3092 Background: Trastuzumab deruxtecan (T-DXd) consists of deruxtecan, a novel topoisomerase 1 (TOP1) inhibitor, covalently bound via a lysosomal protease-cleavable linker to the human epidermal growth factor receptor 2 (HER2)-targeting antibody, trastuzumab. The mechanism(s) underlying the clinical activity seen in multiple tumor types across various HER2 levels is of ongoing, active interest. To investigate the T-DXd mechanism of action, we developed a pilot clinical trial of T-DXd with a detailed pharmacodynamic analysis from TOP1 target engagement to downstream effects of DNA damage in patients with HER2-expressing (IHC 1-3+, HER2 amplified, or HER2 mutated) advanced solid tumors (NCT04294628). Methods: Based on preclinical studies modeling the trial, research tumor biopsies from consenting patients were collected at three time points: pre-treatment, post-dose Cycle 1 (48-96 hours) and pre-dose Cycle 3. The biopsies were evaluated for TOP1 inhibition, induction of stabilized TOP1covalent complexes (TOP1cc) and induction of downstream DNA damage repair (DDR) markers (RAD51, pNBS1, RPA32) using validated, quantitative multiplex immunofluorescence assays on fixed tumor sections with image analysis methodology. In addition, a retrospective analysis of Schlafen 11 expression in baseline biopsies was performed to determine its predictive value of tumor responses to T-DXd. Results: Twenty-one biopsy pairs (pre-treatment and C1, 48-96h post-dose 1) were evaluable for TOP1 molecular response and downstream DDR marker induction. TOP1 target modulation was detected in 15 (71%) of the on-treatment biopsies and markers of DDR were induced in 18 (86%) biopsy pairs. TOP1 target inhibition, robust induction of downstream DNA damage response including stalled replication fork progression, and DNA breaks were observed in HER2-amplified/2+/3+ tumors, and in HER2 1+ tumors. Conclusions: Our results confirm the intended TOP1 molecular mechanism of action of T-DXd in HER2-amplified/2+/3+ and importantly extend that finding to HER2 1+ tumors, resulting in DNA damage in nearly all cases. Pharmacodynamic biomarker studies are poised to yield important insights into the molecular effects of T-DXd in solid tumors.

The proteasome inhibitor bortezomib and purine nucleoside analog clofarabine combination had greater than additive activity in the NCI-ALMANAC preclinical screen. We conducted a phase 1 trial (NCT02211755) to evaluate the combination’s safety and efficacy in patients. We administered bortezomib subcutaneously on days 1 and 4, and clofarabine intravenously on days 1–5 of each 21-day cycle. The primary objective was to establish the safety, tolerability, and maximum tolerated dose (MTD) of bortezomib and clofarabine in patients with refractory solid tumors, lymphomas, or MDS. The secondary objective was to determine the effects of the combination on biomarkers of cell death and DNA damage response (DDR) in tumor biopsies. Of 28 patients enrolled, 11 had a best response of stable disease (median 5 cycles; range 2–10 cycles), including 5 patients (4 from the solid tumor cohort, 2 of which were at MTD) with stable disease for ≥ 6 cycles. The MTD for the solid tumor cohort was 1.3 mg/m2 bortezomib on days 1 and 4, and 1.5 mg/m2 clofarabine on days 1–5 of each cycle. The MDS cohort closed prior to MTD determination, due to low accrual. The most common study drug related adverse events were hematologic. Two out of 3 patients with evaluable biopsies had increased markers of cell death, and 1 patient also had increased DDR markers after treatment. The combination of bortezomib with clofarabine demonstrated limited antitumor effects possibly due to the inability to reach the efficacious doses achieved in preclinical models.

PURPOSE Tissue biopsy specimens, both remnant diagnostic specimens and those collected for ancillary study, are an invaluable resource for clinical oncology research. However, using biopsy specimens for molecular research is associated with innate challenges, such as insufficient tissue and/or tumor content, and low nucleic acid yields as well as analyte degradation due to suboptimal preanalytical workflows. METHODS The National Cancer Institute's Biorepositories and Biospecimen Research Branch convened a meeting that included expert-guided discussions that centered on strategies to mitigate these challenges and their effects on molecular analysis. RESULTS Participants, who included medical oncologists, interventional radiologists, pathologists, and molecular biologists, offered best practice guidance on biopsy collection, preservation, storage, and extraction techniques. Their recommendations were largely based on the optimized workflows that were implemented at their respective institutions, which improved the likelihood of producing reproducible molecular data. Pre- and postcollection techniques, such as clear cross-team communication, prebiopsy scoring based on lesion- and patient-specific criteria, biopsy collection and handling practices, and tumor enrichment options, were also discussed. CONCLUSION The proceedings revealed that increasing awareness of the challenges associated with research use of tissue biopsies is key to developing assay-specific strategies that ensure sufficient tumor specimens are available for molecular oncology research. The lessons shared here from large-scale and multicenter trials will, ideally, inform the design of new cancer research studies, thereby harnessing the full potential of valuable clinical biopsy specimens.

