The use of antibody-drug conjugates (ADCs) specific to human epidermal growth factor receptor 2 (HER2), such as ado-trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (Enhertu, T-DXd), and trastuzumab duocarmazine (SYD985), to treat breast cancer patients has been associated with a variety of toxicities including corneal epithelial keratitis. The anatomic basis for ocular toxicities has not been established. Using immunohistochemistry, HER2 expression was identified in the human corneal, limbal and conjunctival epithelium of fetal and adult human eyes. We also identified HER2 expression in tissues from the Macaca fascicularis (monkey) to confirm a similar distribution of HER2 expression as found in humans and to support the use of this species as a toxicology model in preclinical testing of anti-HER2 ADCs. As in the human, expression of HER2 in monkey was limited to normal epithelium, as illustrated for the eye, kidney, and uterus. Incubation of normal human corneal epithelial cell lines with trastuzumab or T-DXd demonstrated the capacity for receptor-mediated endocytosis of HER2-targeted therapies by epithelia in the eye. Our findings of HER2 expression in normal human corneal epithelium suggest that the ocular adverse events associated with HER2-targeted ADCs may be driven, at least in part, by an on-target effect.

Purpose: HER2 amplification occurs in 18% to 27% of gastric and gastroesophageal junction cancers. Lapatinib, a potent ATP-competitive inhibitor simultaneously inhibits both EGFR and HER2. To explore the role of HER family biology in upper gastrointestinal cancers, we evaluated the effect of lapatinib, erlotinib, and trastuzumab in a panel of molecularly characterized human upper gastrointestinal cancer cell lines and xenografts. Experimental Design: EGFR and HER2 protein expression were determined in a panel of 14 human upper gastrointestinal cancer cell lines and HER2 status was assessed by fluorescent in situ hybridization. Dose-response curves were generated to determine sensitivity to lapatinib, erlotinib, and trastuzumab. In HER2-amplified cells, the combination of trastuzumab and lapatinib was evaluated using the median effects principal. The efficacy of lapatinib, trastuzumab, or the combination was examined in HER2-amplified xenograft models. Results: Lapatinib had concentration-dependent antiproliferative activity across the panel with the greatest effects in HER2-amplified cells. There was no association between EGFR protein expression and sensitivity to any of the HER-targeted agents. Cell cycle analysis revealed that lapatinib induced G1 arrest in sensitive lines and phosphorylated AKT and phosphorylated ERK were decreased in response to lapatinib as well. The combination of lapatinib and trastuzumab was highly synergistic in inhibiting cell growth with a combination index of <1. The combination also induced greater decreases in AKT and ERK activation, G0-G1 cell cycle arrest, and increased rates of apoptosis. In vivo studies showed that the combination of lapatinib and trastuzumab had greater antitumor efficacy than either drug alone. Conclusion: Together, these data suggest that lapatinib has activity in HER2-amplified upper gastrointestinal cancer and supports the ongoing clinical investigation of lapatinib in patients with HER2-amplified disease. Clin Cancer Res; 16(5); 1509–19

