Tumor Specimens Research Articles

Abstract PO5-13-11: Quantitative Assessment of HER2 Expression in Intraductal Neoplasms of the Breast

Abstract HER2 (ERBB2) is an established prognostic and predictive marker for patients with invasive breast cancer and used in HER2-targeted therapy. The clinical and biological significance of expression of HER2 protein in patients with intraductal neoplasms of the breast is not well characterized. In this study, we used our HS-HER2 (High Sensitivity-HER2) which is a CAP/CLIA certified laboratory derived test (LDT) using quantitative immunofluorescence (QIF). We evaluated HER2 expression in DCIS (ductal carcinoma in situ), LCIS (lobular carcinoma in situ) and benign lesions to correlate with their clinicopathologic characteristics. Thirty- eight cases of DCIS, and fourteen cases of LCIS and thirty benign cases were selected from the Yale Pathology department archives and 252 ROIs (regions of interest) were annotated by a board-certified pathologist. DCIS is classified according to the three-tier nuclear grading system: low, intermediate, and high-nuclear grade. LCIS is classified as PLCIS (pleomorphic variant) and CLCIS (classic LCIS) according to the WHO classification of breast tumors. Serial sections of FFPE tumor specimens were used to accurately quantify the HER2 expression by the HS-HER2 assay and the acquisition by QuPath v.04 with Qymia extension. According to the LOD (limit of detection,3 amol/mm2), LOQ (limit of quantification, 9 amol/mm2) and LOL (limit of linearity, 23 amol/mm2) of the optimized HS-HER2 assay as performed in our CLIA lab shows a broad range of HER2 expression in both DCIS and LCIS lesions, ranging from 0.7 amol/mm2 to 111.4 amol/mm2 in DCIS and 3.6 amol/mm2 to 49.9 amol/mm2 in LCIS. High grade DCIS lesions express higher average HER2 levels (111.4 + 0.7 amol/mm2) than combined low and intermediate grade DCIS (77.9 +1.9 amol/mm2) with no significant difference between low/intermediate and high-grade DCIS. CLCIS lesions express higher average HER2 level (49.9 + 3.6 amol/mm2) than PLCIS (35.1 + 5.4amol/mm2) but again this is not statistically significant. HER protein expression is relatively low in benign lesions ranging from 0.8 to 8.4 amol/mm2. Using the HS-HER2 assay, our results show quantitative level HER2 levels in DCIS and LCIS breast lesions. These findings may be important as HER2 targeted therapies that work in gene-unamplified cancer (HER2 conjugated ADCs) gain broader usage in the clinic. Citation Format: Nay Nwe Nyein Chan, Haiying Zhan, Revekka Khaimova, Thazin Aung, Charles Robbins, Patricia Gaule, David Rimm. Quantitative Assessment of HER2 Expression in Intraductal Neoplasms of the Breast [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 PO5-13-11.

Abstract PO3-08-04: Prevalence of BRCA1/2 mutations in an underrepresented population of women with breast cancer: Observations from the City of Hope INSPIRE study