Supplementary Figure S6. Low-, medium-, and high dose modulation of heterodimer levels following a dose of venetoclax

Supplementary Figure S12. In vivo pharmacodynamic changes in Mcl-1, Bim, and Bad levels following cirtuvivint without or with venetoclax in MV4-11 and KG-1a tumors

Supplementary Table S2. STR profiling of lymphoblastoid-like cell lines

Supplementary Table S5. In vivo pharmacokinetics of S63845

Supplementary Figure S1. AMO-1 and MOLT-4 cells were treated with either navitoclax, S63845, or vehicle control

We previously reported initial results of the pivotal phase II trial of atezolizumab for patients with alveolar soft part sarcoma (ASPS; ClinicalTrials.gov identifier: NCT03141684). Here, we report on three additional years of observation. Fifty-three patients with ASPS received atezolizumab. Median duration of response increased to 37.0 months. Objective response rate (ORR) and median progression-free survival (mPFS) remained essentially as previously reported (35.8% [95% CI, 23.1 to 50.2] and 20.8 months [IQR, 7.6-not reached], respectively). ASPSCR1::TFE3 fusion type was determined for 47/53 patients; ORR and mPFS were higher among the 41 patients expressing type 1 (43.9% [95% CI, 28.5 to 60.2] and 28.3 months [IQR, 9.2-not reached], respectively) than the six patients expressing type 2 (0% [95% CI, 0 to 45.9] and 7.5 months [IQR, 3.9-not reached], respectively, PFS HR, 3.2 [95% CI, 1.01 to 10.2]). Eleven patients chose a per-protocol drug holiday (range, 3.5-26.4 months) after ≥2 years of treatment; two experienced disease progression during the holiday. Nine eligible patients elected to receive bevacizumab plus atezolizumab after progressing on monotherapy; ORR was 0% and mPFS was 18.5 months (IQR, 7.9-21.1) in this small cohort. Long-term results support using atezolizumab to treat ASPS, even for several years; a drug holiday with careful monitoring may be an option for some patients.

Supplementary Figure S2. Dose effects of S63845 in vitro

Supplementary Figure S11. Body weights of mice bearing either MV4-11 or KG-1a tumors

Abstract Background: Pidnarulex (CX-5461) is a first-in-class G-quadruplex-stabilizing agent that induces replication-dependent DNA damage, inhibits RNA polymerase I and topoisomerase II (Top2). Pidnarulex has demonstrated safety and preliminary efficacy in a prior phase I study (NCT02719977), with partial responses in 4 of 32 patients and stable disease in 11 patients, including 4 with responses ≥6 months. The National Cancer Institute’s (NCI) preclinical in vitro studies in homologous recombination (HR)-proficient (, OVCAR3) and HR-deficient (HRD) (A2780, IGROV1) cell lines show that pidnarulex (1 µM or 3 µM in vitro;) significantly increased percentage of RPA32, pSer33-RPA32, 53BP1 and Rad51 nuclear foci 24 hours post-treatment, indicating DNA damage response activation. G4 stabilization (BG4 antibody) was observed in all 3 cell lines at both concentrations. This pilot study (NCT06606990) aims to determine whether pidnarulex induces a Rad51 response, defined as the percentage of cells with ≥5 Rad51 positive nuclear foci in post-treatment tumor biopsies. Methods: This single-center pilot study aims to enroll 40 adult patients (age ≥18) distributed equally between 2 cohorts: with or without HRD-associated alterations (deleterious BRCA1/2; Fanconi anemia gene mutations; functional alterations in ARID1A, ATM, ATR, BRIP1, BAP1, BARD1, CDK12, CHK1, CHK2, IDH1/2, MRE11A, NBN, PALB2, RAD50, RAD51, RAD51B/C/D, RAD54L). The primary objective is assessing Rad51 response following pidnarulex treatment. Secondary objectives include safety (CTCAE v5.0), objective response rate (RECIST v1.1), pharmacokinetics (PK) of pidnarulex, and additional tumor DNA-damage and repair markers RPA32, pSer33-RPA32, γH2AX, 53BP1, pSer8-RPA32, pKap1, and pNBS1) associated with clinical response. Eligible adults must have histologically confirmed solid tumors, ECOG ≤2, adequate organ function, and tumor amenable to biopsy. Pidnarulex is administered at 325 mg/m² IV on days 1 and 8 of each 28-day cycle. Dose reductions occur for grade ≥3 non-hematologic toxicities, with a maximum of 2 reductions (DL-1: 250 mg/m²; DL-2: 200 mg/m²). Mandatory tumor biopsies are collected at baseline and on cycle 1 day 2 (24 ± 2 hours post-dose) for pharmacodynamic assessment. Blood samples are collected for PK analyses (mandatory) and ctDNA (optional). Study success requires 16 evaluable biopsies per cohort, with ≥3 patients (19%) showing a Rad51 response, providing 90% power to detect a true 30% Rad51-positive response rate at a 5% false-positive rate. Funded by NCI Contract No. HHSN261201500003I. Citation Format: Jibran Ahmed, Brian Ko, Sarah J. Shin, Murielle Hogu, Lawrence Rubinstein, Himabindu Gali, Deborah F. Wilsker, Ralph E. Parchment, Laura Kuhlmann, Steven Gore, James H. Doroshow, Alice P. Chen. Pilot study of pidnarulex pharmacodynamics in patients with advanced solid tumors [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 CT293.

