Archival Tissue Research Articles

Prospective observational study to explore genes and proteins predicting efficacy and safety of brigatinib for ALK-gene rearranged non-small-cell lung cancer: study protocol for ABRAID study (WJOG11919L).

ALK-tyrosine kinase inhibitors (ALK-TKIs) are effective for treating non-small-cell lung cancer with ALK gene rearrangement; however, resistance is inevitable. Brigatinib is a unique ALK-TKI that is effective against many resistance mutations. However, data on factors associated with its efficacy and resistance mechanisms are limited. This study will evaluate the efficacy and safety of brigatinib in the real world and explore factors related to its efficacy, safety, and resistance mechanisms. Prospective observational study. This study is approved by the Ethics Committee of Wakayama Medical University. Written informed consent will be obtained from all patients before study-related procedures. This study comprises three cohorts. Cohorts A, B, and 0 will enroll patients receiving alectinib as the first ALK-TKI, receiving alectinib as the first ALK-TKI and subsequently cytotoxic agents and/or lorlatinib after alectinib, and without a history of ALK-TKI, respectively. Overall, 100, 30, and 50 patients will be enrolled in Cohorts A, B, and 0, respectively. Circulating tumor DNA before starting brigatinib and at disease progression will be analyzed in all cohorts using a hypersensitive next-generation sequencing (NGS) PGDx Elio plasma resolve panel. Serum protein levels will be analyzed using the Milliplex xMAP assay system with a Luminex 200 (Luminex, Austin, USA). The enrollment period is 31 months and the patients will be observed for 2 years after enrollment. Archived tissues will be collected for NGS analysis, gene expression analysis, and immunohistochemistry staining 1 year after completion of registration. Quality of life and safety evaluation using electronic patient-reported outcomes will be investigated. This study will elucidate predictors of ALK-TKI efficacy and resistance mechanisms and evaluate the efficacy and safety of brigatinib in a real-world setting. The results will provide crucial information for establishing treatment strategies, discovering novel biomarkers, and developing new therapeutic agents. UMIN000042439.

QPCR assay for detection of Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Elements from CAR-T and TCR-T cells in fresh and formalin-fixed tissue.

As adoptive cellular therapies become more commonplace in cancer care, there is a growing need to monitor site-specific localization of engineered cells-such as chimeric antigen receptor T (CAR-T) cells and T-cell receptor T (TCR-T) cells-in patients' tissues to understand treatment effectiveness as well as associated adverse events. Manufacturing CAR-T and TCR-T cells involves transduction with viral vectors commonly containing the WPRE gene sequence to enhance gene expression, providing a viable assay target unique to these engineered cells. Quantitative PCR (qPCR) is currently used clinically in fresh patient tissue samples and blood with target sequences specific to each immunotherapy product. Herein, we developed a WPRE-targeted qPCR assay that is broadly applicable for detection of engineered cell products in both fresh and archival formalin-fixed paraffin embedded (FFPE) tissues. Using both traditional PCR and SYBR Green PCR protocols, we demonstrate the use of this WPRE-targeted assay to successfully detect two CAR-T cell and two TCR-T cell products in FFPE tissue. Standard curve analysis reported a reproducible limit of detection at 100 WPRE copies per 20μL PCR reaction. This novel and inexpensive technique could provide better understanding of tissue abundance of engineered therapeutic T cells in both tumor and second-site toxicity tissues and provide quantitative assessment of immune effector cell trafficking in archival tissue.

Equus caballus papillomavirus type 2 (EcPV2)-associated benign penile lesions and squamous cell carcinomas.

Squamous cell carcinoma is the most common genital, ocular and gastric tumour in horses. Equus caballus papillomavirus type 2 (EcPV2) DNA has been detected in several studies in equine penile squamous cell carcinomas (SCCs) and precursor lesions providing evidence of a causal role of EcPV2 in equine genital SCCs. Recently, EcPV2 E6/E7 nucleic acids were also detected in equine gastric SCCs, but further studies are required to determine the role of EcPV2 infection in the pathogenesis of gastric SCC. EcPV2 nucleic acids have been rarely described in ocular SCCs and precursor lesions. To investigate the presence of EcPV2 nucleic acids with polymerase chain reaction (PCR) and in situ hybridisation (ISH) in penile hyperplasias, papillomas and SCCs in horses and to determine whether EcPV2 nucleic acids can be detected in SCCs affecting other locations, including the stomach, ocular tissues and larynx. Twenty-one archival formalin-fixed paraffin embedded (FFPE) tissue samples, including 12 genital lesions comprising penile hyperplasias, papillomas and SCCs, 6 ocular SCCs, 2 gastric SCCs and 1 laryngeal SCC, were screened by PCR and ISH for EcPV2 E6/E7 DNA and mRNA. Archival FFPE tissue samples (eyelid and penile mucosa and preputium) from six horses without a diagnosis or history of neoplastic or papillomavirus-associated disease were included as controls. EcPV2 nucleic acids were detected by PCR and ISH in all genital lesions (12/12) and gastric SCCs (2/2), in two ocular SCCs (2/6) and in one laryngeal SCC (1/1). In control horses, one eyelid sample was positive in PCR but not in ISH. The remaining control samples were negative for EcPV2 E6/E7 nucleic acids in PCR and ISH. These results further support the role of EcPV2 infection in the development of equine genital SCCs and suggest that EcPV2 infection may also act as a predisposing factor for other SCCs in horses, including gastric, ocular and laryngeal SCCs.

