Distinct Molecular Features Research Articles

ELAVL2 loss promotes aggressive mesenchymal transition in glioblastoma

Glioblastoma (GBM), the most lethal primary brain cancer, exhibits intratumoral heterogeneity and molecular plasticity, posing challenges for effective treatment. Despite this, the regulatory mechanisms underlying such plasticity, particularly mesenchymal (MES) transition, remain poorly understood. In this study, we elucidate the role of the RNA-binding protein ELAVL2 in regulating aggressive MES transformation in GBM. We found that ELAVL2 is most frequently deleted in GBM compared to other cancers and associated with distinct clinical and molecular features. Transcriptomic analysis revealed that ELAVL2-mediated alterations correspond to specific GBM subtype signatures. Notably, ELAVL2 expression negatively correlated with epithelial-to-mesenchymal transition (EMT)-related genes, and its loss promoted MES process and chemo-resistance in GBM cells, whereas ELAVL2 overexpression exerted the opposite effect. Further investigation via tissue microarray analysis demonstrated that high ELAVL2 protein expression confers a favorable survival outcome in GBM patients. Mechanistically, ELAVL2 was shown to directly bind to the transcripts of EMT-inhibitory molecules, SH3GL3 and DNM3, modulating their mRNA stability, potentially through an m6A-dependent mechanism. In summary, our findings identify ELAVL2 as a critical tumor suppressor and mRNA stabilizer that regulates MES transition in GBM, underscoring its role in transcriptomic plasticity and glioma progression.

Ependymomas of the spinal region in adults: Clinical and pathological features and MYCN expression levels in spinal ependymomas and myxopapillary ependymomas

BackgroundEpendymomas (EPNs) of the spinal region are a heterogeneous group of tumors that account for 17.6 % in adults. Four types have been recognized: subependymoma, spinal ependymoma (Sp-EPN), myxopapillary ependymoma (MPE), and Sp-EPN-MYCN amplified, each with distinct histopathological and molecular features. MethodsThis study investigated the clinical and pathological characteristics and MYCN expression levels of 35 Sp-EPN and MPE cases diagnosed at a tertiary university hospital over a decade-long period. ResultsTwenty-five cases were Sp-EPN and 10 cases were MPE, and were graded as WHO grade 2, except for 1 Sp-EPN case with grade 3 features. The most common symptoms were lower back pain and difficulty in walking. Radiology showed different tumor sizes and locations along the spinal cord, with MPEs exclusively in the lumbosacral region. Surgery was the main treatment, and gross total resection was achieved in all cases except for one. Immunohistochemistry showed low Ki-67 proliferation indices in all cases, and no MYCN expression. During follow-up, 3 (8.6 %) cases recurred and/or metastasized and 5 cases (14.3 %) died. No significant difference was found in disease-free survival or overall survival between Sp-EPN and MPE cases. However, 3 cases with grade 2 histology demonstrated recurrence and/or metastasis, despite the lack of MYCN expression. ConclusionOur results underscore the multifactorial nature of tumor aggressiveness in EPNs of the spinal region. This study enhances our knowledge of the clinical and pathological features of Sp-EPNs and MPEs and highlights the need for better diagnostic and prognostic markers in these rare tumors.

Abstract 4269: Effect of PAX3-FOXO1 fusion gene on cell-extracellular matrix interaction of Rhabdomyosarcoma

Abstract Background Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, categorized by two subtypes, fusion protein-positive (FPRMS) and negative (FNRMS), based on the existence of PAX3-FOXO1 fusion gene (P3F). Different features between the two subtypes could guide the use of distinct treatment approaches. We discovered that FNRMS exhibited enrichment of gene sets related to cell-extracellular matrix (ECM) interaction than FPRMS. ECM, providing physical support as a scaffold, regulates cancer development. We hypothesize that a distinct molecular feature, P3F, may cause the difference in cell-ECM interaction between FPRMS and FNRMS. Methods To confirm the higher cell-ECM interaction of FNRMS vs. FPRMS, we used confocal reflection microscopy (CRM), enabling us to monitor the fibrous structures of ECM. We generated the spheroids with FNRMS and FPRMS cell lines by hanging drop method and embedded them into collagen type I. Then, we assessed orientation and dynamic changes in the structure of collagen fibers surrounding spheroids. Also, we analyze the transcriptome of FPRMS cells with P3F knockdown to confirm if P3F regulates cell-ECM interaction negatively via RNA seq. Additionally, we examined ECM surrounding spheroids of P3F knockdown FPRMS cells if P3F modulates fibrous structures of ECM, indicating cell-ECM interaction. Results We showed that collagen fibers are oriented more perpendicular to the FNRMS spheroid surface, indicating the strong pulling and pushing effect of cells. However, fibers surrounding FPRMS spheroids showed parallel alignment to their surfaces, reflecting passively pushed by the spheroids. Additionally, fibers near FNRMS spheroids showed greater displacement than those near FNRMS spheroids, which suggests stronger cell-ECM interaction in FNRMS than in FPRMS. Furthermore, P3F knockdown FPRMS cells exhibited significant enrichment of gene sets related to cell-ECM interaction. Their CRM results showed that collagen fiber orientation was significantly perpendicular to the surfaces of spheroids of P3F knockdown FPRMS cells than those of control cells. P3F knockdown cell spheroids also displayed enhanced displacement of ECM fibers than control cells. These results suggest that P3F suppresses cell-ECM interaction, consistent with higher cell-ECM interaction of FNRMS than FPRMS. Conclusions The PAX3-FOXO1 gene may regulate cell-ECM interaction negatively, causing distinct features between FPRMS and FNRMS, which is crucial for various cellular functions and suggest different treatment between the two RMS subtypes. Citation Format: Chandra Kaladhar Vemula, Ivan Chavez, Anthony Chronopoulos, Jinseok Park. Effect of PAX3-FOXO1 fusion gene on cell-extracellular matrix interaction of Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4269.