501 Background: In the NATALEE clinical trial, RIB + NSAI significantly improved invasive disease–free survival (iDFS) vs NSAI alone in patients (pts) with stage II/III HR+/HER2− EBC. We report on the prognostic and predictive value of baseline gene exp in NATALEE. Methods: Pts were randomized 1:1 to RIB + NSAI or NSAI alone. Men and premenopausal women received goserelin. Eligible pts had anatomical stage IIA (if N0 with additional risk factors [G3, or G2 with Ki67 ≥20% or high genomic risk] or N1 [1-3 axillary lymph nodes]), IIB, or III disease per AJCC (8th ed). Gene exp in surgical samples was profiled by NanoString BC360 panel. PAM50-based intrinsic subtype × treatment (tx) interaction was estimated by likelihood ratio test. Prognostic and predictive effects of PAM50 subtypes and genomic risk/proliferation signature scores created from gene exp data were assessed with Cox proportional hazards models. Differences in genomic risk and proliferation scores were analyzed by Wilcoxon test. Associations between gene exp and RIB benefit were estimated with Cox models. Results: In all, 3022 baseline surgical samples were analyzed. iDFS benefit with RIB + NSAI was consistent in the biomarker (hazard ratio [HR], 0.72) and intent-to-treat populations (HR, 0.71). PAM50 subtype distribution was comparable across tx arms and differed between N0 and N1-N3 in pts with luminal A (LumA; 50% vs 68%), luminal B (lumB; 41% vs 26%), and basal-like (BSL; 6.4% vs 2.5%) disease, with similar percentages for HER2-enriched (HER2E; 2.5% vs 3.0%). PAM50 subtypes were strongly prognostic (HRs vs LumA: LumB, 1.39; HER2E, 2.62; BSL, 3.92). RIB had benefit across all PAM50 subtypes (HRs: LumA, 0.77; LumB, 0.71; HER2E, 0.50; BSL, 0.42), with no significant subtype × tx interaction ( P = .34). Higher genomic risk signature or proliferation signature scores showed a trend for increased RIB benefit, again with no significant interaction. Pts with N0 vs N1-N3 had significantly higher genomic risk scores and proliferation scores. Exploratory analysis of the predictive effect of gene exp on RIB benefit identified several genes for which higher (eg, CEACAM6 , NOD2 , and GPX3) or lower exp (eg, GATA3 , SLC39A6 , and MAPT ) was associated with increased RIB benefit. Conclusions: In this analysis of NATALEE, which examined the largest dataset of surgical tumor samples from any adj CDK4/6i trial in HR+/HER2− EBC, RIB had benefit across all PAM50 subtypes, with a trend for increased benefit in pts with higher genomic risk signature or proliferation signature scores. Baseline exp levels of several genes were associated with differential RIB benefit in EBC, showing potential predictive and prognostic value. The findings reinforce the therapeutic benefit of RIB in combination with ET across HR+/HER2− EBC populations. Clinical trial information: NCT03701334 .

This cohort study evaluates the incidence of cataract surgery among patients receiving mirvetuximab soravtansine to treat ovarian cancer.

Abstract Cadherin 17 (CDH17), is an emerging target for antibody-based therapeutics in colorectal cancer (CRC), with multiple CDH17-directed chimeric antigen receptor-T/natural killer, bispecific antibodies and antibody drug conjugates (ADCs) in preclinical and early-stage clinical development. Success of these molecules depends on their ability to overcome high expression of P-glycoprotein (P-gp) pumps in CRCs and CDH17 in normal gut epithelium. Here, we investigate the therapeutic potential of CDH17-directed ADCs.High frequency expression of CDH17 in patient tumor microarrays and cell line derived xenografts (CDXs) was confirmed by IHC assay. CDH17-directed ADCs were generated through conjugation of a fully humanized CDH17 mAb to either an anti-mitotic (CDH17-MMAE) or topoisomerase 1 inhibitor (CDH17-exatecan) payload via a cleavable linker. Selective ADC binding and internalization were confirmed by flow cytometry and immunofluorescence.CDH17-MMAE treatment induced significant anti-tumor activity in 5/5 CDH17+ CRC CDXs models, with the strongest and most prolonged responses observed in xenografts that were P-gp low/negative. Head-to-head comparison of CDH17-MMAE and CDH17-exatecan payloads demonstrated improved efficacy for CDH17-exatecan over CDH17-MMAE in high P-gp expressing models. The functional role of P-gp in resistance to MMAE-based ADCs was confirmed using cell lines engineered to either overexpress P-gp through transfection or to lose P-gp expression through CRISPR knockdown. Forced overexpression of P-gp in SNUC1 CDH17+ cells conferred resistance to CDH17-MMAE, while sensitivity to CDH17-exatecan remained. Conversely, CRISPR knockdown of P-gp in CDH17+ LS513 cells reversed resistance to CDH17-MMAE. These findings were confirmed in xenograft studies.Due to lack of binding of the anti-human CDH17-ADCs with mouse or rat CDH17, a mouse model expressing human CDH17 extracellular ECD (B-hCDH17 mice) was used in pharmacokinetic (PK) studies. In wild-type mice, both ADCs exhibited PK profiles consistent with those typically observed for most ADCs. However, in humanized B-hCDH17 mice, both CDH17-MMAE and CDH17-exatecan ADCs were completely cleared from mouse serum less than 96 hours post-dosing, indicating that CDH17 expression in normal colon and ileum may act as a clearance sink for the ADCs.Data presented here support use of topoisomerase 1 inhibitor payloads in cancers where P-gp expression is high. Target-mediated clearance driven by normal gut CDH17 expression presents a pharmacokinetic challenge that may limit clinical utility and require dose optimization or alternative dosing strategies to achieve sufficient tumor exposure while managing potential on-target, off-tumor toxicity. Citation Format: Neil A. O'Brien, Jun Zhang, Martina SJ McDermott, Ke Wei Gong, Ming Lu, Benjamin Hoffstrom, Min Liang, Weiping Jia, Tong Luo, Athena M. Madrid, Raul Ayala, John A. Glaspy, Leonard Presta, Dennis J. Slamon.. Preclinical evaluation of target-mediated clearance and alternative payloads in CDH17-directed antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1323.

