Three biomarkers (HER2, PD-L1, and microsatellite status) in a large cohort of metastatic gastroesophageal adenocarcinomas: The MD Anderson Cancer Center experience.

Human epidermal growth factor receptor-2 (HER2), programmed death-ligand 1 (PD-L1), and microsatellite (MS) status are well-established biomarkers in gastroesophageal adenocarcinomas (GEAs). However, it is unclear how the combination of these biomarkers is associated with clinicopathological factors and prognosis. This retrospective study included baseline metastatic GEA patients who were tested for all three biomarkers (HER2, PD-L1, and MS status) at the MD Anderson Cancer Center between 2012 and 2022. Stratification was performed according to the combination of biomarker profiles: triple negative (TN), single positive (SP), and multiple positive (MP). Comparative analyses of clinicopathological factors and survival using combinations of biomarkers were performed. Among the 698 GEA patients analyzed, 251 (36.0%) were classified as TN, 334 (47.9%) as SP, and 113 (16.1%) as MP. The MP group showed a significant association with tumors located in the esophagus (p < .001), well to moderate differentiation (p < .001), and the absence of signet ring cells (p < .001). In the survival analysis, MP group had a significantly longer overall survival (OS) compared to the other groups (MP vs. TN, p < .001 and MP vs. SP, p < .001). Multivariate Cox regression analysis revealed that MP serves as an independent positive prognostic indicator for OS (hazard ratio = 0.63, p < .01). Our findings indicate that MP biomarkers are associated with a favorable prognosis in metastatic GEA. These results are reflective of clinical practice and offer valuable insights into how therapeutics and future biomarkers could influence therapy/prognosis.

mRNA knockdown by single strand RNA is improved by chemical modifications.

While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown. We identify that 2'F ribose modifications coupled with 5'-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo. The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity. While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.

SMARCA4 Mutations in Gastroesophageal Adenocarcinoma: An Observational Study via a Next-Generation Sequencing Panel.

BACKGROUND: The clinical impact of SMARCA4 mutations (SMARCA4ms) in gastroesophageal adenocarcinoma (GEA) remains underexplored. This study aimed to examine the association of SMARCA4ms with clinical outcomes and co-occurrence with other gene mutations identified through a next-generation sequencing (NGS) panel in GEA patients. METHODS: A total of 256 patients with metastatic or recurrent GEA who underwent NGS panel profiling at the MD Anderson Cancer Center between 2016 and 2022 were included. Comparative analyses were performed to assess clinical outcomes related to SMARCA4ms. The frequency and types of SMARCA4ms and their co-occurrence with other gene mutations were also examined. RESULTS: SMARCA4ms were identified in 19 patients (7.4%). These SMARCA4ms were significantly associated with non-signet ring cell subtype (p = 0.044) and PD-L1 positive expression (p = 0.046). No difference in survival between the SMARCA4m and SMARCA4-normal group was observed (p = 0.84). There were significant associations between SMARCA4ms and FANCA, IGF1R, KRAS, FANCL, and PTEN alterations. Notably, 15 of the 19 SMARCA4m cases involved SNV missense mutations, with frequent co-occurrences noted with TP53, KRAS, ARID1A, and ERBB2 mutations. CONCLUSIONS: These results serve as the first comprehensive examination of the relationship between SMARCA4ms and clinical outcomes in GEA.

Therapeutic Advances in the Treatment of Gastroesophageal Cancers.

Gastroesophageal cancers are a group of aggressive malignancies that are inherently heterogeneous with poor prognosis. Esophageal squamous cell carcinoma, esophageal adenocarcinoma, gastroesophageal junction adenocarcinoma, and gastric adenocarcinoma all have distinct underlying molecular biology, which can impact available targets and treatment response. Multimodality therapy is needed in the localized setting and treatment decisions require multidisciplinary discussions. Systemic therapies for treatment of advanced/metastatic disease should be biomarker-driven, when appropriate. Current FDA approved treatments include HER2-targeted therapy, immunotherapy, and chemotherapy. However, novel therapeutic targets are under development and future treatments will be personalized based on molecular profiling. Herein, we review the current treatment approaches and discuss promising advances in targeted therapies for gastroesophageal cancers.

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

A phase Ib study of Oraxol (oral paclitaxel and encequidar) in patients with advanced malignancies.

PURPOSE: Oraxol is an oral formulation of paclitaxel administered with a novel, minimally absorbed P-glycoprotein inhibitor encequidar (HM30181A). This phase Ib study was conducted to determine the maximum-tolerated dose (MTD) of Oraxol administered at a fixed dose for up to 5 consecutive days in patients with advanced malignancies. METHODS: Part 1 of this study utilized a 3 + 3 dose-escalation design to determine the MTD of oral paclitaxel 270 mg plus oral encequidar 15 mg administered daily. Dose escalation was achieved by increasing the number of consecutive dosing days per week (from 2 to 5 days per week). Dosing occurred for 3 consecutive weeks out of a 4-week cycle. Part 2 treated additional patients at the MTD to determine tolerability and recommended phase II dose (RP2D). Adverse events, tumor responses, and pharmacokinetic profiles were assessed. RESULTS: A total of 34 patients (n = 24 in Part 1, n = 10 in Part 2) received treatment. The MTD of Oraxol was determined to be 270 mg daily × 5 days per week per protocol definition and this was declared the RP2D. The most common treatment-related adverse events were fatigue, neutropenia, and nausea/vomiting. Hypersensitivity-type reactions were not observed. Of the 28 patients evaluable for response, 2 (7.1%) achieved partial response and 18 (64.3%) achieved stable disease. Pharmacokinetic analysis showed rapid absorption of paclitaxel when administered orally following encequidar. Paclitaxel daily exposure was comparable following 2-5 days dose levels. CONCLUSION: The oral administration of encequidar with paclitaxel was safe, achieved clinically relevant paclitaxel levels, and showed evidence of anti-tumor activity.

