Basal-like Subtype Research Articles

Abstract B054: Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of 10%. Dense desmoplasia results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a deficiency in unsaturated fatty acids (uFAs), as the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger subsequent ER stress responses under uFA limited conditions in vitro. ER stress responses are governed by three major stress sensors: Inositol-requiring enzyme 1𝛼 (IRE1𝛼), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6). ER stress responses are also activated in PDAC murine and patient samples. Interestingly, the expression of ER stress markers is much higher in malignant lesions than benign pancreatic intraepithelial neoplasia. In contrast to tumor promoting, we found that PERK, ATF6 and IRE1𝛼 - XBP1 are cytotoxic to PDAC cells in vitro, as their knockout improved viability under stress conditions. Surprisingly, pharmacological inhibitor of XBP1 splicing, B-I09, didn’t recapitulate the results of the genetic approach. While B-I09 has cytoprotective effects in some PDAC lines, it is cytotoxic in others. In addition to inhibiting XBP1 splicing, B-I09 also inhibits regulated IRE1𝛼 dependent decay (RIDD). We speculate that RIDD might have opposite roles to XBP1s, leading to the discrepancy in the cellular phenotypes we observed. Most B-I09 responders are reported to be the basal-like transcriptional subtype. Therefore, we hypothesize that the basal-like subtype is more susceptible to B-I09. So far, we have shown that basal-like patient tumors are significantly enriched in the ER stress response pathways from bulk patient RNA-seq datasets, and we are currently validating this result in the publicly available scRNA-seq datasets from patients which contain basal-like and classical subtypes. We have generated isogenic models with basal-like features, such as epithelial to mesenchymal transition and deltaNP63 overexpression. However, our results suggest that these basal-like features did not sensitize classical PDAC cells to B-I09. Our lab has previously shown that B-I09 is selectively lethal to the MYC-transformed tumors. We are currently testing whether MYC overexpression would sensitize the non-responders to B-I09. It is exciting to see that some of the PDAC lines such as PANC1 and KPC murine line 4662 have reduced viability under B-I09 treatment. However, in our subcutaneous xenograft and allograft experiments, B-I09 only leads to a mild tumor growth suppression, which is unlikely to be therapeutically beneficial by itself. We are currently testing whether XBP1 inhibition could result in an enhanced tumor suppression using OVA antigen stimulated system, as XBP1 inhibition in tumor cells was shown to have tumor-repressing roles via immune system. Citation Format: Yanqing (Christine) Jiang, Xu Han, Nathan Coffey, Mai Wang, John Tobias, Raymond Ng, Carson Poltorack, M. Celeste Simon. Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B054.

Abstract B070: Histone deacetylation inhibition promotes bi-directional subtype switch in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with 5-year survival of just 11%. Recent large-scale transcriptomic studies have identified two prognostically relevant neoplastic tumor subtypes, i.e. the well-differentiated classical (CLA) subtype with better prognosis, characterized by epithelial gene expression, as well as the dedifferentiated, invasive basal-like (BL) subtype, which displays mesenchymal stemness-related gene signatures. These subtypes have shown to be highly plastic and responsive to extrinsic signals, e.g. from the microenvironment or treatment-induced. Here, we investigate master transcription factor networks governing lineage determination in the different subtypes and the role of histone deacetylation (HDAC) co-regulators in their maintenance. We utilized established, subtype-defined PDAC cell lines in vitro and in orthotopic transplantation models to determine subtype plasticity in response to HDAC inhibition by the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). By integration of RNA-seq, ATAC-seq as well as HiChIP for the activating mark histone 3 lysine 27 acetylation (H3K27ac), we show that the subtype-dependent chromatin organization and regulatory networks can be influenced by pharmacological inhibition of chromatin remodelers, in turn promoting changes in transcription of lineage-determining factors. Targeting these transcription factor networks by HDAC inhibition can lead both to a favorable switch from BL to CLA identity, as well as a detrimental CLA to BL switch. Functional assay such as trans-well invasion further supported the changes in subtype identity following SAHA treatment. This bi-directional subtype switch was additionally validated in patient-derived xenograft cell lines. Together, our findings highlight the importance of understanding the underlying transcriptional and epigenetic mechanisms of the pancreatic cancer subtypes, as they may exhibit opposing responses to targeted therapies. Citation Format: Lukas Klein, Mengyu Tu, Eva K. Schmitt, Frederike Penz, Athanasia Mizi, Stefan Küffer, Elisabeth Hessmann, Argyris Papantonis, Volker Ellenrieder, Shiv K. Singh. Histone deacetylation inhibition promotes bi-directional subtype switch in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B070.