BACKGROUND:Epigenetic modulators may sensitize platinum-resistant ovarian cancer (PROC) to immune checkpoint inhibition by reprogramming the tumor microenvironment. METHODS:We report clinical and translational findings from a phase II non-randomized study of pembrolizumab and oral azacitidine in 34 women with PROC (NCT02900560). Key eligibility criteria included age 18 years or older, performance status of 0-1, measurable disease, platinum-resistant disease and histologically confirmed epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma. Primary endpoints included safety, tolerability, overall response rate (ORR) and disease control rate (DCR). Secondary endpoints included CA-125 response. The effect of combined epigenetic and immunotherapy was evaluated by transcriptomic analyses of 72 serially biopsied tumors. RESULTS:We show that the combination is moderately well tolerated and most common grade 3-4 adverse events are gastrointestinal side effects and anemia. ORR is 2.9% and DCR is 50%; with 3 of the 27 evaluable patients attaining a CA-125 response. Differential gene expression analyses reveal an upregulation of inflammatory and cytolytic genes and co-inhibitory checkpoints 6 weeks on-therapy. Upregulation of interferon signaling, antigen presentation and immune cell adhesion and migration gene sets are prominent on-therapy, together with an increase in density of CD8 + T-cells. Patients with a CA-125 and/or clinical response show an enrichment of adaptive and conserved immune response gene sets on-therapy. CONCLUSIONS:Our findings highlight the potential of epigenetic modulators to re-shape the tumor microenvironment of PROC toward a more inflammed phenotype and may point to approaches to augment immunotherapy response.

Abstract Introduction: Epigenetic modulators may sensitize platinum-resistant ovarian cancer (PROC) to immune checkpoint inhibition by reprogramming the tumor microenvironment. Methods: We report clinical and translational findings from a phase II non-randomized study of pembrolizumab and oral azacitidine in 34 women with PROC (NCT02900560). Key eligibility criteria included age ≥ 18 years, ECOG performance status of 0-1, measurable disease and platinum-resistant, histologically confirmed epithelial ovarian cancer, fallopian tube carcinoma or primary peritoneal carcinoma. Primary endpoints included safety, tolerability, overall response rate (ORR), and disease control rate (DCR). Secondary endpoints included CA-125 response. The effect of combined epigenetic modulation and immunotherapy was evaluated by transcriptomic analyses of 72 serially biopsied tumors. Target gene expression analyses were used to identify differentially expressed genes and pathways, and bulk RNA sequencing was used for gene set enrichment analyses (GSEA) and to infer T cell receptor (TCR) assemblies. Results: The combination of azacitidine and pembrolizumab was moderately well tolerated, and the most common grade 3-4 adverse events were gastrointestinal side effects and anemia. ORR was 2.9% and DCR was 50%; with 3 of the 27 evaluable patients attaining a CA-125 response. Differential gene expression analyses revealed an upregulation of inflammatory and cytolytic genes (FDR-adjusted p=0.005) and co-inhibitory checkpoints (p=0.005) 6 weeks on-therapy. Upregulation of interferon signaling (p=0.006), antigen presentation (p=0.003), costimulatory signaling (p=0.002), and immune cell adhesion and migration (p=0.005) pathways were prominent on-therapy, together with an increase in CD8+ T-cell density (p=0.05). GSEA analyses were concordant with these findings, showing enrichment of immune and inflammatory gene sets on-therapy and upregulation of gene sets linked to IFN-γ response, natural killer cell-mediated cytotoxicity, and immunoregulatory interactions (p < 10e-13). Findings were not confounded by histological subtype. TCR abundance analyses evaluating significant TCR clonotypic expansions and regressions showed that intra-tumoral TCR repertoires were reshaped on-therapy. Furthermore, patients with a CA-125 and/or clinical response showed enrichment of hallmark inflammatory gene sets at baseline (p < 10e-10) and of adaptive and conserved immune response gene sets on therapy (p < 10e-09). Conclusions: Our findings highlight the potential of epigenetic modulators to reshape the tumor microenvironment of PROC toward a more inflammatory phenotype and may point to approaches to augment immunotherapy response. Citation Format: Blair V. Landon, Julia L. Boland, Andrea E. Wahner Hendrickson, Deborah K. Armstrong, Boris Winterhoff, Jaime Wehr, Akshaya V. Annapragada, Christopher Cherry, Archana Balan, Guneet Kaleka, Victor E. Velculescu, Stephen B. Baylin, Cynthia A. Zahnow, Dennis J. Slamon, Gottfried E. Konecny, John A. Glaspy, Valsamo (Elsa) Anagnostou. Pembrolizumab and epigenetic modification with azacitidine reprogram the tumor microenvironment of platinum-resistant epithelial ovarian cancer: clinical and translational findings from a phase II study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT260.

