High-risk Subtypes Research Articles

COL10A1 expression distinguishes a subset of cancer-associated fibroblasts present in the stroma of high-risk basal cell carcinoma.

Basal cell carcinoma (BCC) is the most frequently diagnosed skin cancer and the most common malignancy in humans. Different morphological subtypes of BCC are associated with a low or high risk of recurrence and aggressiveness, but the underlying biology of how the individual subtypes arise remains largely unknown. As the majority of BCCs appear to arise from mutations in the same pathway, we hypothesized that BCC development, growth and invasive potential is also influenced by the tumour microenvironment and, in particular, by cancer-associated fibroblasts (CAFs) and the factors they secrete. To characterize the stroma of the different BCC subtypes with a focus on CAF populations. To investigate the stromal features of the different BCC subtypes, we used laser capture microdissection (LCM) followed by RNA sequencing (RNA-Seq). Fifteen BCC samples from five different 'pure' subtypes (i.e. superficial, nodular, micronodular, sclerosing and basosquamous; n = 3 each) were selected and included in the analysis. Healthy skin was used as a control (n = 6). The results were confirmed by immunohistochemistry (IHC). We validated our findings in two independent public single-cell RNA-Seq (scRNA-Seq) datasets and by RNAscope. The stroma of the different BCC subtypes were found to have distinct gene expression signatures. Nodular and micronodular appeared to have the most similar signatures, while superficial and sclerosing the most different. By comparing low- and high-risk BCC subtypes, we found that COL10A1 is overexpressed in the stroma of sclerosing/infiltrative and basosquamous but not in micronodular high-risk subtypes. Those findings were confirmed by IHC in 93 different BCC and 13 healthy skin samples. Moreover, scRNA-Seq analysis of BCCs from two independent datasets found that the COL10A1-expressing population of cells is associated with the stroma adjacent to infiltrative BCC and shows extracellular matrix remodelling features. We identified COL10A1 as a marker of high-risk BCC, in particular of the sclerosing/infiltrative and basosquamous subtypes. We demonstrated at the single-cell level that COL10A1 is expressed by a specific CAF population associated with the stroma of infiltrative BCC. This opens up new, tailored treatment options, and suggests COL10A1 as a new prognostic biomarker for BCC progression.

Dysplasia Detected in Patients With Serrated Epithelial Change Is Frequently Associated With an Invisible or Flat Endoscopic Appearance, Nonconventional Dysplastic Features, and Advanced Neoplasia.

The significance of serrated epithelial change (SEC), defined as endoscopically invisible hyperplastic polyp (HP)-like mucosal change identified in patients with inflammatory bowel disease (IBD), remains unclear. Although some studies reported an increased risk of synchronous and/or metachronous colorectal neoplasia in patients with SEC, including advanced neoplasia (high-grade dysplasia or colorectal cancer), the development of SEC is not significantly associated with increased colonic inflammation. This contrasts with the reported positive correlation between increased colonic inflammation and the risk of colorectal neoplasia in ulcerative colitis, arguing against the notion that SEC may represent a form of dysplasia. As such, this study aimed to characterize the features of synchronous and metachronous dysplasia detected in patients with SEC to identify factors contributing to the increased risk of colorectal neoplasia, including advanced neoplasia, observed in a subset of these patients. Clinicopathologic features of 46 IBD patients with SEC (n=109) and synchronous and/or metachronous dysplasia (n=153) were analyzed. All dysplastic lesions were subtyped as either conventional or nonconventional dysplasia. As controls, 45 IBD patients with endoscopically visible or polypoid HP (n=75) and synchronous and/or metachronous dysplasia (n=87) were analyzed. The SEC group included 28 (61%) men and 18 (39%) women with a mean age of 58 years and a long history of IBD (mean duration: 23 years). The majority of patients (n=34; 74%) had ulcerative colitis, and 12 (26%) had Crohn's disease. Thirty-nine (85%) patients had a history of pancolitis, and 2 (4%) had concomitant primary sclerosing cholangitis. Twenty-seven (59%) patients had multifocal SEC. SEC was predominantly found in the left colon (n=52; 48%) and rectum (n=34; 31%). Dysplasia in the SEC group was often endoscopically invisible or flat (n=42; 27%) and demonstrated nonconventional dysplastic features (n=49; 32%). Six nonconventional subtypes were identified in the SEC group, including 17 (11%) dysplasia with increased Paneth cell differentiation, 12 (8%) hypermucinous dysplasia, 8 (5%) crypt cell dysplasia, 7 (5%) goblet cell deficient dysplasia, 3 (2%) sessile serrated lesion-like dysplasia, and 2 (1%) traditional serrated adenoma-like dysplasia. Advanced neoplasia was detected in 11 (24%) patients. The SEC group was more likely to have nonconventional dysplasia (32%, P <0.001), invisible/flat dysplasia (27%, P <0.001), and advanced neoplasia (24%, P <0.001) than the control group (7%, 2%, and 0%, respectively). High-risk nonconventional subtypes (ie, hypermucinous, crypt cell, and goblet cell deficient dysplasias) accounted for 18% of all dysplastic lesions in the SEC group, which were not seen in the control group ( P <0.001). The SEC group (n=35; 76%) also had a higher rate of concordance between the location of SEC and the area of synchronous/metachronous dysplasia than the control group (n=22; 49%) ( P =0.007). In conclusion, dysplasia detected in patients with SEC is often endoscopically invisible/flat (27%), nonconventional (32%, including the high-risk subtypes), and found in the same colonic segment as SEC (76%), which may in part explain why some patients with SEC are associated with an increased risk of colorectal neoplasia, including advanced neoplasia. The finding of SEC may warrant a careful follow-up colonoscopy with increased random biopsy sampling, especially in the segment of colon with SEC.

