randomized clinical trial and phase 2 OLE study enrolled patients from 43 hospitals or clinical centers across 19 countries between July 2015 and August 2017, with follow-up until November 2022. Of 212 eligible patients with hATTR who completed the phase 3 APOLLO or phase 2 OLE parent studies, 211 enrolled in and 138 completed the global OLE. Intervention Patisiran, 0.3 mg/kg, intravenously once every 3 weeks for up to 5 years. Main Outcomes and Measures Outcomes evaluated at year 5 of the global OLE included disability (polyneuropathy disability [PND] score); polyneuropathy severity (Neuropathy Impairment Score [NIS]), nutritional status (modified body mass index [mBMI]), quality of life (Norfolk Quality of Life–Diabetic Neuropathy [Norfolk QOL-DN]), and Rasch-Built Overall Disability Scale (R-ODS), with no statistical hierarchy. Safety, survival probability, and mortality were also assessed. Results At the global OLE baseline, the mean (SD) age was 61.3 (12.3) years, and 156 patients (73.9%) were male. In 138 patients completing the study, PND scores remained stable or improved in 89 patients (65.0%), NISs showed a mean (SD) change of 10.9 (14.7), and mean (SD) mBMI (calculated as weight in kilograms divided by height in meters squared times serum albumin in grams per liter) increased by 46.4 (120.7) over 5 years from baseline. Norfolk QOL-DN and R-ODS scores showed mean (SD) changes of 4.1 (16.7) and –3.7 (6.2), respectively. Adverse events (AEs) leading to study withdrawal occurred in 47 patients (22.3%). Infusion-related reactions were the most common treatment-related AE (n = 34 [16.1%]). Overall, 41 patients (19.4%) died during the study. Patisiran treatment in the parent study and low familial amyloid polyneuropathy score at parent study baseline were associated with significantly improved survival. Conclusions and Relevance In the longest study of an RNAi therapeutic for any disease, patisiran treatment resulted in modest changes for patients with hATTR-PN with an acceptable safety profile. These results highlight the importance of initiating early treatment for hATTR and the potential of RNAi therapeutics in medicine. Trial Registration ClinicalTrials.gov Identifier: NCT02510261

Abstract Introduction: Estrogen receptor-positive (ER+)/HER2-negative (HER2-) breast cancers commonly acquire constitutively active mutations in the ERα-encoding gene (ESR1), resulting in endocrine therapy (ET) resistance. In the phase 2, ELAINE 2 study (NCT04432454), LAS (a breast ER antagonist) combined with Abema provided a median progression-free survival (PFS) of ~13 months (mos) and clinical benefit rate (CBR) of 66% in patients with endocrine-resistant, ER+/HER2- mBC and ESR1 mutations (mESR1; Damodaran. J Clin Oncol 2023;41:suppl16;1057). Our objective was to describe baseline genomic alterations co-occuring with mESR1 and treatment responses to LAS plus Abema in patients with these concurrent genomic alterations. Methods: Women (age ≥18 yrs) with ER+/HER2- mBC that progressed after prior ET and CDK4/6 inhibitor (CDK4/6i) with an mESR1 detected in blood using the Sysmex-Inostics SafeSeq circulating tumor DNA (ctDNA) test were enrolled in ELAINE 2. Oral LAS 5 mg/day and Abema (provided by Eli Lilly and Co) 150 mg BID were taken until disease progression, death, unacceptable toxicity, or withdrawal. The Guardant360 CDx test was used to identify and describe baseline genomic alterations in ctDNA (including mESR1). Median PFS and CBR were assessed in patients with mESR1 and co-existing baseline genomic alterations. For patients who withdrew before disease progression, their last observation time was used as the progression time for PFS estimation. Data were summarized descriptively with no formal hypothesis testing. Results: Of the 29 patients (median age 60 yrs) enrolled, mutations in ESR1 were identified in 26 by Guardant360; all these 26 patients had received prior CDK4/6i, 21 (81%) prior fulvestrant, and 12 (46%) prior chemotherapy for mBC. Alterations in 39 genes were concurrently identified with mESR1, including TP53 mutations (n=11), PIK3CA mutations (n=8), CCND1 amplifications (n=6), and FGFR1 amplifications (n=5); co-amplification of CCND1 and FGFR1 was identified in 3 patients. LAS plus Abema treatment was associated with a CBR of 73% and a median PFS of 12.9 mos in the 26 patients with mESR1 (Table). CBRs were 63% in the co-occurring PIK3CA mutation subgroup and 64% in the TP53 mutation subgroup; median PFS times were 7.8 and 8.3 mos, respectively. All patients in the CCND1 and FGFR1 amplification subgroups achieved clinical benefit (CBR 100%) and the median PFS was 16.6 mos for both subgroups. In patients with mESR1 and co-alterations in ≥2 other genes of interest (PIK3CA, FGFR1, CCND1, TP53, ERBB2, CCNE1, or RB1), CBR was 83% and median PFS was 16.6 mos. Conclusion: Using Guardant360 ctDNA profiling from patients in ELAINE 2, we demonstrate that other baseline genomic alterations are frequently detected concurrently with mESR1 in the endocrine resistant setting, but without apparent compromise on the efficacy of LAS plus Abema. These results should