Abstract 5100: Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade

Abstract Background: Why some patients fail or have short lived response to immune checkpoint blockade (ICB) immunotherapy remains largely unknown. While baseline molecular assessments have provided clues to prognostic factors, insights into resistance drivers remains elusive. This is partially due to the difficulty in getting access to post progression samples from patients that were either primary resistant or developed acquired resistance after an initial response to ICB. Thus, the tumor-intrinsic and -extrinsic features that are selected for during progression and potentially drive primary and acquired resistance to immunotherapy remain underexplored. Methods: To compare clinical features and immunogenomic drivers of acquired and primary resistance to ICB across major cancers, we analysed and annotated de-identified patient records in the Tempus real-world database. We built an immuno-oncology cohort consisting of >2500 multimodal (DNA, RNA and clinical outcome data) pre-treatment baseline with >1500 post-treatment tumour biopsy samples from mainly NSCLC, TNBC, HNC and Bladder cancer patients. We used bulk RNA-seq data to estimate activation of the hallmark oncogenic pathways and immune cell composition and used panel DNA-seq data (>500 genes) to quantify mutation selection at the gene and pathway levels using dndscv. Results: Compared to acquired, primary resistant patients tended to have a higher observation of liver lesions at progression. Post-ICB, acquired resistant NSCLC and HNC patients showed a significantly inflamed tumour microenvironment (TME) characterised by higher estimation of infiltration of T cells and myeloid cells and higher activation of interferon gamma (IFNg) signalling as compared to primary resistant patients. In addition, in post-ICB acquired resistance in NSCLC we observed selection for mutations in genes involved in known immunomodulatory pathways, including loss-of-function mutations in B2M. Consistently, acquired resistance patients showed stronger selection for mutations in Hedgehog and Notch pathways as compared to primary resistance patients across NSCLC, HNC and TNBC post-ICB. Conclusions: Acquired and primary ICB resistant patients have distinct clinical and molecular features at progression. Their tumours’ TME is fundamentally different with acquired resistance TMEs being infiltrated with immune cells albeit escaped post progression. In addition, ICB selects mutations that potentially activate pathways such as Notch and Hedgehog. This multi-modal Real-World Data with post therapy biopsies has given insights for patient selection strategies and provides rational into combination treatment options for acquired resistant patients. Citation Format: Mohamed Reda Keddar, Sebastian Carrasco Pro, Kathleen Burke, Ana Camelo Stewart, Mark Cobbold, Ross Stewart, Sajan Khosla, Ben Sidders, Scott Hammond, Douglas C. Palmer, Jonathan Dry, Martin L. Miller. Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5100.

Abstract 7500: Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC)

Abstract Background: Invasive lobular carcinoma (ILC) has distinctive clinical and molecular features compared with invasive ductal carcinoma (IDC). CTCs and tdEVs are independent prognostic factors in MBC, however, liquid biopsy studies focusing on ILC are to date scarce. Higher CTC levels have been observed in ILC than IDC. One study including 28 ILC pts suggested the use of higher CTC cutoff for prognosis stratification. The clinical significance of tdEVs in ILC is unexplored. This study aimed to assess differences in the distribution and prognostic value of CTCs and tdEVs between ILC and IDC. Methods: Blood samples were collected from 304 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). Blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Association of CTCs and tdEVs with overall survival (OS) was tested in ILC and IDC pts. A count of CTCs ≥ 5 was considered high. The cutoff levels for tdEVs were < 20 (low), 20-79 (intermediate), and ≥ 80 (high), as previously reported. Additional exploratory cutoffs for CTCs (≥ 20, ≥ 50, ≥ 80, and ≥ 100) and tdEVs (based on quartiles of tdEV distribution in the overall population and ILC) were also evaluated. Results: Of the 304 pts, 47 had ILC while 257 had IDC. 56% of pts received first-line therapy. Median CTC count was 8 [interquartile range (IQR) 2-35] in ILC and 1 (IQR 0-8) in IDC pts (P <.001). A significantly higher median tdEVs count was observed in ILC (36; IQR 18-115) than in IDC (11; IQR 2-86) pts (P =.002). High levels of CTCs (≥5) were not associated with OS in ILC (P =.88), even when higher cutoffs (≥ 20, ≥ 50, and ≥ 80) were used. Only the detection of ≥ 100 CTCs was significantly associated with OS in ILC pts (median OS 36 vs 10 months; HR 3.4; P =.028). In contrast, the prognostic effect of CTCs ≥ 5 was strong in IDC and maintained for all cutoffs. Similarly, while the prognostic value for tdEVs was confirmed for IDC using both 20-80 and exploratory cutoffs (3-105 and 18-155), no association between tdEV levels and OS was observed in ILC regardless of the cutoffs used. Conclusion: ILC is associated with higher CTCs and tdEVs compared to IDC, probably due to the impaired cell-cell adhesion that characterizes ILC. Unlike IDC, the prognostic effect for the established cutoff of ≥ 5 CTC was not observed in ILC. A higher CTC cutoff could be considered for this subtype of BC. For tdEV no association with OS was observed, regardless of the cutoff used. These findings emphasize that ILC is a distinct entity also in the liquid biopsy features. Because of the low prevalence of ILC, research efforts should be directed at combining data from ILC pts to better characterize this subtype of BC and understand the reason behind the different associations with OS as compared to IDC. Citation Format: Eleonora Nicolo, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz-Arcos, Mara S. Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda K. Strickland, Caterina Gianni, Youbin Zhang, Paolo D'Amico, Andrew A. Davis, Jeannine Donahue, Huiping Liu, William J. Gradishar, Ami N. Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7500.

