Cervical cancer is largely driven by human papillomavirus (HPV) infection, yet clinically actionable molecular subtypes and effective targeted therapies remain limited. Here, we define biologically and clinically relevant subtypes and evaluate targeted therapeutic strategies. Analysis of public datasets, coupled with functional studies in cervical cancer cell lines and immune assays, identified three subtypes: (I) PIK3CA wild-type without YAP1 amplification, (II) PIK3CA-mutant without YAP1 amplification, and (III) PIK3CA wild-type with YAP1 amplification. Notably, YAP1 amplification is associated with poorer patient survival. The PI3Kα-specific inhibitors, Alpelisib (BYL-719) and Inavolisib (GDC-0077), selectively inhibited proliferation in multiple PIK3CA-mutant cervical cancer cell lines but had minimal effect in PIK3CA wild-type cells. Alpelisib further reduced expression of HPV16 E7, PD-L1 (CD274), YAP1, and EGFR specifically in PI3Kα-mutant models. In an HPV16-positive, HLA-A2-positive, PIK3CA-mutant cell line (CaSki), antigen-specific donor T cells (NexImmune) induced cytotoxicity in a dose-dependent manner. Importantly, combining BYL-719 with T-cell therapy enhanced tumor cell killing, with maximal effects observed following drug pretreatment prior to T-cell exposure. These findings identify actionable molecular subtypes of cervical cancer and support targeting PI3Kα in PIK3CA-mutant tumors. Moreover, combining PI3K inhibition with antigen-specific immunotherapy represents a promising strategy to improve outcomes in advanced HPV16-associated cervical cancer.

e17522 Background: Cervical cancer is driven by human papillomavirus (HPV) infection yet lacks molecularly defined subtypes and approved targeted therapies. We defined molecular subtypes of cervical cancer and identify actionable therapeutic vulnerabilities. Methods: Whole-exome sequencing on cervical tumors from Guatemala and Venezuela, with validation in TCGA, AACR Genie, and Caris cohorts. Functional studies were conducted in cervical cancer cell lines using targeted inhibitors, immune co-culture assays, and proliferation analyses. Results: We identified four somatic mutation subtypes of cervical cancer: (I) No mutation ( PIK3CA / STK11 wild-type (wt) without YAP1 amplification); (II) PIK3CA -mutant, wt for STK11 , YAP1 ; (III) YAP1 -amplified, wt for PIK3CA , STK11 ; and (IV) STK11 -mutated or deleted, wt for PIK3CA , YAP1 . STK11 alterations were significantly enriched in adenocarcinomas compared with squamous cell carcinomas (23%, X 2 =4.4; p = 0.0037) and were confirmed in the AACR Project GENIE (p = 0.035) and Caris cohorts (p = 2.5e-06). YAP1 amplification and STK11 alterations were associated with younger age at diagnosis and poorer overall survival compared to subtype I. STK11 alterations also correlated with inferior outcomes following immunotherapy compared to subtype I. PI3Kα-specific inhibitors (alpelisib and inavolisib) selectively suppressed proliferation in PIK3CA -mutant cervical cancer cell lines but not in PIK3CA wt cell lines. However, the pan-AKT inhibitor capivasertib showed variable activity, inhibiting some but not all PIK3CA -mutated cell lines and the SiHa (PIK3CA wt) cell line. Alpelisib reduced the expression of HPV16 E7, CD274/PD-L1 , YAP1 , and EGFR exclusively in PIK3CA -mutant cell lines. We tested for synergy between PI3Kα inhibition and immune-directed therapy. In HPV16-positive, HLA-A2, PIK3CA -mutant cells, alpelisib enhanced antigen-specific T cell–mediated cytotoxicity, with maximal effect observed following drug pretreatment and washout prior to T cell exposure. Conclusions: Our findings define clinically relevant molecular subtypes of cervical cancer and identify PIK3CA -mutant tumors as sensitive to PI3Kα inhibition. In published clinical studies, 7 patients with PIK3CA -mutant cervical cancer treated with alpelisib achieved partial response or stable disease. Combining PI3K inhibitors with immunotherapy represents a promising strategy for advanced cervical cancer. Distribution of cervical cancer subtypes by histology. Type I WT Type II PIK3CA Type III YAP1 Type IV STK11 SCC AD SCC AD SCC AD SCC AD Sum 1425 641 724 241 174 23 105 83 % Adeno in type 31% 25% 12% 44% Total (%) 2068 (60%) 965 (28%) 197 (5.7%) 188 (5.5%) Pooled data from Guatemala, TCGA, AACR Genie, and Caris datasets. SCC, squamous cell carcinoma; AD, adenocarcinoma.

