Chromosome 9p21 Research Articles

Abstract 4636: Genome-wide drug anchor screens identify CAAP1 and AKAP17A as regulators of PRMT5 inhibitor sensitivity

Abstract MTA-cooperative PRMT5 inhibitors are an emerging treatment option for patients with one of the 10-15% of all human cancers harboring MTAP homozygous deletion. To identify potential regulators of sensitivity to PRMT5 inhibitors, we performed genome-wide CRISPR knockout screens in the presence and absence of an MTA-cooperative PRMT5 inhibitor. Knockout of CAAP1 and AKAP17A were among the strongest sensitizing hits across multiple MTAP-deleted cancer cell lines representing different histologies. Strikingly, the CAAP1 gene co-localizes with MTAP and CDKN2A on chromosome 9p21. Co-deletion of CAAP1 is reported in 20 percent of MTAP-deleted cancers in the TCGA PanCancer Atlas. CAAP1 or AKAP17A knockout in MTAP-deleted cancer cell lines sensitized the cells to PRMT5 inhibitors including the clinical stage MTA-cooperative inhibitors, TNG908 and TNG462, and the non-MTA-cooperative inhibitor, GSK3326595. Moreover, we discovered that CAAP1 and AKAP17A protein levels are interdependent, as knockout of either gene caused decreased protein levels for the other. Consistent with this finding, CAAP1 reconstitution in CAAP1-deleted cell lines led to increased AKAP17A levels. Endogenous CAAP1 and AKAP17A protein levels are positively correlated across a panel of cancer cell lines and MTAP-deleted patient-derived xenograft models. Consistent with a previous report (Ni et al., 2023), exogenous CAAP1 and AKAP17A co-immunoprecipitation studies suggest that the proteins form a protein complex. AKAP17A and CAAP1 are not well-characterized proteins, but PRMT5 inhibitors induce global alternative splicing events (ASEs) in cancer cells, and based on preliminary studies a possible function for the CAAP1/AKAP17A complex could be to mitigate ASEs induced by PRMT5 inhibition. Collectively, these data indicate that CAAP1 and AKAP17A exist interdependently and mediate sensitivity to PRMT5 inhibitors. The colocalization and 20 percent incidence of CAAP1 deletion in the setting of MTAP deletion may suggest that such patients will have improved responses to PRMT5-targeted therapy. S. Yoda and M. R. Tonini contributed equally. Citation Format: Satoshi Yoda, Matthew R. Tonini, Hilary E. Nicolson, Samuel Poleretzky, Silvia Fenoglio, Steven Lombardo, Lauren Grove, Samuel R. Meier, Ashley Choi, Yi Yu, Kevin M. Cottrell, John P. Maxwell, Teng, Jannik N. Andersen, Kimberly J. Briggs. Genome-wide drug anchor screens identify CAAP1 and AKAP17A as regulators of PRMT5 inhibitor sensitivity [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 4636.

Abstract 3634: Copy number alterations in chromosome 19 genes associate with reduced immune infiltrate and adverse outcomes in lung cancer

Abstract Deleterious mutations in STK11/LKB1, KEAP1, and SMARCA4 have been shown to be associated with adverse response to immunotherapy in several lung cancer cohorts. However, the biological and clinical implications of deletions of these genes, which all reside on chromosome 19p (chr19p), has not been extensively described. We sought to first characterize the association between immune infiltration and these copy number changes in chr19p. To do this, we analyzed mutation, copy number, and gene expression data from non-small cell lung cancer cohorts in the Cancer Genome Atlas (TCGA). Chr19p is frequently deleted across cancers in the TCGA dataset, with higher rates in lung adenocarcinoma. In both lung adenocarcinoma and squamous cell carcinoma, nearly half of TCGA tumors have deletion of chr19p including STK11, KEAP1, and SMARCA4, and these tumors are enriched for STK11 mutation. Immune infiltrate (estimated by methylation patterns) was not significantly associated with STK11 mutation but was significantly reduced in tumors with chr19 deletion when controlling for tumor type and overall copy number load. In addition, low-level deletion of any of these genes correlated with decreased expression of immune signaling pathways. Based on these findings, we next wanted to characterize the association between chr19p copy number and immunotherapy response. We previously reported that low-level deletion of STK11 correlated with an immunosuppressive, treatment-refractory phenotype in samples collected from non-small cell lung cancer (NSCLC) patients with resectable disease in a phase II study of neoadjuvant atezolizumab + chemotherapy. To confirm these findings in a larger cohort, we examined the AACR GENIE NSCLC biopharma collective (BPC) dataset. A subset of this cohort included patients treated with immunotherapy (atezolizumab, nivolumab, and/or pembrolizumab) and with copy number data available or GISTIC analysis of individual genes (n = 204). Here, deletion of any of these three genes correlated with worse overall survival, and this was statistically significant for STK11 deletion. Overall, our data suggests that a broader set of patients, defined by copy number changes in these genes, may experience this adverse immunobiology. Further study to elucidate the mechanistic implications of this association is warranted. Citation Format: Brian S. Henick, Yohanna Georgis, Benjamin O. Herzberg, Carla P. Concepcion-Crisol, Alison M. Taylor. Copy number alterations in chromosome 19 genes associate with reduced immune infiltrate and adverse outcomes in 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 3634.