Comprehensive characterization of genomic features and clinical outcomes following targeted therapy and secondary cytoreductive surgery in OCCC: a single center experience.

Ovarian clear cell carcinoma (OCCC) is associated with chemoresistance. Limited data exists regarding the efficacy of targeted therapies such as immune checkpoint inhibitors (ICI) and bevacizumab, and the role of secondary cytoreductive surgery (SCS). We retrospectively analyzed genomic features and treatment outcomes of 172 OCCC patients treated at our institution from January 2000 to May 2022. Next-generation sequencing (NGS) was performed where sufficient archival tissue was available. 64.0% of patients were diagnosed at an early stage, and 36.0% at an advanced stage. Patients with advanced/relapsed OCCC who received platinum-based chemotherapy plus bevacizumab followed by maintenance bevacizumab had a median first-line progression-free survival (PFS) of 12.2 months, compared with 9.3 months for chemotherapy alone (hazard ratio=0.69; 95% confidence interval [CI]=0.33, 1.45). In 27 patients who received an ICI, the overall response rate was 18.5% and median duration of response was 7.4 months (95% CI=6.5, 8.3). In 17 carefully selected patients with fewer than 3 sites of relapse, median PFS was 35 months (95% CI=0, 73.5) and median overall survival was 96.8 months (95% CI=44.6, 149.0) after SCS. NGS on 58 tumors revealed common mutations in ARID1A (48.3%), PIK3CA (46.6%), and KRAS (20.7%). Pathogenic alterations in PIK3CA, FGFR2, and NBN were associated with worse survival outcomes. Median tumor mutational burden was 3.78 (range, 0-16). All 26 patients with available loss of heterozygosity (LOH) scores had LOH <16%. Our study demonstrates encouraging outcomes with bevacizumab and ICI, and SCS in select relapsed OCCC patients. Prospective trials are warranted.

Expression levels of genes involved in lipogenesis and cholesterol synthesis in adenomyosis

Abstract Objectives Adenomyosis is a benign uterine disease that occurs with the invasion of the endometrial gland and stoma into the myometrium. The etiology and molecular pathology of adenomyosis are not yet fully understood. Tissue samples of patients diagnosed with adenomyosis and healthy endometrial tissues were investigated for the lipogenesis and cholesterol synthesis pathways. It was aimed to determine the difference between adenomyosis and healthy endometrial tissues in terms of lipid metabolism and to investigate the mechanism of adenomyosis in this context. Methods Formalin-fixed paraffin-embedded archival tissues were used in the current retrospective study. A total of 76 patient samples and 3 groups were used. Group 1: adenomyotic tissue (n=28), Group 2: eutopic endometrial tissue (n=30), and Control Group (n=18). In these groups, Sterol regulatory element binding protein 1 (SREBP1) molecule, fatty acid synthase (FASN), acetyl-CoA carboxylase (ACACA), ATP-citrate lyase (ACLY), HMG-CoA reductase (HMGCR), and HMG-CoA synthase (HMGCS) markers were evaluated by using RT-PCR method. Results Statistically significant differences (p&lt;0.05) were found between the groups regarding expression levels of HMGCR, HMGCS, ACLY, ACACA, and SREBP1. HMGCR, HMGCS, ACLY, and SREBP1 gene expression levels between Group 1 and Group 2 and HMGCS, ACACA, ACLY, and SREBP1 gene expression levels between Group 1 and Control Group were determined as statistically different. A significant difference was detected only in HMGCR gene expression levels between Group 2 and the Control Group. Conclusions These results show that genes involved in lipid metabolism may be associated with the molecular pathogenesis of adenomyosis.

The Immunohistochemical expressions and diagnostic values of E-cadherin and β-Catenin in suspected cervical lesion or cervical cancer