be interpreted with caution considering the small numbers of patients and the exploratory nature of the analysis. The ELAINE 2 study suggests the potential of LAS plus Abema for treating ESR1-mutated, ER+/HER2- mBC in the post-CDK4/6i setting. This will be further evaluated in the larger, registrational, phase 3, ELAINE 3 study (NCT05696626) that compares LAS plus Abema with fulvestrant plus Abema. Table. Treatment response by genomic alterations concurrent with ESR1 mutations (n=26). aFor patients who withdrew before disease progression, the last observation time was used as the progression time for PFS estimation. bOther genes of interest include PIK3CA, FGFR1, CCND1, TP53, ERBB2, CCNE1, or RB1. CBR, clinical benefit rate; mPFS, median progression-free survival. Citation Format: Senthil Damodaran, Massimo Cristofanilli, Matthew Goetz, Ciara O'Sullivan, Gary Riordan, Hope Rugo, Daniel Stover, Seth Wander, Dominic Carroll, Paul V. Plourde, David J Portman, Einav Nili-Gal Yam. Baseline genomic alterations and the activity of lasofoxifene (LAS) plus abemaciclib (Abema) in patients with ER+/HER2- metastatic breast cancer (mBC): the ELAINE 2 study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-09.

Abstract Introduction: Use of long-term endocrine therapy (ET) for ER+ breast cancer often leads to acquired ESR1 mutations (mutESR1), causing endocrine resistance, tumor progression, and poor prognosis. An unmet clinical need exists for treating ER+ mBC patients with mutESR1, particularly after progression on CDK4/6 inhibitors (CDK4/6i). ELAINE 2 is an open-label, phase 2, multicenter trial evaluating safety and efficacy of lasofoxifene (LAS [selective estrogen receptor modulator]) plus abemaciclib (Abema [CDK4/6i], provided by Eli Lilly) in patients with ER+/HER2- and mutESR1 mBC who progressed after prior ET. Preliminary data with LAS plus Abema showed median progression-free survival of 55.7 wks, objective response rate of 50%, and 24-wk clinical benefit (CB) rate of 69%, with an acceptable safety and tolerability profile. Here, we report ESR1 ctDNA mutant allele frequency (MAF) and correlations of ESR1 MAF changes with CB. Methods: ELAINE 2 patients with detectable ctDNA mutESR1 at baseline (BL) were analyzed. Oral LAS 5 mg/day and Abema 150 mg BID were taken until disease progression, death, unacceptable toxicity, or withdrawal from the study. ctDNA was assessed by the Sysmex-Inostics SafeSeq assay—which detects mutESR1 at low allele fractions—at BL, every 4 wks, and end of treatment. MAF changes from BL to wk 4 were characterized as decreased (decrease in ESR1 MAF or none detected [ND]), increased (increase in MAF), or equivocal (in polyclonal patients [>1 mutESR1] with some MAF increasing and decreasing trends). Correlations of MAF change at 4 wks with CB at 24 wks were explored. Results: 29 patients (median of 2 prior metastatic therapies: 97% CDK4/6i, 79% fulvestrant, 48% chemotherapy) had BL mutESR1 of Y537S (66%), D538G (45%), Y537N (28%), and other less frequently detected mutations; 14 (48.3%) patients were polyclonal. 26 of 29 patients had evaluable BL and wk-4 ctDNA results: 21 patients had decreased MAF (81% [54% with ND]), 3 (12%) had increased, and 2 (8%) had equivocal ESR1 MAF changes (Table). CB at 24 wks was seen in 17 patients with a decrease, 2 with an increase, and 1 with equivocal MAF change. A sensitivity of 89.5% and specificity of 20% were calculated for predicting CB based on direction of ESR1 MAF change. The positive predictive value (PPV) for CB with decreased MAF was 81% and the negative predictive value (NPV) for an increased MAF was 33%. Of the 14 (54%) patients with ND ESR1 MAF, 13 had CB resulting in 87% sensitivity, 50% specificity, 93% PPV, and 33% NPV. Conclusion: In ELAINE 2, 81% of patients had decrease/cleared (ND) mutESR1 after 4 wks of LAS plus Abema, which correlated with clinical benefit. All mutESR1 detected appear targeted with this therapy. High sensitivity and favorable PPV were observed in patients with decreased MAF, and even more so in those with ND MAF; however, increased MAF was less specific and not as predictive of treatment failure. Our results indicate that ESR1 liquid biopsy evaluation may be an adequate non-invasive surrogate marker for monitoring patients on treatment. Further study in a larger population of women with endocrine-resistant mBC and acquired mutESR1 is warranted to explore this potential for monitoring treatment response or resistance to this novel LAS-Abema combination. Table. Change from baseline to week 4 in ESR1 MAF and clinical benefit at 24 weeks. CI, confidence interval; MAF, mutant allele fraction; ND, none detected; NPV, negative predictive value; PPV, positive predictive value. *Sensitivity and specificity analyses do not include equivocal results. Citation Format: Senthil Damodaran, Halle Moore, Ciara C. O’Sullivan, Paul V. Plourde, Gary Riordan, Hillary S. Sloane, Debu Tripathy, Dominic Carroll, David J Portman. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene plus abemaciclib in the ELAINE 2 study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-02.