Abstract 253: Investigating key molecular players in putative stem cell subpopulations of prostate cancer

Abstract Background: Androgen deprivation therapy is the standard treatment for prostate cancer (PCa). Nevertheless, despite initial effectiveness, pre-existing cancer stem cell (CSC) populations invariably lead to incurable castration-resistant prostate cancer. CSCs are a subset of cancer cells possessing self-renewal properties, driving tumor progression and regrowth. CD44+ PCa cells exhibit more stemness features and are enriched in tumorigenic and metastatic progenitor cells. Here, we aim to explore different subpopulations in tumors based on levels of CD44 expression and investigate whether they have distinct molecular properties and functional characteristics. Material and Method: The number of CD44+ cells was evaluated in a tissue microarray from primary prostate cancer patient samples (EMPACT cohort) with clinical follow-up. Flow cytometry sorted the CRPC model LAPC9 tumor into CD44 high (CD44-H) and CD44 low (CD44-L) cells. RNA sequencing was performed on these subpopulations to explore their transcriptomic profiles. CD44 expression was validated at the cDNA and protein levels using qPCR and Western blot (WB) analysis. Sorted cells were maintained as organoids in vitro. To trace the dynamics of CD44-H and CD44-L subpopulations in vivo, LAPC9 tumor was labeled by fluorescent and luciferent markers. The CD44 status was determined by flow cytometry when tumor growth. Results: Patients with elevated expression of CD44 (higher number of CD44+ cells) at the time of surgery exhibited a greater propensity for clinic progression (5-year progression-free survival: 75% vs 95%, P=0.03). Furthermore, CD44-H cell ratio increased in the LAPC9 after castration, indicating that CD44-H cells can survive after treatment. Subsequent WB analysis confirmed that CD44 was highly expressed in sorted CD44-H cells, validating the flow cytometry results. Analysis of the CD44 RNA-seq data showed CD44 transcript variants CD44v10 (CD44-201) and CD44v7-10 (CD44-209) were notably upregulated in CD44-H cells. In organoid culture, sorted CD44-H cells displayed enhanced formation, indicating they possessed higher proliferative capacity and clonogenicity than CD44-L cells. Furthermore, when GFP-labeled CD44-H cells and RFP-labeled CD44-L cells were recombined in vivo, CD44-H cells exhibited robust proliferation, while CD44-L cells transitioned to a CD44hi state to facilitate tumor growth. Moreover, those GFP and RFP subpopulations displayed the same CD44hi cell ratio at the endpoint. Conclusion: Elevated CD44 expression is related to an increased risk in primary PCa. In addition, CD44-H cells exhibit high tumorigenicity in vitro and in vivo and can convert to each other. Moreover, the ratio of CD44-H cells in tumor at endpoint is consistent despite the starting CD44 expression status. Furthermore, the upregulation of CD44v10 and v7-10 in the CD44-H subpopulation may promote tumor formation and metastasis. Citation Format: Wanli Cheng, Marta De Menna, Francesco Bonollo, Panagiotis Chouvardas, George N. Thalmann, Sofia Karkampouna, Marianna Kruithof Julio. Investigating key molecular players in putative stem cell subpopulations of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 253.

Abstract 2942: Deciphering histological subtype-associated cellular and molecular characteristics of lung adenocarcinoma using single-cell RNA sequencing and spatial transcriptomics

Abstract Lung adenocarcinoma (LUAD) is the most frequently observed subtype found in non-small cell lung cancer (NSCLC). Despite its high prevalence, the molecular and histological heterogeneity in LUAD pose a significant challenge for providing effective patient care and aiming to reduce the high mortality rate. The histopathological classification proposed by WHO in 2015 describes five histologic patterns of lung adenocarcinoma (lepidic, acinar, papillary, micropapillary, and solid), and it has been shown that the existence of high-grade subtypes (micropapillary and solid) is associated with poorer survival outcomes. Nevertheless, the specific cellular and molecular mechanisms responsible for these clinical outcome differences based on distinct histological patterns remain unclear. Here, we conducted single-cell RNA sequencing (scRNA-seq) to unveil insights into the tumor microenvironment (TME) and identify cancer cell biomarkers associated with distinct histological patterns. Our study involved 18 specimens from primary LUAD patients who underwent surgical resection, all of whom were previously untreated. These specimens were classified into three groups: acinar/papillary (A/P), micropapillary (MP), and solid. Remarkably, patients with solid-type tumors showed a notable enrichment of CD8+ T cell exhaustion, alongside a significant abundance of GPNMB-high macrophages in the myeloid compartment. Additionally, macrophages from the solid-type demonstrated cholesterol-efflux activation, particularly in alveolar macrophages. It is well-known that cholesterol efflux-activated macrophages display a tumor-associated macrophage phenotype, contributing to immunosuppression in the TME. In contrast, the A/P group exhibited a higher enrichment of mast cells compared to the solid-type. This suggests that the solid-type tumor landscape is more immunosuppressive than the A/P group. In the context of cancer cells, we identified that A/P cancer cells displayed a well-differentiated pattern with a high alveolar epithelial type II cell (AT2) signature. In contrast, solid-type cancer cells showed poorly differentiated states, coupled with a significant activation of the epithelial-mesenchymal transition (EMT) process. Remarkably, even within A/P patients, those with a small portion of solid patterns in the tumor tissue showed higher clonal complexity compared to patients without any solid pattern. Furthermore, we found that cancer cell plasticity can be regulated by cell-cell interactions in the TME. Collectively, this study unveils the relationship between histological patterns and the TME, encompassing distinct immune responses and molecular characteristics of cancer cells in LUAD. These findings provide valuable insights for potential targeted therapeutic strategies. Citation Format: Ju Sung Lee, Ji Yun Jeong, Mi Jeong Hong, Hyo-Gyoung Kang, Jin Eun Choi, Ju Young Kim, Yoon Ha Choi, Jong Kyoung Kim, Shin Yup Lee. Deciphering histological subtype-associated cellular and molecular characteristics of lung adenocarcinoma using single-cell RNA sequencing and spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2942.