Drug-tolerant, high-grade serous ovarian cancer (HGSOC) cells that persist after first-line chemotherapy and subsequently relapse often retain sensitivity to secondary treatment, suggesting a therapeutic window before stable chemoresistance emerges. We performed single-cell RNA sequencing (scRNA-seq) on seven matched pairs of tumors, collected pre- and post-chemotherapy, to define vulnerabilities in these reversibly-resistant cells. Treatment induced a marked enrichment of tumor and stromal cell populations expressing correlated interferon (IFN) and inflammatory (IFM) gene signatures, with a concurrent depletion of proliferation-related and MYC-associated states in the tumor cells. Cross-cohort single cell sequencing analysis of >130 treatment-naive tumors revealed heterogeneity in the abundance of IFN/IFM-expressing cells. Multiplex immunofluorescence imaging of IFN-stimulated gene (ISG) products confirmed the presence of spatially clustered ISG-positive tumor cells in all cases, as well as in serous tubal intraepithelial carcinomas (STIC lesions), the presumptive HGSOC precursors. ISG expression correlated strongly with ORF1P, a protein encoded by the endogenous retrotransposon LINE1. These data suggest that early oncogenic events drive LINE1 derepression and innate immune activation, establishing an IFN-rich transcriptional state that persists in tumor subpopulations and is strongly enhanced by chemotherapy.

Supplemental Figure S1: Expression of CLDN6 by transcript in the GTEX dataset of normal tissue samples.

Supplemental Figure S2: Amino acid sequence alignment of CLDN6 (P56747) and CLDN9 (O95484) with the extracellular loops highlighted

KRAS is a commonly mutated oncogene long considered undruggable. Recent discoveries found compounds capable of binding to KRASG12C that inhibit KRAS-dependent signaling by locking the protein in its inactive state. However, clinical efficacy of these molecules has been limited by high rates of innate and acquired therapeutic resistance. Here, we developed cell line models of acquired resistance to the KRAS-G12C inhibitor MRTX849 (adagrasib) to study the mechanisms that underlie resistance which may help in the identification of novel therapeutic strategies to overcome resistance. KRASG12C-mutant H358 and H1373 lung cancer cell lines were treated through long-term exposure of increasing doses of the MRTX849 to generate the resistant clones, H358-R and H1373-R. Antiproliferative IC50s were determined by treating cells with serially diluted drug for 6 days and cells were quantified using CellVista® at Day 1 and Day 6. Resistant cells were genotyped by PCR amplification. Changes in protein signaling were determined by immunoblotting and changes in mRNA signaling were measured using RNA-seq analysis. A 15-fold shift in IC50 was observed between H358-R and parental H358 (p-value = 0.029), and between H1373-R and parental H1373 (p-value = 0.022). Genotyping of the cell lines indicated that mutant KRASG12C was preserved in both resistant cell lines. RNA-seq analysis revealed a significant log-fold increase in RPS6 expression between H358-R and parental cells treated with MRTX849 (p-value = 6.06E-6). Treatment with MRTX849 inhibited downstream signaling of ERK1/2 and RSK in both parental and resistant cell lines. However, signaling downstream of mTORC1 through pS6 only remained active in the presence of MRTX849 in the resistant cells. Treatment with the mTORC1 inhibitor, RAD001, induced partial inhibition of pS6 in the resistant cells, whereas combined treatment with MRTX849 and RAD001 induced complete and sustained inhibition of resistant pS6 signaling. Combination treatment with MRTX849 and RAD001 also produced significantly improved inhibition of proliferation in both the acquired resistant clones relative to treatment with either single agent alone; H358-R (p-value = 0.0099) and H1373-R (p-value = 0.018). In this study, we confirmed mTOR/S6 signaling as a potential escape pathway for KRAS-G12C inhibition. Targeting mTORC1 in combination with MRTX849 induced a sustained knockdown of S6-signaling and reversed resistance to MRTX849. Further genomic, transcriptomic and proteomic profiling of the generated resistant cell lines may identify additional mechanisms responsible for acquired KRAS-G12C resistance. Athena M. Madrid, Gabriel Choi, Jenny J. Hong, Sahel Hazeghsa, Jenny Z. Zhang, Kenny Castro, Neil A. O'Brien, Dennis J. Slamon. Targeting mTOR/S6 signaling reverses acquired resistance to MTRX849 in KRASG12C-mutant lung adenocarcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 561.