Dissecting gastric cancer heterogeneity and exploring therapeutic strategies using bulk and single-cell transcriptomic analysis and experimental validation of tumor microenvironment and metabolic interplay.

Gastric cancer, the fifth most prevalent cancer worldwide, is often diagnosed in advanced stages with limited treatment options. Examining the tumor microenvironment (TME) and its metabolic reprogramming can provide insights for better diagnosis and treatment. This study investigates the link between TME factors and metabolic activity in gastric cancer using bulk and single-cell RNA-sequencing data. We identified two molecular subtypes in gastric cancer by analyzing the distinct expression patterns of 81 prognostic genes related to the TME and metabolism, which exhibited significant protein-level interactions. The high-risk subtype had increased stromal content, fibroblast and M2 macrophage infiltration, elevated glycosaminoglycans/glycosphingolipids biosynthesis, and fat metabolism, along with advanced clinicopathological features. It also exhibited low mutation rates and microsatellite instability, associating it with the mesenchymal phenotype. In contrast, the low-risk group showed higher tumor content and upregulated protein and sugar metabolism. We identified a 15-gene prognostic signature representing these characteristics, including CPVL, KYNU, CD36, and GPX3, strongly correlated with M2 macrophages, validated through single-cell analysis and an internal cohort. Despite resistance to immunotherapy, the high-risk group showed sensitivity to molecular targeted agents directed at IGF-1R (BMS-754807) and the PI3K-mTOR pathways (AZD8186, AZD8055). We experimentally validated these promising drugs for their inhibitory effects on MKN45 and MKN28 gastric cells. This study unveils the intricate interplay between TME and metabolic pathways in gastric cancer, offering potential for enhanced diagnosis, patient stratification, and personalized treatment. Understanding molecular features in each subtype enriches our comprehension of gastric cancer heterogeneity and potential therapeutic targets.

Diffusion kurtosis imaging-based habitat analysis identifies high-risk molecular subtypes and heterogeneity matching in diffuse gliomas.

High-risk types of diffuse gliomas in adults include isocitrate dehydrogenase (IDH) wild-type glioblastomas and grade 4 astrocytomas. Achieving noninvasive prediction of high-risk molecular subtypes of gliomas is important for personalized and precise diagnosis and treatment. We retrospectively collected data from 116 patients diagnosed with adult diffuse gliomas. Multiple high-risk molecular markers were tested, and various habitat models and whole-tumor models were constructed based on preoperative routine and diffusion kurtosis imaging (DKI) sequences to predict high-risk molecular subtypes of gliomas. Feature selection and model construction utilized Least absolute shrinkage and selection operator (LASSO) and support vector machine (SVM). Finally, the Wilcoxon rank-sum test was employed to explore the correlation between habitat quantitative features (intra-tumor heterogeneity score，ITH score) and heterogeneity, as well as high-risk molecular subtypes. The results showed that the habitat analysis model based on DKI performed remarkably well (with AUC values reaching 0.977 and 0.902 in the training and test sets, respectively). The model's performance was further enhanced when combined with clinical variables. (The AUC values were 0.994 and 0.920, respectively.) Additionally, we found a close correlation between ITH score and heterogeneity, with statistically significant differences observed between high-risk and non-high-risk molecular subtypes. The habitat model based on DKI is an ideal means for preoperatively predicting high-risk molecular subtypes of gliomas, holding significant value for noninvasively alerting malignant gliomas and those with malignant transformation potential.

Do HPV 16 positive/ASC-H cervical cancer screening results predict CIN 2+ better than other high-risk HPV subtypes?

To determine whether patients with atypical squamous cells, cannot exclude high grade squamous intraepithelial neoplasia (ASC-H) cytology have a correlation between high-risk human papillomavirus (HPV) type and CIN 2+1 lesion in final pathology. The study was conducted retrospectively, using data from three tertiary gynecologic oncology centers located in various regions of Turkey. Data from 5,271 patients who had colposcopy between January 2003 and January 2021 were analyzed. A total of 163 patients who had ASC-H cervical cytology test results, based on the Bethesda 2014 classification were eligible, and of these 83 (50.9%) who tested positive for HPV were included in the study. There was no correlation between the occurrence of CIN 2+ lesions and age (p=0.053). If there was any HPV 16 positivity (only HPV 16, HPV 16 and 18, HPV 16 and others) the presence of CIN 2+ lesions in the final pathology increased significantly. In HPV 16 positive ASC-H patients, the probability of CIN 2+ lesions in the final pathology were 72.5% while this rate was 48.1% in HPV 16 negative group (p=0.033). The guidelines do not provide a comprehensive definition of the role of the HPV test in managing ASC-H. Positive high-risk HPV types, especially HPV 16, together with an ASC-H smear result should bring to mind the possibility of high-grade dysplasia.