Abstract Background: Because African American and Latina women have been less likely to undergo germline testing, it has been difficult to determine the prevalence of hereditary breast and ovarian cancer predisposition in these populations. Here we report the prevalence of BRCA1/2 mutation in patients with a breast cancer diagnosis from City of Hope’s (COH) INSPIRE (Implementing Next-generation Sequencing for Precision Intervention and Risk Evaluation) study. Methods: Patients with a history of any stage breast cancer (stage 0-IV), who were seen by a City of Hope physician at the Duarte or Upland campuses, were approached by a clinical research coordinator for participation in an institutional tissue biorepository project. Demographic information included age, race and ethnicity. Patients consent for this IRB-approved study to store tumor specimens for future research use and Invitae germline testing for 155 predisposition genes which is performed at no cost to the patient. Results: From 7/9/20 until 4/2023, 2413 women consented for participation and underwent germline testing. Mutations in BRCA1 were identified in 53 and BRCA2 in 65. Table 1 summarizes the prevalence of BRCA1/2 mutations according to race and ethnicity. Pathogenic variants in BRCA1 were identified in 22/742 (3.0%) of Hispanic women compared to 16/1552 (1.7%) non-Hispanics (p=0.046). BRCA2 mutations were comparable 16/742 (2.2%) Hispanics and 47/1505 (3%) non-Hispanics. Hispanic women with breast cancer were 2.49 times as likely as non-Hispanics to carry a pathogenic germline BRCA1 mutations than BRCA2 (p=0.026) Conclusion: The prevalence of BRCA1/2 mutations in the African American and Latina population is higher than what was observed in White women. These data provide additional support for the recommendation to perform germline testing in all women with breast cancer, regardless of race or ethnicity Prevalence of BRCA 1 and 2 mutations by Self-Reported Race and Ethnicity # Tested BRCA1 BRCA2 BRCA1 or 2 VUS/Negative Race Native American 15 0 1 (6.7%) 1 (6.7%) 14 (93.3%) Pacific Islander 8 0 0 0 8 (100%) Asian 367 5 (1.4%) 11 (3%) 16 (4.4%) 351 (95.6%) Black 136 7 (5.1%) 4 (2.9%) 11 (8.1%) 125 (91.9%) White 1675 37 (2.2%) 44 (2.6%) 81 (4.8%) 1594 (95.2%) Other 86 1 (1.2%) 4 (4.7%) 4* (4.7%) 82 (95.3%) Unknown 126 3 (2.4%) 1 (0.8%) 5 (3.2% 122 (96.8%) TOTAL 2413 53 (2.2%) 65 (2.7%) 117 (4.8%) 2296 (95.2%) Ethnicity Hispanic 742 22(3%) 16 (2.2%) 37* (5%) 705(95%) Non-Hispanic 1552 26(1.7%) 47(3%) 73(4.7%) 1479 (95.3%) Unknown 119 53(2.2%) 2(1.7%) 7(5.9%) 112(94.1%) TOTAL 2413 53(2.2%) 65(2.7%) 7(5.9%) 112(94.2%) * Patient had both BRCA1 and BRCA2 mutations Citation Format: Joanne Mortimer, Sydney Lindsey, Ilana Solomon, Wai Park, Duveen Sturgeon, Kathleen Blazer, Stacy Gray, Joseph Bonner, Xiaoyu Xia, Stephen Gruber. Prevalence of BRCA1/2 mutations in an underrepresented population of women with breast cancer: Observations from the City of Hope INSPIRE study [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 PO3-08-04.

Immunogenomics and spatial proteomic mapping highlight distinct neuro-immune architectures in melanoma vs. non-melanoma-derived brain metastasis.

Brain metastases (BrMs) are a devastating complication of solid tumours. A better understanding of BrMs biology is needed to address their challenging clinical management. Immunogenomic and digital spatial analyses were applied to interrogate the peripheral blood and tumour specimens derived from 53 unique patients with BrMs originating from different solid tumours. At craniotomy time, patients with melanoma-derived brain metastasis (MBM) displayed in the periphery lower neutrophil-lymphocyte ratio (NLR) compared to non-melanoma-derived brain metastasis (non-MBM). Regardless of the primary tumour source, higher NLR was associated with reduced overall survival (OS). Tumour MicroEnviroment genomic evaluations revealed higher expression of genes identifying NK, CD8 and B cells in MBM vs. non-MBM. Moreover, MBM patients with longer OS displayed increased CD8+ cell infiltration. Spatial proteomic analysis further highlighted enriched infiltration of CD8+ cells, antigen-presenting cells, T-cell agonists and B cells in MBM. Conversely, increased expression of genes and proteins associated with neurodevelopment, cell-cell adhesion and neutrophil infiltration were observed in non-MBM. These findings reveal an increased immunogenicity of MBM vs non-MBM and highlight the presence of a unique neuro-immune interplays in MBM vs non-MBM, suggesting that a balance between neuro-immune architectures might be associated with diverging clinical outcome of patients with BrMs.

Abstract PO5-05-11: The effect of eribulin treatment for patients with HER2-low breast cancer