Abstract 509: The MPS1/TTK inhibitor, NMS-01940153, synergizes with tisotumab vedotin in colorectal cancer

Abstract Background: MPS1 kinase plays a crucial role in maintaining genomic integrity by controlling the spindle assembly checkpoint (SAC). MPS1 is highly expressed in tumors of different origins and overexpression is associated with poor prognosis. By inhibiting MPS1, cancer cells are forced to enter anaphase before chromosomes are aligned, leading to mitotic catastrophe and apoptosis. NMS-01940153 (NMS-153) is a potent and selective MPS1 inhibitor, with antitumor activity in vitro and in vivo. NMS-153, under investigation in a clinical trial, shows early signs of clinical activity, an acceptable safety profile, with manageable and reversible neutropenia as the main adverse effect (EudraCT 2020-001002-26; Reig et al, Eur J Cancer 174S1, 2022). Microtubule targeting agents (MTAs), such as taxanes and vinca alkaloids, cause mitotic arrest and activate the SAC. Concomitant SAC disruption by MPS1 inhibition synergizes with MTAs, but the combination is limited by neutropenia in the clinic. Purpose and Methods: The goal is to show synergism between NMS-153 and MTA and to find a strategy to overcome the limiting neutropenia of this combination. For this, Tisotumab vedotin (TV), an anti-Tissue Factor (TF) with Monomethyl auristatine E (MMAE) payload, was used. TF is aberrantly expressed in a variety of tumors compared to normal tissue counterpart. In colorectal cancer (CRC), TF is overexpressed in patients with distinct molecular features including BRAF mutation, a colon cancer population that hardly responds to chemotherapy or BRAF inhibitors. At therapeutic doses, TV induces low rate of grade ≥3 neutropenia. The combination of NMS-153 and TV is expected to synergize and have an acceptable safety profile. LS411N CRC model expressing TF and harboring a BRAFV600E mutation was selected for testing NMS-153 and TV in combination. In vivo, NMS-153 was tested at 13 or 20 mpk every 4 days for 7 doses. TV was tested at 1 or 4 mpk weekly for 2 doses. The 2 therapeutic doses of both agents were tested in combination. Results: In vitro, synergism of NMS-153 with TV in LS411N cells was observed. In vivo, NMS-153 and TV as single agents were efficacious in LS411N xenograft with TGIs up to 53% and 89%, respectively. The median survival time (MST) for the vehicle group was 35.5 days. NMS 153 and TV single agents prolonged survival with MSTs up to 53 and 73.3 days, respectively. When combined, a dose-dependent therapeutic benefit was observed, with an MST of >137 days, and 6 out of 10 mice profiting of long-term tumor regressions at the highest doses tested. The combination benefit was statistically significantly different from single agents and more than additive. Treatments were well tolerated. Conclusions: The combination of NMS-153 with TV is synergistic in the LS411N CRC model. These data strongly suggest the potential for clinical efficacy of NMS-153 with TV and the combinability of NMS-153 with TV and other MMAE-based ADCs in different tumor contexts. Citation Format: Gemma Texido, Fabio Gasparri, Antonella Ciavolella, Laura Gianellini, Anders E. Pedersen, Alberto Ocaña, Riccardo Colombo, Domenico Roberti, Lisa Mahnke, Claudia Perrera. The MPS1/TTK inhibitor, NMS-01940153, synergizes with tisotumab vedotin in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 509.

Abstract 409: Deciphering chromosomal instability in consensus molecular subtypes (CMS) in CRC: Insights from an integrative multi-omics approach