Background: Cervical lesions are the cellular changes of a pathologic process with a bacterial and viral etiology ranging from cervicitis, cervical intraepithelial neoplasm and invasive cancer. The integrity of the cervical epithelium is maintained by complex cell adhesion molecules. This work is aimed at determining the expression pattern diagnostic value of the adhesion molecules to enhance early detection of cancer. Methods: The study population was from 10 years retrospective archival tissue blocks of cervical cancer among women in Abuja metropolis from 2005 to 2015 Histopathological diagnosis made, from the 80 cases examined 20(25%) cases were cervicitis (CC), 15(18.8%) were cervical intraepithelial neoplasia (CINI), 15(18.8%) were cervical intraepithelial neoplasia (CIN2) while 30(37.3%) cases were squamous cell carcinoma SCC. They were examined immunohistochemically by Avidin Biotin Complex (ABC) method. E-Cadherin and Beta-Catenin, (cell adhesion molecules). Results: Statistically significant rate of positivity were found in the expression of β-catenin 100% among all the categories of the studied cases. β-catenin expression was significantly higher in CINI than in SCC, while in E-cadherin, therewere no statistically significant difference (p&lt;0.05) between the distribution of high expression and low expression of E-cadherin in both CINI and SCC. In expression of E-cadherin among the cases studied, there was a significant difference (p&lt;0.05) between rate of positivity and negativity for the expression of E-cadherin. A statistically significant rate of positivity was found in the expression of β-catenin among all the categories of the studied cases. In the expression E-cadherin, there was a gradual decrease in high expression from cervicitis to CINI, CIN2 and SCC. There was Significant (p&lt;0.05) high degree of expression of E-cadherin in cervicitis and CINI against CIN2 and SCC, while a significant (p&lt;0.05) low degree expression of E-cadherin in CIN2 and SCC against Cervicitis and CINI. In β-catenin expression, there as a gradual decrease in high expression from cervicitis to CIN2 and SCC expect in CINI were all had high expression. Conclusion: This study showed that most of the immunological markers examined can be used for identification of different stages involved in cancer progression, also for diagnosis of cancer, predictive prognosis and can be explore as therapy targets for cervicitis and Squamous cell carcinoma.

The Brazilian national prospective active surveillance (AS) cohort of patients with low-risk prostate cancer in the public health system: vigiaSUS study protocol

BackgroundProstate cancer exhibits a very diverse behaviour, with some patients dying from the disease and others never needing treatment. Active surveillance (AS) consists of periodic PSA assessment (prostate-specific antigen), DRE (digital rectal examination) and periodic prostate biopsies. According to the main guidelines, AS is the preferred strategy for low-risk patients, to avoid or delay definitive treatment. However, concerns remain regarding its applicability in certain patient subgroups, such as African American men, who were underrepresented in the main cohorts. Brazil has a very racially diverse population, with 56.1% self-reporting as brown or black. The aim of this study is to evaluate and validate the AS strategy in low-risk prostate cancer patients following an AS protocol in the Brazilian public health system.MethodsThis is a multicentre AS prospective cohort study that will include 200 patients from all regions of Brazil in the public health system. Patients with prostate adenocarcinoma and low-risk criteria, defined as clinical staging T1–T2a, Gleason score ≤ 6, and PSA < 10 ng/ml, will be enrolled. Archival prostate cancer tissue will be centrally reviewed. Patients enrolled in the study will follow the AS strategy, which involves PSA and physical examination every 6 months as well as multiparametric MRI (mpMRI) every two years and prostate biopsy at month 12 and then every two years. The primary objective is to evaluate the reclassification rate at 12 months, and secondary objectives include determining the treatment-free survival rate, metastasis-free survival, and specific and overall survival. Exploratory objectives include the evaluation of quality of life and anxiety, the impact of PTEN loss and the economic impact of AS on the Brazilian public health system.DiscussionThis is the first Brazilian prospective study of patients with low-risk prostate cancer under AS. To our knowledge, this is one of the largest AS study cohort with a majority of nonwhite patients. We believe that this study is an opportunity to better understand the outcomes of AS in populations underrepresented in studies. Based on these data, an AS national clinical guideline will be developed, which may have a beneficial impact on the quality of life of patients and on public health.Trial registrationClinicaltrials registration is NCT05343936.

Abstract LB_A16: DareonTM-5: An open-label Phase II trial of BI 764532, a DLL3-targeting T cell engager, in patients with relapsed/refractory small cell lung cancer or other neuroendocrine carcinomas

