Barozzi, Iros

Abstract Background. The phase 3 PALLAS trial (NCT02513394) compared two years of the CDK4/6 inhibitor palbociclib with endocrine therapy of provider choice, versus endocrine therapy alone, as adjuvant treatment for patients with Stage II-III hormone receptor-positive HER2-negative (HR+/HER2-) breast cancer. Genomic subtype (PAM50 intrinsic subtype) measured from whole-transcriptome RNA sequencing data was defined in the protocol of the PALLAS trial as the primary biomarker for analysis of prediction and prognosis. Clinical data have been previously presented (Gnant et al, JCO 2022), and the trial now has 5-year median follow-up. Methods. As part of trial eligibility, all participants in PALLAS provided a tumor tissue block prior to randomization (surgical if primary resection, core biopsy if neoadjuvant treatment) for translational analyses (TRANS-PALLAS). The biorepository and laboratory were blinded to identity and processed samples in random order, to minimize bias. Nucleic acids were extracted from samples with sufficient tumor tissue and cellularity (>25 mm2 with ≥20% cancer nuclei). The Genome Sequencing Center at Washington University St. Louis performed whole-transcriptome RNA sequencing. Libraries were prepared from 1 µg DNase-1 treated total RNA, if total RNA DV200 >28 (Agilent Bioanalyzer), using an unbiased library protocol of RNA HyperPrep kit with RiboErase (HMR) (Kapa Biosystems, Wilmington, MA). 100 bp paired-end sequencing was performed on NovaSeq 6000 using S4 Reagent Kit (Illumina, San Diego, CA), with 48 libraries pooled per lane. Intrinsic subtype was determined using Bioclassifier package (Research PAM50 script, Parker et al.) only for the analysis population of primary breast cancer samples that had not been exposed to prior neoadjuvant therapy. Invasive disease-free survival (IDFS) will be visualized using Kaplan-Meier plots, with log-rank test between groups. Cox models of proportional hazards will be developed to evaluate prognosis adjusted for known clinical covariates, or for predictive interactions. The pre-defined level of significance is a two-sided 0.05. Results. From the total study population of 5796 enrolled patients, 4655 tissue blocks had sufficient tumor content to process for RNA, with 3931 yielding sufficient RNA for sequencing, and 2669/4655 (57.3%) submitted tissue blocks had DV200 ≥28 and were successfully sequenced. Clinical unblinding revealed 2370 unique patients (1182 in the palbociclib treatment arm and 1188 in the control arm) with intrinsic subtype defined from their untreated primary tumor: 1555 (65.6%) luminal A, 287 (12.1%) luminal B, 167 (7.0%) HER2-enriched, 310 (13.1%) basal-like, 51 (2.2%) normal-like. We will report the results for association of molecular subtype, proliferation score, and Risk of Recurrence (ROR) scores with invasive disease-free survival (IDFS) by treatment arm at the meeting. Conclusions. The TRANS-PALLAS cohort represents one of the largest biorepositories of HR+/HER2- early breast cancer reflecting contemporary systemic management in the framework of a prospectively randomized global trial. Required tumor block submission in this phase 3 trial yielded data from unbiased whole-transcriptome RNA sequencing of the primary tumor prior to treatment from 41% of the PALLAS participants. The proportion of luminal A cancers was unexpectedly high (66%), indicating a lower-risk distribution of cancers in this population. The planned analyses of prediction and prognosis are ongoing and those results will be presented at the time of the meeting. Support: AFT, ABCSG, Pfizer, ECOG-ACRIN, NSABP Foundation, GBG, BIG; Clinicaltrials.gov Identifier: NCT02513394; https://acknowledgments.alliancefound.org Citation Format: Daniel Stover, Dominik Hlauschek, Erica Mayer, W. Fraser Symmans, Mark Watson, Iros Barozzi, Martin Filipits, Karla Ballman, Meritxell Bellet- Ezquerra, Justin Balko, Gabor Rubovszky, Nicholas Zdenkowski, Adam Brufsky, Guenther Steger, Claudine Isaacs, Sibylle Loibl, Fernando Henao, Meredith Regan, Yuan Liu, Christian Fesl, Patrick O'Brien, Angela DeMichele, Michael Gnant, Otto Metzger. Protocol-defined biomarker analysis in the PALLAS (AFT-05) adjuvant trial: Genomic subtype derived from RNA sequencing of HR+/HER2- early breast cancer [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 GS03-07.

The activation of the receptor tyrosine kinase Axl by Gas6 is a major driver of tumorigenesis. Despite recent insights, tumor cell-intrinsic and -extrinsic Axl functions are poorly understood in hepatocellular carcinoma (HCC). Thus, we analyzed the cell-specific aspects of Axl in liver cancer cells and in the tumor microenvironment. We show that tumor-intrinsic Axl expression decreased the survival of mice and elevated the number of pulmonary metastases in a model of resection-based tumor recurrence. Axl expression increased the invasion of hepatospheres by the activation of Akt signaling and a partial epithelial-to-mesenchymal transition (EMT). However, the liver tumor burden of Axl+/+ mice induced by diethylnitrosamine plus carbon tetrachloride was reduced compared to systemic Axl−/− mice. Tumors of Axl+/+ mice were highly infiltrated with cytotoxic cells, suggesting a key immune-modulatory role of Axl. Interestingly, hepatocyte-specific Axl deficiency did not alter T cell infiltration, indicating that these changes are independent of tumor cell-intrinsic Axl. In this context, we observed an upregulation of multiple chemokines in Axl+/+ compared to Axl−/− tumors, correlating with HCC patient data. In line with this, Axl is associated with a cytotoxic immune signature in HCC patients. Together these data show that tumor-intrinsic Axl expression fosters progression, while tumor-extrinsic Axl expression shapes an inflammatory microenvironment.