Abstract 7295: A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR

Abstract The Janus kinase 2 (JAK2) gene is located on chromosome 9p24.1 and encodes a non-receptor tyrosine kinase that plays a central role in cytokine and growth factor signaling. The JAK2 protein is especially important for controlling the production of blood cells from hematopoietic stem cells. In fact, the JAK2 mutation at amino acid 617 (Valine 617 to Phenylalanine, V617F) is the most frequently detected mutation in myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). This dominant gain-of-function V617F mutation results in constitutive tyrosine phosphorylation activity, leading to uncontrolled blood cell production, including increased numbers of erythrocytes, neutrophils, and platelets. To detect the JAK2 V617F mutation in DNA from patient blood samples, we developed a unique real-time PCR assay using our proprietary XNA technology that enables the assay to selectively amplify the mutant sequence by using a synthetic DNA analog XNA (Xenonucleic Acid). The JAK2 V617F mutation detection assay is designed to detect a valine-to-phenylalanine mutation at amino acid 617 (V617F). PCR primers, TaqMan probes, and XNA were designed for the JAK2 V617F mutation, along with primers and probes for an internal control gene in the duplex setting. The analytical performance of the assay was verified and validated using a variety of control samples. The results revealed that the XNA completely suppressed the amplification of wildtype sequence while only the V617F mutant sequence was amplified. The JAK2 V617F mutation detection assay is highly reproducible with intra- and inter-assay coefficients of variation of less than 10%. Results for the assay’s analytical sensitivity indicated that V617F could be detected with about 0.25% mutant allele frequency in 10 ng DNA input. In addition, no significant cross-amplification between V617F and V617I was observed. Thus, the JAK2 V617F mutation detection assay is specific for the V617F mutation. The JAK2 V617F mutation detection assay, using XNA-based technology, provides high sensitivity and specificity to detect the JAK2 V617F mutation. Thus, this assay would be a useful tool for clinical decision-making in determining the presence of the JAK2 V617F mutation in DNA from patient blood samples. Citation Format: Shuo Shen, Robert Brown, Daniel Kim, Larry Pastor, Andrew Y. Fu, Pushpinder Kaur, Alisha Babu, Wei Liu, Aiguo Zhang, Hiromi Tanaka, Michael Y. Sha. A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR [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 7295.

Abstract 3364: PELO is a synthetic lethal target in chromosome 9p21-deleted or MSI cancers

Abstract Homozygous deletion of chromosomal region 9p21.3 is one of the most frequently observed somatic copy number alterations in cancers, occurring in approximately 15% of all cancers. Many of the 9p21.3-deleted cancers are further characterized by poor clinical outcomes including subsets of glioblastoma, pancreatic, esophageal, and urothelial cancers. Large deletions of chromosomal regions may also provide unique therapeutic opportunities. Specifically, cancer-specific therapeutic opportunities can arise from co-deletions of passenger genes neighboring tumor suppressors. As such, we sought to identify novel therapeutic targets associated with Ch9p21.3 loss in cancers and identified the pelota mRNA surveillance and ribosome rescue factor (PELO) as the top preferential dependency in 9p21.3-deleted cancers. Intriguingly in another analysis, we identified PELO to be amongst the top preferential dependencies in cancers with microsatellite instability (MSI), which accounts for subsets of colorectal, endometrial, gastric, and ovarian cancers. Given the strength of PELO as a candidate synthetic lethal target in two distinct cancer subsets, we performed focused validation with CRISPR interference across multiple cell lines. Knockdown of PELO preferentially impaired the viability of MSI or 9p21-deleted as compared to control (9p21-proficient and microsatellite stable) cells. Further validation with patient-derived organoid and in vivo xenograft models confirmed these findings. We next sought to refine the predictive biomarkers for PELO dependency and identified loss of FOCAD (a 9p21.3 gene and an interactor of the SKI mRNA surveillance pathway) as necessary and sufficient for PELO dependency in the 9p21.3-deleted context. Our findings implicate splicing alterations involving TTC37, a key protein in the SKI mRNA surveillance complex, as the underlying cause of PELO dependency in MSI cancers. Together, we found that loss of large deletions of 9p21.3 and MSI independently impair the SKI complex, uncovering a dependency upon PELO and highlighting a novel functional interaction between PELO and the SKI complex. Furthermore, the strength of this synthetic lethal interaction in a broad range of cancers with poor outcomes underscores the potential of PELO as a promising drug target for patients with 9p21.3-deleted or MSI cancers. Citation Format: Edmond M. Chan, Patricia Borck, Nolan Bick, Kristina Jankovic, Isabella Boyle, Lucy Parker-Burns, Kyla Foster, Anthony Lau, Ashir Borah, Joshua Dempster, Francisca Vazquez. PELO is a synthetic lethal target in chromosome 9p21-deleted or MSI cancers [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 3364.