Between 10 and 20% of cervical cancers are adenocarcinomas with poorer five-year survival and higher recurrence. To identify somatic alterations driving cervical cancer, we performed whole-exome sequencing of 308 subjects with invasive disease from Guatemala and Venezuela. Consistent with other studies, there is a higher rate of TP53 mutations in adenocarcinomas versus squamous cell carcinomas (SCC), especially in HPV-negative tumors. We identified a higher rate of mutations and deletions (23%) in the STK11 tumor suppressor gene in adenocarcinomas versus SCC. This result was confirmed in the AACR Project Genie and Caris cohorts. Whole-genome sequencing and SNP-array data identified significant numbers of focal deletions on chr19p that disrupt STK11, undetected by exome sequencing. In one tumor, HPV integration disrupts STK11. Chr19p is commonly deleted in cervical cancer, and we document a high rate of independent inversions, chromosomal translocations, and breakage-fusion-bridge events that provide the second hit to STK11. Significantly, STK11 alterations are associated with a younger age of onset and poorer overall survival and survival on immunotherapy. Apart from STK11, PIK3CA mutations and YAP1 amplification are prevalent cervical cancer drivers. STK11 mutations and deletions co-occur significantly with YAP1 amplifications, suggesting an interaction between these pathways. In contrast, STK11 alterations are mutually exclusive to PIK3CA mutation, suggesting redundancy. Cervical adenocarcinomas exhibit significantly lower CD274 (PD-L1) expression and a poorer response to immune checkpoint inhibitors (ICIs). STK11 mutations are associated with poor responses to ICI in other cancers, and elucidating the role of STK11 in cervical cancer may improve targeted and immunotherapies.

Objective:Cervical cancer is caused by human papillomavirus (HPV) infections; however, there are no molecularly defined subtypes, and few approved targeted therapies. We defined molecular subtypes and tested targeted agents. Methods:Public datasets were analyzed; cell lines were treated with drugs; and donor T cells and their proliferation were measured. Results:We define three molecular subtypes: I, Wild type for PIK3CA/no YAP1 amplification; II, PIK3CA mutation/no YAP1 amplification; III, PIK3CA WT/YAP1 amplification. Patients with YAP1-amplified cervical cancer have poorer survival. The PI3K-specific inhibitors Alpelisib (BYL-719) and Inavolisib (GDC0077) inhibit the proliferation of multiple PIK3CA-mutated cervical cancer cell lines, but not a PIK3CA wild-type (WT) line. The pan-AKT inhibitor, Capivasertib (AZD5363), suppressed some but not all tested PIK3CA-mutated cell lines and one PIK3CA-wt cell line (SiHa). Alpelisib inhibits the expression of the HPV16 E7 oncoprotein, CD274/PD-L1, YAP1, and EGFR genes, only in PI3K-mutated cell lines. Treatment of an HPV16-positive, HLA-A2, PIK3CA mutant cell line (CaSki) with T cells (NexImmune), specific to HPV16 tumor antigens inhibited in a T cell: target cell ratio-dependent manner. BYL-719, in combination with donor T cells, enhances cytotoxicity against CaSki cells. Furthermore, pretreatment with BYL-719 and removing the drug, followed by treatment with donor T cells, had the maximum effect. Conclusions:Our study revealed molecular inhibitors targeting mutant PIK3CA cervical cancer. When combined with immune therapies, these agents may improve outcomes of advanced HPV16 cancers. Further research on targeted therapies will improve the prognosis of patients with cervical cancer.