Abstract Background: Small cell lung cancer (SCLC), extra-pulmonary neuroendocrine carcinomas (epNECs) and large cell NECs (LCNECs) of the lung are difficult-to-treat, aggressive tumors with limited treatment (Tx) options. Standard of care for metastatic disease is platinum-based chemotherapy ± immunotherapy. However, nearly all patients (pts) relapse, and outcomes are poor. Hence, alternative therapies are needed. Delta-like ligand 3 (DLL3), a Notch ligand, is highly expressed on the cell surface of SCLCs, epNECs and LCNECs. BI 764532, a humanized IgG-like T cell engager, binds to DLL3-positive tumor cells and CD3 on T-cells and promotes T cell-mediated cytotoxicity. In an ongoing first-in-human trial, BI 764532 exhibited promising efficacy and manageable tolerability in pts with DLL3-positive SCLC, epNEC and LCNEC of the lung (NCT04429087). Here we describe a planned Phase II dose optimization trial to assess safety/efficacy of two different doses of BI 764532 monotherapy in pts with progressive/recurrent SCLC, epNEC or LCNEC of the lung (NCT05882058). Methods: The trial will recruit adult pts (n = ~120; ~70 sites; ~15 countries) with histologically/cytologically confirmed relapsed/refractory SCLC, epNEC or LCNEC of the lung who have received ≥2 (SCLC) or ≥1 (epNEC/LCNEC) prior lines of therapy, including ≥1 platinum-based regimen. Pts with mixed tumor histology are permitted if the SCLC/NEC component is predominant (≥50% of tumor tissue). Pts will be randomized 1:1 to receive one of two dose levels of intravenous BI 764532 (3-week cycles) stratified by tumor type (n ~ 60/arm). Tx will continue until disease progression, undue toxicity, withdrawal of consent or any other documented criteria for stopping Tx (maximum Tx duration: 36 months). Interim analyses for dose selection are planned after 30 pts have been followed for ≥6 and ≥12 weeks or have stopped Tx. A third interim efficacy analysis will be conducted when 60 pts per dose have been followed for ≥12 weeks or have stopped Tx. Key exclusion criteria: ECOG PS ≥2; untreated/symptomatic brain metastases or leptomeningeal disease; active/previous history of interstitial lung disease/pneumonitis; previous Tx with DLL3-targeting therapies; Tx with other anti-cancer drug &amp;lt;4 weeks prior to first administration of BI 764532; unresolved toxicity from prior anti-tumor Tx; diagnosis of immunodeficiency, or intake of immunosuppressive therapy ≤7 days prior to first administration of BI 764532. Primary endpoints: objective response (RECIST v1.1; investigator assessment); occurrence of Tx-emergent adverse events (TEAEs) during the on-Tx period. Secondary endpoints: duration of objective response; progression-free survival; disease control; overall survival; pt-reported outcomes; TEAEs leading to discontinuation during the on-Tx period. Further endpoints: pharmacokinetics (PK); prespecified and exploratory biomarker analysis (archival tumor tissue is required). The comparison of the two target doses in this trial will be based on the totality of data, including safety, efficacy, PK and biomarker data. Citation Format: Valentina Gambardella, Alastair Greystoke, Martin Reck, Meiruo Liu, Martha Mueller, Ulrich Duenzinger, Emily B Bergsland, Taofeek Owonikoko. DareonTM-5: An open-label Phase II trial of BI 764532, a DLL3-targeting T cell engager, in patients with relapsed/refractory small cell lung cancer or other neuroendocrine carcinomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A16.

Correlation of Clinical and Histopathological Features of Salivary Pleomorphic Adenoma.

Salivary gland tumors represent about 3% of the head and neck tumors. Pleomorphic adenoma (PA) is the most common benign salivary gland tumor. This study was conducted to investigate and describe some clinical and histopathological aspects of salivary pleomorphic adenomas with special reference to the epithelial and mesenchymal components. In this retrospective study, one hundred tissue samples diagnosed as PA were sourced from archival tissue blocks between 2009 and 2019 in this retrospective study. Some clinical and demographic features, including age, sex, tumor size, and tumor location were recorded. This study included only samples taken by excisional biopsy. Then, the samples were histologically classified into three subtypes according to the proportion of epithelial and stromal components. The demographic and clinicopathological variables were statistically analyzed using Chi-square test or Fisher's exact test, considering a significance level of 5% (p< .05). In the present study, most cases (61%) were found in females, representing a female-male ratio of 1.6:1. The peak incidence was seen in the 4th and 5th decades of life. In 87% of cases, PA occurred in major salivary glands. There was a significant difference between the age of the patient and squamous metaplasia (p= 0.036). There was also a significant difference between the size of tumor and the amount of myxoid stroma (p= 0.021). Extensive myxoid stromawas mostly seen in tumors larger than 3.37cm (p= 0.001). In addition, there was a statistically significant difference between capsular invasion and the development of squamous metaplasia (p= 0.001). In this study, there was a significant correlation between the gland type and capsular features and between the size of tumor and rate of squamous metaplasia. A detailed clinical and histopathological analysis of PAs may provide a better insight to the pathophysiology of the lesion, tumor cell differentiation, and prognostic factors.

Major changes in AD brain gene expression caused by the T9861C mtDNA mutation

AbstractBackgroundEvidence of mitochondrial dysfunction in Alzheimer’s Disease (AD) has accumulated over the years. We have previously identified a mutation in mitochondrial DNA (mtDNA) that is strongly associated with AD. This T9861C mutation changes a phenylalanine into a leucine at amino acid position 219 of cytochrome c oxidase subunit 3, resulting in a significant reduction of cytochrome oxidase activity. We report the effect of this mutation on the expression of genes involved in mitochondrial function in AD brains.MethodTotal RNA sequencing with ribosome depletion was performed on frozen archival tissue from parietal cortex of three control and eight sporadic AD (four AD, and four AD+ (AD patients possessing the T9861C mtDNA mutation)) brains. Reads were aligned to a transcriptome reference (Ensemble, GCRh38) containing all annotated mRNA and noncoding RNA using kallisto bus (v0.48). Read counts were calculated for genes using bustools (v0.41). Count data were analyzed using RNAfuzzyApp. Read count normalization was performed using TMM and differential gene expression was determined using edgeR. Pathway enrichment was performed using Gene Ontology (GO).ResultThe heatmap shown identifies a total of 472 deferentially expressed genes (DEGs, p value &lt;0.05, FDR &lt;0.05) in the comparison of control vs AD vs AD+ samples. Principal component analysis shows that gene expression in AD samples is significantly different from that in controls and that a more striking difference exists between the AD+ samples and both control and AD samples. The 472 DEGs can be grouped into five clusters. These clusters impact a variety of mitochondrial functions related to synaptic signaling, cell junctions, neuron projections, the HIF‐1 signaling pathway, voltage‐gated channel activity and carbohydrate metabolism.ConclusionThe presence of the T9861C mutation in the brains of AD patients has a significant effect on expression of the brain transcriptome in the parietal cortex. There is a general and robust decrease in expression of the majority of DEGs relative to control and non‐mutation containing AD brains. There is also a smaller number of DEGs that are upregluated compared to control and non‐mutation containing AD brains. These gene expression changes impact mitochondrial function resulting in neuronal failure and neurodegeneration.