Abstract A078: Response and resistance to KRAS inhibition in PDAC mouse models

Abstract Pancreatic ductal adenocarcinoma (PDAC), among the most lethal cancers with a 5-year survival rate of 11%, is a KRAS-driven disease. Studies have shown that a single glycine (G) to aspartic acid (D) substitution on codon 12 of KRAS protein (known as KRASG12D mutation) appears in 41% of all PDAC cases, highlighting the necessity of targeting KRASG12D as a therapeutic approach. MRTX1133, a recently developed potent, non-covalent, direct, and selective KRASG12D small molecule inhibitor demonstrates a high-affinity interaction with KRASG12D. Previous studies have shown striking efficacy of MRTX1133 on growth inhibition of mouse/human PDAC tumor cells carrying KRASG12D mutation in various in vivo and in vitro models, during short-term treatment. Here in our study, we investigated the efficacy of continuous long-term treatment with MRTX1133 in two different PDAC mouse models: KPC (a genetically engineered mouse model harboring a KRASG12D mutation) and its syngeneic orthotopic model. In both models, tumor sizes were detected by ultrasound imaging and after reaching 100-300 mm3 in volume, mice were enrolled in either MRTX1133 or vehicle treatment (30mg/kg, IP, BID). Tumor sizes were monitored by ultrasound regularly and the treatment continued until mice reached clinical endpoint. At endpoint, mice were sacrificed, and tumors were harvested for a series of downstream experiments. Our results showed that while, in both models, vehicle treated tumors grew aggressively and mice reached endpoint in average 6 days post treatment, drug treated tumors regressed rapidly and survival rate increased significantly to as late as 222 days post treatment in KPC model. However, in both models, drug treated tumors ultimately relapsed, developed resistance to the drug, and resulted in death. Further analysis of KPC mice ultrasound images evidenced that the relapsed tumors occurred at the same sites of the primary tumors. This suggests the possibility of relapsed tumors arising from the regrowth of the dormant residual cells from the primary tumors. Re-implantation of the resistant tumors either in the flank or pancreata of the new wildtype mice resulted in the formation of drug-resistant tumors in hosts. In addition, histopathology analysis of the tumor sections demonstrated a significant increase in the ratio of invasive PDAC to PanIN areas in the drug resistant tumors. Furthermore, multiplex immunofluorescence of both MRTX1133 resistant and vehicle treated tumors from KPC mice demonstrated co-expression of markers representing basal-like and classical PDAC subtypes. Moreover, flow cytometry analysis indicated an increase in the myeloid population of the resistant tumors comparing to the vehicle treated group. In the future directions, through applying a broad range of cellular and molecular experiments we will characterize the kinetics of KRAS inhibitor drug resistance and define possible combination therapy startegies to overcome drug resistance mechanisms. Citation Format: Laleh Abbassi, Jullien Dilly, Annan Yang, Giselle Uribe, Branden Parent, Connor Hennessey, Kevin Kapner, Alexander Jordan, Shatavisha Dasgupta, Micaela Morgado, Taimour Baslan, Li Qiang, Peter Winter, Seema Chugh, Alex Shalek, Ben Stanger, Srivastan Raghavan, Scott Lowe, Brian Wolpin, Stephanie Dougan, Jonathan Nowak, Andrew Aguirre. Response and resistance to KRAS inhibition in PDAC mouse models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A078.

Abstract A118: Chromatin accessibility profiling of human pancreatic tumors reveals epigenetic features of malignancy and rapid recurrence

Abstract Pancreatic ductal adenocarcinoma (PDAC) disease progression involves complex cell state transitions in both malignant and non-malignant populations within the tumor microenvironment. To uncover PDAC cell states and epigenetic features associated with clinical outcome, we studied 42 PDAC samples (35 primary tumors and 7 metastases) from 41 patients with single cell ATAC-seq, along with 10 sample-matched single cell RNA-seq profiles. Our work uncovered thousands of chromatin regions with altered accessibility in malignant cells relative to normal ductal and acinar cells. Numerous epigenetic changes were universal to all PDAC clusters regardless of patient-of-origin and occurred proximal to genes involved in glycolysis, regulation of MYC, hypoxic response, extracellular matrix remodeling, and repression of transposable elements. Topic modeling of high dimensional chromatin data revealed networks of co-accessible cis-regulatory regions active in malignant cells, as well as networks specific to basal-like and classical PDAC molecular subtypes. Furthermore, our epigenomic data also suggest alternate paths of lineage infidelity across the classical-basal continuum: while basal-like PDAC cells acquire chromatin accessibility mimicking that of skin or mammary epithelial tissue, several classical subtype tumors featured a subpopulation of malignant cells with increased accessibility of neural-associated chromatin regions genome-wide. Within the immune compartment, we also identified a cis-regulatory network with increased activity in T-cells sampled from rapidly recurring PDAC tumors. Motif analysis of this network revealed enrichment for SMAD and FOXP1 motifs, suggesting widespread modification of T-cells by TGF-β. To our knowledge, our work is the largest single-cell resolution study of chromatin state in human PDAC to date, revealing key epigenetic features associated with PDAC subtype and clinical outcome. Citation Format: Kevin MacPherson, Jason M. Link, Patrick Worth, Brett Sheppard, Andrew Fields, Colin Daniel, Andrew Nishida, Carl Pelz, Trent Waugh, Ethan Agritelle, John Muschler, Andrew Adey, Rosalie Sears. Chromatin accessibility profiling of human pancreatic tumors reveals epigenetic features of malignancy and rapid recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A118.