Abstract CRC is a heterogeneous disease with different molecular subtypes, which can have a profound impact on treatment response and patient outcomes. CMS classification based on gene expression profiles has been shown to differentiate patients into clinically relevant sub-groups. However, deep multi-omics characterization of the CMS classes based on high quality patient data is lacking. Through the integration of multi-omics data, we aim to enhance our understanding of CRC’s molecular heterogeneity and thereby facilitate the development of personalized therapeutic strategies. We performed a multi-omics analysis of CRC patients to identify potential new features of CMS groups. 890 fresh-frozen, surgically resected CRC tumor and adjacent normal samples were used to analyze whole genome and RNA sequencing data. DNA and RNA was prepared using Qiagen AllPrep Universal and KAPA Hyper Prep kit (DNA), as well as TruSeq Stranded Total mRNA kits and sequenced on a NovaSeq6000 system. We identified CMS subtypes based on RNA-Seq data and performed all statistical analyses using R. Our analysis revealed distinct molecular characteristics across the four CMS subtypes, including unique driver mutations, signaling cascades, and immune cell infiltration profiles. The poor prognosis associated with CMS4 patients was corroborated by our clinical data. Notably, we observed high global chromosomal instability (CIN) profiles for both CMS2 and CMS4 and identified recurrent global aneuploidy patterns within these subtypes that link alterations of known cancer genes to these large-scale structural events. We found higher levels of CIN in CMS2 compared to CMS4, which appears to be predominantly driven by numerical, rather than structural CIN. Specifically, in CMS2, SMAD2/4 is commonly impacted by deletion on chromosome arm 18q, while PLCG1 is more influenced by amplifications on 20q respectively, suggesting potential values as CRC CMS biomarkers. In conclusion, our integrative multi-omics analysis offers a comprehensive understanding of CRC molecular subtypes, which can inform personalized treatment strategies. Our findings underscore the importance of considering not only specific driver mutations but also large-scale structural rearrangements in the context of CMS subtypes. Citation Format: Julia Bischof, Jonathan Woodsmith, David Church. Deciphering chromosomal instability in consensus molecular subtypes (CMS) in CRC: Insights from an integrative multi-omics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 409.

Abstract 6459: Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data

Abstract Background: Up to 85% of lung cancer patients have non-small cell lung cancer (NSCLC). Standard of care for NSCLC includes immune checkpoint inhibitors (ICI) and/or platinum-based chemotherapy (CT). However, therapy success is limited with up to 70% of patients not responding to ICI. Mechanisms of resistance across treatments are not fully understood. In this study, we explore real-world data to characterize molecular and genomic changes in advanced NSCLC treated with ICI, ICI+CT, or CT. Methods: Stage III/IV NSCLC patients receiving first-line (1L) ICI, ICI+CT, or CT were identified from Tempus Labs, Inc.’s de-identified, multimodal real-world database. Patients’ solid tissue biopsies taken pre- or post-1L underwent next generation sequencing with the Tempus xT assays (DNAseq of 648 genes at 500x coverage and whole transcriptome RNAseq). Patients’ clinical and outcome data were abstracted from electronic health records. We analyzed changes in gene expression, pathway enrichment, estimated immune cell components, and somatic mutations in responders (complete/partial response) and non-responders (progression/stable disease) upon 1L treatment. Results: 1L non-responders (70%, 51%, and 47%) were identified across cohorts (143 ICI, 764 ICI+CT, and 228 CT, respectively). 58% of pre-1L biopsies were taken from distal metastatic tissues. Distinct pathways altered post-1L include the downregulated E2F target pathway in CT cohort, and upregulated TNFα/NFκB signaling and the hypoxia and protein secretion pathways in ICI cohort. Significant (FDR < 0.05) augmented expression was observed for 90, 23, and 65 genes in non-responders vs. responders post ICI, ICI+CT, and CT, respectively. RNAseq deconvolution reveals decreased plasma and CD4+ activated T cell fraction in metastatic biopsies, whereas increased CD4+ activated T cell and monocyte fraction was seen after treatment only in ICI cohort. Genes with oncogenic driver mutations reported in NSCLC were detected including TP53 (68%), KRAS (30%), STK11 (13%), EGFR (11%), CDKN2A (10%), KEAP1 (9%), and NF1 (7%). Of the 250 mutated genes, 72 (29%) were commonly not detected post-1L, whereas 38, 3, and 8 unique genes with loss of detectable mutations were identified post-1L of ICI, ICI+CT, and CT, respectively. Since loss of detectable mutations is a sign of loss of subpopulations of cancer cells hosting such mutations that succumb to therapy, these genetic mutations could be used to select patients who can benefit from such therapy. Conclusions: Distinct molecular characteristics including tumor immune components and genomic alterations are associated with 1L response in advanced NSCLC. Real-world data analyses provide opportunity to explore distinct mechanisms of therapy resistance and identify potential biomarkers of therapy response for further validation in clinical studies. Citation Format: Xiaoyong Fu, Jordan Kardos, Paola Correa, Min Wang, Li, Jackson Egen, Shahed Iqbal. Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6459.

Abstract 6466: Multi-omics analysis of molecular characteristics and transformation mechanisms of stage I-III micropapillary lung adenocarcinoma