ABSTRACT Cervical adenocarcinoma accounts for 15%–20% of cervical cancers and is associated with poorer survival and reduced response to screening and immunotherapy compared with squamous cell carcinoma (SCC). The genomic drivers underlying this molecular subgroup remain incompletely characterized. Whole‐exome sequencing was performed on 302 invasive cervical cancers from Guatemala and Venezuela. Structural variation analysis was conducted using SNP‐array and whole‐genome sequencing data. Findings were replicated in more than 4600 additional cervical cancer samples from TCGA, AACR Project GENIE, MSKCC, and Caris datasets. TP53 mutations were more frequent in adenocarcinoma than SCC, particularly in HPV‐negative tumors. STK11 alterations, including mutations and focal deletions, were significantly enriched in HPV‐positive adenocarcinomas compared with SCC and affected 23% of adenocarcinomas overall. Whole‐genome analyses identified recurrent focal deletions, inversions, chromosomal rearrangements, and breakage‐fusion‐bridge events involving chromosome 19p and STK11 that were not detected by exome sequencing alone. STK11 alterations were associated with younger age at diagnosis, poorer overall survival, and inferior outcomes following immune checkpoint inhibitor (ICI) therapy. STK11 alterations significantly co‐occurred with YAP1 amplification but were largely mutually exclusive with PIK3CA mutation. Cervical adenocarcinomas also demonstrated significantly lower CD274 (PD‐L1) expression than SCC. STK11 alterations define a distinct molecular subgroup of cervical adenocarcinoma characterized by structural disruption of chromosome 19p, younger age at onset, and poorer clinical outcomes. These findings have implications for molecular classification and future targeted therapeutic approaches in cervical cancer.

The metabolite composition of fresh tobacco leaves determines cured tobacco quality, yet the metabolic diversity among flue-cured tobacco varieties and its underlying regulatory mechanisms remain poorly understood. Here, we performed integrated widely targeted metabolomics (UPLC-MS/MS) and transcriptomics (RNA-seq) on fresh leaves of five major flue-cured tobacco cultivars (YY87, ZC208, KRK26, NC55, and YY301) grown in Shandong, China, to decipher the molecular basis of inter-varietal metabolic variation. We identified 2,522 metabolites, revealing significant metabolic divergence among varieties, with the most pronounced differences between NC55 and YY87 (748 differentially accumulated metabolites). Multi-omics integration highlighted phenylpropanoid and flavonoid biosynthesis as the core pathways driving this differentiation. Weighted gene co-expression network analysis (WGCNA) and K-means clustering identified gene modules strongly associated with specific metabolic phenotypes. Gene–metabolite association analysis identified key associations: the peroxidase 43-like gene LOC107786050 showed a strong positive correlation with the aroma precursor sinapaldehyde, whereas the cytochrome P450 98A2-like gene LOC107797681 was primarily associated with the accumulation of the antioxidant taxifolin. This study reveals distinct metabolic and transcriptional patterns among representative flue-cured tobacco cultivars and identifies candidate gene–metabolite associations related to aroma-precursor and flavonoid antioxidant metabolism. These findings provide potential targets for future validation and may support quality-oriented improvement of flue-cured tobacco cultivars.

Invasive cervical cancer (ICC) is the leading cause of cancer-related deaths among women in low-income regions, with approximately 70% of cases caused by high-risk human papillomavirus (HPV) strains—primarily HPV 16 and HPV 18. HPV infection triggers the production of viral oncoproteins E6 and E7, which interfere with the tumor suppressor proteins p53 and pRb, respectively, leading to disrupted cell cycle control and uncontrolled cellular proliferation. Moreover, only a few targeted therapies are approved for treatment. To address this gap, we conducted exome sequencing on 400 ICC fresh frozen samples from Guatemala, the country with one of the highest incidences of cervical cancer globally. We also performed long-read whole genome sequencing (WGS) on 29 cervical cancer cell lines with a coverage depth ranging from 50 to 100x. Additionally, ICC data from public database like TCGA was included in the analysis. Our lab previously identified that YAP1 oncogene amplification (11%) is associated with earlier diagnosis (12 years earlier on average), poorer survival, and a novel aggressive cervical cancer subtype, more prevalent in minority populations. Treatment with the PIK3CA inhibitor Alpelisib reduced proliferation in YAP1 amplified cervical cancer cell lines, like CaSki, SNU1000, SNU1299, QG-U, C4I and CERV-215 but had no effect on PIK3CA wildtype cells. We also observed that STK11, a tumor suppressor gene, is frequently altered in ICC. Exome sequencing data from Guatemala revealed that 32% of tumors (25% in adenocarcinoma (ASC) and 7% in squamous cell carcinoma (SCC)) have STK11 alterations, consistent with our WGS data showing alterations in 35% of cervical cancer cell lines. These mutations are associated with a 10-year earlier age of diagnosis in minority populations. Furthermore, we noticed that STK11 and YAP1 are both functionally involved in the same Hippo signaling pathway. Interestingly, we found in TCGA that 24% of STK11 wild-type cases have YAP1 amplifications while only 8% of STK11 altered cases have YAP1 amplification (p=0.009), exhibiting a co-occurrence of STK11 alterations with YAP1 amplification. We propose a model that HPV E7 oncoprotein and STK11 alterations activate YAP1, promoting oncogenesis. Therefore, co-occurrence of STK11 alterations and HPV infection may synergize to promote more aggressive cancers through hyper-activation of YAP1. Presently, we are validating our observations in highly expressed STK11 cervical cancer cell lines (direct RNA sequencing data) through siRNA transfection and protein expression. This suggests that evaluating YAP1 expression in STK11-deleted cell lines could uncover additional opportunities for targeted therapies and screening invasive cervical cancer cases for STK11 and YAP1 gene alterations may help identify women who are more likely to respond favorably to targeted therapies. Sonam Tulsyan, Emma Robinson, Hong Lou, Wen Luo, Yi Xie, Qian Yang, Isabele Rodriguez, Eduardo Gharzouzi, Enrique Alvirez, Michael Dean. Long-read whole genome sequencing identifies STK11 and YAP1 genes as novel biomarkers for treating invasive cervical cancer [abstract]. In: Proceedings of the 18th AACR Conference on the Science of Cancer Health Disparities; 2025 Sep 18-21; Baltimore, MD. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2025;34(9 Suppl):Abstract nr A085.