Abstract A085: A comparison of the recruitment and enrollment of racially and ethnically minoritized patients in two non-interventional genetic testing studies in Florida

Abstract Objectives: To compare participation rates of minority individuals in two non-interventional genetic studies. Background: Enrollment of participants into non-interventional studies is important in understanding cancer development in minority patients. Black, Hispanic, and Latin men and women are diagnosed with aggressive forms of cancer at more advanced stages however, the underlying mechanisms leading to poor outcomes of disease is still not well understood. To understand disease development, two studies were launched. Study 1 focused on the recruitment of mutation carriers not yet diagnosed with cancer. Participants of this study were of any race and consented to the collection of fresh tissue. Participants were physician-referred and approached by the study team on the day of surgery. The patients received an explanation of the objectives and conditions of participation. Study 2 was focused on recruiting minority participants in clinic with a confirmed diagnosis of breast or prostate cancer. Participants consented to the collection of archival tissue and participants were called prior to their clinic visit and explained the objectives and conditions of participation. We compared the rates of enrollment of minority patients into these non-interventional genetic studies. Methods: Participants were Black, Hispanic/Latino(a), or White, Non-Hispanic who were mutation carriers without cancer or had an active diagnosis of ovarian, breast, or prostate cancer. The study team was composed of Latin, South, and Central American Spanish-speakers and African American Caribbeans Haitian-Creole speakers. All study materials were available in English, Haitian Creole, and Spanish. Matched recruitment was completed between the patient population and study team members who spoke English, Haitian Creole, and Spanish. The studies were assessed and compared for their methods of recruitment, participation rates, and demographics of their enrolled patient populations between December 2022 and May 2023. Results: A total of 121 men and women are included in this data analysis. 81.8% of patients approached for both studies consented to participate. 92.9% of those who consented were minorities. In Study 1, 68.6% eligible patients agreed to participate (24/35). Of the 24 patients enrolled, 41.7% (10/24) were White Hispanic/Latina, 20.8% (5/24) were Black Hispanic and non-Hispanic, 29.2% (7/24) were White non-Hispanic, and 2.7% (2/36) declined race. Of those who rejected participation, 63.6% were Black non-Hispanic. In Study 2, 87.2% (75/86) of the 86 eligible patients consented to the study. 72% (54/75) were female and 28% (21/75) were male. Of the female participants, 31.5% (17/54) were Black non-Hispanic and 68.5% (37/54) were White Hispanic/Latino. Of the male participants, 47.6% (10/21) were Black and 52.3% (11/21) were Hispanic/Latino. 63.6% of those who rejected participation were White Hispanic. Conclusions: Our results indicate that study acceptance by racially and ethnically minoritized individuals is dependent on recruitment approach and current health status. Citation Format: Destiny B. Burnett, Sandy St-Hilaire, Melissa N. Castillo, Daniela M. Zuniga Carlier, Osmaray Morales Casanova, Ashlee J. Sealy, Alex P. Sanchez, Matthew P. Schulmbrecht, Camille Ragin, Sophia George, Judith Hurley, Janaki Sharma, Alejandra Perez. A comparison of the recruitment and enrollment of racially and ethnically minoritized patients in two non-interventional genetic testing studies in Florida [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr A085.

Abstract A054: Interleukin-16 is a novel target to prevent age-associated epigenetic changes leading to malignant transformation associated with ovarian high-grade serous carcinoma (HGSOC)