Abstract 7610: Predictive biomarkers for immuno-chemotherapy response in biliary tract malignancies

Abstract Background: Biliary tract cancers (BTCs) refer to invasive malignancies of the biliary tree. Lack of specific symptoms in early-stages frequently leads to late-stage diagnosis, reducing therapeutic options. Surgical resection remains the only curative option for early-stage disease; instead, patients with advanced disease usually receive chemotherapy (cisplatin plus gemcitabine) in combination with durvalumab as first-line treatment and mFOLFOX-6 as second-line, but effects are limited and only a small proportion of patients respond to treatment. Recent advances in molecular characterization of BTCs allowed the identification of several alterations (e.g. IDH1/2 mutations, FGFR2 rearrangement) for which targeted agents are now available in second line setting but only for a small subset of patients. However, prognosis for patients with advanced stage disease remains poor. Treatment failure depends on several factors such as intra- and inter-tumor heterogeneity and the complex immunological landscape. Therefore, the identification of predictive biomarkers able to individuate patients that will benefit from immuno-chemotherapy will be crucial. Methods: BTC patients at any stage were enrolled in two arms: A) BTCs undergoing surgery; B) advanced/relapsed BTCs. For this study, patients belonging to arm B were selected, in particular those who received first-line immuno-chemotherapy. Blood samples were collected before and after three months of therapy. Ascites samples were collected for patients with advanced disease who underwent paracentesis during therapy. DEPArray NxT (Menarini Silicon Biosystems) was used for phenotypic analysis and single ascites-derived cell isolation. The Ampli1 Lowpass kit was used for copy number aberration (CNA) profiling. Extracellular vesicles (EVs) were isolated from plasma at baseline and after three months of immuno-chemotherapy by size-exclusion chromatography and analyzed for size distribution and concentration using Nanosight NS300. Surface EV markers were analyzed by flow cytometry using the MACSPlex Exosome Kit human (MiltenyiBiotec). Results: We enrolled 43 patients starting from August 2022: 14 in arm A and 29 in B. 23 patients from arm B received immuno-chemotherapy and 3 underwent paracentesis during therapy. Patient-derived organoids (PDOs) were successfully established from tumor cells recovered from ascites of these patients. Epithelial cells recovered from ascites displayed aberrant CNA profiles with gains in chromosomes 1q, 8q, 10, 12p, 20, and losses in 1q, 3q, 4, 8q, 9 and X. EV size distribution and concentration were similar before and after treatment as well as surface markers exposure. Further analyses in correlation with clinical features of patients will be performed. Conclusions: Our approach will contribute to identify potential predictive factors associated with response/resistance to immuno-chemotherapy in BTCs. Citation Format: Michele Zanoni, Tania Rossi, Martina Valgiusti, Giuliano La Barba, Michela Cortesi, Sara Bandini, Giulia Bartolini, Alessandra Dubini, Paolo Di Gioia, Chiara Gallio, Luca Esposito, Alessandra Virga, Giorgia Gurioli, Sara Bravaccini, Giovanni Martinelli, Paola Ulivi, Giovanni Luca Frassineti, Giorgio Ercolani, Ilario Giovanni Rapposelli. Predictive biomarkers for immuno-chemotherapy response in biliary tract malignancies [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 7610.

Abstract 5211: Somatic copy number variation as prognostic marker for recurrence in never-smokers with early-stage lung adenocarcinoma