Abstract Background: The micropapillary pattern (MIP) is recognized as a high-grade histological subtype in lung adenocarcinoma (LUAD), with its presence often indicative of a poorer prognosis. Despite distinctive molecular characteristics of MIP, there is a research gap regarding the molecular mechanisms underlying micropapillary transformation and prognosis factors for micropapillary LUAD. Methods: Surgically resected tumor samples from 101 patients with stage I-III LUAD and MIP components exceeding 30% were collected. The tumor samples were microdissected to separate MIP components from non-MIP components, and both fractions underwent RNA and DNA whole-exome sequencing (WES). The molecular profiles between MIP and non-MIP components, along with MIP-naïve LUAD tissues from an external cohort, were compared. Associations between molecular characteristics and recurrence-free survival were also assessed. Results: The genomic landscapes of MIP and non-MIP components within tumor tissues featuring MIP patterns exhibited remarkable similarity. At the transcriptomic level, MIP components displayed elevated PRB4 expression (FDR < 0.001), an up-regulated cell cycle pathway (P = 0.004), and reduced natural killer cell-mediated cytotoxicity (P = 0.066). Nevertheless, in comparison to MIP-naïve LUAD tissues, the MIP components showed higher chromosomal instability (P < 0.001) and revealed eighteen enriched somatic alterations, encompassing EGFR mutations, EGFR amplifications, and CDKN2A/CDKN2B deletions, all linked to up-regulations in cell proliferation pathways and down-regulated immune pathways. In the context of MIP transformation, shared mutations were observed in 97.8% (91/93) of patients between MIP and non-MIP components within the same tissues, suggesting a common origin. The subclonal fractions were comparable between paired MIP and non-MIP components, while a significant co-occurrence of EGFR amplification, CDKN2A deletion, and CDKN2B deletion was identified in MIP components. A comprehensive analysis combining differential gene expression and Cox analysis, employing a both-direction stepwise Akaike Information Criteria selection, identified the high expressions of five genes (GALNT4, MIAT, FOSL1, LAT2, SMN2) as adverse factors associated with the recurrence of micropapillary LUAD. Conclusions: We conducted an in-depth analysis of the molecular characteristics and transformation mechanisms of micropapillary lung adenocarcinoma, employing microdissection techniques to investigate at both genomic and transcriptomic levels within a substantial cohort, providing insights for the precision medicine of this aggressive cancer subtype. Citation Format: Kang Shao, Shugeng Gao, Fengwei Tan, Anqi Shao, Xiaoli Feng, Qi Xue, Yang Qu, Bo Zheng, Wei Zheng, Hanlin Chen, Qiuxiang Ou, Haimeng Tang. Multi-omics analysis of molecular characteristics and transformation mechanisms of stage I-III micropapillary lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6466.

Abstract 6247: Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) and linitis-plastica type gastric cancer (STAD) are the most representative refractory cancers even in the era of molecularly targeted therapy and immunotherapy. Extensive characterization of these tumors, especially regarding the genomic and epigenomic characteristics of pure cancer cells, has been hampered by the high stromal content in the primary tumor samples. Here we took advantage of cancer cells purified from malignant ascites and established corresponding cell lines to overcome the limitations associated with the analysis and to discover their vulnerabilities. Methods: We developed cell lines from malignant ascitic fluid samples obtained from 60 patients with PDAC and 59 patients with linitis-plastica type STAD. We performed comprehensive multi-omic analyses of these cell lines using long-read whole-genome sequencing (WGS) for complex somatic genomic alterations and for methylation aberration. We also conducted long-read RNA sequencing (iso-seq) for full-length isoform exploration, and chromatin immunoprecipitation coupled with sequencing (ChIP-seq) for enhancer analysis. Complementary short-read whole-genome sequencing and RNA sequencing were also performed. Results: Comprehensive transcriptome analyses identified a cluster with high epithelial-mesenchymal transition (EMT) characteristics, consisting of the majority of the PDAC cell lines and a half of the STAD cell lines. ChIP-seq analysis against H3K27ac revealed the distinct transcriptional circuit that generated EMT cluster-specific super-enhancers governed by SMAD3, independent of cancer type. Most of the transcriptional activity was controlled by the gene methylation. We also identified the differential expression levels of the splicing factors between the EMT-activated and the non-activated groups. Comprehensive isoform analysis by iso-seq revealed that the isoform usage was completely different between the EMT group and the other, especially in genes of the actomyosin pathway which confer characteristic features of the EMT cluster in the cell morphology and cell motility. Using the CHM13 reference genome, long-read WGS analyses revealed the recurrent structural alterations specific to the EMT group such as those involving CDKN2A. We also identified PDAC-specific structural alterations. Conclusion: We identified the distinct molecular characteristics of the EMT-activated group in PDAC and linitis-plastica type STAD which may confer the therapeutic resistance. Our findings provide an interesting insight for the further innovation of the treatment strategy for these intractable cancers. Citation Format: Yosuke Tanaka, Hiroyuki Mano. Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6247.

Abstract 5825: Non-genetic determinants driving the evolutional trajectories of drug-tolerant-persisters in KRAS Non-small cell lung cancer

Abstract Rare cell populations manage to evade the effects of cancer therapies, ultimately leading to drug resistance and subsequent cancer relapse. These resilient populations, often referred to as "persisters," exhibit distinct molecular characteristics when compared to the broader tumor population. In the current standard of clinical care for non-small cell lung cancer (NSCLC), patients typically undergo a series of standard treatments, such as surgery followed by chemotherapy/immunotherapy, and then targeted therapy, based on the disease stages. This continues until drug resistance emerges, with little attention given to the state of these persister cells in response to these treatments. In the case of drug resistance in KRAS-mutated NSCLC, ongoing clinical investigations are exploring the combined efficacy of KRAS G12Ci inhibitors with drugs that target mitogen-activated protein kinase (MAPK) feedback reactivation, immunotherapy, and chemotherapy. Despite the myriad of combination therapies, persister populations invariably emerge. The manner in which these persisters evolve to withstand therapies remains elusive. We employed a DNA barcode lineage tracing analysis coupled with mathematical modeling to decode persister evolution to therapies targeting different pathways, e.g., KRAS+ SHP2, KRAS+CDK4/6, and KRAS+MCL-1. Our model reveals that rapidly emerging persister populations are predetermined and do not exhibit clear genetic alterations. In some cells, common persisters exist in response to different therapies. Single-cell ATAC-RNA sequencing showed that these persisters are enriched in chromatin-binding of transcription factors that regulate classic non-homologous end-joining (cNHEJ) DNA repair (ZNF384), interferon response (IRF1), and stem-like characteristics (FOXP1), along with the activation of an IFN-γ transcriptional signature. These findings underscore the connection between specific DNA repair processes, chromosomal instability, and resistance to KRAS G12Ci. In other cells, however, distinct persister populations exist to different therapies, leading to the hypothesis that these diverse persister populations can be targeted orthogonally or sequentially to achieve tumor regression. In summary, our study unveils the heterogeneity in persister evolution trajectories in KRAS NSCLC. Our platform identifies targetable persister populations in NSCLC models and offers a proof-of-concept for non-standard strategies to combat drug resistance based on persister cell states. Citation Format: Chendi Li, Christopher J. Graser, Qian Qin, Anahita Nimbalkar, Usman Syed, Barbara Karakyriakou, Sarah Clark, Anne Y. Saiki, Paul E. Hughes, Christopher Ott, Luca Pinello, Franziska Michor, Aaron Hata. Non-genetic determinants driving the evolutional trajectories of drug-tolerant-persisters in KRAS Non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5825.