Supplementary Figure S10. Bcl-xL–Bim levels in response to S63845

The HGF/SF (hepatocyte growth factor/scatter factor) receptor tyrosine kinase MET is overexpressed and/or activated in many tumors, providing therapeutic targets for antibody-drug conjugates and tyrosine kinase inhibitors. Reliable measurement of activated MET is fundamental for pharmacodynamic assessment of MET-targeted therapies and for expanded and proper use of such therapies in patients with tumors driven by activated MET with or without associated MET amplification or known activating mutations. To address the paucity of tools for directly measuring MET activation in tumor cells within patient biopsy specimens, we developed a robust, quantitative immunofluorescence microscopy assay to measure levels of pY(1235)MET and total MET in in vitro, in vivo, and patient tumor specimens. We validated this assay through assessment of MET inhibitor-treated preclinical models, peptide blocking experiments to demonstrate specificity, and concordance with corresponding measurements from the same specimens using a previously validated sandwich immunoassay of tumor lysates. Given the importance of plasma membrane-associated MET in initiating its canonical signaling cascades, as well as the demonstrated non-canonical signaling from nuclear localized MET in different tumor cell types and in response to various environmental stimuli, we developed assay capability to measure levels of pY(1235)MET and total MET within the plasma membrane or nucleus; these assays enable future explorations of the biological and clinical relevance of MET subcellular localization patterns. Finally, using tissue microarrays of over 50 resected tumor specimens from patients with colorectal carcinoma or non-small cell lung cancer, we demonstrated that tumor levels of pY(1235)MET do not always track total MET expression, suggesting that measurement of activated MET in tumor could hold potential as an independent biomarker to identify additional patients who might benefit from MET-directed targeted therapy-beyond those with tumor MET amplification, MET overexpression, or established MET-activating mutations.

Supplementary Figure S4. Plasma and tumor pharmacokinetics of S63845 and venetoclax

3031 Background: Trastuzumab deruxtecan (T-DXd) is a HER2-directed antibody conjugated to topoisomerase 1 inhibitor, deruxtecan, payload that is an effective treatment strategy across several tumor types and various HER2 expressions. The relative contributions of each of the underlying mechanisms driving the broad clinical activity require further elucidation for the ongoing rational development of this promising agent. To this end, our pilot study (NCT04294628) evaluates the pharmacodynamics (PD) of T-DXd in patients (pts) with solid tumors displaying a variety of HER2 expression levels. Methods: This multicenter pilot study enrolled pts with HER2-expressing advanced solid tumors as defined by HER2 immunohistochemistry (IHC) score of 1+ or greater or ERBB2 amplifications (amp) or mutations (mut). Baseline HER2 expression was evaluated by Ventana PATHWAY immunohistochemical (IHC) analysis. T-DXd was administered at 5.4 mg/kg intravenously once every 3 weeks, in 21-day cycles (C), with mandatory tumor biopsies collected at baseline, post-dose C1, and pre-dose C3. Blood samples for biomarker analyses were collected throughout the study. PD biomarkers for topoisomerase 1 (TOP1) target engagement, consequent DNA damage repair (DDR), and tumor immune microenvironment changes were analyzed. Overall response was also evaluated. Results: Sixty-one pts received T-DXd. Eligibility HER2 (eHER2) status was determined from pt records: 21 IHC 1+, 22 IHC 2+, 7 IHC 3+, 8 ERBB2 amp, 3 ERBB2 mut. Of the 41 pts with baseline biopsies centrally assessed for HER2 (bHER2) by IHC, 13 had bHER2 scores discordant with eHER2, including 8 patients who were eHER2 1-2+ but were bHER2 null. No new safety signals were observed. One pt had a complete response (cervical, bHER2 3+), 14 pts had confirmed partial responses (PR), including 3 who were bHER2 null (2 ovarian, 1 uterine), and 5 pts had unconfirmed PR (uPR). Of the 21 paired biopsies assessable for PD response, 15 demonstrated TOP1 target modulation and 14 of those 15 also demonstrated DDR induction. Substantial tumor infiltration and activation of CD8+ T cells at baseline and/or following T-DXd administration occurred in several patients and were particularly prevalent in those with response. In the one responding patient where lesion-specific analyses were possible, we observed significant diameter reduction (3.9 cm to 1.9 cm) and PD responses in a bHER2 null lesion. Conclusions: Clinical responses and target modulation were observed in pts irrespective of bHER2 expression. Lesion-specific analyses provide evidence of antitumor activity and target engagement even in a bHER2-null lesion. Genomic analyses to identify additional molecular determinants of response or resistance to T-DXd are ongoing. This project was funded in part by the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024. Clinical trial information: NCT04294628 .

Supplementary Figure S5. Low-, medium-, and high dose modulation of heterodimer levels following a dose of S63845

Supplementary Figure S3. Dose effects of navitoclax in vitro