Abstract Objective: Lung cancer in never-smokers has a distinct genomic profile with low tumor mutation burden, more tumor promotor mutations and fewer somatic copy number variations (CNV) than smokers. CNVs have shown to be associated with poor outcome in advanced stage disease. However, the relationship between CNV and clinicopathologic features on prognosis in early-stage lung cancer is undefined. To address this knowledge gap, we assessed the impact of CNV on recurrence in never-smokers who underwent curative resection for pathological stage I-II lung adenocarcinoma using an integrated genomic and clinicopathological analysis. Methods: We analyzed a cohort of 210 never-smokers with stage I-II lung adenocarcinoma treated from 2014 to 2022 who met the inclusion criteria (no neoadjuvant therapy, pathological stage I-II, complete resection, next-generation sequencing available). The patient cohort was stratified using unsupervised hierarchical clustering of arm-level CNVs. We assessed cumulative incidence of recurrence (CIR) between CNV groups using competing risk regression analysis, adjusting for SUVmax, pathologic stage, lymphovascular invasion, IASLC grade, and tumor mutation burden. Results: Of the 210 patients, 159 patients were female (76%), and patients were primarily from Caucasian (n=140, 67%) or Asian decent (n=50, 24%). Based on arm-level CNV clustering, the cohort was stratified into three groups: CNV low (n=86, 41%), CNV moderate (n=75, 36%) and CNV high (n=49, 23%). The CNV low tumors were characterized by a low CNV burden. Conversely, the CNV moderate tumors primarily exhibited gains in chromosomes 1q, 5p and 7p, along with loss of heterozygosity in chromosome 9p, 9q and 13. CNV high tumors were characterized by whole-genome doubling. Whole-genome doubling was significantly more common in CNV high tumors (n=33, 66%) compared to CNV low (n=2, 2.3%) and CNV moderate tumors (n=2, 2.7%) (p<.001). EGFR (n=145, 69%) and TP53 (n=61, 29%) were the most frequently altered genes, with EGFR alterations being significantly more prevalent in CNV high tumors (n=41, 84%) compared to CNV low tumors (n=46, 53%) (p<.001). In total, 42 patients (20%) developed recurrences. The CIR was lower in the CNV low group (5y-CIR, 14%, 95%CI: 6.5%-25%) compared to the CNV moderate (5y-CIR, 31%, 95%CI: 18%-44%) and CNV high group (5y-CIR, 48%, 95%CI: 27%-66%) (p=.004), with adjusted hazard ratios of 2.3 (95%CI: 0.93-5.71; p=0.074) and 2.65 (95%CI: 1.07-6.60; p=.037), respectively. The CIR for locoregional (n=11, 5%) and distant recurrence (n=31, 15%) remained lower in the CNV low group compared to the combined CNV moderate & high groups (Gray test p=.020 and p=.049, respectively). Conclusion: In never-smokers who underwent curative treatment of pathological stage I-II lung adenocarcinoma, copy number status seems a predictor of recurrence, with CNV low tumors associated with the best prognosis. Citation Format: Stijn Vanstraelen, Allison Reiner, Brooke H. Mastrogiacomo, Kay See Tan, Joseph Dycoco, Bernard J. Park, Prasad S. Adusumilli, Matthew J. Bott, David R. Jones, Gaetano Rocco. Somatic copy number variation as prognostic marker for recurrence in never-smokers with early-stage 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 5211.

Beckwith–Wiedemann syndrome with juvenile fibrous nodules and lobular breast tumors: a case report and review of the literature

BackgroundBeckwith–Wiedemann syndrome (BWS) is a genomic imprinting disorder caused by diverse genetic and/or epigenetic disorders of chromosome 11p15.5. BWS presents with a variety of clinical features, including overgrowth and an increased risk of embryonal tumors. Notably however, reports of patients with BWS and breast tumors are rare, and the association between these conditions is still unclear. Insulin-like growth factor-2 (IGF2) expression is known to be associated with the development of various cancers, including breast cancer, and patients with BWS with specific subtypes of molecular defects are known to show characteristic clinical features and IGF2 overexpression.Case presentationA 17-year-old girl who had been diagnosed with BWS based on an umbilical hernia, hyperinsulinemia, and left hemihypertrophy at birth, visited our department with a gradually swelling left breast. Her left breast was markedly larger than her right breast on visual examination. Imaging examinations showed two tumors measuring about 10 cm each in the left breast, and she was diagnosed with juvenile fibroadenoma following core needle biopsy. The two breast tumors were removed surgically and the patient remained alive with no recurrence. The final diagnosis was juvenile fibroadenoma without malignant findings. Immunohistochemical staining using IGF2 antibody revealed overexpression of IGF2 in the cytoplasm of ductal epithelial cells. Because of her clinical features and IGF2 overexpression, molecular defects of 11p15.5 including a possible genetic background of paternal uniparental disomy of chromosome 11 or hypermethylation of imprinting center 1 was suspected.ConclusionsIn this case, overexpression of IGF2 suggested a possible relationship between BWS and breast tumors. Moreover, the characteristic clinical features and IGF2 staining predicted the subtype of 11p15.5 molecular defects in this patient.