Culture media composition influences patient-derived organoid ability to predict therapeutic responses in gastrointestinal cancers.

BACKGROUNDA patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO's ability to predict clinical response to gastrointestinal (GI) cancers.METHODSWe generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments.RESULTSWe report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures.CONCLUSIONWhile we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers.TRIAL REGISTRATIONNot applicable.FUNDINGThe Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).

Nucleoside optimization for RNAi: a high-throughput platform.

The RNA induced silencing complex (RISC) contains at its core the endonuclease Argonaute (Ago) that allows for guide strand (GS)-mediated sequence-specific cleavage of the target mRNA. Functionalization of the sugar/phosphodiester backbone of the GS, which is in direct contact with Ago, presents a logical opportunity to affect RISC's activity. A systematic evaluation of modified nucleosides requires the synthesis of phosphoramidites corresponding to all four canonical bases (A, U, C, and G) and their sequential evaluation at each position along the 21-nucleotide-long GS. With the use of a platform approach, the sequential replacement of canonical bases with inosine greatly simplifies the problem and defines a new activity baseline toward which the corresponding sugar-modified inosines are compared. This approach was validated using 2'-O-benzyl modification, which demonstrated that positions 5, 8, 15, and 19 can accommodate this large group. Application of this high-throughput methodology now allows for hypothesis-driven rational design of highly potent, immunologically silent and stable siRNAs suitable for therapeutic applications.

Expression of oncogenic HRAS in human Rh28 and RMS-YM rhabdomyosarcoma cells leads to oncogene-induced senescence.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The two predominant histologic variants of RMS, embryonal and alveolar rhabdomyosarcoma (eRMS and aRMS, respectively), carry very different prognoses. While eRMS is associated with an intermediate prognosis, the 5-year survival rate of aRMS is less than 30%. The RMS subtypes are also different at the molecular level-eRMS frequently has multiple genetic alterations, including mutations in RAS and TP53, whereas aRMS often has chromosomal translocations resulting in PAX3-FOXO1 or PAX7-FOXO1 fusions, but otherwise has a "quiet" genome. Interestingly, mutations in RAS are rarely found in aRMS. In this study, we explored the role of oncogenic RAS in aRMS. We found that while ectopic oncogenic HRAS expression was tolerated in the human RAS-driven eRMS cell line RD, it was detrimental to cell growth and proliferation in the human aRMS cell line Rh28. Growth inhibition was mediated by oncogene-induced senescence and associated with increased RB pathway activity and expression of the cyclin-dependent kinase inhibitors p16 and p21. Unexpectedly, the human eRMS cell line RMS-YM, a RAS wild-type eRMS cell line, also exhibited growth inhibition in response to oncogenic HRAS in a manner similar to aRMS Rh28 cells. This work suggests that oncogenic RAS is expressed in a context-dependent manner in RMS and may provide insight into the differential origins and therapeutic opportunities for RMS subtypes.

A Review: Molecular Aberrations within Hippo Signaling in Bone and Soft-Tissue Sarcomas.

The Hippo signaling pathway is an evolutionarily conserved developmental network vital for the regulation of organ size, tissue homeostasis, repair and regeneration, and cell fate. The Hippo pathway has also been shown to have tumor suppressor properties. Hippo transduction involves a series of kinases and scaffolding proteins that are intricately connected to proteins in developmental cascades and in the tissue microenvironment. This network governs the downstream Hippo transcriptional co-activators, YAP and TAZ, which bind to and activate the output of TEADs, as well as other transcription factors responsible for cellular proliferation, self-renewal, differentiation, and survival. Surprisingly, there are few oncogenic mutations within the core components of the Hippo pathway. Instead, dysregulated Hippo signaling is a versatile accomplice to commonly mutated cancer pathways. For example, YAP and TAZ can be activated by oncogenic signaling from other pathways, or serve as co-activators for classical oncogenes. Emerging evidence suggests that Hippo signaling couples cell density and cytoskeletal structural changes to morphogenic signals and conveys a mesenchymal phenotype. While much of Hippo biology has been described in epithelial cell systems, it is clear that dysregulated Hippo signaling also contributes to malignancies of mesenchymal origin. This review will summarize the known molecular alterations within the Hippo pathway in sarcomas and highlight how several pharmacologic compounds have shown activity in modulating Hippo components, providing proof-of-principle that Hippo signaling may be harnessed for therapeutic application in sarcomas.

In vivo activity and duration of short interfering RNAs containing a synthetic 5'-phosphate.

Endogenous and exogenous short interfering RNAs (siRNAs) require a 5'-phosphate for loading into Ago2 and cleavage of the target mRNA. We applied a synthetic 5'-phosphate to siRNA guide strands to evaluate if phosphorylation in vivo is rate limiting for maximal siRNA knockdown and duration. We report, for the first time, an in vivo evaluation of siRNAs with a synthetic 5'-phosphate compared to their unphosphorylated versions. siRNAs that contained a 5'-phosphate had the same activity in vivo compared with unphosphorylated siRNAs, indicating phosphorylation of an siRNA is not a rate limiting step in vivo.

Differential mass spectrometry of rat plasma reveals proteins that are responsive to 17beta-estradiol and a selective estrogen receptor modulator PPT.

Estrogens are a class of steroid hormones that interact with two related but distinct nuclear receptors, estrogen receptor (ER) alpha and beta. To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17beta-estradiol (E2) or an ERalpha-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPT-responsive proteins, such as serine protease inhibitor family members.