Abstract Background: Eribulin, a nontaxane, synthetic microtuble dynamics inhibitor, induces longer overall survival (OS) in patients with advanced or metastatic breast cancer (MBC). Interestingly, it also improves the tumor immune microenvironment via vascular remodeling. DESTINY-Breast04 trial showed that trastuzumab deruxtecan resulted in significantly longer OS of human epithelial growth receptor 2 (HER2)-low breast cancer patients, and this mechanism may also be related to tumor microenvironment. In this study, we investigated the effect of eribulin for patients with HER2-low breast cancer. Materials and Methods: This retrospective study comprised 97 MBC treated with eribulin from August 2011 to April 2019 at our institute. HER2 levels were determined through tumor biopsy specimen at first medical examination. We evaluated the correlations between HER2 levels and clinicopathological features, including the prognosis and immune related markers in each breast cancer subtype. We also classified the progressive disease (PD) into progression due to pre-existing lesion (PPL) and progression due to new metastasis (PNM) based on systemic computed tomography findings, and investigated the relationships between HER2 expression and PD types. Results: HER2-high group had significantly better OS than other groups (p=0.021, log-rank). Hormone receptor (HR)-negative/HER2-low patients had significantly better progression-free survival (PFS) than HR-negative/HER2-negative (p=0.024, log-rank). Though we also examined HER2 levels of recurrent tumor specimen, it was not a significantly useful prognostic marker in patients with HR-negative breast cancer (p=0.085, log-rank). There was no significant correlation between HER2 expression and any tested clinicopathological parameter, including absolute lymphocyte count, neutrophil-to-lymphocyte ratio and tumor infiltrating lymphocytes. PD were observed in 64 patients (PPL: n=47, PNM: n=17). PD type of all HR-positive/HER2-high patients was PNM, whereas all HR-negative/HER2-high patients was PPL (p=0.035). HER2-low group tended to have a higher PPL rare than negative group (83.3% vs 69.2%). Conclusions: In HR-negative/HER2-low group, PFS was improved by eribulin treatment, which might be due to unique anticancer effect of eribulin, suppression of epithelial-mesenchymal transition. Further study of this mechanism through basic research is important. Also, assessment of HER2 from primary tumor may be more useful than recurrent specimen. Citation Format: Wataru Goto, Saeko Henmi, Mariko Nishikawa, Asuka Kouchi, Rika Kouhashi, Akimichi Yabumoto, Koji Takada, Yuka Asano, Yukie Tauchi, Kana Ogisawa, Tamami Morisaki, Shinichiro Kashiwagi. The effect of eribulin treatment for patients with HER2-low 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 PO5-05-11.

Individualized Cell-Free DNA Monitoring With Chromosomal Junctions for Mesothelioma

IntroductionThe spatially complex nature of mesothelioma and interventions like pleurodesis, surgery, and radiation often complicate imaging-based assessment. Further, cell-free DNA (cfDNA) based monitoring strategies are inadequate for mesothelioma, given the presence of a few recurring nonsynonymous somatic variants. However, patient-specific chromosomal rearrangements are commonly found in mesothelioma. Our study objective was to develop an individualized cfDNA assay to enable blood-based monitoring using circulating tumor DNA (ctDNA) in mesothelioma. We hypothesized that the unique chromosomal rearrangement junctions found in mesothelioma could be employed for individualized ctDNA detection and disease monitoring. MethodsDNA was extracted from tumor specimens for whole genome sequencing. Chromosomal junctions, prioritized by highest allele frequency and low homology to the rest of the genome, were selected for detection. Primers and Taqman probes were designed to span the junctions, forming personalized junction panels. Patient plasma obtained before therapy and at response assessment was tested for the presence of personalized junctions via quantitative PCR. ResultsOur study included nine patients, four with peritoneal and five with pleural mesothelioma. 763 chromosomal junctions were identified in the tumors of all cases. We selected three to five junctions per sample for quantitative PCR. We detected 25/30 (83%) of selected junctions in the plasma of seven out of nine patients (78%). Cell-free junction detection at follow-up was concordant with disease status: cfDNA junctions were detected in three patients with persistent disease, and not detected in a patient with no evidence of disease after surgery. ConclusionsWith further validation, individualized ctDNA junction assays could supplement imaging for disease monitoring in mesothelioma.

Direct Implantation of Patient Brain Tumor Cells into Matching Locations in Mouse Brains for Patient-Derived Orthotopic Xenograft Model Development.

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

The clinical significance of mitochondrial calcium uniporter in gastric cancer patients and its preliminary exploration of the impact on mitochondrial function and metabolism.

The objective of this study is to elucidate the influence of MCU on the clinical pathological features of GC patients, to investigate the function and mechanism of the mitochondrial calcium uptake transporter MCU in the initiation and progression of GC, and to explore its impact on the metabolic pathways and biosynthesis of mitochondria. The ultimate goal is to identify novel targets and strategies for the clinical management of GC patients. Tumor and adjacent tissue specimens were obtained from 205 patients with gastric cancer, and immunohistochemical tests were performed to assess the expression of MCU and its correlation with clinical pathological characteristics and prognosis. Data from TCGA, GTEx and GEO databases were retrieved for gastric cancer patients, and bioinformatics analysis was utilized to investigate the association between MCU expression and clinical pathological features. Furthermore, we conducted an in-depth analysis of the role of MCU in GC patients. We investigated the correlation between MCU expression in GC and its impact on mitochondrial function, metabolism, biosynthesis, and immune cells. Additionally, we studied the proteins or molecules that interact with MCU. Our research revealed high expression of MCU in the GC tissues. This high expression was associated with poorer T and N staging, and indicated a worse disease-free survival period. MCU expression was positively correlated with mitochondrial function, mitochondrial metabolism, nucleotide, amino acid, and fatty acid synthesis metabolism, and negatively correlated with nicotinate and nicotinamide metabolism. Furthermore, the MCU also regulates the function of the mitochondrial oxidative respiratory chain. The MCU influences the immune cells of GC patients and regulates ROS generation, cell proliferation, apoptosis, and resistance to platinum-based drugs in gastric cancer cells. High expression of MCU in GC indicates poorer clinical outcomes. The expression of the MCU are affected through impacts the function of mitochondria, energy metabolism, and cellular biosynthesis in gastric cancer cells, thereby influencing the growth and metastasis of gastric cancer cells. Therefore, the mitochondrial changes regulated by MCU could be a new focus for research and treatment of GC.