Abstract C109: Nanostring-based subtyping of pancreatic ductal adenocarcinoma is strongly influenced by the stromal compartment

Abstract The prognostic significance of basal-like and classical subtypes of pancreatic ductal adenocarcinoma (PDAC) is well-established, and ongoing clinical trials are poised to validate the predictive utility of such subtypes in guiding first-line treatment decision-making. While the Purity-independent (PurIST)-based subtyping strategy provides a method for PDAC subtype determination based on the Nanostring gene expression quantification platform, the utility of this platform has not been tested in a real-world patient setting. Here, we leverage a retrospective dataset of patients with resectable PDAC (n=299) that received Nanostring sequencing in combination with comprehensive clinical metadata curation and histopathological analysis. All sequencing analyses were performed using formalin-fixed paraffin-embedded (FFPE) tumor whole sections. The proportion of basal-like cases (15%) was comparable with other resectable PDAC cohorts, and patients with basal-like tumors showed reduced overall survival (p=3.9e-4). Histopathological quantification of epithelial, stromal and normal components in each sample revealed significantly (p=0.007) reduced classical gene expression in stroma-high samples. Out of 29 patients with repeat sampling from different FFPE blocks derived from the same tumor and surgery, 31% of tumors received discordant subtypes calls and increases in the amount of stroma sequenced between two samples from the same tumor was negatively correlated with expression of classical subtype genes (p=0.02). Taken together, these data demonstrate the presence of intra-tumoral subtype heterogeneity, highlight the quantity of stroma sequenced as a major contributing factor in PurIST-based subtype determination and generate insight into the reproducibility of this subtyping strategy in a real-world clinical setting. Citation Format: James T. Topham, Steve E. Kalloger, Joanna M. Karasinska, Jenny E. Chu, Hassan Ali, Dongxia Gao, Christine Chow, Andrew Metcalfe, Jonathan M. Loree, David F. Schaeffer, Daniel J. Renouf. Nanostring-based subtyping of pancreatic ductal adenocarcinoma is strongly influenced by the stromal compartment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C109.

Abstract A109: Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis

Abstract Introduction The major driver for pancreatic ductal adenocarcinoma (PDAC) is oncogenic KRAS. However, adult acinar cells, a probable origin of PDAC, are largely refractory to KrasG12D-mediated oncogenic transformation. With the concomitant loss of transcription factors that regulate acinar cell differentiation, such as Pdx1 (Pancreatic and Duodenal Homeobox 1), acinar cells undergo a rapid cell identity switch, known as acinar-to-ductal metaplasia (ADM). How loss of cell identity cooperates with oncogenic Kras to induce pancreatic transformation is largely unclear. Methods To elucidate mechanisms responsible for the accelerated cellular reprogramming in KrasG12D;Pdx1f/f animals, single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) from frozen pancreatic bulk tissue was performed. Chromatin accessibility states were captured at early stages of carcinogenesis and correlated to RNA-seq data. Differentially regulated genes were validated by multiplex RNAscope and immunohistochemistry staining and functionally studied in pancreatic cancer cell lines. Results Single-cell ATAC-seq proved a powerful tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. Notably, acinar cells of KrasG12D;Pdx1f/f animals as well as a proportion of metaplastic lesions in both, KrasG12D and KrasG12D;Pdx1f/f mice, showed elevated accessibility and expression of the Ror2 (Receptor Tyrosine Kinase Like Orphan Receptor 2) gene. As a receptor tyrosine kinase, Ror2 controls noncanonical Wnt signaling and other essential signaling pathways, such as PI3K/AKT or Ras-MAPK. Genetic ablation of Ror2 in a mouse model of pancreatic neoplasia resulted in a shift in ADM cell identity, enriching ADM lesions with a senescent duct cell phenotype. In PDAC, ROR2 expression correlates with the more aggressive basal-like subtype. Overexpression of ROR2 in pancreatic cancer cell lines with a classical differentiation induced epithelial-to-mesenchymal transition, characterized by the downregulation of multiple epithelial markers and upregulation of mesenchymal genes. Knockout of ROR2 in pancreatic cancer cells significantly decreased cell proliferation. Conclusions Our in-depth sequencing data revealed that expression of KrasG12D with the concomitant loss of Pdx1 leads to vast alterations of acinar cell identity. We identified the receptor kinase Ror2 as a regulator of pancreatic cancer initiation and driver of pancreatic cancer cell aggressiveness. Citation Format: Simone Benitz, Malak Nasser, Alexander Steep, Jonathan Preall, Ujjwal Mahajan, Ian Loveless, Erick Davis, Hui-Ju Wen, Daniel Long, Michaela Louw, Samuel Zwernik, Donald Rempinski, Jacee Moore, Daniel Salas-Escabillas, Thomas Metzler, Ling Huang, Nina Steele, Ivonne Regel, Filip Bednar, Howard Crawford. Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A109.

Pathologic and immunohistochemical prognostic markers in residual triple-negative breast cancer after neoadjuvant chemotherapy.