Asciminib stands out as the superior tyrosine kinase inhibitor to combine with anti-CD20 monoclonal antibodies for the treatment of CD20+ Philadelphia-positive B-cell precursor acute lymphoblastic leukemia in preclinical models.

Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCPALL) is a high-risk acute lymphoblastic leukemia subtype characterized by the presence of BCR::ABL1 fusion gene. Tyrosine kinase inhibitors (TKIs) combined with chemotherapy are established as the first-line treatment. Additionally, rituximab (RTX), an anti-CD20 monoclonal antibody (mAb) is administered in adult BCP-ALL patients with ≥20% of CD20+ blasts. In this study, we observed a marked prevalence of CD20 expression in patients diagnosed with Ph+ BCP-ALL, indicating a potential widespread clinical application of RTX in combination with TKIs. Consequently, we examined the influence of TKIs on the antitumor effectiveness of anti-CD20 mAbs by evaluating CD20 surface levels and conducting in vitro functional assays. All tested TKIs were found to uniformly downregulate CD20 on leukemic cells, diminishing the efficacy of RTX-mediated complement-dependent cytotoxicity. Interestingly, these TKIs displayed varied effects on NK cell-mediated antibody-dependent cytotoxicity and macrophage phagocytic function. While asciminib demonstrated no inhibition of effector cell functions, dasatinib notably suppressed the anti-CD20-mAb-mediated NK cell cytotoxicity and macrophage phagocytosis of BCP-ALL cells. Dasatinib and ponatinib also decreased NK cell degranulation in vitro. Importantly, oral administration of dasatinib, but not asciminib, compromised NK cell activity within patients' blood, determined by ex vivo degranulation assay. Our results indicate that asciminib might be preferred over other TKIs for combination therapy with anti-CD20 mAbs.

Phase II/III study of paclitaxel/carboplatin alone or combined with either trastuzumab and hyaluronidase-oysk or pertuzumab, trastuzumab, and hyaluronidase-zzxf in HER2 positive, stage I-IV endometrial serous carcinoma or carcinosarcoma (NRG-GY026).

TPS5641 Background: Uterine serous carcinoma and uterine carcinosarcoma are rare, high-risk uterine cancer subtypes that overexpress HER2 in up to 40% and 10% of cases, respectively. Phase II data demonstrated a benefit in adding trastuzumab in HER2 positive advanced and recurrent uterine serous carcinoma. Dual anti-HER2 therapy has the potential for further improvement in survival. Methods: This is a non-blinded, three-arm randomized phase II/III study. The primary objective is to evaluate the efficacy of trastuzumab and hyaluronidase-oysk (HERCEPTIN HYLECTA) or a fixed dose combination of pertuzumab, trastuzumab, and hyaluronidase-zzxf (PHESGO) in combination with paclitaxel/carboplatin in patients with HER2 positive uterine serous carcinoma or carcinosarcoma. The primary outcome of the phase II component is PFS. Depending on the phase II results, the study will progress to a phase III study of either two or three study arms. Patients in the control arm (Arm 1) receive carboplatin and paclitaxel every 3 weeks for 6 cycles. Patients on Arm 2 receive carboplatin and paclitaxel with trastuzumab and hyaluronidase-oysk every 3 weeks for 6 cycles plus one year of maintenance trastuzumab and hyaluronidase-oysk. Patients enrolled on Arm 3 receive carboplatin and paclitaxel with trastuzumab, pertuzumab, and hyaluronidase-zzxf every 3 weeks for 6 cycles plus one year of maintenance trastuzumab, pertuzumab, and hyaluronidase-zzxf . Those with an ongoing partial response may continue maintenance therapy for up to three years. Patients are eligible if they are diagnosed with chemo-naïve, non-recurrent stage IA-IVB myoinvasive uterine serous or carcinosarcoma. All tumors must be HER2 positive as defined by the ASCO/CAP Breast Cancer 2018 guidelines (testing performed locally). Tumors may also be HER2 positive based on next generation sequencing testing. Patients may have measurable, non-measurable, or no measurable disease and may have a plan for vaginal brachytherapy . Key exclusion criteria include severe heart disease or interstitial lung disease. The phase II portion of this study is open and enrolling. Clinical trial information: NCT05256225 .

The incidence of brain metastases in breast cancer according to molecular subtype and stage: a 10-year single institution analysis.