Deciphering the role of alternative splicing in neoplastic diseases for immune-oncological therapies.

Alternative splicing (AS) is an important molecular biological mechanism regulated by complex mechanisms involving a plethora of cis and trans-acting elements. Furthermore, AS is tissue specific and altered in various pathologies, including infectious, inflammatory, and neoplastic diseases. Recently developed immuno-oncological therapies include monoclonal antibodies (mAbs) and chimeric antigen receptor (CAR) T cells targeting, among others, immune checkpoint (ICP) molecules. Despite therapeutic successes have been demonstrated, only a limited number of patients showed long-term benefit from these therapies with tumor entity-related differential response rates were observed. Interestingly, splice variants of common immunotherapeutic targets generated by AS are able to completely escape and/or reduce the efficacy of mAb- and/or CAR-based tumor immunotherapies. Therefore, the analyses of splicing patterns of targeted molecules in tumor specimens prior to therapy might help correct stratification, thereby increasing therapy success by antibody panel selection and antibody dosages. In addition, the expression of certain splicing factors has been linked with the patients' outcome, thereby highlighting their putative prognostic potential. Outstanding questions are addressed to translate the findings into clinical application. This review article provides an overview of the role of AS in (tumor) diseases, its molecular mechanisms, clinical relevance, and therapy response.

A comparative study of INSM1 (clone MRQ70) immunoreactivity on CytoLyt® pretreated cytology and resection specimens of pancreatic neuroendocrine tumors.

Insulinoma-associated protein 1 (INSM1) is a newly characterized sensitive and specific immunohistochemical marker for neuroendocrine (NE) tumors. Whereas more traditional NE markers, such as chromogranin A and synaptophysin, are cytoplasmic, INSM1 is uniquely nuclear and thus could serve as a useful addition to NE tumor workup. While application of immunohistochemical studies to cytology specimens is becoming increasingly relevant, knowledge of the effects of the certain fixatives as well as the pattern and intensity of immunoexpression are important considerations. Sixteen cases of pancreatic neuroendocrine tumor (PanNET) diagnosed between 2015 and 2021 underwent both fine-needle aspiration, which was subsequently prepared in CytoLyt®-fixed cytology cell block (CCB), and surgical resection, in which specimens were prepared into formalin-fixed paraffin embedded blocks (FFPE). For all samples, INSM1 immunoreactivity was classified according to staining intensity and extent, then compared between CCBs and matched FFPEs. All 16 FFPE specimens demonstrated strong and diffuse INSM1 immunoreactivity, while only 10/16 (62.5%) CCBs were positive. Of those 10, only 2/10 (20%) demonstrated strong and diffuse reactivity. The choice of fixative has a demonstrable effect on the immunoreactivity of INSM1 in PanNET. Even though the sensitivity is lower in CytoLyt®-fixed cell block specimens, the addition of INSM1 is useful, especially in challenging cases that may be negative for one or more of the traditional NE markers.

Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients

BackgroundGlioblastoma is a malignant and aggressive type of central nevous system malignancy characterized by many distinct biological features including extensive hypoxia. Hypoxia in glioblatoma associates with complex signaling patterns including activation of several pathways such as MAPK, PI3K-AKT/mTOR and IL-6/JAK/STAT3 with the master regulator HIF-1, which in turn drive particular tumor behaviors determining, in the end, treatment outcomes and patients fate. Thus, the present study was designed to investigate the expression of selected hypoxia related factors including STAT3 in a small set of long-term surviving glioma patients.MethodsThe expression of selected hypoxia related factors including STAT3 was evaluated in a time series of formalin fixed paraffin embedded and cryopreserved glioma samples from repeatedly resected patients. In addition, comparative studies were also conducted on primary glioma cells derived from original patient samples, stabilized glioma cell lines and tumor-xenograft mice model. Obtained data were correlated with clinical findings too.ResultsGlioblastoma samples of the analyzed patients displayed heterogeneity in the expression of hypoxia- related and EMT markers with most interesting trend being observed in pSTAT3. This heterogeneity was subsequently confirmed in other employed models (primocultures derived from glioblastoma tissue resections, cryopreserved tumor specimens, stabilized glioblastoma cell line in vitro and in vivo) and concerned, in particular, STAT3 expression which remained stable. In addition, subsequent studies on the role of STAT3 in the context of glioblastoma hypoxia demonstrated opposing effects of its deletion on cell viability as well as the expression of hypoxia and EMT markers.ConclusionsOur results suport the importance of STAT3 expression and activity in the context of hypoxia in malignant glioblastoma long-term surviving glioma patients while emphasizing heterogeneity of biological outcomes in varying employed tumor models.