Background and Objectives:Cancer continues to be a predominant cause of mortality worldwide, underscoring the critical need to identify and develop novel biomarkers to improve prognostic accuracy and therapeutic approaches. The dysregulation of ELAVL1 is linked to various diseases, including cancer. Nevertheless, its role across different cancer types remains insufficiently investigated. Methods:We conducted a systematic investigation into the expression patterns, prognostic significance, genomic alterations, modifications, and functional implications of ELAVL1 in pan-cancer types. Besides, we performed in vitro and in vivo experiments to confirm the role of ELAVL1 in nasopharyngeal carcinoma (NPC). Results:By utilizing multi-omics datasets, we found obvious overexpression of ELAVL1 in various cancer types at both the mRNA and protein levels, with predominant expression in malignant cells. Survival analysis revealed that increased ELAVL1 expression was linked to unfavorable outcomes in certain cancers; however, its effect difers among various cancer types. Additionally, we found that the genomic alterations and modifications of ELAVL1 were related to tumor progression. We discovered that ELAVL1 was elevated in NPC tissues. In addition, survival analysis indicated that NPC patients with higher ELAVL1 expression had worse prognoses. Functional assays demonstrated that ELAVL1 suppression led to decreased proliferation and migration in NPC cell lines. Moreover, ELAVL1 knockdown effectively inhibited NPC progression in the lymph node and lung metastasis models. Conclusions:In summary, ELAVL1 exhibits diverse and complex involvement in tumor progression. Targeting it might inhibit tumor progression, making it a promising biomarker and therapeutic target for enhancing cancer treatment outcomes.