Crosstalk of cell death pathways unveils an autophagy-related gene AOC3 as a critical prognostic marker in colorectal cancer

The intricate crosstalk of various cell death forms was recently implicated in cancers, laying a foundation for exploring the association between cell death and cancers. Recent evidence has demonstrated that biological networks outperform snapshot gene expression profiles at discovering promising biomarkers or heterogenous molecular subtypes across different cancer types. In order to investigate the behavioral patterns of cell death-related interaction perturbation in colorectal cancer (CRC), this study constructed the interaction-perturbation network with 11 cell death pathways and delineated four cell death network (CDN) derived heterogeneous subtypes (CDN1-4) with distinct molecular characteristics and clinical outcomes. Specifically, we identified a subtype (CDN4) endowed with high autophagy activity and the worst prognosis. Furthermore, AOC3 was identified as a potential autophagy-related biomarker, which demonstrated exceptional predictive performance for CDN4 and significant prognostic value. Overall, this study sheds light on the complex interplay of various cell death forms and reveals an autophagy-related gene AOC3 as a critical prognostic marker in CRC.

Clinical and Molecular Insights into Hypertrophic Scars and Keloids: A Literature Review

Background: Hypertrophic scars and keloids represent two distinct forms of abnormal wound healing, characterized by excessive fibrous tissue formation. Despite their prevalence, the pathophysiological mechanisms underlying their development and persistence remain incompletely understood. This literature review synthesizes current clinical and molecular insights into hypertrophic scars and keloids, aiming to highlight differences in their etiology, pathogenesis, and therapeutic responses. Methods: A comprehensive review of the literature was conducted, focusing on articles that provided significant insights into the clinical manifestations, genetic predispositions, molecular pathways, and treatment strategies related to hypertrophic scars and keloids. Experimental studies and clinical trials were included to encompass a wide range of data sources. Results: Hypertrophic scars are confined to the original wound boundary and may regress over time, whereas keloids extend beyond the wound margins and do not regress. Clinically, hypertrophic scars and keloids differ in their appearance, texture, and predilection sites. At the molecular level, these differences are underscored by distinct profiles of cytokine expression, growth factor activity, and extracellular matrix composition. Genetic studies have identified several predisposing factors, including specific gene mutations and polymorphisms. Treatment strategies vary; however, intralesional corticosteroids remain the first-line treatment for both conditions. Emerging therapies targeting specific molecular pathways offer potential for improved outcomes. Conclusion: Hypertrophic scars and keloids are complex conditions with distinct clinical and molecular characteristics. Understanding these differences is crucial for developing targeted and effective therapies. Future research should focus on unraveling the genetic basis of these conditions and exploring novel therapeutic targets. Enhanced knowledge of the pathogenic mechanisms will facilitate the advancement of personalized medicine approaches in the management of hypertrophic scars and keloids.

Abstract A026: Identifying mechanisms to target in combination with trametinib to improve therapeutic efficacy in low-grade serous ovarian cancer

Abstract A026: Identifying mechanisms to target in combination with trametinib to improve therapeutic efficacy in low-grade serous ovarian cancer

Image-Based Subtype Classification for Glioblastoma Using Deep Learning: Prognostic Significance and Biologic Relevance.

To apply deep learning algorithms to histopathology images, construct image-based subtypes independent of known clinical and molecular classifications for glioblastoma, and produce novel insights into molecular and immune characteristics of the glioblastoma tumor microenvironment. Using whole-slide hematoxylin and eosin images from 214 patients with glioblastoma in The Cancer Genome Atlas (TCGA), a fine-tuned convolutional neural network model extracted deep learning features. Biclustering was used to identify subtypes and image feature modules. Prognostic value of image subtypes was assessed via Cox regression on survival outcomes and validated with 189 samples from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) data set. Morphological, molecular, and immune characteristics of glioblastoma image subtypes were analyzed. Four distinct subtypes and modules (imClust1-4) were identified for the TCGA patients with glioblastoma on the basis of the image feature data. The glioblastoma image subtypes were significantly associated with overall survival (OS; P = .028) and progression-free survival (P = .003). Apparent association was also observed for disease-specific survival (P = .096). imClust2 had the best prognosis for all three survival end points (eg, after 25 months, imClust2 had >7% surviving patients than the other subtypes). Examination of OS in the external validation using the unseen CPTAC data set showed consistent patterns. Multivariable Cox analyses confirmed that the image subtypes carry unique prognostic information independent of known clinical and molecular predictors. Molecular and immune profiling revealed distinct immune compositions of the tumor microenvironment in different image subtypes and may provide biologic explanations for the patterns in patients' outcomes. Our image-based subtype classification on the basis of deep learning models is a novel tool to refine risk stratification in cancers. The image subtypes detected for glioblastoma represent a promising prognostic biomarker with distinct molecular and immune characteristics and may facilitate developing novel, individualized immunotherapies for glioblastoma.