The persistence of residual tumour after neoadjuvant chemotherapy (NAC) in localised triple-negative breast cancer (TNBC) is known to have a negative prognostic value. However, different degrees of expression of some immunohistochemical markers may correlate with different prognoses. The expression of biomarkers with a known prognostic value, i.e., cytokeratin 5/6 (CK5/6), androgen receptor (AR), epidermal growth factor receptor (EGFR) proliferation-related nuclear antigen Ki-67, human epidermal growth factor receptor 2 (HER2), protein 53 (p53), forkhead box protein 3 (FOXP3), and cluster differentiation 8 (CD8), was analysed by immunohistochemistry in 111 samples after NAC in non-metastatic TNBC patients addressed to Georges-François Leclerc Cancer Centre Dijon, France. Clinical and pathological variables were retrospectively collected. Cox regression was used to identify immunohistochemical (IHC) and clinicopathological predictors of event-free survival (EFS) (relapse or death). Median age was 50.4 years (range 25.6-88.3), 55.9% (n = 62) were non-menopausal, 70 (63.1%) had stage IIA-IIB disease. NAC was mostly sequential anthracycline-taxanes (72.1%), and surgical intervention was principally conservative (51.3%). We found 65.7% ypT1, 47.2% lymph node involvement (ypN+), and 29.4% lymphovascular invasion (LVI). Most residual tumours were EGFR >110 (H-score) (60.5%, n = 66), AR ≥4% (53.2%, n = 58), p53-positive mutated (52.7%, n = 58), CD8 ≥26 (58.1%, n = 61), FOXP3 ≥7 (51.4%, n = 54), more than half in the stroma, and 52.3% (n = 58) HER2 score 0. After a median follow-up of 80.8 months, 48.6% had relapsed. Median EFS was 62.3 months (95% CI, 37.2-not reached (NR)). Factors independently associated with poor EFS were AR-low (p = 0.002), ypN+ (p < 0.001), and LVI (p = 0.001). Factors associated with lower overall survival (OS) were EGFR-low (p = 0.041), Ki-67 high (p = 0.024), and ypN+ (p < 0.001). Post-NAC residual disease in TNBC showed biomarkers specific to a basal-like subtype and markers of lymphocyte infiltration mostly present in the stroma. Prognostic markers for EFS were AR, LVI, and ypN and warrant further validation in a prognostic model.

The Accuracy of Breast Cancer Diagnostic Tools.

Background Breast cancer (BC)remains a significant health concern,leadingtoillnessanddeathamong womenglobally. Itisessential to detectBCearly usingimagingtechniquesthataccurately reflectthe final pathology, guiding suitable intervention strategies. Objectives This study aimed toevaluatetheagreementbetween radiological findings and histopathological resultsinBCcases. Methods We conducted aretrospective reviewofbreast core needle biopsies (CNBs)inwomenover a six-year period (2017-2022) at Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia. The pathologicaldiagnoses were compared with thefindingsfrompreceding radiological investigations.We also compared the tumour sizes inthe resection specimenswiththeir radiologicalcounterparts. Results A total of 641 cases were includedin the study.Ultrasound (US),mammography,andmagnetic resonance imaging (MRI)yieldeddiagnostic accuracies of 85%, 77.9%, and 86.9%,respectively. MRIhadthe highest sensitivityat72.2%, while US hadthe lowestat61%. MRI provided the best agreementwiththefinalresected tumor size. By contrast,mammographytended to overestimate the size (41.9%), and US most frequently underestimated it(67.7%).The connectionbetween basal-like molecular subtypes and the Breast Imaging Reporting and Data System (BIRADS)-5 classifications wasonlystatistically significantfor MRI (p=0.04). The luminal subtypewas more likely to showspeculation in mammography.Meanwhile,BIRADS-4revealedaconsiderablenumber of benign pathologiesacrossall the threemodalities. Conclusions MRIdemonstrated thehighestaccuracy,sensitivity, specificity,andpositive predictive value (PPV) fordiagnosingandestimating the tumorsize.Mammographyoutperformed US in terms of sensitivityandyielded thehighestnegative predictive value (NPV). US, meanwhile, offered superiorspecificity, PPV,and accuracy. Therefore, combiningthese diagnostic methods could yield significant benefits.

S100A8/A9 as a risk factor for breast cancer negatively regulated by DACH1

BackgroundS100A8 and S100A9 are members of Ca2+-binding EF-hand superfamily, mainly expressed by macrophages and neutrophils. Limited by the poor stability of homodimers, they commonly exist as heterodimers. Beyond acting as antibacterial cytokines, S100A8/A9 is also associated with metabolic and autoimmune diseases such as obesity, diabetes, and rheumatoid arthritis. While the involvement of S100A8/A9 in breast cancer development has been documented, its prognostic significance and the precise regulatory mechanisms remain unclear.MethodsS100A8/A9 protein in breast cancer samples was evaluated by immunohistochemistry staining with tumor tissue microarrays. The serum S100A8 concentration in patients was measured by enzyme-linked immunosorbent assay (ELISA). The S100A8 secreted by breast cancer cells was detected by ELISA as well. Pooled analyses were conducted to explore the relationships between S100A8/A9 mRNA level and clinicopathological features of breast cancer patients. Besides, the effects of S100A8/A9 and DACH1 on patient outcomes were analyzed by tissue assays. Finally, xenograft tumor assays were adopted to validate the effects of DACH1 on tumor growth and S100A8/A9 expression.ResultsThe level of S100A8/A9 was higher in breast cancer, relative to normal tissue. Increased S100A8/A9 was related to poor differentiation grade, loss of hormone receptors, and Her2 positive. Moreover, elevated S100A8/A9 predicted a worse prognosis for breast cancer patients. Meanwhile, serum S100A8 concentration was upregulated in Grade 3, basal-like, and Her2-overexpressed subtypes. Additionally, the results of public databases showed S100A8/A9 mRNA level was negatively correlated to DACH1. Stable overexpressing DACH1 in breast cancer cells significantly decreased the generation of S100A8. The survival analysis demonstrated that patients with high S100A8/A9 and low DACH1 achieved the shortest overall survival. The xenograft models indicated that DACH1 expression significantly retarded tumor growth and downregulated S100A8/A9 protein abundance.ConclusionS100A8/A9 is remarkedly increased in basal-like and Her2-overexpressed subtypes, predicting poor prognosis of breast cancer patients. Tumor suppressor DACH1 inhibits S100A8/A9 expression. The combination of S100A8/A9 and DACH1 predicted the overall survival of breast cancer patients more preciously.