Breast cancer (BC) is the second most common etiology of brain metastases (BrM). We aimed to examine the incidence of BrM among all BC patients presenting to a large tertiary cancer centre over one decade. We included all BC patients presenting consecutively between 2009 and 2019 and cross referenced that cohort to a radiotherapy database, identifying patients treated for BrM at any time following their initial presentation. Cumulative incidences (CI) of BrM diagnoses were calculated using death as a competing risk and compared using the Fine-Gray method. Overall survival was estimated using the Kaplan Meier method. We identified 12,995 unique patients. The CI of BrM in patients who initially presented with Stage 0-4 disease was 2.1%, 3.7%, 9.4%, 10.6%, and 28.7%, respectively at 10years. For 8,951 patients with available molecular subtype data, 6,470 (72%), 961 (11%), 1,023 (11%), and 497 (6%) had hormone-receptor (HR)-positive/ERBB2-, HR-negative/ERBB2-, HR-positive/ERBB2 + , and HR-negative/ERBB2 + disease, respectively; the CI of BrM in each was 7.6%, 25.3%, 24.1%, and 26.6%, at 10years following BC diagnosis, respectively. Median overall survival (OS) following BC diagnosis and BrM diagnosis was 28years 95% CI [25, 32] and 10months 95% CI [9, 12], respectively. From a large, registry-based study, we observed that patients with ERBB2 + and triple negative BC have the highest incidence of BrM. Our data supports prospective surveillance brain MRI studies. Given advancements in BrM treatment, clinicians should have a low threshold for brain imaging in BC patients with high risk subtypes.

Role of mitophagy in head and neck squamous cell carcinoma: Prognosis and immune insights

ObjectiveTo explore the correlation between mitophagy and the tumor microenvironment (TME) in patients with head and neck squamous cell carcinoma (HNSCC), with an aim to enhance therapeutic efficacy for HNSCC. MethodsA machine learning-based multigene prognostic signature was developed based on mitophagy-related differentially expressed genes (MRGs) identified in The Cancer Genome Atlas cohort. This signature was correlated with the TME using gene set enrichment analysis. The association between this prognostic signature and various immunological features of the TME was explored, including status of tumor-infiltrating immune cells, expression of immune checkpoint molecules, and the immunoscore. Immunohistochemistry validated the expression of hub gene CSNK2A2 and assessed its relationship with immunomarker expression. Quantitative PCR validated CSNK2A2 knockdown in HNSCC cell lines. Functional experiments including Transwell assays to determine cell migration and invasion, Cell Counting Kit 8 assay, and 5-ethynyl-2-deoxyuridine assay were performed to confirm the role of CSNK2A2 in HNSCC. Finally, a subcutaneous xenograft model was generated in C3H mice to validate our findings. ResultsThe MRG-based prognostic signature showed excellent predictive performance. High-risk patients had significantly shorter progression-free and overall survival (P ​< ​0.0001) than low-risk patients. CD8+ T cell infiltration was lower in high-risk groups, whereas low-risk groups showed higher immunological marker expression. Thus, the low-risk HNSCC subtype may benefit from immune therapy, while high-risk subtypes may benefit from chemotherapy (P ​< ​0.001). CSNK2A2 was highly expressed and strongly correlated with CD8 and PD-L1 based on immunohistochemistry of the HNSCC tissue microarray. CSNK2A2 knockdown reduced cell migration, invasion, and proliferation, and arrested cells in G1 phase. In vivo, it led to slower tumor growth and smaller tumor volumes. ConclusionWe established a potential prognostic signature that could improve HNSCC management in the future. CSNK2A2 may be a new biomarker to predict immunotherapy efficacy in HNSCC.

Identification of immunogenic cell death-related damage-related molecular patterns (DAMPs) to predict outcomes in patients with head and neck squamous cell carcinoma

PurposeHead and neck cancer is the sixth most common type of cancer worldwide, wherein the immune responses are closely associated with disease occurrence, development, and prognosis. Investigation of the role of immunogenic cell death-related genes (ICDGs) in adaptive immune response activation may provide cues into the mechanism underlying the outcome of HNSCC immunotherapy.MethodsICDGs expression patterns in HNSCC were analyzed, after which consensus clustering in HNSCC cohort conducted. A 4-gene prognostic model was constructed through LASSO and Cox regression analyses to analyze the prognostic index using the TCGA dataset, followed by validation with two GEO datasets. The distribution of immune cells and the response to immunotherapy were compared between different risk subtypes through multiple algorithms. Moreover, immunohistochemical (IHC) analyses were conducted to validate the prognostic value of HSP90AA1 as a predictor of HNSCC patient prognosis. In vitro assays were performed to further detect the effect of HSP90AA1 in the development of HNSCC.ResultsA novel prognostic index based on four ICDGs was constructed and proved to be useful as an independent factor of HNSCC prognosis. The risk score derived from this model grouped patients into high- and low-risk subtypes, wherein the high-risk subtype had worse survival outcomes and poorer immunotherapy response. IHC analysis validated the applicability of HSP90AA1 as a predictor of prognosis of HNSCC patients. HSP90AA1 expression in tumor cells promotes the progression of HNSCC.ConclusionsTogether, these results highlight a novel four-gene prognostic signature as a valuable tool to assess survival status and prognosis of HNSCC patients.