Abstract Low-grade chronic inflammation and persistent oxidative stress are hallmarks of malignant transformation. High-grade serous ovarian carcinoma (HGSOC) is a fatal malignancy of women, and the median age of its incidence is 60-65 years. Our earlier studies showed enhanced expression of interleukin 16 (IL-16, an inflammatory cytokine), its receptor CD9, and glucose-regulated protein (GRP78, a marker of cellular stress and inhibitor of DNA damage repair mechanism) in postmenopausal ovaries and patients with HGSOC as compared with premenopausal subjects. It is possible that these age-associated increases in IL-16, CD9, and GRP78 expression may contribute to HGSOC-related epigenetic modifications of gH2A histone family member X (gH2AX, a marker for double-stranded DNA breaks), and histone deacetylase 1 (HDAC1), involved in DNA repair. The goal of this study was to examine if age-associated such changes lead to HGSOC-associated malignant transformation and if so, to determine mechanisms involved in such transformation. Materials and Methods: Exploratory study: Archived tissue specimens were selected for immunohistochemical, immunoblotting, genomic studies, and immunoassays based on the review of their hematoxylin and eosin staining and final pathological reports. Specimens were grouped as normal premenopausal women (30-50 years old), normal postmenopausal women (55-85 years old), and ovarian high-grade serous carcinoma (HGSC). In vitro study: human ovarian surface epithelium (HOSE) cells were treated with or without recombinant human IL-16 (rhIL-16) and recombinant human GRP78 (rhGRP78), and small interfering RNA-GRP78 (siGRP78) for 24 hours or 48 hours. Nuclear fractionations from treated- or untreated HOSE cells or OVCAR3 (HGSOC cell line) cells were extracted. Results: Compared to premenopausal women, a subset of postmenopausal women exhibited significantly elevated expression (P&amp;lt;0.01) of inflammatory markers IL-16, and its receptor CD9, as well as a marker of oxidative stress (GRP78). Additionally, markers of epigenetic changes, including gH2AX and HDAC1, presented similar patterns of increase in their expression. These observations were also consistent in HGSOC patients. Enhanced expression of GRP78, gH2AX, HDAC1, and CD9 in rhIL-16 treated HOSE cells like OVCAR3 cells and their reduction in siGRP78 treated HOSE cells suggests that age-associated increase in IL-16 in postmenopausal women may be a risk factor for malignant transformation and GRP78 may be a mechanism for IL-16-induced HGSOC development. Thus, IL-16 and GRP78 represent novel targets that may be used to develop novel targeted therapies to prevent HGSOC. Expression of gH2AX and HDAC1 increased in rhGRP78-treated HOSE cells. Conclusion: The results of this study suggest that age-associated increases in IL-16 (a marker of chronic inflammation) and GRP78 (a marker of oxidative stress) may contribute to the predisposition for HGSOC-associated malignant transformation. IL-16 and GRP78 offer potential targets for the prevention of HGSOC in women. Support: The Portes Foundation Citation Format: Jessica Ramirez, Animesh Barua. Interleukin-16 is a novel target to prevent age-associated epigenetic changes leading to malignant transformation associated with ovarian high-grade serous carcinoma (HGSOC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A054.

CTNI-52. SURVIVAL OUTCOMES IN RECURRENT GLIOBLASTOMA (RGBM) PATIENTS TREATED WITH A SINGLE INTRA-TUMORAL ADMINISTRATION OF BIZAXOFUSP, AN IL-4R-TARGETING TOXIN, IN A PHASE IIB TRIAL

Abstract BACKGROUND Bizaxofusp (MDNA55) is an IL4R-targeting toxin in development for recurrent (r)GBM, a universally fatal disease with mOS of 6-9 months. IL-4R is overexpressed in GBM and surrounding tumor microenvironment, with high expression associated with poor clinical outcome. Method: Efficacy and safety were evaluated in a single-arm, open-label, multi-center Ph2 study using convection-enhanced delivery of Bizaxofusp. The primary endpoint of mOS was compared to results from a blinded eligibility-matched external control arm (ECA). OS was defined as time from relapse to death/censor. IL-4R expression was determined by immunohistochemistry of available archival tumor tissues from both arms. RESULTS Bizaxofusp showed an acceptable safety profile at doses of up to 240 μg. Forty-four subjects were treated with Bizaxofusp per protocol; 81 ECA subjects met the eligibility criteria of the study. The Bizaxofusp group had a significantly longer mOS (12.85 months compared to 7.7 months for ECA; HR, 0.62; 95% CI, 0.42-0.89). Amongst the unmethylated MGMT and high IL-4R sub-groups, subjects treated with Bizaxofusp had significantly longer mOS when compared to the ECA. Eleven patients (25%) treated with Bizaxofusp survived &amp;gt;24 months; 3 patients were still alive (29.9, 32.8 and 40.1 months) at last follow-up. High IL-4R expression was associated with improved survival (mOS, 15.8 versus 9.8 months; HR, 0.7; 95% CI, 0.39-1.46) at all doses. However at high doses (180 μg) subjects with low IL-4R expression had significantly longer survival versus low dose Bizaxofusp (mOS, 15.4 versus 9.1 months; HR, 0.28; 95% CI, 0.09-0.91). CONCLUSION In patients with rGBM, a single treatment of Bizaxofusp resulted in significantly improved OS compared to eligibility-matched ECA. High-dose Bizaxofusp was equally effective irrespective of IL4R expression. A Phase 3 registration trial will use a novel hybrid design with a propensity-matched ECA comprising two-thirds of the control arm, setting a new precedent for GBM clinical trials.

CTNI-23. SINGLE CELL TRANSCRIPTOMICS, PHARMACOKINETICS, AND PHARMACODYNAMICS OF COMBINED CDK4/6 AND MTOR INHIBITION IN A PHASE 0 TRIAL OF RECURRENT HIGH-GRADE GLIOMA