Association between pathological characteristics and recurrence score by OncotypeDX in resected T1-3 and N0-1 breast cancer: a real-life experience of a North Hungarian regional center.

Introduction: The 21-gene analysis (OncotypeDX) is validated test for pT1-3, pN0-1 with hormone receptor (HR) positive and normal expression of human epidermal growth factor receptor-2 (HER2) breast cancer (BC) to determine the aggressiveness of the disease based on the calculation of Recurrence Score (RS). Methods: In this retrospective study the authors correlated pathological characteristics and Recurrence Score (RS) by traditional statistical methods and Observed Oriented Modeling (OOM) in a realistic cohort of BC patients. Results: OncotypeDX tests were performed in 94 tumour specimens of 90 BC patients. >83% of node-negative (pN0) and >72% of node-positive (pN1) cases could avoid chemotherapy. For pN0 cases, non-parametric correlation and tests demonstrated significant association in eight types of characteristics [progesterone receptor (PR) expression, Ki-67 value, Ki-67 group, PR group, grade, estrogen receptor (ER) expression, Nottingham Prognostic Index (NPI) and Clinical Risk]. For pN1 cases, parametric correlation and tests showed significant association in six characteristic types (number of positive nodes, ER and PR expression, PR group, Ki-67 group and NPI). Based on OOM for pN0 cases, significant associations were established in three characteristics (Ki-67 group, grade and NPI group). For pN1 cases OOM found significant associations in seven characteristics (PR group, PNI, LVI, Ki-67 group, grade, NPI group and number of positive nodes). Conclusion: First in oncology, OOM was applied, which found some other significant characteristics associated with RS than traditional statistical methods. There were few patients, where no clinical associations were found between characteristics and RS contrary to statistically significant differences. Therefore, the results of these statistical analyses can be neither applied for individual cases nor able to provide the bases for screening patients, i.e., whether they need for OncotypeDX testing or not. OncotypeDX still provides a personalised approach in BC.

Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens

We present a rigorous validation strategy to evaluate the performance of Ultivue multiplex immunofluorescence panels. We have quantified the accuracy and precision of four different multiplex panels (three human and one mouse) in tumor specimens with varying levels of T cell density. Our results show that Ultivue panels are typically accurate wherein the relative difference in cell proportion between a multiplex image and a 1-plex image is less than 20% for a given biomarker. Ultivue panels exhibited relatively high intra-run precision (CV ≤ 25%) and relatively low inter-run precision (CV >> 25%) which can be remedied by using local intensity thresholding to gate biomarker positivity. We also evaluated the reproducibility of cell–cell distance estimates measured from multiplex images which show high intra- and inter-run precision. We introduce a new metric, multiplex labeling efficiency, which can be used to benchmark the overall fidelity of the multiplex data across multiple batch runs. Taken together our results provide a comprehensive characterization of Ultivue panels and offer practical guidelines for analyzing multiplex images.

Comprehensive bioinformatics analysis unveils THEMIS2 as a carcinogenic indicator related to immune infiltration and prognosis of thyroid cancer.

The aim of this study was to identify biomarkers associated with the initiation and prognosis of thyroid cancer and elucidate the underlying pathogenic mechanisms. We obtained expression profiles and clinical information from the Cancer Genome Atlas (TCGA)-THCA and three datasets (GSE53157, GSE82208, and GSE76039). The three microarray datasets were combined using Perl and the sva package in R and termed 'merged dataset'. Weighted gene co-expression network analysis (WGCNA) identified 15 gene co-expression modules in the merged dataset and 235 hub genes. Venn diagram analysis revealed 232 overlapping genes between the merged and THCA datasets. Overlapping genes were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The least absolute shrinkage and selection operator (LASSO) regression identified THEMIS2 as a candidate hub gene. Cox, Kaplan-Meier (K-M) survival and gene set enrichment analysis (GSEA) confirmed the correlation of THEMIS2 with overall survival, its enrichment in immunologic processes, and its association with the p53 and JAK-STAT signaling pathways. Its expression was positively correlated with those of immune checkpoints and the infiltration level of immune cells. Receiver operating characteristic curve (ROC) analysis confirmed that THEMIS2, a diagnostic biomarker, could distinguish between tumor and normal specimens. The nomogram (ROC or DCA) model containing THEMIS2, age, and stage predicted favourable prognoses. Thus, THEMIS2 was a biomarker of immune infiltration and prognosis in thyroid cancer.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