Cervical cancer (CC) is the fourth most common cancer in women worldwide, with approximately 660,000 new cases and 350,000 deaths each year. The burden of CC falls disproportionately on low- and middle-income countries. In Guatemala, the incidence of CC is high due to limited access to prevention and screening. Persistent infection with human papillomavirus (HPV) is the primary cause of CC. Over 200 HPV types have been identified, thirteen are classified as high-risk (hr) or oncogenic. Among these, HPV16, 18, and 45 account for approximately 75% of CC cases globally. However, little is known about the role of rare hrHPV types, HPV31,33,35,39,51,52,56,58,59,68 in the development of CC. To address this gap, we evaluate the genetic variation of rare hrHPV types in cervical tumors from Guatemala by determining the structure of HPV and genomic changes linked to tumorigenesis. Between 2011 and 2013, 700 cervical tumor tissue samples were collected from patients aged 18 and older at the Instituto de Cancerología in Guatemala. 58 tumors were sequenced using Oxford Nanopore long-read whole-genome sequencing (WGS), achieving a minimum coverage of 30X per sample. This method generates DNA reads up to one million base pairs in length, allowing for detailed analysis of HPV structure, including both integration and episomal forms. Sequences were aligned to the human genome (hg38) and HPV genomes using EPI2ME labs. Each tumor sequence was analyzed by bioinformatic tools including IGV, BLAT, BLAST, RStudio, and MEGA to characterize HPV type, subtype and pattern. Of the 58 tumors, 51 (88%) had one of 10 rare hrHPV types or a probable hrHPV type (HPV26, HPV30) infection. The most common types found among the tumors were HPV52 (20%) and HPV58 (18%), followed by HPV39 (12%), HPV31 (10%), and HPV35 (8%). Among all tumors, we identified slightly higher proportion of episomal HPV forms (53%) compared to integrated forms (47%). When tumors were sub-grouped by HPV alpha genera, we detected alpha 5, 6, 7, and 9, with alpha 7 and 9 being the most prevalent. Episomal HPV tumors were significantly more frequent in alpha 9 HPVs (42%) compared to alpha 5,6,7 combined (11%) (P=0.047). Notably, one tumor exhibited extrachromosomal DNA (ecDNA) containing human and HPV sequences, suggesting an alternative mechanism of HPV oncogene activation. In our study, we identified rare HPV types in cervical cancer tumor tissues and is the first to study the mechanisms of rare types using Oxford Nanopore long read WGS. This study advances the understanding of the molecular mechanisms of the rare HPV types contributing to CC in patients in Guatemala. Ongoing analyses include bioinformatic evaluation of viral and host mutations, along with phylogenetic and statistical methods to characterize HPV sublineages and their role in carcinogenesis. Tawnjerae Joe, Sonam Tulsyan, Hong Lou, Yi XIe, Michael Dean. Understanding the molecular mechanisms of cervical carcinogenesis caused by rare human papillomavirus types [abstract]. In: Proceedings of the 18th AACR Conference on the Science of Cancer Health Disparities; 2025 Sep 18-21; Baltimore, MD. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2025;34(9 Suppl):Abstract nr A048.

Invasive cervical cancer (ICC) is the most common cause of death due to cancer for women living in poverty worldwide. However, there are no approved targeted therapies or schemes to divide cervical cancer for treatment. To address this, we have performed exome and long-read whole genome sequencing (WGS) of 450 ICC tumors from Guatemala. In addition, we performed 50-100X long-read WGS on 29 cervical cancer cell lines. This includes nearly all publicly available cell lines. We extended these findings using 807 ICC samples from cBioPortal (TCGA, AACR-Genie, MSKCC met). The two most commonly activated oncogenes in ICC are PIK3CA (mutated in 35% of tumors) and YAP1 (amplified in 10-14% of ICC). PIK3CA mutations and YAP1 amplifications are mutually exclusive and together define three subtypes. I-wild-type (WT) for both genes, II-PIK3CA-mutated, III-YAP1 amplified. Alpelisib is approved for PIK3CA-mutated breast cancer, and in the initial first-in-human trial, all five ICC patients responded. We treated three PIK3CA-mutated cervical cancer cell lines. Alpelisib causes a dramatic decrease in proliferation at the lowest dose (5 uM) but not in PIK3CA WT cell lines. Responsive cells displayed reduced expression of the HPV E7 oncoprotein and the checkpoint inhibitor CD274/PD-L1 protein. As PD-L1 inhibits cytotoxic T cells, we tested Alpelisib in combination with patient-activated T cells to HPV antigens and found the combined treatment showed optimum cell killing. We demonstrated that YAP1 amplification defines an aggressive subtype of ICC with a 14-year earlier age of onset and poorer survival, even in stage 1 ICC. YAP1 amplification is 3-fold more common in African American and twice as common in Asian ICC. WGS of cell lines and tumors demonstrates that 95% of YAP1 amplification occurs through breakage-fusion-bridge events that co-amplify the anti-apoptotic BIRC2/3 genes. Currently, there are no approved YAP1 inhibitors, but in cervical cells, YAP1 acts to upregulate epidermal growth factor (EGF) ligands and the EGF receptor (EGFR). We tested the EGFR inhibitor Lapatinib on YAP1-amplified CaSki cells and found a five-fold reduction in proliferation. Additional recurrent driver genes in ICC that may be targeted for therapy include MYC, PTEN, KRAS, FBXW7, and STK11. We conclude that oral PI3K inhibitors such as Alpelisib are a viable therapeutic option for up to 200,000 cervical patients annually and may enhance immunotherapies. Initial results suggest that EGFR pathway inhibitors may be beneficial in the most aggressive subtype of ICC. Our cell line panel can allow the testing of additional therapeutics to the YAP1 and other pathways to expand treatment further. Michael C. Dean, Sonam Tulsyan, Hong Lou, Emma Robinson, Ayse Keskus, Tanveer Ahmad, Isabel Rodriguez, Yi Xie, Jia Liu, Wen Luo, Herbert Higson, David Langan, Sojung Kim, Mathias Oelke, Mikhail Kolmogorov. Long-read whole genome sequencing identifies genes for targeted therapy to treat cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3747.