Abstract 6144: A polymorphic TERT tandem repeat creating an expandable intronic G4 quadruplex is associated with TERT expression, relative leukocyte telomere length and cancer risk

Abstract A multi-cancer GWAS region at human chromosome 5p15 encodes telomerase reverse transcriptase (TERT). One of the GWAS signals is rs10069690-T, which creates an alternative intron 4 splicing site, co-producing full-length (FL) and truncated INS1b transcript for dominant-negative telomerase-nonfunctional TERT. A variable number tandem repeat (VNTR6-1, 38 bp repeat unit) within intron 6 remains uncharacterized in relation to GWAS signals and cancer risk. We hypothesized that VNTR6-1 might contribute to GWAS signals represented by rs10069690. We scored VNTR6-1 and rs10069690 in short-read WGS data (n=3,202) from the 1000 Genomes Project with a machine learning model using the GLMnet engine, and in phased long-read assemblies (n=544). We defined two VNTR6-1 groups: Short (24-27 copies) and Long (40.5-66.5 copies); the VNTR6-1-Long group was preferentially linked with rs10069690-T compared to C allele (p=2.53E-13). We show that haplotypes of rs56345976 and rs33961405 alleles can predict VNTR6-1 groups. We imputed VNTR6-1 in UK Biobank controls (n=351,634), PLCO cancer cases and controls (n=100,445), and African Burkitt lymphoma cases (aBL, n=79). Functional studies in aBL tumors revealed a suggestive association between reduced total TERT expression, rs10096690-T (p=0.035) and VNTR6-1-Long (p=0.053). VNTR6-1 forms a G4 quadruplex structure sensitive to G4 stabilizing ligands, altering the ratio between FL-TERT and a dominant-negative TERT-β isoform in isogenic cell lines with/without VNTR6-1. In GTEx and TCGA, several metrics related to cell proliferation and telomerase activity correlated positively with FL-TERT expression and negatively with TERT-β expression. We created a composite marker with 0, 1 or 2 copies of the FL-TERT-associated haplotype (VNTR6-1-Short/rs10069690-C) to capture the effects of these variants that negatively affect TERT expression and telomerase activity. The number of copies of the VNTR6-1/rs10069690 haplotype was associated with relative leukocyte telomere length in UK Biobank controlling for sex and age (β=0.049, p=8.75E-78). This marker was also associated with a reduced risk of bladder and prostate cancer but an increased risk of breast, endometrial, ovarian, thyroid cancer, and glioma in PLCO. Our findings reveal that germline variants VNTR6-1-Long/rs10069690-T underlie the multi-cancer GWAS signal and jointly decrease TERT expression and telomerase activity, potentially reducing the risk of some cancers by limiting cell growth. However, decreased telomerase activity could deregulate stem cell-driven tissue regeneration and genome integrity, increasing the risk of other cancers. Our findings unveil novel mechanisms of cancer susceptibility and propose therapeutic avenues for targeting TERT isoform ratios to modulate telomerase activity and its cellular effects. Citation Format: Oscar Florez-Vargas, Michelle Ho, Max Hogshead, Chia-Han Lee, Kaitlin Forsythe, Brenen Papenberg, Kristine Jones, Wen Luo, Kedest Teshome, Cornelis Blauwendraat, Mikhail Kolmogorov, Stephen J. Chanock, Mitchell J. Machiela, John Schneekloth, Shahinaz Gadalla, Sharon A. Savage, Sam M. Mbulaiteye, Ludmila Prokunina-Olsson. A polymorphic TERT tandem repeat creating an expandable intronic G4 quadruplex is associated with TERT expression, relative leukocyte telomere length and cancer risk [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 6144.

Abstract 6929: Inter- and intratumoral PIK3CA subclonal diversity in breast cancer contextualized by single-cell multiomics