Abstract PL03-02: From microniches to single cells: Understanding the impact of intratumoral microbes on spatial and cellular heterogeneity in cancer

Abstract The tumor microenvironment (TME) is a dynamic network that includes a variety of non-malignant cells such as endothelial cells, fibroblasts, and immune cells, which play a crucial role in tumor behavior. Advances in molecular analysis have recently identified the presence of intratumoral microbiota in several solid cancer types. However, the specific ways these microorganisms interact with human cells within the TME and contribute to disease progression are not yet fully understood. Our recent research provides new insights into how bacterial components of the intratumoral microbiota contribute to tumor heterogeneity, particularly in head and neck and colorectal cancers. To investigate these interactions, we have adapted and applied emerging technologies in spatial transcriptomics, spatial proteomics, and single-cell sequencing (INVADEseq). Our spatial analyses show that the intratumoral microbiota are not distributed randomly within human tumors; instead, they localize to distinct microniches that promote immune and epithelial cell functions supportive of cancer progression. These bacterial-colonized microniches are characterized by myeloid cell infiltration, upregulation of MAPKs and immune checkpoint proteins, and reduced T-cell levels. Furthermore, INVADEseq single-cell RNA sequencing has enabled us to identify the dominant host cell-associated bacteria, the human cell types they infect, and their impact on the transcriptional programs of infected cells from patient tumor specimens. In oral squamous cell carcinoma, Fusobacterium and Treponema species have been identified as the dominant cell-associated bacterial taxa, primarily infecting subsets of macrophages and aneuploid cancer epithelial cells and upregulating pathways associated with cancer progression. Our findings offer insights into the complex interplay between intratumoral microbiota and human components of the TME, emphasizing their role in supporting intratumoral heterogeneity and influencing cancer progression. Understanding these dynamics holds significant promise for the development of targeted therapeutic strategies aimed at disrupting bacterial-human spatial and cellular interactions within the TME. Citation Format: Susan Bullman. From microniches to single cells: Understanding the impact of intratumoral microbes on spatial and cellular heterogeneity in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr PL03-02.

Abstract LB224: Neoplastic immune mimicry potentiates breast cancer progression

Abstract Dedifferentiation programs are commonly enacted during breast cancer progression where they enable novel cellular phenotypes. Increased cellular plasticity within the neoplastic compartment of tumors correlates with disease aggressiveness, often manifesting as greater resistance to cytotoxic therapies or increased ability to metastasize to distant organs. Here we report that subpopulations of ostensibly stem-like neoplastic breast epithelial cells express canonical leukocyte cell surface receptor proteins, and have named this unique cellular program ‘immune mimicry’ (IM). We have documented neoplastic cells engaging in IM by examination of histopathological breast tumor specimens and their derived cell lines, public human breast tumor single-cell RNA-seq datasets, and in murine transgenic and cell line-derived mammary cancer models. Immune-mimicked neoplastic cells harbor hallmarks of dedifferentiation and appear enriched for developing aggressive and high-grade tumors. Experimental studies with breast cancer cell lines revealed that neoplastic cells expressing leukocyte surface features are elicited by growth-arresting conditions, such as conventional cytotoxic chemotherapeutic treatments and during metastatic dissemination in mice. In addition, using proof-of-concept studies, we leveraged the canonical leukocyte activation marker CD69, to demonstrate how its expression by neoplastic epithelial cells confers a proliferative advantage under low-density conditions. We conclude that neoplastic breast epithelial cells expressing leukocyte surface receptors may signify a particularly malignant and tumor-initiating cell state. Moving forward, neoplastic IM will be evaluated for prognostic utility in breast cancer to determine whether this unique cell state stratifies patients with increased risks for tumor recurrence and metastasis. The authors acknowledge NIH/NCI (K00 CA212132, T32 CA254888, P30 CA069533), the Collins Medical Trust, the Susan G Komen Foundation, and the National Foundation for Cancer Research for funding. Citation Format: Eric B. Berens, Sokchea Khou, Elaine Huang, Amber Hoffman, Briana Johnson, Zena Werb, Laura M. Heiser, Lisa M. Coussens. Neoplastic immune mimicry potentiates breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB224.