To better understand cervical cancer progression, we analyzed RNA from 262 biopsies from women referred for colposcopy. We determined the HPV type and analyzed the expression of 51 genes. HPV31 was significantly more prevalent in precancer than stage 1 cancer and invasive cancer (p < 0.0001), and HPV16 increased in invasive disease (p < 0.0001). CCNE1, MELTF, and ULBP2 were significantly increased in HPV16-positive compared to HPV31 precancers, while NECTIN2 and HLA-E expression decreased. Markers of the innate immune system, DNA repair genes, and cell cycle genes are significantly increased during cancer progression (p = 0.0001). In contrast, the TP53 and RB1 tumor suppressor gene expression is significantly decreased in cancer cells. The T cell markers CD28 and FLT3LG expression decreased in cancer while FOXP3, IDO1, and ULBP2 expression increased. There is a significantly higher survival rate in individuals with increased expression of CD28 (p = 0.0005), FOXP3 (p = 0.0002), IDO1 (p = 0.038), FLT3LG (p = 0.026), APOBEC3B (p = 0.0011), and RUNX3 (p = 0.019), and a significantly lower survival rate in individuals with increased expression of ULBP2 (p = 0.035). These results will help us elucidate the molecular factors influencing the progression of cervical precancer to cancer. Understanding the risk of progression of specific HPV types and sublineages may aid in the triage of positive patients, and better knowledge of the immune response may aid in developing and applying immunotherapies.

Pediatric cancers are a diverse set of both hematologic and solid malignancies. Approximately 5% of pediatric cancer cases are caused by known genetic conditions, but little is known about genetic susceptibility in Central American children. Most pediatric leukemia and lymphoma cases have high response and cure rates, and many solid malignancies have better outcomes than similar adult cancers. We evaluated the germline genetic variation of pediatric solid tumor cases in Guatemala and Nicaragua in reference hospitals, capturing most of the cancer cases nationwide. We performed exome sequencing on 1360 subjects (957 with solid tumors and 405 with leukemias) and classified variants using an automated pipeline using ClinVar and InterVar and by manual review. The most prevalent cancer types were acute lymphocytic leukemia (357), Hodgkin's lymphoma (229), osteosarcoma (119), and retinoblastoma (93). Pathogenic mutations were identified in 25 pediatric cancer susceptibility genes. Excluding retinoblastoma, 6.5% of cases had a Pathogenic or Likely Pathogenic germline mutation in a cancer-predisposing gene. As expected, RB1 mutations were frequent in retinoblastoma patients, with 81% of confirmed bilateral cases having a Pathogenic or Likely Pathogenic mutation. Mutations in the NF1 gene were observed in neurofibromatosis cases and non-rhabdomyosarcoma. TP53 mutations were observed in osteosarcoma and rhabdomyosarcoma cases, and WT1 mutations were observed in Wilms tumor and renal cancer cases. A single case of pheochromocytoma was found to have a novel VHL gene mutation. Higher rates of pediatric Hodgkin's lymphoma (HL) are found in Central American countries, and the cause of this is unknown. We did not find a significantly mutated gene in the HL cases, suggesting that a common germline mutation is not a significant factor. In conclusion, 6.5% of Central American solid tumor cases have a germline mutation in genes known or suspected to influence cancer risk. Jesica M. Godinez Paredes, Claudia Garrido, Patricia Calderón, Isabel Rodríguez, Yi Xie, Jia Liu, Wen Luo, Herbert Higson, Kristine Jones, Hong Lou, Lisa Garland, Dulilio Leytón, Lesly Chapman, Edmundo Torres-González, Julie Sawitzke, Matthew Gianferante, Sandra Luna-Fineman, Veronica Girón, Mauricio Castellanos, Federico Antillón-Klussman, Lisa Mirabello, Gerardo Mejia, Michael Dean. The spectrum of germline cancer gene mutations in Central American pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2273.