Abstract Rare clonotypes within pre-cancerous tissues can drive progression to cancer. However, the evolution of rare clonotypes in tumors or normal tissue cannot be defined in the absence of single-cell resolution. At this single-cell level, multiomic interrogation across the Central Dogma of Biology provides enhanced power to reconstruct such evolutionary trajectories, defining the mutational profile, cell identify, and receptor expression within each subpopulation. Leveraging a multiomic approach, we aimed to define how different mutations in the same oncogenic driver observed in the same tumor resection associate with copy number variation (CNV) across the genome. We analyzed individual ductal carcinoma in situ/invasive ductal carcinoma cells using a unified whole-genome and full-transcript RNAseq workflow (ResolveOME™, BioSkryb Genomics) coupled with panel-level extracellular protein information through oligo-conjugated antibodies (BioLegend). We sequenced the exomes and transcriptomes of ResolveOME-amplified single cells from mastectomy samples from twelve patients. At the single nucleotide variant (SNV) level, we identified an allelic series of PIK3CA oncogenic driver mutations in the same tumor resection. A single amino acid, in-frame deletion of E109 dominated the sample, followed by H1047R and K111E in decreasing subclonal abundance. A fourth mutation, E345T, not present in the first sample, was detected as the sole PIK3CA variant in the second tumor sample. Intriguingly, each respective PIK3CA mutation class was associated with a distinct copy number alteration profile revealed by low-coverage whole-genome sequencing: Cells harboring the predominant ΔE109 mutation displayed chromosome 8p,16p, and 17 loss while the less abundant H1047R mutation was in single cells harboring 1q gain, 4q loss, and 22 loss in addition to the 8p and 16q loss present in the ΔE109 cells. PIK3CA K111E had a quiescent, 2n copy number profile. The transcriptomic arm of ResolveOME, containing an oligo-conjugated antibody readout of surface protein expression, jointly confirmed the epithelial identity for the cells harboring the oncogenic PIK3CA mutations. A subpopulation of cells harboring prototypical breast cancer CNV were typed as non-epithelial with increased stemness characteristics, indicative of the ability to resolve phenotypic cellular states. These results suggest a tight interrelationship between CNV and SNV influencing the relative rate of clonal expansion. They also provide the opportunity to explore CNV:SNV signature association with loci exclusive of PIK3CA, and to exploit the power of multiomic integration for lineage reconstruction and for defining common oncogenic signatures of the evolving tumors. Citation Format: Jon Zawistowski, Isai Salas-Gonzalez, Tia Tate, Tatiana Morozova, Katherine Kennedy, Durga Arvapalli, Jamie Remington, Jeffrey Marks, E. Shelley Hwang, Gary Harton, Victor Weigman, Jay A. West. Inter- and intratumoral PIK3CA subclonal diversity in breast cancer contextualized by single-cell multiomics [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 6929.

Abstract 5047: Identifying genomic features associated with pathologic lymph node metastasis in lung adenocarcinoma patients

Abstract Introduction: Lymph node metastasis evaluation in solid tumors is mainly complicated by the diagnostic yield of standard imaging modalities and tumor size contradictions. Exploring clinical/molecular features that complement the use of radiologic and bronchoscopy findings may provide significant values for a more accurate pathologic determination of nodal metastasis. Methods: We analyzed surgery-resected tumor samples from 72 treatment-naïve lung adenocarcinoma (LUAD) patients via whole-exome sequencing (WES). Mutational profiles of patients categorized based on their pathologic lymph node metastasis (pN) status were assessed, and the association between metastatic-driving features and overall survival (OS) was then explored in both the study cohort and the Cancer Genome Atlas Program (TCGA) external cohort. Results: Among all 72 patients, 41 (56.9%) were pathologically confirmed as positive for lymph node metastasis (pN-positive), while 31 (43.1%) were categorized as pN-negative. The most frequently mutated gene was TP53 in both subgroups (54.8% and 53.7%, respectively), and the aberrant KEAP1-NRF2 signaling pathway was found significantly more enriched in pN-negative patients. Copy-number variant analysis showed that chromosome 7p amplifications (X7p_amp) at the arm level and EGFR amplifications at the focal gene level were more frequently identified in pN-positive patients (P=0.008 and P=0.004, respectively). Importantly, these amplifications were associated with worse OS in both the study cohort and the TCGA external dataset. Consistent with the unfavorable prognosis, pN-positive patients demonstrated a higher chromosome instability score (CIS) (median: 0.32 vs. 0.14, P=0.14) and a lower tumor mutation burden (TMB) (median: 1.83 vs. 3.85 muts/Mb, P=0.048) compared to pN-negative patients. Conclusion: Our findings suggest that X7p_amp, particularly EGFR amplification, might be associated with pathologic involvement of lymph nodes and an unfavorable prognosis in LUAD patients. Our study highlights the importance of integrating clinical and genomic data into conventional radiologic modalities for a more accurate determination of pathologic nodal involvement. Citation Format: Shijie Wang, Zhiwei Zhou, Song Wang, Rongyun Guo, Zeming Ma, Dachuan Zhao, Liang Wang, Yinan Liu, Yuanyuan Ma, Jianzhi Zhang, Sha Wang, Yedan Chen, Haimeng Tang, Qiuxiang Ou, Jinfeng Chen. Identifying genomic features associated with pathologic lymph node metastasis in lung adenocarcinoma patients [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 5047.