Abstract CT137: Efficacy and immunological outcomes of non-fucosylated anti-CTLA-4 (BMS-986218) + degarelix acetate vs. degarelix acetate alone in men with high-risk localized prostate cancer (Neo-Red-P)

Abstract Background: Men with high-risk localized prostate cancer are at significant risk for disease recurrence and there is currently no role for neoadjuvant therapy. Although androgen deprivation therapy (ADT) is immunomodulatory and can increase CD8 T cell infiltration, these effects are counterbalanced by the expansion of suppressive regulatory T cells (Tregs). We therefore designed a correlative-rich clinical trial for men with high-risk localized prostate cancer to test whether BMS-986218 - a novel afucosylated anti-CTLA-4 antibody optimized for antibody-dependent cell-mediated cytotoxicity (ADCC) - depletes Tregs and augments response to ADT. Methods: Neo-Red-P is a single-center, two-arm, open-label pilot study assessing degarelix (ADT) versus ADT + anti-CTLA-4-NF (BMS-986218) in patients with treatment naïve high-risk localized prostate cancer. Key eligibility criteria included histologically confirmed adenocarcinoma with Gleason sum of ≥4+3 and clinical stage T1c-T3bN0M0. All patients received degarelix (ADT; 240mg SQ) 2 weeks before radical prostatectomy (RP). Patients were randomized to Arm A (ADT only), or Arm B (ADT + BMS-986218 [20mg IV] 2 doses Q2w starting 3 weeks before RP). The primary endpoint was the safety and feasibility of neoadjuvant BMS-986218 in prostate cancer. Secondary endpoints included the pathologic complete response rate, rate of undetectable PSA at 12 months, PSA response rate (>50% decrease pre-RP), and time-to-PSA recurrence. Fresh tumor specimens were collected at RP for single-cell RNA-sequencing, cytometry by time-of-flight, next-generation sequencing, and multiplex immunofluorescence. A treatment naïve cohort of matching demographic and pathological characteristics was collected for comparative analysis (n=14). Results: Between February 26, 2020, and November 15, 2022, 24 patients were enrolled. Severe treatment-related adverse events (TRAE; Grade ≥ 3) were rare in both arms; 0% in the ADT alone arm and 4% in the combination arm, suggesting a tolerable safety profile with the addition of BMS-986218. In the combination arm, 54% of subjects achieved a 50% reduction in PSA versus 33% in the ADT alone arm. In the ADT alone arm, 80% of patients had an undetectable PSA at 12 months, with a median recurrence-free survival of 1.61 years (IQR: 1.50-1.99 years). In the combination group, 75% had an undetectable PSA at 12 months, with a median recurrence-free survival of 1.82 years (IQR: 0.89-2.07 years). The overall recurrence rate in patients treated with combination therapy was 25% (3 of 12 patients) versus 20% (2 of 10 patients) in the ADT alone arm. Correlative studies suggest that aCTLA4-NF mitigates ADT-induced Treg accumulation through enhanced Fc receptor activity and augmented CTL activation, indicating an enhanced anti-tumor response with combination therapy. Conclusions: The neoadjuvant combination of ADT + aCTLA-4-NF (BMS-986218) is feasible, well tolerated, and induces significant remodeling of the immune TME through enhanced Fc receptor and CTL activity. Rates of disease recurrence following surgery were low in this high-risk population, warranting further investigation of combination immuno-hormonal therapy in the neoadjuvant setting for high-risk prostate cancer. Clinical Trial Information: NCT04301414 Citation Format: Patrick J. McCann, Casey R. Ager, Aleksandar Z. Obradovic, Matthew G. Chaimowitz, Samuel Pan, Caroline J. Laplaca, Parin Shah, Renu K. Virk, Christopher B. Anderson, Catherine S. Spina, Karie D. Runcie, Mark N. Stein, Charles G. Drake, Matthew C. Dallos. Efficacy and immunological outcomes of non-fucosylated anti-CTLA-4 (BMS-986218) + degarelix acetate vs. degarelix acetate alone in men with high-risk localized prostate cancer (Neo-Red-P) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT137.