Pathogenic Mechanisms Activated by NSD2 Translocations in Multiple Myeloma

The NSD2 gene is overexpressed in multiple cancers and is associated with cancer cell proliferation, invasion, and metastasis. NSD2 gene is universally present in multiple myeloma (MM) patients with t(4;14) (p16;q32) translocation, a high-risk subtype. NSD2 is characterized by different breakpoints. NSD2Δ3 breakpoint is associated with shorter survival than other breakpoints. NSD2Δ3 contains non-coding RNA ACA11 within intron 18. Additionally, 80% of t(4;14) MM patients lack the retinoblastoma tumor suppressor gene (RB1). We hypothesize that ACA11 cooperates with RB1 loss in MM pathogenesis. Also, we hypothesize that NSD2Δ3 increases protein expression driving myeloma growth. To overexpress NSD2WT, snoACA11, and NSD2Δ3 in cells, we cloned NSD2WT, and NSD2Δ3 plasmid constructs in a murine stem cell virus (MSCV)-IRES- GFP vector. We isolated primary B cells from RB1 wild-type and knock-out mice by CD43 microbeads and transduced those cells using a virus containing ACA11 and NSD2WT. ACA11 overexpression significantly increased B cell proliferation in Rb1dKO mice. This hyperproliferative phenotype is induced by reactive oxygen species (ROS) levels. However, we did not find a further increase in B cell proliferation with co-overexpression of NSD2WT and ACA11 in Rb1dKO mice. We demonstrated increased protein stability by finding higher NSD2Δ3 protein expression compared to NSD2WT protein expression in HEK293T cells. These results support our hypothesis and demonstrate oncogenic role of ACA11 in t(4;14) MM. Future work will be done to determine the cell biological effects of NSD2Δ3 and establish its role in myeloma pathogenesis. Completion of this study will advance understanding of this epigenetic NSD2 gene and potentially develop targeted drugs for NSD2, providing more individualized treatment options for these patients. University of Iowa Department of Internal Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enhancing NSCLC recurrence prediction with PET/CT habitat imaging, ctDNA, and integrative radiogenomics-blood insights

While we recognize the prognostic importance of clinicopathological measures and circulating tumor DNA (ctDNA), the independent contribution of quantitative image markers to prognosis in non-small cell lung cancer (NSCLC) remains underexplored. In our multi-institutional study of 394 NSCLC patients, we utilize pre-treatment computed tomography (CT) and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to establish a habitat imaging framework for assessing regional heterogeneity within individual tumors. This framework identifies three PET/CT subtypes, which maintain prognostic value after adjusting for clinicopathologic risk factors including tumor volume. Additionally, these subtypes complement ctDNA in predicting disease recurrence. Radiogenomics analysis unveil the molecular underpinnings of these imaging subtypes, highlighting downregulation in interferon alpha and gamma pathways in the high-risk subtype. In summary, our study demonstrates that these habitat imaging subtypes effectively stratify NSCLC patients based on their risk levels for disease recurrence after initial curative surgery or radiotherapy, providing valuable insights for personalized treatment approaches.

Incidence of oncogenic HPV infection in women with and without mental illness: A population-based cohort study in Sweden.

Women with mental illness experience an increased risk of cervical cancer. The excess risk is partly due to low participation in cervical screening; however, it remains unknown whether it is also attributable to an increased risk of infection with human papillomavirus (HPV). We aimed to examine whether women with mental illness had an increased infection rate of HPV compared to women without mental illness. Using a cohort design, we analyzed all 337,116 women aged 30 to 64 and living in Stockholm, who had a negative test result of 14 high-risk HPV subtypes in HPV-based screening, during August 2014 to December 2019. We defined women as exposed to mental illness if they had a specialist diagnosis of mental disorder or had a filled prescription of psychotropic medication. We identified incident infection of any high-risk HPV during follow-up and fitted multivariable Cox models to estimate hazard ratios (HR) with 95% confidence intervals (CI) for HPV infection. A total of 3,263 women were tested positive for high-risk HPV during follow-up (median: 2.21 years; range: 0 to 5.42 years). The absolute infection rate of HPV was higher among women with a specialist diagnosis of mental disorder (HR = 1.45; 95% CI [1.34, 1.57]; p < 0.001) or a filled prescription of psychotropic medication (HR = 1.67; 95% CI [1.55, 1.79]; p < 0.001), compared to women without such. The increment in absolute infection rate was noted for depression, anxiety, stress-related disorder, substance-related disorder, and ADHD, and for use of antidepressants, anxiolytics, sedatives, and hypnotics, and was consistent across age groups. The main limitations included selection of the female population in Stockholm as they must have at least 1 negative test result of HPV, and relatively short follow-up as HPV-based screening was only introduced in 2014 in Stockholm. Mental illness is associated with an increased infection rate of high-risk HPV in women. Our findings motivate refined approaches to facilitate the WHO elimination agenda of cervical cancer among these marginalized women worldwide.