Exploring the molecular and immune-landscape of lung cancer associated with cystic airspaces

To explore the molecular biological characteristics of lung cancer associated with cystic airspaces (LCCA) and its potential roles on prognosis. A total of 165 LCCAs and 201 non-LCCAs were enrolled in this study. Bulk RNA sequencing was implemented in eight LCCAs and nine non-LCCAs to explore the differentially expressed genes. TCGA data were used to analyze LCCA-specific genes that associated with overall survival (OS). The median age was 60 (IQR 53 to 65) years in LCCA cohort. We found LCCA were predominant in men and had less visceral pleura invasion (VPI) or lympho-vascular invasion (LVI). Moreover, LCCA presented with higher histological heterogeneity. Kaplan-Meier analysis showed that patients of age more than 60 and positive VPI had significantly less PFS in LCCA. Cox regression suggested that LCCA, micropapillary subtype proportion and VPI were the independent risk factors for PFS. LCCA had up-regulated pathways associated with EMT, angiogenesis and cell migration. In addition, LCCA displayed higher levels of immunosuppressor infiltration (M2 macrophages, CAFs and MDSCs) and distinct cell death and metabolic patterns. BCR/TCR repertoire analysis revealed less BCR richness, clonality and high-abundance shared clonotypes in LCCA. Finally, Cox regression analysis identified that four cystic-specific genes, KCNK3, NRN1, PARVB and TRHDE-AS1, were associated with OS of lung adenocarcinoma (LUAD). And cystic-specific risk scores (CSRSs) were calculated to construct a nomogram, which performance well. Our study for the first time indicated significantly distinct molecular biological and immune characteristics between LCCA and non-LCCA, which provide complementary prognostic values in early-stage non-small cell lung cancer (NSCLC).

Integration of CD4+ T cells and molecular subtype predicts benefit from PD-L1 blockade in muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is a disease characterized by molecular and clinical heterogeneity, posing challenges in selecting the most appropriate treatment in clinical settings. Considering the significant role of CD4+ T cells, there is an emerging need to integrate CD4+ T cells with molecular subtypes to refine classification. We conducted a comprehensive study involving 895 MIBC patients from four independent cohorts. The Zhongshan Hospital (ZSHS) and The Cancer Genome Atlas (TCGA) cohorts were included to investigate chemotherapeutic response. The IMvigor210 cohort was included to assess the immunotherapeutic response. NCT03179943 was used to evaluate the clinical response to a combination of immune checkpoint blockade (ICB) and chemotherapy. Additionally, we evaluated genomic characteristics and the immune microenvironment to gain deeper insights into the distinctive features of each subtype. We unveiled four immune-molecular subtypes, each exhibiting distinct clinical outcomes and molecular characteristics. These subtypes include luminal CD4+ Thigh, which demonstrated benefits from both immunotherapy and chemotherapy; luminal CD4+ Tlow, characterized by the highest level of fibroblast growth factor receptor 3 (FGFR3) mutation, thus indicating potential responsiveness to FGFR inhibitors; basal CD4+ Thigh, which could benefit from a combination of ICB and chemotherapy; and basal CD4+ Tlow, characterized by an immune suppression microenvironment and likely to benefit from transforming growth factor-β (TGF-β) inhibition. This immune-molecular classification offers new possibilities for optimizing therapeutic interventions in MIBC.

Molecular mechanisms in MASLD/MASH-related HCC.

Liver cancer is the third leading cause of cancer-related deaths and ranks as the sixth most prevalent cancer type globally. NAFLD or metabolic dysfunction-associated steatotic liver disease, and its more severe manifestation, NASH or metabolic dysfunction-associated steatohepatitis (MASH), pose a significant global health concern, affecting approximately 20%-25% of the population. The increased prevalence of metabolic dysfunction-associated steatotic liver disease and MASH is parallel to the increasing rates of obesity-associated metabolic diseases, including type 2 diabetes, insulin resistance, and fatty liver diseases. MASH can progress to MASH-related HCC (MASH-HCC) in about 2% of cases each year, influenced by various factors such as genetic mutations, carcinogen exposure, immune microenvironment, and microbiome. MASH-HCC exhibits distinct molecular and immune characteristics compared to other causes of HCC and affects both men and women equally. The management of early to intermediate-stage MASH-HCC typically involves surgery and locoregional therapies, while advanced HCC is treated with systemic therapies, including anti-angiogenic therapies and immune checkpoint inhibitors. In this comprehensive review, we consolidate previous research findings while also providing the most current insights into the intricate molecular processes underlying MASH-HCC development. We delve into MASH-HCC-associated genetic variations and somatic mutations, disease progression and research models, multiomics analysis, immunological and microenvironmental impacts, and discuss targeted/combined therapies to overcome immune evasion and the biomarkers to recognize treatment responders. By furthering our comprehension of the molecular mechanisms underlying MASH-HCC, our goal is to catalyze the advancement of more potent treatment strategies, ultimately leading to enhanced patient outcomes.