Abstract LB190: Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer

Abstract In the context of the tumor microenvironment (TME), a substantial increased secreted protein, chitinase-3 like-protein-1 (CHI3L1) has been observed, emanating from various cell types such as cancer cells, macrophages, and T cells. The development of an immunosuppressive and fibrotic TME has been implicated in CHI3L1-mediated cancer progression. Despite recognizing the potential for CHI3L1 blockade to facilitate cancer regression, there remains a limited understanding of the regulatory mechanisms governing the expression of this tumor-promoting protein within cancer cells. Our investigations, along with others, have identified low levels of CHI3L1 protein in normal epithelial cells, in stark contrast to its abundant presence in pancreatic cancer tumor specimens. We propose that the interaction between cancer and stromal cells in the TME induces CHI3L1 expression within cancer cells. To test this hypothesis, we established a co-culture system involving cancer and stromal cells including pancreatic stellate cells, macrophages and T cells to emulate the TME of pancreatic cancer. Subsequently, we observed a ten-fold increase in CHI3L1 expression in cancer cells upon exposure to co-culture media. Through secretomic analysis, C-X-C motif chemokine ligand 10 (CXCL10) emerged as a potential inducer of CHI3L1 expression in response to co-culture media. CXCL10/CXCR3 signaling has been reported to correlate with poor prognosis in pancreatic cancer, while their role in disease development is still unclear. Concurrently, Gene Set Enrichment Analysis (GSEA) identified the activation of TNFα signaling via NF-κB and TGF-β pathways in pancreatic cancer patients with high CHI3L1 expression. Notably, chromatin-immunoprecipitation and promoter activity assays validated the direct binding and transcriptional regulatory role of NF-κB, a downstream transcription factor of CXCL10, on CHI3L1 gene in cancer cells. In addition, epigenetic reprogramming occurred during co-culture media education. Our observations indicated demethylation of the CHI3L1 promoter during co-culture media exposure, a phenomenon confirmed to be mediated by STAT3 activation. In summary, this study elucidates, for the first time, the intricate crosstalk between cancer and stromal cells within the TME of pancreatic cancer as a pivotal factor for the transcriptional and epigenetic dysregulation of CHI3L1, which then contributes to TME remodeling and malignant pancreatic cancer progression. Citation Format: Pei-Chia (Peggy) Su, Yi-Ching Wang. Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB190.

A single-cell strategy for the identification of intronic variants related to mis-splicing in pancreatic cancer.

Most clinical diagnostic and genomic research setups focus almost exclusively on coding regions and essential splice sites, thereby overlooking other non-coding variants. As a result, intronic variants that can promote mis-splicing events across a range of diseases, including cancer, are yet to be systematically investigated. Such investigations would require both genomic and transcriptomic data, but there currently exist very few datasets that satisfy these requirements. We address this by developing a single-nucleus full-length RNA-sequencing approach that allows for the detection of potentially pathogenic intronic variants. We exemplify the potency of our approach by applying pancreatic cancer tumor and tumor-derived specimens and linking intronic variants to splicing dysregulation. We specifically find that prominent intron retention and pseudo-exon activation events are shared by the tumors and affect genes encoding key transcriptional regulators. Our work paves the way for the assessment and exploitation of intronic mutations as powerful prognostic markers and potential therapeutic targets in cancer.

Efficacy of Personalized Postoperative Epilepsy Management in Patients with Glioblastoma Utilizing IDH1 Gene Assessment.

We explored the correlation between the presence of isocitrate dehydrogenase-1 (IDH1) mutations and the incidence of postoperative epilepsy in patients with glioblastoma, as well as assessed the efficacy of preemptive administration of antiepileptic medications in mitigating the occurrence of postoperative epilepsy. Fifty-three patients who received a postoperative pathological diagnosis of glioblastoma, were enrolled in this study. Tumor specimens were subjected to IDH1 gene analysis. The patient cohort was stratified based on their IDH1 mutation status and the administration of prophylactic antiepileptic drugs during the postoperative phase. We subsequently conducted a comparative analysis of postoperative epileptic complications within each patient subgroup. In the cohort of 53 patients under study, the occurrence of epilepsy was observed in 10 out of 21 patients carrying IDH1 mutations, while 5 out of 32 patients with wild-type IDH1 also experienced epilepsy, revealing a statistically significant difference (P < 0.05). Among the 27 patients who received prophylactic antiepileptic drugs, 6 of them developed epilepsy, whereas 9 out of 26 patients who did not receive prophylactic antiepileptic drugs exhibited concurrent epilepsy, with no statistically significant difference (P > 0.05). However, when performing a subgroup analysis, it was found that 3 out of 12 patients with IDH1 mutations who received prophylactic antiepileptic drugs experienced epilepsy, whereas 7 out of 9 patients who did not receive prophylactic antiepileptic drugs developed epilepsy, demonstrating a statistically significant difference (P < 0.05). Furthermore, within the group of 15 patients with wild-type IDH1, 3 patients who received prophylactic antiepileptic drugs developed epilepsy, while 2 cases of epilepsy occurred among the 17 patients who did not receive prophylactic antiepileptic drugs, with no statistically significant difference (P > 0.05). In individuals with IDH1 mutant glioblastoma who have undergone surgical resection, the implementation of preventive antiepileptic therapy demonstrates a potential to diminish the occurrence of postoperative epilepsy.