Abstract 4947: Uncovering the molecular basis for clinically relevant sphingolipidomic subtypes in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) has been extensively studied at the genomic level, resulting in various genomic classifications. Despite the progress, three key challenges remain: 1) a significant proportion of patients lack identified genomic features, 2) a discrepancy exists between genomic risk classification and clinical outcomes, and 3) many mutated targets remain non-druggable. There is an urgent need for an enhanced AML risk classification that goes beyond genomic aberrations to guide effective therapeutics. Recent research, including our own, has increasingly linked AML pathogenesis and therapeutic resistance with dysfunctional sphingolipid metabolism, a family of bioactive molecules crucial for cellular functions. In a recent manuscript, we identified two robust sphingolipidomic clusters in AML, characterized by reciprocal abundances of hexosylceramide (Hex) and sphingomyelin (SM) species (HexloSMhi, HexhiSMlo). These clusters correlate with latent transcriptional states and stratify patient groups in multiple independent datasets such as TCGA, and BeatAML, with the HexloSMhi cluster representing a high-risk subgroup associated with poor clinical outcomes. These findings underscore the role of sphingolipids in refining AML risk assessment and creating avenues for discovery and therapeutic intervention. However, the signaling states driving these sphingolipidomic clusters and the mechanisms elevating risk in the HexloSMhi cluster require further investigation. To identify regulatory genes for the AML subtypes, we constructed gene regulatory networks using differentially expressed genes. Focusing on the high-risk subtype, we identified highly connected genes ranked by connectivity, including KDM1A, GSK3β, LCK, and STAT5A. We found that higher STAT5A abundances in the HexloSMhi subtype, particularly correlated with pro-survival Bcl-xL proteins. Furthermore, we identified STAT5 protein abundance as a key variable associated with drug sensitivity from pharmacological screening with sphingolipid pathway inhibitors and AML therapeutics 30 AML cell lines and patient samples. This is encouraging as STAT5 is druggable as evidenced in the NIH-Pharos druggable genome database, and several small-molecule inhibitors are currently in clinical trials. Analysis of gene expression data suggest that STAT5 inhibition, through genetic or pharmacological means, downregulates cell cycle-related genes and significantly alters sphingolipid gene expression. Recent work by others indicates that sphingolipid-catalyzing enzymes regulate STAT5 activity in leukemias, with ceramides acting as a second messenger in cells. Taken together, this suggests a possibility of a positive feedback loop between sphingolipid homeostasis and activity of STAT5; the details of this interaction require further investigation. Citation Format: B Bishal Paudel, Su-Fern Tan, Johnson Ung, David Claxton, David J. Feith, Thomas P. Loughran, Kevin A. Janes. Uncovering the molecular basis for clinically relevant sphingolipidomic subtypes in acute myeloid leukemia [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 4947.

Abstract 363: Genetic or pharmacological inactivation of CREBBP sensitizes B-cell acute lymphoblastic leukemia to ferroptotic cell death upon BCL2 inhibition

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological malignancy of B lineage progenitors. It remains a leading cause of death in childhood, while outcomes in adults are dismal. There is therefore a need to better understand drivers of high-risk B-ALL and to develop novel therapeutic approaches targeting these challenging patient cohorts. Loss-of-function mutations affecting CREBBP are recurrent second-hit mutations across multiple genetic subtypes of B-ALL and are associated with adverse features, including high-risk genetic subtypes and persistent measurable residual disease. In addition, they have been mechanistically associated with chemoresistance and are more frequently found in relapse. We sought to identify novel treatment options for CREBBP-mutated high-risk B-ALL. CREBBP-mutated isogenic human B-ALL cell lines were genome-engineered to provide a platform for synthetic lethal drug screening. We subjected these cell lines to a targeted drug screen focused on clinically-actionable drugs in classes that have either been implicated or hypothesized to show differential sensitivity patterns in published models of B cell lymphoma and other CREBBP-mutated malignancies. Unexpectedly, CREBBP-mutated cells were not differentially sensitive to traditional cytotoxic chemotherapy, and paradoxically showed a degree of sensitization to the glucocorticoid Dexamethasone, used in current ALL induction regimens. As anticipated, and validating our screen design, inhibitors of the CREBBP paralogue EP300 (the CREBBP/EP300-specific bromodomain inhibitor Inobrodib and the CREBBP/EP300 acetylase inhibitor A485) exhibited synthetic lethality, consistent with previous reports in B-cell lymphoma. The most potent hit was the BCL2 inhibitor Venetoclax, which we show acts through a non-canonical, but BCL2-dependent mechanism resulting in ferroptotic programmed cell death. CREBBP-mutated cell lines were transcriptionally and functionally characterized, revealing underlying differences in cell-cycle, metabolism and response to oxidative stress. Acquisition of resistance to Venetoclax further dysregulated these pathways and resulted in a transcriptionally-convergent state. Lastly, we demonstrate that small-molecule inhibition of CREBBP sensitizes B-ALL cells, regardless of genotype, to Venetoclax-induced ferroptosis in vitro and in vivo, providing a potential novel drug combination for broader clinical translation in B-ALL. In summary, we have identified a number of actionable compounds that specifically target CREBBP-mutated high-risk B-ALL, demonstrate a novel mechanism-of-action for the BCL2 inhibitor Venetoclax in B-ALL and propose CREBBP-inhibitors and Venetoclax as a novel treatment combination for B-ALL across genotypes. Citation Format: Alicia Garcia Gimenez, Jonathan Ditcham, Dhoyazan M. Azazi, Eshwar Meduri, Ryan Asby, Nathalie Sakakini, Cecile K. Lopez, Nisha Narayan, Jaana Bagri, Tumas Beinortas, Shuchi Agrawal Singh, George Giotopoulos, Michael P. Murphy, Sarah J. Horton, Brian J. Huntly, Simon E. Richardson. Genetic or pharmacological inactivation of CREBBP sensitizes B-cell acute lymphoblastic leukemia to ferroptotic cell death upon BCL2 inhibition [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 363.