Abstract 4360: Integrative analysis of multiplex interphase FISH copy number with whole exome sequencing identifies tumor evolution in Stage II colon cancer

Abstract Colon cancer ranks as the second leading cause of cancer-related death worldwide. Although standard therapies are highly effective in stage II lymph-node negative colon cancers, about 10-15% of these patients show disease relapse within the next 5-year period after intended curative surgery. Extensive prior research has highlighted the role of intratumor heterogeneity, arising from genomic instability, as a leading contributor to metastasis in colon cancer. In the current study, we pioneered the integration of Multiplex Interphase FISH (miFISH) and Whole Exome Sequencing (WES) data aiming to depict intratumor heterogeneity and delineate the landscape of genetic aberrations that underlie the early metastatic potential in stage II colon cancer patients. To this aim, we have showcased the genome-wide copy-number alterations and single nucleotide mutations derived from WES analysis of samples obtained from primary stage II colon tumors and their patient-matched liver metastases of nine patients using multi-region sampling. miFISH was employed for the concurrent quantification of copy-numbers for nine genes relevant to colon cancer and a centromeric control probe in intact tumor nuclei derived from archival patient material allowing us to establish ploidy baselines and gain and loss patterns for the genes analyzed. Our study was revealing of several copy number alterations, such as gains of chromosomes 1q, 7, 8q, 13q and 20, and losses affecting chromosomes 17p and 18, which were confirmed by miFISH. Based on our SNV data, most frequently mutated genes were APC, TP53, KRAS. We were able to show several mutations in genes belonging to specific pathways most commonly altered in colorectal cancer, such as APC, TCF7L2, AXIN2, FBXW7 in WNT signaling, SMAD4 in TGF-ß signaling, PIK3CA and PTEN in PI3K signaling and KRAS in RTK-RAS signaling pathways. In nearly all cases, subclones that led to metastasis were already present in the primary cancer, indicating a direct progression from the primary to metastatic disease. However, the divergence from the primary tumor to metastasis enabled us to uncover several potential genetic aberrations present in the metastasis but not observed in the primary tumor. Moreover, in one of our cases, despite harboring APC and TP53 mutations in both the primary and metastatic tumor, unique mutations for these genes along with several distinct mutations in the metastasis were identified. These observations suggest that the metastasis might not be related to the primary tumor analyzed and may have arisen from an independent lesion. In conclusion, applying miFISH and WES to decipher the mutational and copy number landscape in metastatic stage II colon cancer provided comprehensive insights into the complex and heterogenous nature of primary colon cancer and the clonal evolution leading to subsequent liver metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Jack Zhu, Diba Yaghoubi, Sara Lahoz, Veronica Pablo-Fontecha3, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Integrative analysis of multiplex interphase FISH copy number with whole exome sequencing identifies tumor evolution in Stage II colon 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 4360.

Evolution of chromosome-arm aberrations in breast cancer through genetic network rewiring

The basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and displays consistent large chromosomal deletions. Here, we characterize evolution and maintenance of chromosome 4p (chr4p) loss in basal breast cancer. Analysis of The Cancer Genome Atlas data shows recurrent deletion of chr4p in basal breast cancer. Phylogenetic analysis of a panel of 23 primary tumor/patient-derived xenograft basal breast cancers reveals early evolution of chr4p deletion. Mechanistically we show that chr4p loss is associated with enhanced proliferation. Gene function studies identify an unknown gene, C4orf19, within chr4p, which suppresses proliferation when overexpressed-a member of the PDCD10-GCKIII kinase module we name PGCKA1. Genome-wide pooled overexpression screens using a barcoded library of human open reading frames identify chromosomal regions, including chr4p, that suppress proliferation when overexpressed in a context-dependent manner, implicating network interactions. Together, these results shed light on the early emergence of complex aneuploid karyotypes involving chr4p and adaptive landscapes shaping breast cancer genomes.

An updated advancement of bifunctional IL-27 in inflammatory autoimmune diseases.

Interleukin-27 (IL-27) is a member of the IL-12 family. The gene encoding IL-27 is located at chromosome 16p11. IL-27 is considered as a heterodimeric cytokine, which consists of Epstein-Barr virus (EBV)-induced gene 3 (Ebi3) and IL-27p28. Based on the function of IL-27, it binds to receptor IL-27rα or gp130 and then regulates downstream cascade. To date, findings show that the expression of IL-27 is abnormal in different inflammatory autoimmune diseases (including systemic lupus erythematosus, rheumatoid arthritis, Sjogren syndrome, Behcet's disease, inflammatory bowel disease, multiple sclerosis, systemic sclerosis, type 1 diabetes, Vogt-Koyanagi-Harada, and ankylosing spondylitis). Moreover, in vivo and in vitro studies demonstrated that IL-27 is significantly in3volved in the development of these diseases by regulating innate and adaptive immune responses, playing either an anti-inflammatory or a pro-inflammatory role. In this review, we comprehensively summarized information about IL-27 and autoimmunity based on available evidence. It is hoped that targeting IL-27 will hold great promise in the treatment of inflammatory autoimmune disorders in the future.

Chromosomal mapping of a major genetic locus from Agropyron cristatum chromosome 6P that influences grain number and spikelet number in wheat.