Adjuvant anthracyclines and taxanes reduce recurrence and death in early‐stage breast cancer (EBC) patients, but toxicity is a concern. Studies show conflicting results on the correlation between body mass index (BMI) and outcomes. Limited data exist on the efficacy of adjuvant taxanes among BMI categories and the impact of different taxane‐based chemotherapies (paclitaxel vs. docetaxel) on disease recurrence. Here, we present a pooled analysis of 13,486 EBC patients treated with adjuvant anthracyclines ± taxanes from seven GEICAM and TRIO trials (1996–2008) conducted. Patients were classified into four BMI categories: normal (<25.0), overweight (25.0–29.9), obese (30.0–34.9), and severely obese (≥35.0). BMI was evaluated as a predictive factor for the efficacy and toxicity of taxane‐based chemotherapy. Our results show the following findings: patients' distribution by BMI was 44% normal, 33% overweight, 16% obese, and 8% severely obese. Seventy‐nine percent received taxane‐based chemotherapy. Ten‐year invasive disease‐free survival (iDFS) was 71%, 70%, 68%, and 64% for normal, overweight, obese, and severely obese patients, respectively. Obese and severely obese patients had significantly worse outcomes (HR 1.15 and 1.29, respectively). Invasive disease‐free survival with docetaxel vs. non‐docetaxel was significant in the normal BMI group, while iDFS with paclitaxel was significant in the obese group. Relevant toxicity was observed in 5%, 5.5%, 5.9%, and 9.3% of normal, overweight, obese, and severely obese patients who received docetaxel. In conclusion, heavier EBC patients had a worse prognosis with adjuvant taxane‐based chemotherapy. Normal BMI patients benefited more from docetaxel, while obese patients benefited more from paclitaxel.

Supplemental Figure S5: Induction of apoptosis following 48 hr treatment of CLDN6 positive OVCA429 and CLDN6 negative M202 cells with a range of concentrations (50 μg/ml - 0.3658μg/ml) of CLDN6-23-ADC compared to control IgG ADC.

File containing supplemental data

IMPORTANCE AND OBJECTIVE:Several definitions of efficacy endpoints have been proposed for clinical trials in early breast cancer (EBC). Invasive disease-free survival (IDFS) and other endpoints have been used as surrogates for overall survival (OS) in adjuvant trials. The aim was to evaluate these endpoints in a comparative way in a large cohort of patients to assess whether they are appropriate surrogates for OS. PARTICIPANTS AND DESIGN:Individual patient data from 8 phase III randomized adjuvant chemotherapy (CT) trials for EBC were analyzed. Trial-level surrogacy analyses were conducted using weighted least squares regression to assess the association between treatment effects on surrogates at different time points-IDFS and other defined endpoints (by STEEP v2.0)-and OS. Correlation between the log hazard ratio (HR) of the endpoints and OS were quantified using R2 values. Preplanned subgroup analyses by tumor subtype were also performed. RESULTS:Results censoring at 5 years and at longer follow-up (median: 9.6 years) were similar. HR of IDFS and invasive BC-free survival (IBCFS) at 5 years showed a R2 > 0.7 relative to HR of OS. All other surrogates revealed correlations with R2 > 0.5. For patients with hormone receptor-positive/HER2-negative intermediate/high-risk tumors, correlation showed higher R2 for IDFS (R2 = 0.89; 95% confidence interval [CI] = [0.80-0.98]) and IBCFS (R2 = 0.84; 95% CI = [0.71-0.97]) than distant-recurrence endpoints, while for those with triple negative tumors the highest correlation was seen with distant disease-free survival (R2 = 0.86; 95% CI = [0.75-0.98]). CONCLUSIONS AND RELEVANCE:Although all studied endpoints are appropriate surrogates for OS when evaluating CT-based adjuvant interventions, investigators should consider the BC subtype to select the primary endpoint. Trial registration number: ClinTrials.gov: GEICAM/9805: NCT00121992; GEICAM/9906: NCT00129922; GEICAM/2003-02: NCT00129389; GEICAM/2003-10: NCT00129935; CIBOMA/2004-01_GEICAM/2003-11: NCT00130533; BCIRG 001: NCT00688740; BCIRG 005: NCT00312208 and BCIRG 006: NCT00021255.