Abstract 3952: Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia

Abstract Detection of minimal residual disease (MRD) after initial diagnosis (IDX) and therapy is a strong prognostic marker for relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have explored clonal genetic and gene expression changes between initial diagnosis and relapse. However, studying tumor cells present at MRD is challenging due to their low abundance. Examining the genomic profiles of leukemia cells that resist induction chemotherapy and understanding their evolutionary relationship to cells present at diagnosis may lead to a better understanding of the cells that eventually seed relapse. We analyzed somatic point mutations, indels, and copy number variants from 41 pediatric B-ALL patients with high-risk B-ALL. We specifically focused on patients from the following four common high-risk subtypes: KMT2A-rearranged, Ph+, Ph-like, and iAMP21, who had detectable disease at the end of induction. Leukemic burden at end-induction (day 29) ranged from 0.03% to 66.00%. Data were generated in partnership with the National Cancer Institute’s Human Tumor Atlas Network and the Children’s Oncology Group. Our data set of 41 patients includes 39 with paired initial diagnostic and end-induction samples that we used to create tumor evolution models based on single cells across disease stages. We produced single-cell multiome gene expression (scRNA) and chromatin accessibility (scATAC) profiles (10x Genomics) with a median of 7428 leukemic blast cells at IDX (range: 2490-14219) and 1907 at MRD (range: 240-10167) after filtering. We called somatic mutations from scRNA and scATAC using SComatic and Monopogen, and we detected copy number changes using Numbat and EpiAneufinder. We also designed a targeted DNA sequencing panel including 335 variants based on B-ALL literature and database searches to produce scDNA (Mission Bio) for 24 patients (16 IDX-MRD pairs) with a median of 2346 cells profiled at IDX (range: 340-8849) and 1828 at MRD (range: 596-5133). We built tumor phylogenies based on somatic SNVs and CNVs with COMPASS. Using multiple tools and complementary single-cell omics data types to detect somatic changes improved our confidence in the events used for tumor evolution tree construction. Our approach allowed us to define recurrent genomic events and patterns across pediatric B-ALL subtypes and to describe the clonal evolution of cells measured at initial diagnostic and end-induction disease stages. We were also able to link cell-specific mutation events to their transcriptional and epigenomic signatures, including mapping mutation events along the B-cell development trajectory and within differentially expressed gene pathways. Some cases showed dramatic shifts in subclonal structure between IDX and MRD that may be related to therapy-induced clonal selection, such as a loss of NRAS hotspot mutation. Citation Format: Steven M. Foltz, Avi Loren, Changya Chen, Rushabh Mehta, Elizabeth Li, Jason Xu, Fatemeh Alikarami, Kathrin M. Bernt, Kai Tan. Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia [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 3952.

Abstract 141: A human induced pluripotent stem cell model of KMT2A rearranged leukemia

Abstract Acute lymphoblastic leukemia in infants (iALL) is a high-risk subtype of childhood leukemia with poor survival outcomes despite intensive therapies. Rearrangement of KMT2A (KMT2A-r) occurs in 70% of cases and is associated with refractoriness to therapy, early relapse, and rapid leukemia progression. KMT2A-r generates a driver fusion oncogene, most commonly KMT2A::AFF1 in iALL, which leads to epigenetic dysregulation of target gene transcription. iALL with KMT2A::AFF1 is an unusual cancer in that other somatic mutations are uncommon, there are no known genetic risk factors, and phenotypic switch from lymphoid to myeloid, mixed, or undifferentiated phenotype is common during treatment and particularly resistant to therapies. The cell of origin is thought to be a very early hematopoietic precursor, with transcriptomic studies of iALL blasts showing similarities to hematopoietic stem and progenitor cells (HSPCs), multipotent progenitors and early lymphoid progenitors (ELPs). Unfortunately, research into this rare but devastating disease has been hindered by a lack of appropriate, representative models. Interestingly, KMT2A-r originates in HSPCs in utero and the age of onset of iALL is a strong prognostic factor, with age younger than six months associated with the highest risk of relapse. However, little is known regarding how KMT2A-r subverts early hematopoiesis or drives the severe disease phenotype. In an effort to understand the role of the developmental state of the cell of origin in iALL, we have created a highly controlled induced pluripotent stem (iPS) cell model system of KMT2A::AFF1 leukemia. Specifically, we engineered human iPS cell lines with doxycycline regulatable expression of KMT2A::AFF1 fusion and we are employing CRISPR gene editing technology to introduce clinically identified variants of interest. Utilizing directed differentiation, we have produced functional HSPCs from iPS cells. Notably, this model recapitulates hematopoietic ontogeny, with the ability to control iALL induction at specific developmental stages. Following doxycycline treatment, we demonstrate that the cells express KMT2A::AFF1 fusion transcripts. We are utilizing single cell genomics to investigate transcriptomic changes during hematopoietic differentiation of our KMT2A::AFF1 iPS cells. We will compare these results to published data sets of single cell RNA sequencing data from healthy fetal HSPCs and to our existing iALL and pediatric KMT2A-r ALL single cell sequencing data sets. Our iPS cell based iALL model system provides the opportunity to investigate a range of critical and outstanding questions of iALL disease initiation, progression, and treatment. Citation Format: Erin Guest, Meagan Vacek, Jacqelyn Nemechek, Irina Pushel, Bradley T. Thornton, Molly Leyda, Midhat S. Farooqi, John M. Perry, Jay L. Vivian. A human induced pluripotent stem cell model of KMT2A rearranged leukemia [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 141.