Purpose of the study: YAP1 is the most frequently amplified oncogene in cervical cancer. The Cancer Genome Atlas Research Network study showed that the YAP1 (11q22) oncogene is amplified in 11% of cervical cancer cases. Our lab recently identified that YAP1 oncogene amplification is associated with a 12-year earlier age of diagnosis, poorer survival, and defies a novel aggressive cervical cancer subtype that is more frequent in minority populations. The present study aims to characterize 19 YAP1 amplified cervical cancers from Guatemala and Venezuela via Oxford Nanopore technology (ONT) whole genome long-read sequencing. Methods: In this study, 380 cervical cancer patients were assessed for YAP1 amplification using a TaqMan assay, with 45 samples identified as positive for YAP1 amplification. These 45 samples were then subjected to whole genome sequencing with 5x coverage using Oxford Nanopore Technologies (ONT) to confirm the amplification status. A subset of 19 YAP1-amplified samples underwent deeper sequencing via ONT-LSK114 library preparation. The HPV integration status in these samples was determined by uploading the sequencing data (fastq_pass files) to the Cancer Genomics Cloud, where breakpoints were manually examined using the Integrated Genome Viewer (IGV). HPV and human DNA segments were further analyzed and identified using bioinformatics tools such as BLAT and BLAST. Results: In our study, we confirmed YAP1 amplification in 24 out of 45 tumor samples using barcode sequencing, and due to limited DNA availability (1 µg), only 19 of these samples underwent deep sequencing with 25-30x coverage. Notably, we observed a higher frequency of HPV integration (11/19) in YAP1-amplified tumors compared to those with episomal HPV status, suggesting a potential link between HPV integration and YAP1 amplification. We noticed that YAP1 amplification is driven by Breakage-Fusion-Bridge (BFB) events, which also lead to the co-amplification of the nearby BIRC2/3 genes, contributing to genomic instability and immune escape. This mechanism was further supported by a similar pattern observed in the TCGA whole genome sequence data, where 27 out of 31 YAP1-amplified cervical tumors also exhibited BFB events. Finally, treatment with the PIK3CA inhibitor Alpelisib resulted in a decrease in YAP1 protein levels in CaSki cells, a cervical cancer cell line with mutated PIK3CA and amplified YAP1 expression. Conclusions: These findings highlight a possible interplay between HPV integration and BFB-driven amplification of YAP1 and its neighboring genes, offering insights into potential therapeutic targets for this novel aggressive subtype of cervical cancer. Citation Format: Sonam Tulsyan, Hong Lou, Yi Xie, Emma Robinson, Danielle Kayembe, Eduardo Gharzouzi, Roberto Orozco, Enrique Alvirez, Michael Dean. Identifying a novel aggressive subtype of cervical cancer predominantly affecting minority populations through long-read whole genome sequencing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B018.

Burkitt lymphoma (BL) is a B-cell malignancy that disproportionately affects children in sub-Saharan Africa. We performed a genome-wide association study (GWAS) in a combined set of 800 childhood cases and 3865 controls in East Africa, controlling for age, sex, country, population-specific principal components, and a genetic relationship matrix. This analysis identified a BL-protective region within chromosome 21q22.12 tagged by the rs111457485-T allele (odds ratio [OR] = 0.57; p = 5.7 × 10−9). The results were robust in standard meta-analysis (OR = 0.57, p < 1.6 × 10−8), sensitivity analyses (removing genomic outliers and related individuals), and after adjustment for Epstein-Barr virus (EBV) status. Genomic analyses revealed long-range (over ~700 kb) chromatin interactions between the chr21q22.12 locus and the RUNX1-P1 promoter region. The African-specific rs2242780-C allele (r2 = 0.69 with the rs111457485-T allele in the study controls) showed increased enhancer activity in in-vitro Luciferase reporter assays (p = 4.5 × 10−10), nominating it as the likely functional variant for the BL-associated loci. In addition to the association with reduced BL risk in GWAS (OR = 0.62, p = 2.24 × 10−8), the rs2242780-C allele was also associated with better survival in patients with abdominal-only BL in exploratory analyses (hazard ratio = 0.39, p = 0.038, 106 patients, 59 deaths). Our GWAS uncovered novel BL-protective loci near RUNX1, offering insights into the genetic etiology of BL in African children.