Women diagnosed with high grade serous ovarian cancer (HGSOC) often respond to initial rounds of neoadjuvant chemotherapy, but then undergo multiple rounds of relapse and regression before acquiring resistance. This pattern of response and relapse suggests that transient, non-genetic resistance mechanisms are engaged by a subpopulation of tumor cells allowing them to survive and re-form a tumor. To elucidate the mechanisms underlying this transient resistance, we performed scRNASeq and cyclic immunofluorescence (CyCIF) on matched pre- and post-treatment tissues from seven patients. Our transcriptomics data revealed that the interferon (IFN) pathways, IFNα and IFNγ, and the tumor necrosis factor alpha (TNFα) pathway were enriched following therapy in six out of the seven patients. The enrichment of the IFN and TNFα pathways following chemotherapy exposure suggested that tumor cells expressing these programs may survive treatment and thus contribute to relapse, prompting us to study this population further. Spatial analysis of three interferon-stimulated genes (ISGs) revealed that a large proportion of ISG+ tumor cells appear to occupy specific niches containing ISG+ immune cells, while other ISG+ tumor cells were not adjacent to ISG+ immune cells, suggesting both intrinsic and extrinsic mechanisms of IFN pathway activation. It has been reported that IFN is activated in the early lesions of HGSOC known as serous tubule intraepithelial carcinomas (STICs). To investigate whether the IFN pathway activity early in tumorigenesis may be associated with reactivation of retroviral DNA, we performed CyCIF on samples containing STICs and found correlated expression of ISGs and the LINE1 protein product, ORF1P. These STIC areas were typically not associated with immune cells. Similarly, we detected correlated expression of ORF1P with ISGs in invasive tumor cells. Together, our findings suggest that endogenous retroviral reactivation may activate the cGAS/STING pathway, turning on IFN and establishing an IFN+ tumor cell population. To further investigate the cellular heterogeneity of HGSOC and identify pathways that are co-expressed with the inflammatory pathways, we sub-clustered individual patients’ tumors. This analysis revealed two distinct tumor cell states: one associated with high expression of the inflammatory pathways with additional stress-related programs and EMT, and the other with MYC pathway, cellular proliferation, and metabolic pathways. We were able to confirm this coordinated expression in an additional 40 pre-treatment samples. Using an independent cohort from the TRIO14 ovarian cancer trial, we found the same pattern in HGSOC tumors from 46 patients. Future studies of this population aim to understand the functional importance of the IFN/inflammatory/stress state and to identify targetable vulnerabilities. Nomeda Girnius, Muyi Liu, Laura M. Selfors, Jaeyoung Kang, Linna Lahmadi, Mayura Thomas, Aylin Z. Henstridge, Morgan Gruner, Jordan Mattson, Dennis J. Slamon, Gottfried E. Koneckny, Andrew C. Nelson, Timothy K. Starr, Ronny Drapkin, Boris J. Winterhoff, Joan S. Brugge. Chemotherapy enriches one of two pre-existing coordinated cellular programs characterized by inflammatory and stress pathways in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1249.