Single-cell transcriptome profiling of primary tumors and paired organoids of pancreatobiliary cancer

Single-cell RNA-seq (scRNA-seq) and cancer organoid model have shown promise in investigating tumor microenvironment heterogeneity and facilitating chemotherapeutic drug testing to inform treatment selection. It is still unknown whether the scRNA-seq results based on organoid can faithfully reflect the heterogeneity of primary pancreatobiliary cancer. To reveal the similarities and differences between primary tumors and their matched organoids at transcriptome level, we conducted scRNA-seq for paired primary tumors and organoids from one cholangiocarcinoma (CCA) and two pancreatic ductal adenocarcinoma (PDAC) patients. We identified inter-patient and intra-tumor heterogeneity and found that the organoids retained copy number variation (CNV) patterns of primary tumors. There was no significant difference in cancer stem cell (CSC) properties between the primary tumors and the organoids, whereas organoid from one PDAC case had increased mesenchymal-score and decreased epithelial-score compared with the primary tumors. All organoids showed a transition tendency from the classical subtype to the basal-like subtype in the transcriptional level. Organoids and primary tumors differed in metabolic and unfolded protein response (UPR) signatures. In addition, we revealed the heterogeneity of cancer associated fibroblasts (CAFs) and T cells, and explored the developmental trajectory of T cells. Our findings facilitate further understanding of organoid model and confirm its application prospects in pancreatobiliary cancer.

Overexpression of MTHFD2 represents an inflamed tumor microenvironment and precisely predicts the molecular subtype and immunotherapy response of bladder cancer.

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), whose aberrant expression is common in cancers, has recently been identified as a potential regulator of immune response. However, its immune-related role in bladder cancer (BLCA) and its association with immunotherapy efficacy remain unclear. RNA sequencing data from The Cancer Genome Atlas (TCGA) was applied to analyze the immunological roles and prognostic value of MTHFD2 in pan-cancers. The association of MTHFD2 with several immunological features of tumor microenvironment (TME), including cancer-immunity cycle, immune cells infiltration, immune checkpoints expression, and T cell inflamed score was analyzed in TCGA-BLCA cohort. The predictors of cancer treatments effectiveness, including the expression and mutation of certain genes, molecular subtypes, and several signatures were evaluated as well. These results were validated by another independent cohort (GSE48075). Finally, the predictive value of MTHFD2 for TME and immunotherapy efficacy were validated using immunohistochemistry assay and RNA sequencing data from IMvigor210 cohort, respectively. MTHFD2 was found to be positively associated with several immunological features of an inflamed tumor microenvironment (TME) in various cancers and could predict BLCA patients' prognosis. In BLCA, high expression of MTHFD2 was observed to be positively related with the cancer-immunity cycle, the infiltration of several immune cells, and the expression of immunoregulators and T-cell inflamed scores, indicating a positive correlation with the inflamed TME. Moreover, patients with high MTHFD2 expression were more likely to be basal-like subtypes and respond to BLCA treatments, including immunotherapy, chemotherapy, and target therapy. The clinical data of the IMvigor210 cohort confirmed the higher response rates and better survival benefits of immunotherapy in high-MTHFD2-expression patients. Collectively, high MTHFD2 predicts an inflamed TME, a basal-like subtype, and a better response to various therapeutic strategies, especially the ICB therapy, in bladder cancer.

A ten long noncoding RNA-based prognostic risk model construction and mechanism study in the basal-like immune-suppressed subtype of triple-negative breast cancer.

According to the Fudan University Shanghai Cancer Center (FUSCC) system, triple-negative breast cancer (TNBC) is divided into four stable subtypes: (I) luminal androgen receptor, (II) immunomodulatory, (III) basal-like immune-suppressed (BLIS), and (IV) mesenchymal-like. However, the treatment outcomes of the corresponding targeted therapies are unsatisfactory, especially for the BLIS subtype. Therefore, we aimed to identify the key long noncoding RNAs (lncRNAs) to construct a prognostic model for BLIS subtype and discover potential targets to explore potential therapeutic strategies in this study. The FUSCC cohort was used to establish a prognostic risk model via least absolute shrinkage and selection operator (LASSO) and Cox regression analysis. The Cancer Genome Atlas (TCGA) cohort was then used to evaluate and verify the model. To understand the functional aspects of the model, functional, immune landscape, mutation, and drug sensitivity analyses were performed between high- and low-risk groups. Ten prognostic-related lncRNAs identified, including C5ORF66-AS2, DIO3OS, FZD10-DT, LINC00393, LNC-ERI1-32, LNC-FOXO1-2, LNC-SPARCL1-1, HCG23, LNC-MMD-4 and LNC-TMEM106C-6, were selected for risk score system construction. The results showed that the model constructed could divide the patients with BLIS subtype into two groups of high and low risk, and patients with higher risk scores had shorter recurrence-free survival. In addition, drug sensitivity analysis identified 3 compounds, including BMS-754807, cytochalasin b, and linifanib, that could have a potential therapeutic effect on patients with the BLIS subtype. The risk prognosis model showed good prognostic value for the BLIS subtype patients, and the ten lncRNAs may be potential therapeutic targets.