Severe corneal injuries often result in corneal scarring, leading to visual impairment and corneal blindness. Currently, there is a lack of effective anti-corneal fibrosis drugs in clinical practice. MicroRNA-based therapies hold significant potential in combating fibrosis. However, the barrier function of the cornea and the fluid environment of the ocular surface reduce drug permeability and bioavailability, presenting significant challenges for local drug application. This study employs microfluidic technology to encapsulate miRNA29b in lipid nanoparticles (LNP) to create an LNP-miRNA29b delivery system (LNP-mir29b) for treating corneal mechanical injuries. In vitro experiments show that LNP-mir29b significantly inhibits the expression of α-smooth muscle actin (α-SMA) in an induced corneal stromal cell fibrosis model. In vivo experiments using rabbit corneal mechanical injury models indicate that LNP-mir29b effectively reduces fibrosis in the corneal stroma, promotes organized rearrangement of stromal collagen fibers, and decreases the expression of fibrosis-related genes, including Col1A2, Col3A1, Fn, and α-SMA. Additionally, LNP-mir29b accelerates the migration of corneal epithelial cells, promotes wound healing of the epithelium, restores the structural integrity of the corneal epithelium. The LNP system proposed in this study offers a novel approach with anti-fibrotic functionality, providing a new strategy for reducing scarring during the corneal injury repair process.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

To better understand cervical cancer progression, we analyzed RNA from 262 biopsies from women referred for colposcopy We determined HPV type and analyzed the expression of 51 genes. HPV31 was significantly more prevalent in precancer than stage 1 cancer and invasive cancer (p < 0.0001) and HPV16 increased in invasive disease (p < 0.0001). CCNE1, MELTF, and ULBP2 were significantly increased in HPV16-positive compared to HPV31 precancers while NECTIN2 and HLA-E expression decreased. Markers of the innate immune system, DNA repair genes, and cell cycle genes are significantly increased during cancer progression (p = 0.0001). In contrast, the TP53 and RB1 tumor suppressor gene expression is significantly decreased in cancer cells. TheT cell markers CD28 and FLT3LG expression decreased in cancer while FOXP3, IDO1, and ULBP2 expression increased. There is a significantly higher survival rate in individuals with increased expression of CD28 (p = 0.0005), FOXP3 (p = 0.0002), IDO1 (p = 0.038), FLT3LG (p = 0.026), APOBEC3B (p = 0.0011), and RUNX3 (p = 0.019), and a significantly lower survival rate in individuals with increased expression of ULBP2 (p = 0.035). These results will help us understand the molecular factors influencing the progression of cervical precancer to cancer. ### Competing Interest Statement The authors have declared no competing interest.

Abstract HPV16 is the most oncogenic type of human papillomaviruses (HPV). Integration of HPV into the human genome is an important mechanism of carcinogenesis but is absent in at least 30% of HPV16+ tumors. We applied long-read whole-genome sequencing (WGS) to cervical cancer cell lines and tumors to characterize HPV16 carcinogenesis in the absence of integration. Large tandem arrays of full-length and unique truncated viral genomes integrated into multiple chromosomes were identified in two HPV16+ cell lines. The dispersion of characteristic viral variants to multiple integration sites indicates that viral deletions formed as extrachromosomal DNA (a phenomenon we term HPV superspreading). In addition, we identified an HPV16+ cell line with unintegrated (episomal) DNA that has tandem arrays of full-length, truncated, and rearranged HPV16 genomes (multimer episomes). Cytogenetic analysis of this cell line shows intense extrachromosomal HPV staining, including structures resembling double-minute chromosomes. WGS of HPV16+ cervical tumor samples from Latin America revealed that 11 of 20 tumors with only episomal HPV (EP) had intact monomer episomes. The remaining nine EP tumors had multimer and rearranged HPV genomes. The majority (80%) of HPV rearrangements and deletions disrupted the E1 and E2 genes, and EP tumors overexpressed the E6 and E7 viral oncogenes, a similar profile to tumors with HPV integration. Tumors with putative multimer HPV integrations display HPV multimers and concatemers of human and viral sequences. Our data uncovered a novel mechanism for HPV16 to cause cancer without integration through aberrant episomal replication, forming rearranged, mutated, and multimer episomes. Significance: Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.