Abstract Adult High-grade gliomas (HGGs) often exhibit deregulation of RB-CDK4/6 and mTOR signaling pathways. This study investigated tumor single cell transcriptomics, pharmacokinetics, and pharmacodynamics immediately following combined CDK4/6 (ribociclib) and mTOR (everolimus) inhibition in recurrent HGG. 24 patients with recurrent gliomas harboring RB+, CDKN2A/B deletion or CDK4/6 amplification, and PTEN loss or PIK3CA mutations were enrolled in a phase 0 trial. Patients received ribociclib treatment for 5 days before surgery, including six who received ribociclib (400mg QD) plus everolimus (2.5mg QD), and six dose-escalation cohorts (n = 3 each) reaching ribociclib 600mg QD plus everolimus 70mg QW. No dose-limiting toxicities were observed. Validated LC-MS/MS methods determined total and unbound drug concentrations. Preoperative MRI revealed a marked decrease in gadolinium-enhancement in all patients. In gadolinium-nonenhancing tumor regions, the median unbound concentration of ribociclib was 561 nM, while no unbound everolimus concentrations were detected. Ribociclib’s pharmacodynamic effects in surgical specimens were compared with matched archival tissue using IHC. Across IDH-wild-type tumors, there was significant decrease in MIB1 levels between the matched archival and surgical specimens (p &amp;lt; 0.05) suggesting drug-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) analyses were performed on 17 IDH-wild-type recurrent samples treated with ribociclib plus everolimus and 29 IDH-wild-type recurrences treated with standard therapies. Cellular state analysis based on snRNAseq data demonstrated a decreased neural progenitor-like (NPC-like) malignant cell state compared with standard therapies (p &amp;lt; 0.01), indicating inhibition of the CDK4-driven neural progenitor-like cell state. This observation was validated using snRNA and patient-derived glioma cell lines exposed to ribociclib, showing a consistent decrease in NPC-like proportions and cycling cells (p &amp;lt; 0.05). This study highlights the value of integrating pharmacokinetics/pharmacodynamics and snRNA data to assess treatment effects in phase 0 surgical tissues. These findings support further development of ribociclib, but not everolimus, for the treatment of glioma patients.

CTNI-42. PHASE I CLINICAL TRIAL OF PEPOSERTIB PLUS RADIATION IN NEWLY DIAGNOSED MGMT-UNMETHYLATED GLIOBLASTOMA

Abstract BACKGROUND DNA-dependent protein kinase (DNA-PK) is a pivotal component of DNA damage repair (DDR) pathways and is an attractive target in glioblastoma because its expression renders tumors less vulnerable to radiotherapy. Peposertib is a small molecule DNA-PK inhibitor that has been shown to potentiate radiotherapy and regress glioblastoma tumors pre-clinically. We conducted a two-stage phase I trial of peposertib plus radiation in newly-diagnosed MGMT-unmethylated glioblastoma (NCT04555577). Method: In stage 1, patients received peposertib plus standard-of-care radiotherapy to determine the maximum-tolerated dose (MTD) based on the Bayesian Optimal Interval design (maximum n = 24). Stage 2 is a window-of-opportunity expansion cohort and will include 5 surgical patients to evaluate tumor drug concentration. Both groups receive adjuvant temozolomide for 6-cycles. RESULTS Fifteen completed the 10-week dose-limiting toxicity (DLT) period; 3-50mg, 3-100mg, 3-200mg, 6-300mg. One DLT (G3 radiation necrosis [RN] at 300mg) was observed. Most notable toxicity was 3 patients with transient G3 dermatitis of the scalp (not DLT per protocol) (2-200mg, 1-300mg). Therefore, radiation dose constrains to the skin were incorporated and subsequent patients did not experience this toxicity. Next-generation sequencing (NGS) was available for 13 archival tissue. Two patients had pathology-proven RN (one within and one outside of the DLT period). Both tumors had mutations in DDR genes (i.e. ATRX, DICER1). Recurrent tissue was available for one patient after treatment with peposertib showed gain of two ATM mutations. CONCLUSION The initial safety data of peposertib plus radiation in newly-diagnosed MGMT unmethylated glioblastoma is favorable. The cases with RN in tumors with DDR mutations, the gain of two ATM mutations in a recurrent tumor and the cases of scalp dermatitis suggest that peposertib is active and possibly potentiates the effect of radiation. Safety and survival data and correlations with genomics will be presented. The study was supported by EMD Serono (CrossRef Funder ID: 10.13039/100004755).

Cathepsin W, T-cell receptor-associated transmembrane adapter 1, lymphotactin and killer cell lectin like receptor K1 are sensitive and specific RNA biomarkers of canine epitheliotropic lymphoma.

Cutaneous T-cell lymphoma (CTCL) is an uncommon type of lymphoma involving malignant skin-resident or skin-homing T cells. Canine epitheliotropic lymphoma (EL) is the most common form of CTCL in dogs, and it also spontaneously arises from T lymphocytes in the mucosa and skin. Clinically, it can be difficult to distinguish early-stage CTCLs apart from other forms of benign interface dermatitis (ID) in both dogs and people. Our objective was to identify novel biomarkers that can distinguish EL from other forms of ID, and perform comparative transcriptomics of human CTCL and canine EL. Here, we present a retrospective gene expression study that employed archival tissue from biorepositories. We analyzed a discovery cohort of 6 canines and a validation cohort of 8 canines with EL which occurred spontaneously in client-owned companion dogs. We performed comparative targeted transcriptomics studies using NanoString to assess 160 genes from lesional skin biopsies from the discovery cohort and 800 genes from the validation cohort to identify any significant differences that may reflect oncogenesis and immunopathogenesis. We further sought to determine if gene expression in EL and CTCL are conserved across humans and canines by comparing our data to previously published human datasets. Similar chemokine profiles were observed in dog EL and human CTCL, and analyses were performed to validate potential biomarkers and drivers of disease. In dogs, we found enrichment of T cell gene signatures, with upregulation of IFNG, TNF, PRF1, IL15, CD244, CXCL10, and CCL5 in EL in dogs compared to healthy controls. Importantly, CTSW, TRAT1 and KLRK1 distinguished EL from all other forms of interface dermatitis we studied, providing much-needed biomarkers for the veterinary field. XCL1/XCL2 were also highly specific of EL in our validation cohort. Future studies exploring the oncogenesis of spontaneous lymphomas in companion animals will expand our understanding of these disorders. Biomarkers may be useful for predicting disease prognosis and treatment responses. We plan to use our data to inform future development of targeted therapies, as well as for repurposing drugs for both veterinary and human medicine.