Abstract C021: Clinicopathological features and basal-like phenotype expression in triple negative breast cancer at Uyo, Niger-Delta region of Nigeria

Abstract (A) AN INTRODUCTORY SENTENCE INDICATING THE PURPOSES OF THE STUDY: Patients of African Ancestry (AA) experience higher rates of TNBC and mortality than European Ancestry (EA) patients, despite lower overall breast cancer (BC) incidence. BC is the most frequently diagnosed cancer in Nigeria and USA as well as being the second most common cause of cancer-related death in women, although there have been significant advances in treatment strategies over the past few decades. In addition to conventional therapies, in the molecular era, breast cancer patients often receive targeted treatments based on their unique cancer biology in both adjuvant and neoadjuvant settings. There is limited data on the prevalence of Basal-like breast carcinomas in Nigeria owing to the high cost of gene expression profiling which is the gold standard for molecular classification and immunohistochemistry (which can be used as a surrogate). This study aimed to analyze the clinico-pathological features and immunohistochemical expression of basal-like phenotype in TNBC at Uyo, as well as stratifying of invasive breast carcinomas into main molecular subtypes and correlating the Basal-like breast carcinoma subtype with the size of tumour, grade and age of patient. (B) A BRIEF DESCRIPTION OF PERTINENT EXPERIMENTAL PROCEDURES: One hundred and fifteen formalin fixed, paraffin embedded blocks of Invasive breast carcinomas were assembled, stained and analysed for ER, PR, HER-2, Ki67, CK5/6 and EGFR proteins using immunohistochemistry. Stratification of invasive breast carcinomas into four (4) main molecular groups (Luminal, HER-2, Basal-like and normal breast subtypes) was done. Patient’s record and immunohistochemical results were reviewed. The data was analyzed and their results were presented as tables, graphs and photomicrographs. (C) A SUMMARY OF THE NEW, UNPUBLISHED DATA: The mean age of the TNBC patients was 39.0 years (range: 25–74 years). Invasive carcinoma (no special type) was the predominant histologic subtype (90.4%). Out of the 115 cases, ER, PR and HER-2 were positive in 53.0 %, 33.0 % and 12.2 % of cases respectively. CK5/6 and EGFR were also positive in 16.5% and 11.3 % of cases respectively. Ki67 was high (&amp;gt;14 %) in 26.1 % of cases. Our study illustrated that majority of TNBC expressed basal-like markers (51.3 %) and occurred at the 35-44 years age group (33.3%) with a substantial proportion being grade 3 (poorly differentiated) carcinomas (48.7%). There was a significant association between basal-like breast carcinoma and tumour grade.(D) CONCLUSION: Our study illustrated that majority of TNBC expressed basal - like markers and occurred at 35-44 years age group with a substantial proportion being grade 3 (poorly differentiated) carcinomas. There was a positive correlation between basal-like breast carcinoma and tumour grade. It is hoped that research in unraveling biology of basal-like carcinomas among patients of African Ancestry (AA) and Caucasian Ancestry (CA) will be carried out in the future. Citation Format: Emmanuel Kunle Abudu, Obioha Kanu, Ikwo Jonathan Kudamnya, Emem Imo Akpanudo, Oluwasayo Omolara Abudu. Clinicopathological features and basal-like phenotype expression in triple negative breast cancer at Uyo, Niger-Delta region of Nigeria [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 C021.

Association of clinicopathologic and molecular factors with the occurrence of positive margins in breast cancer

PurposeTo explore the association of clinicopathologic and molecular factors with the occurrence of positive margins after first surgery in breast cancer.MethodsThe clinical and RNA-Seq data for 951 (75 positive and 876 negative margins) primary breast cancer patients from The Cancer Genome Atlas (TCGA) were used. The role of each clinicopathologic factor for margin prediction and also their impact on survival were evaluated using logistic regression, Fisher’s exact test, and Cox proportional hazards regression models. In addition, differential expression analysis on a matched dataset (71 positive and 71 negative margins) was performed using Deseq2 and LASSO regression.ResultsAssociation studies showed that higher stage, larger tumor size (T), positive lymph nodes (N), and presence of distant metastasis (M) significantly contributed (p ≤ 0.05) to positive surgical margins. In case of surgery, lumpectomy was significantly associated with positive margin compared to mastectomy. Moreover, PAM50 Luminal A subtype had higher chance of positive margin resection compared to Basal-like subtype. Survival models demonstrated that positive margin status along with higher stage, higher TNM, and negative hormone receptor status was significant for disease progression. We also found that margin status might be a surrogate of tumor stage. In addition, 29 genes that could be potential positive margin predictors and 8 pathways were identified from molecular data analysis.ConclusionThe occurrence of positive margins after surgery was associated with various clinical factors, similar to the findings reported in earlier studies. In addition, we found that the PAM50 intrinsic subtype Luminal A has more chance of obtaining positive margins compared to Basal type. As the first effort to pursue molecular understanding of the margin status, a gene panel of 29 genes including 17 protein-coding genes was also identified for potential prediction of the margin status which needs to be validated using a larger sample set.