Lenvatinib (LEN), a front-line treatment for advanced liver cancer, has a short benefit of 13.6 months median overall survival. Understanding how resistance emerges may provide insight into LEN-combined therapies that might boost its clinical efficacy. LEN-sensitive liver cell lines were conditioned with LEN in culture to establish this study's three LEN-resistant (LR) models. All LR models exhibited a stable right shift in the LEN dose-response curve. The LR models were cross-resistant to other FGFR inhibitors and expressed markers linked to cancer aggressiveness. Pro-apoptosis signals, induced by LEN treatment in Hep3B parental cells, were no longer induced in Hep3B LR cells. Compared to parental cells, Hep3B LR and JHH4 LR cells were no longer Annexin-V positive with LEN treatment, supporting their ability to evade apoptosis. A validated HTS method, using Cell-Titer Glo to measure the cell viability of Hep3B and JHH4 models tested across 440 small molecule inhibitors and LEN at a single concentration, identified nine synergistic combinations. Vetting these leads using a multidose titration assay showed synergy with LEN across all three LR models for two drugs: MRTX0902 targeting SOS1, an intracellular RTK signaling protein, and S63845 targeting MCL1, an anti-apoptosis protein. Multiple RTKs were upregulated in the LR models, thus making SOS1 an attractive target for blocking convergent signals. LEN treatment of parental models increased the expression of pAKT, pERK, and MCL1. LEN with S63845 in Hep3B parental cells reduced pAKT, pERK, and MCL1 and increased γ-H2AX. LEN with S63845 induced more apoptosis than either single agents in parental models or Hep3B LR cells. Enhanced apoptosis was not observed using MRTX0902 alone or with LEN. MRTX0902 alone reduced pAKT and pERK in Hep3B parental cells and was able to offset LEN's accumulation of these proteins in combination. We performed RNA-seq analysis of our model systems at baseline and with LEN treatment. LEN-treated parental cells exhibited a statistically significant increase in MCL1 and SOS1 transcripts. GSEA analysis of LEN-treated parental cells revealed positive MAPK pathway enrichment consistent with SOS1 function. It also identified negative enrichment of genes involved in DNA replication and G2M checkpoint, a p53-regulated process. This novel finding suggested that LEN induces genotoxic stress. Indeed, immunofluorescent foci staining of γ-H2AX increased with LEN treatment of parental Hep3B cells and remained elevated in Hep3B LR cells. Therefore, SOS1 and MCL1 may have a role in overcoming genotoxic stress induced by LEN. Overall, our research has generated multiple LR models and used them to identify resistance components. We also showed that targeting these components (SOS1 and MCL1) synergizes with LEN and inhibits cancer growth. These findings may become relevant patient treatments to extend patient survival against liver cancer. Kevin Chau, Martina S. McDermott, Dennis J. Slamon. Targeting MCL1 or SOS1 can overcome lenvatinib resistance in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 559.

BACKGROUND:The ability to predict ovarian function loss after anticancer treatment is important for appropriate oncofertility counseling and to aid in therapy decision-making for young women with early breast cancer (eBC). METHODS:This biomarker analysis of the BETH (NCT00625898) and KAITLIN (NCT01966471) randomized trials investigated anti-Müllerian hormone (AMH) use, alone and combined with follicle stimulating hormone (FSH) and estradiol (E2), for predicting ovarian function loss following currently adopted chemotherapy and anti-HER2 therapy in premenopausal women with HER2-positive eBC. Serum samples were centrally tested measuring AMH, FSH, and E2 using Roche Elecsys assays. RESULTS:Among 194 included patients (BETH: n = 62; KAITLIN: n = 132), AMH values declined from baseline median 8.44 pmol L-1 to undetectable levels (<0.07 pmol L-1) at the end of therapy, with partial recovery at 36 months (median 0.14 pmol L-1). AMH measured at baseline was predictive of ovarian loss (area under the ROC curve [AUC] = 0.784). Addition of age to AMH slightly improved AUC to 0.800. AMH measured at the end of therapy had AUC 0.741, which increased to 0.785 with addition of age. The combination of AMH at baseline and end of therapy increased prediction to 0.808 and with addition of age to 0.820. Addition of baseline FSH and E2 did not improve prediction in any analysis. CONCLUSIONS:These results support the use of pretreatment measurement of AMH in predicting ovarian function loss in premenopausal women with HER2-positive eBC receiving chemotherapy and anti-HER2 therapy. Measurement of AMH at the end of treatment had reduced accuracy than pretreatment but in combination added slightly to the value of pretreatment sampling.