Exploring the relationship between novel MSN exon mutations and altered expression of other cytoskeleton genes in archival FFPE meningioma tissues

Meningioma, a prevalent brain tumor, can severely impact patients' health and quality of life. However, there is a scarcity of data on Ezrin/Radixin/Moesin (ERM) and the quantification of mutations in the Moesin gene. In an effort to bridge this knowledge gap, we undertook an extensive study to measure the expression of ERM genes in meningioma patients, with a concurrent assessment of mutations in the moesin gene. We analyzed the expression patterns of these genes in 50 formalin-fixed paraffin-embedded meningioma tissues, using the xylene method to deparaffinize the tissues and the TRIzol technique to isolate total RNA for qPCR expression analysis. Our results indicated that Ezrin and Radixin expression levels were significantly upregulated (p < 0.05), while Moesin expression was downregulated (p < 0.05). In addition, we analyzed Moesin exons 2–6 for mutations using sanger sequencing and identified 10 novel variants, including 5 missense, 3 synonymous, and 2 frameshift mutations. Our findings suggests that a potential role of ERM genes in meningioma pathogenesis, and our identification of MSN gene mutations may contribute to understanding their harmful or gain-of-function effects. These results offer new insights into the mechanisms underlying meningioma carcinogenesis, which may facilitate the development of targeted therapies in the future.

EP06.03-10 HER3 Expression in Archived Tissue Samples from Patients with NSCLC Across Various Genomic Subtypes and Characteristics

EP06.03-10 HER3 Expression in Archived Tissue Samples from Patients with NSCLC Across Various Genomic Subtypes and Characteristics

Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes as a predictive biomarker for axitinib in adenoid cystic carcinoma.

This study analyzed the predictive value of artificial intelligence (AI)-powered tumor-infiltrating lymphocyte (TIL) analysis in recurrent or metastatic (R/M) adenoid cystic carcinoma (ACC) treated with axitinib. Patients from a multicenter, prospective phase II trial evaluating axitinib efficacy in R/M ACC were included in this study. H&E whole-side images of archival tumor tissues were analyzed by Lunit SCOPE IO, an AI-powered spatial TIL analyzer. Twenty-seven patients were included in the analysis. The best response was stable disease, and the median progression-free survival (PFS) was 11.1 months (95% CI, 9.2-13.7 months). Median TIL densities in the cancer and surrounding stroma were 25.8/mm2 (IQR, 8.3-73.0) and 180.4/mm2 (IQR, 69.6-342.8), respectively. Patients with stromal TIL density >342.5/mm2 exhibited longer PFS (p = 0.012). Cancer and stromal area TIL infiltration were generally low in R/M ACC. Higher stromal TIL infiltration was associated with a longer PFS with axitinib treatment.

Enhanced Risk Stratification in Early-Stage Endometrial Cancer: Integrating POLE through Droplet Digital PCR and L1CAM.

In order to enhance risk stratification in early-stage endometrial cancer (EC), we conducted molecular classification using surrogate markers, including the POLE droplet digital polymerase chain reaction (ddPCR) and L1CAM immunohistochemistry (IHC). We analyzed archival tumor tissue from 183 early-stage EC patients. POLE pathogenic mutations of P286R, V411L, S297F, A456P, and S459F within exons 9, 13, and 14 were detected using a ddPCR, while the mismatch repair (MMR) status was determined by MMR protein IHC and MSI tests. Additionally, we conducted IHC for p53 and L1CAM. The 183 ECs were categorized into four subgroups: POLE-mutated (15.9%), MMR-deficient (29.0%), p53-abnormal (8.7%), and non-specific molecular profile (NSMP, 46.4%). We further subcategorized the NSMP subgroup into NSMP-L1CAMneg (41.5%) and NSMP-L1CAMpos (4.9%), which we refer to as the molecular L1CAM classification. The molecular L1CAM classification was an independent prognostic factor for recurrence-free survival (RFS) and overall survival (OS) (p < 0.001, each). Integrating molecular L1CAM classification can enhance risk stratification in early-stage EC, providing valuable prognostic information to guide treatment decisions and improve patient outcomes. POLE ddPCR might be a cost-effective and easy-to-perform test as an alternative to POLE NGS.