Abstract C012: Integration of social variables resolves subclasses of tumor microenvironment

Abstract Intro: Recent work has identified subclasses of the tumor microenvironment, with most work emphasizing a robust adaptive immune response associated with ER-negative and/or basal-like tumors. Since adaptive immune responses may influence response to chemotherapy and may be targetable by immunotherapy, understanding population-level heterogeneity in immune response is important. Methods: We leveraged the Carolina Breast Cancer Study (CBCS), a large population-based study that oversampled young (≤50 years) and Black women with invasive breast cancer (BC). We performed NanoString gene expression profiling on tissue from 1,329 BC patients, including 704 (53%) Black and 625 (47%) non-Black women, and assayed genes representative of at least 10 individual immune cell types from both adaptive and innate arms of the immune system, homologous recombination deficiency signature and TP53 mutation status. Using a latent class approach that included variables for self-reported race, triple negative status, individual measures of socioeconomic status and community-level measures of advantage and disadvantage, we identified latent classes that integrated tumor microenvironment with tumor biology, individual and community level exposures under a Cells-to-Society conceptual model of breast cancer outcomes. Results: Latent Class Analysis identified 6 integrated classes of the tumor microenvironment, further subdividing a previously identified and validated adaptive immune class into two subgroups. The two adaptive classes were distinguished by different expression levels of B-cells, CD8+ T cells, cytotoxic cells and PD-L1, and differing frequency of basal-like subtype (Adaptive Class 1 RFD [95% CI]: 19.0 [11.9, 26.2]; Adaptive Class 2 RFD [95% CI]: 32.8 [23.3,42.3]) and presence of genomic instability gene expression subtypes (Adaptive Class-1 RFD [95% CI]: 22.1 [14.0, 30.1]; Adaptive Class-2 RFD [95% CI]: 37.5 [28.1,46.2]) relative to the immune-quiet tumors. Discussion: Social variables have complex effects on tumor biology and microenvironment, but latent class models and other model-based approaches may help to integrate biological data with other social factors to identify susceptible subgroups and to optimize precision medicine in diverse populations. Citation Format: Alina M. Hamilton, Kara McCormack, Marc A. Emerson, Melissa A. Troester, Terry Hyslop. Integration of social variables resolves subclasses of tumor microenvironment [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 C012.

Exploring ceRNA networks for key biomarkers in breast cancer subtypes and immune regulation

Breast cancer is a major global health concern, and recent researches have highlighted the critical roles of non-coding RNAs in both cancer and the immune system. The competing endogenous RNA hypothesis suggests that various types of RNA, including coding and non-coding RNAs, compete for microRNA targets, acting as molecular sponges. This study introduces the Pre_CLM_BCS pipeline to investigate the potential of long non-coding RNAs and circular RNAs as biomarkers in breast cancer subtypes. The pipeline identifies specific modules within each subtype that contain at least one long non-coding RNA or circular RNA exhibiting significantly distinct expression patterns when compared to other subtypes. The results reveal potential biomarker genes for each subtype, such as circ_001845, circ_001124, circ_003925, circ_000736, and circ_003996 for the basal-like subtype, circ_00306 and circ_00128 for the luminal B subtype, circ_000709 and NPHS1 for the normal-like subtype, CAMKV and circ_001855 for the luminal A subtype, and circ_00128 and circ_00173 for the HER2+ subtype. Additionally, certain long non-coding RNAs and circular RNAs, including RGS5-AS1, C6orf223, HHLA3-AS1, circ_000349, circ_003996, circ_003925, circ_002665, circ_001855, and DLEU1, are identified as potential regulators of T cell mechanisms, underscoring their importance in understanding breast cancer progression in various subtypes. This pipeline provides valuable insights into cancer and immune-related processes in breast cancer subtypes.

Biological processes associated with breast cancer subtypes: A meta-analysis study