Research and future directions of HPV-related vaccines

Human papillomavirus (HPV) infection is a major cause of cervical lesions such as cervical cancer in women. Prophylactic vaccines developed against HPV have been widely used, but these vaccines are only effective against certain types of HPV and do not work in people who are already infected. Therefore, the development of therapeutic vaccines is also particularly important. The use of the modified poxvirus (MVA) as a vector for HPV vaccinations has garnered attention in recent years. It has been found that modified poxvirus vaccines are able to combat human papillomavirus infection and have significant safety and efficacy profiles. Based on the study of the distribution of HPV types in different regions and populations, this paper finds that there are differences in the prevalence of high-risk HPV subtypes in different regions, and that a large number of infected people need more effective treatments. At the same time, by comparing the advantages and disadvantages of preventive and therapeutic vaccines, we recognize that both have roles that cannot be replaced by the other in the task of fighting HPV, and that the two vaccines need to be developed together and complement each other in order to minimize the harm of HPV. Based on these findings, this paper provides a constructive reference for future targeted prevention and treatment of HPV in different regions of the world with different HPV distributions.

Correlation of 1p/16q loss of heterozygosity and 1p gain with clinicopathological characteristics and prognosis in Wilms tumor

Objective: To investigate the relationship between 1p/16q loss of heterozygosity (LOH) and 1p gain in Wilms tumor and their clinicopathologic characteristics and prognosis. Methods: A total of 175 Wilms tumor samples received from the Department of Pathology, Beijing Children's Hospital from September 2019 to August 2022 were retrospectively analyzed. The histopathologic type and presence of lymph node involvement were evaluated by two pathologists. The clinical data including patients'gender, age, tumor location, preoperative chemotherapy, and tumor stage were summarized. Fluorescence in situ hybridization (FISH) was done to detect 1p/16q LOH and 1p gain and their correlation with the clinicopathological features and prognosis were analyzed. Results: Among the 175 samples, 86 cases (49.1%) were male and 89 (50.9%) were female. The mean age was (3.5±2.9) years, and the median age was 2.6 years. There were 26 (14.9%) cases with 1p LOH, 28 (16.0%) cases with 16q LOH, 10 (5.7%) cases of LOH at both 1p and 16q, and 53 (30.3%) cases with 1q gain. 1q gain was significantly associated with 1p LOH (P<0.01) and 16q LOH (P<0.01). There were significant differences (P<0.01) between 1q gain, 1p LOH and 16q LOH among different age groups. The rate of 16q LOH in the high-risk histopathological subtype (50.0%) was significantly higher than that in the intermediate-risk subtype (13.6%, P<0.05). The frequency of 1q gain, 1p LOH, and 16q LOH in children with advanced clinical stages (Ⅲ and Ⅳ) was significantly higher than that in children with early clinical stages (Ⅰ and Ⅱ). 1q gain, 1p LOH, and 16q LOH showed no significant correlation with gender, unilateral or bilateral disease, chemotherapy, or lymph node metastasis. The progression-free survival (PFS) time for patients with 1q gain and 1p LOH was significantly shorter than those without these aberrations (P<0.05). Additionally, the PFS time of patients with 16q LOH was slightly shorter than those with normal 16q, although the difference was not statistically significant. Patients with stage Ⅲ to Ⅳ disease exhibiting 1q gain or 1p LOH had a significantly higher relative risk of recurrence, metastasis, and mortality. Conclusions: 1p/16q LOH and 1q gain are associated with age, high-risk histological type, and clinical stage in Wilms tumor. 1q gain and 1p LOH are significantly correlated with the prognosis of Wilms tumor.

Abstract P30: Therapeutic activation of pre-B-cell receptor signaling in high-risk pediatric ALL: exploiting TCF3 and MYC-linked vulnerabilities

Abstract P30: Therapeutic activation of pre-B-cell receptor signaling in high-risk pediatric ALL: exploiting TCF3 and MYC-linked vulnerabilities