Abstract Introduction: Acquired ESR1 mutations (mutESR1) after long-term endocrine therapy drive treatment resistance, metastasis, and poor prognosis for patients (pts) with ER+/HER2- metastatic breast cancer (mBC). Lasofoxifene (LAS), a selective estrogen receptor modulator, alone or with a CDK4/6 inhibitor (CDK4/6i) reduced tumor growth better than fulvestrant (Fulv) in mutESR1 BC xenograft models. ELAINE 1 is a randomized trial of LAS vs Fulv in pts with mutESR1 and prior progression on aromatase inhibitor and CDK4/6i. Preliminary results (ESMO 2022) showed that LAS prolonged median progression-free survival (mPFS) compared with Fulv with a favorable safety profile. Here, we report changes in ESR1 ctDNA mutant allele frequency (MAF) from baseline to 8 wks and their associations with clinical benefit (CB) and mPFS. Methods: ELAINE 1 pts were randomized to oral LAS 5 mg daily or IM Fulv 500 mg on days 1, 15, and 29, then every 4 wks, until disease progression or severe toxicity. ctDNA mutESR1 mutations (baseline and 8 wks) were assessed using the Sysmex Inostics OncoBeam or SafeSeq assays—which detect mutESR1 at low allele fractions. MAF changes from baseline to wk 8 were characterized as decreased (decrease in ESR1 MAF or fully cleared), increased (increase in MAF), or equivocal (polyclonal patients [>1 mutESR1] with some increasing and decreasing MAF trends); correlations with PFS and CB were explored. Efficacy measures included objective response rate (ORR), PFS, and CB at 24 wks (CB defined as response or stable disease ≥24 wks). Results: 103 pts received LAS (n=52) or Fulv (n=51). Most common baseline ESR1 variants detected were D538G (56%), Y537S (39%), Y537N (29%), E380Q (22%); 56 (54%) pts were polyclonal. Of the 61 pts with evaluable baseline and wk 8 ctDNA, LAS decreased mutESR1 MAF in 29/35 pts (83% [11 complete clearance]) while Fulv decreased mutESR1 MAF in 16/26 pts (61.5% [6 complete clearance]) (Table). mPFS with LAS was 8 and 4 mos for pts with decreased/cleared MAF and increased MAF, respectively, and with Fulv was 4.5 and 2.8 mos, respectively (Table). LAS decreased the common mutESR1 variants more frequently than Fulv (median relative change -87.1% vs -14.7%). In pts with decreased MAF, CB was observed in 16/29 LAS pts (55%) and 4/16 Fulv pts (25%). The predictiveness of ESR1 MAF clearance for CB was also explored. Of 11 pts with ESR1 MAF clearance taking LAS, 10 achieved CB, yielding a positive predictive value (PPV) of 90.9%. In contrast, 2/6 pts with ESR1 MAF clearance taking Fulv had CB for a PPV of 33.3%. Sensitivity for predicting CB based on direction of ESR1 MAF change was 94% with LAS and 80% with Fulv. In pts with Y537S MAF (n=33), LAS decreased Y537S in 13/15 (87%), with a median relative MAF decrease of 89%. In marked contrast, Fulv increased Y537S MAF in 11/18 pts (61%), corresponding to an MAF relative increase of 82%. LAS and Fulv resulted in complete clearance of Y537S MAF in 33% and 6% of pts, respectively. Conclusion: Our data demonstrate that LAS more effectively decreased or cleared mutESR1 than Fulv. Further, mutESR1 clearance was associated with prolonged PFS and more CB in LAS but not Fulv pts, suggesting that LAS results in robust mutESR1 target engagement. Taken together, our data suggest mutESR1 as a potential liquid biomarker for predicting response to LAS in mutESR1, endocrine-resistant mBC pts. Table. Change from baseline to week 8 in ESR1 MAF and clinical benefit at 24 weeks. CI, confidence interval; MAF, mutant allele fraction; ND, none detected; PFS, progression-free survival. Citation Format: Matthew P. Goetz, Einav Gal-Yam, Daniel Stover, Sarah L. Sammons, Stephanie L. Graff, Grace Wang, Massimo Cristofanilli, Gary Riordan, Hillary S. Sloane, Dominic Carroll, Paul V. Plourde, David J Portman. Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) from patients with ER+/HER2- metastatic breast cancer (mBC) treated with lasofoxifene or fulvestrant in the ELAINE 1 study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-04.