This study delves into the modular mechanisms underlying various breast cancer subtypes, including Basal-like, HER2, Luminal A, Luminal B, Normal-like, and the differences between Luminal A and B. Through microarrays meta-analyses, the research identifies potential biomarkers for these subtypes by comparing each with the normal state, revealing 408, 429, 531, 346, 113, and 1085 differentially expressed genes associated with Basal-like, HER2, Luminal A, Luminal B, Normal-like, and Luminal A vs Luminal B, respectively. Significant enrichment of top GO terms like 'nuclear-transcribed mRNA catabolic process nonsense-mediated decay', 'SRP-dependent cotranslational protein targeting to membrane', 'translational initiation', 'rRNA processing', and 'viral transcription and response to corticosteroid' was observed in different breast cancer subtypes. Specifically, in the comparison between Luminal A and B cancers, 'tumor necrosis factor-mediated signaling' was the most enriched pathway. The most differentially expressed genes in this comparison were 'TOP2A, AURKA, RRM2, CDK1, and MDA2L1' (up-regulated), and 'LTF and MYBPC1' (down-regulated). These insights could be pivotal in developing new clinical-genomic models and identifying novel therapeutic strategies for specific molecular subgroups of breast cancer. The present study aims to investigate the modular mechanisms underlying different breast cancer subtypes and identifies potential biomarkers for Basal-like subtype (Normal vs Basal-like), HER2 subtype (Normal vs HER2), Luminal A subtype (Normal vs Luminal A), Luminal B subtype (Normal vs Luminal B), Normal-like subtype (Normal vs Normal-like) and between Luminal A and B (Luminal A vs Luminal B) using microarrays meta-analyses. 408, 429, 531, 346, 113, and 1085 differentially expressed genes were associated with Basal-like, HER2, Luminal A, Luminal B, Normal-like subtypes, and ‘Luminal A vs Luminal B’, respectively. Top GO terms significantly enriched for different breast cancer subtypes include ‘nuclear-transcribed mRNA catabolic process nonsense-mediated decay’, ‘SRP-dependent cotranslational protein targeting to membrane’, ‘translational initiation’, ‘rRNA processing’, and ‘viral transcription and response to corticosteroid’. The comparison between Luminal A and B cancers found that ‘tumor necrosis factor-mediated signaling’ was the most enriched pathway and the most differentially expressed genes included ‘sTOP2A, AURKA, RRM2, CDK1 and MDA2L1 (up-regulated)’ and ‘LTF and MYBPC1 (down-regulated)’. These findings may contribute to defining new clinical-genomic models and identifying new therapeutic strategies in the specific molecular subgroups.

MicroRNAs as Molecular Biomarkers for the Characterization of Basal-like Breast Tumor Subtype.

Breast cancer is a heterogeneous disease highlighted by the presence of multiple tumor variants and the basal-like breast cancer (BLBC) is considered to be the most aggressive variant with limited therapeutics and a poor prognosis. Though the absence of detectable protein and hormonal receptors as biomarkers hinders early detection, the integration of genomic and transcriptomic profiling led to the identification of additional variants in BLBC. The high-throughput analysis of tissue-specific micro-ribonucleic acids (microRNAs/miRNAs) that are deemed to have a significant role in the development of breast cancer also displayed distinct expression profiles in each subtype of breast cancer and thus emerged to be a robust approach for the precise characterization of the BLBC subtypes. The classification schematic of breast cancer is still a fluid entity that continues to evolve alongside technological advancement, and the transcriptomic profiling of tissue-specific microRNAs is projected to aid in the substratification and diagnosis of the BLBC tumor subtype. In this review, we summarize the current knowledge on breast tumor classification, aim to collect comprehensive evidence based on the microRNA expression profiles, and explore their potential as prospective biomarkers of BLBC.

Intrinsic Subtype and Overall Survival of Patients with Advanced HR+/HER2- Breast Cancer Treated with Ribociclib and ET: Correlative Analysis of MONALEESA-2, -3, -7.

The MONALEESA-2, -3, -7 trials demonstrated statistically significant and clinically meaningful progression-free survival and overall survival (OS) benefits with ribociclib plus endocrine therapy (ET) versus ET alone in hormone receptor-positive, HER2-negative (HR+/HER2-) advanced breast cancer (ABC). Understanding the association of intrinsic subtypes with survival outcomes could potentially guide treatment decisions. Here, we evaluated the association of intrinsic subtypes with OS in MONALEESA-2, -3, -7. Tumor samples from MONALEESA-2, -3, -7 underwent PAM50-based subtyping. The relationship between subtypes and OS was assessed using univariable and multivariable Cox proportional hazards models. Multivariable models were adjusted for clinical prognostic factors. Overall, 990 tumors (among 2,066 patients) from ribociclib (n = 580) and placebo (n = 410) arms were profiled. Subtype distribution was luminal A, 54.5%; luminal B, 28.0%; HER2-enriched (HER2E) 14.6%; and basal-like, 2.8%; and was consistent across treatment arms. The luminal A subtype had the best OS outcomes in both arms, while basal-like had the worst. Patients with HER2E (HR, 0.60; P = 0.018), luminal B (HR, 0.69; P = 0.023), and luminal A (HR, 0.75; P = 0.021) subtypes derived OS benefit with ribociclib. Patients with basal-like subtype did not derive benefit from ribociclib (HR, 1.92; P = 0.137); however, patient numbers were small (n = 28). The prognostic value of intrinsic subtypes for OS was confirmed in this pooled analysis of the MONALEESA trials (largest dataset in HR+/HER2- ABC). While basal-like subtype did not benefit, a consistent OS benefit was observed with ribociclib added to ET across luminal and HER2E subtypes.

A basal-like pancreatic cancer molecular subtype can be identified on EUS-acquired tissue and is associated to current smoking, lower Ca19.9 expression and worse prognosis

A basal-like pancreatic cancer molecular subtype can be identified on EUS-acquired tissue and is associated to current smoking, lower Ca19.9 expression and worse prognosis