A novel locus on Agropyron cristatum chromosome 6P that increases grain number and spikelet number was identified in wheat-A. cristatum derivatives and across 3 years. Agropyron cristatum (2n = 4x = 28, PPPP), which has the characteristics of high yield with multiple flowers and spikelets, is a promising gene donor for wheat high-yield improvement. Identifying the genetic loci and genes that regulate yield could elucidate the genetic variations in yield-related traits and provide novel gene sources and insights for high-yield wheat breeding. In this study, cytological analysis and molecular marker analysis revealed that del10a and del31a were wheat-A. cristatum chromosome 6P deletion lines. Notably, del10a carried a segment of the full 6PS and 6PL bin (1-13), while del31a carried a segment of the full 6PS and 6PL bin (1-8). The agronomic characterization and genetic population analysis confirmed that the 6PL bin (9-13) brought about an increase in grain number per spike (average increase of 10.43 grains) and spikelet number per spike (average increase of 3.67) over the three growing seasons. Furthermore, through resequencing, a multiple grain number locus was mapped to the physical interval of 593.03-713.89Mb on chromosome 6P of A. cristatum Z559. The RNA-seq analysis revealed the expression of 537 genes in the del10a young spike tissue, with the annotation indicating that 16 of these genes were associated with grain number and spikelet number. Finally, a total of ten A. cristatum-specific molecular markers were developed for this interval. In summary, this study presents novel genetic material that is useful for high-yield wheat breeding initiatives to meet the challenge of global food security through enhanced agricultural production.

Whole F8 gene sequencing identified pathogenic structural variants in the remaining unsolved patients with severe hemophilia A

BackgroundNo F8 genetic abnormality is detected in approximately 1% to 2% of patients with severe hemophilia A (HA) using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. ObjectivesThe study aimed to identify the causal variation in families with a history of severe HA for whom genetic investigations failed. MethodsWe performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or quantitative polymerase chain reaction. ResultsA structural variant disrupting F8 was found in each propositus, so that all the 815 families with a history of severe HA registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, another was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of an Xq28 349 kb gained in F8 intron 5. ConclusionAll the genetically unsolved cases of severe HA in this cohort were due to structural variants disrupting F8. This study highlights the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach fails.

Prevalence of chromosome 8p11.2 translocations and correlation with myeloid and lymphoid neoplasms associated with FGFR1 abnormalities in a consecutive cohort from nine institutions in Japan.

Myeloid and lymphoid neoplasms associated with FGFR1 abnormalities (MLN-FGFR1 abnormalities) are rare hematologic malignancies associated with chromosome 8p11.2 abnormalities. Translocations of 8p11.2 were detected in 10 of 17,039 (0.06%) unique patient cytogenetic studies performed at nine institutions in Japan. No inversions or insertions of 8p11.2 were detected. Among the 10 patients with 8p11.2 translocations, three patients were diagnosed with MLN-FGFR1 abnormalities, which were confirmed by FISH analysis. Peripheral blood eosinophilia was observed in all three patients, and all progressed to AML or T-lymphoblastic lymphoma/leukemia. The prevalence of 8p11.2 translocations in clinical practice and the proportion of MLN-FGFR1 abnormalities in patients with 8p11.2 translocations in Japan were consistent with those in previous reports from Western countries.

Variations in the genomic profiles and clinical behavior of meningioma by racial and ethnic group.

The influence of socioeconomic factors on racial disparities among patients with sporadic meningiomas is well established, yet other potential causative factors warrant further exploration. The authors of this study aimed to determine whether there is significant variation in the genomic profile of meningiomas among patients of different races and ethnicities and its correlation with clinical outcomes. The demographic, genomic, and clinical data of patients aged 18 years and older who had undergone surgery for sporadic meningioma between September 2008 and November 2021 were analyzed. Statistical analyses were performed to detect differences across all racial/ethnic groups, as were direct comparisons between Black and non-Black groups plus Hispanic and non-Hispanic groups. This study included 460 patients with intracranial meningioma. Hispanic patients were significantly younger at surgery (53.9 vs 60.2 years, p = 0.0006) and more likely to show symptoms. Black patients had a higher incidence of anterior skull base tumors (OR 3.2, 95% CI 1.7-6.3, p = 0.0008) and somatic hedgehog mutations (OR 5.3, 95% CI 1.6-16.6, p = 0.003). Hispanics were less likely to exhibit the aggressive genomic characteristic of chromosome 1p deletion (OR 0.28, 95% CI 0.07-1.2, p = 0.06) and displayed higher rates of TRAF7 somatic driver mutations (OR 2.96 95% CI 1.1-7.8, p = 0.036). Black patients had higher rates of recurrence (OR 2.6, 95% CI 1.3-5.2, p = 0.009) and shorter progression-free survival (PFS; HR 2.9, 95% CI 1.6-5.4, p = 0.002) despite extents of resection (EORs) similar to those of non-Black patients (p = 0.745). No significant differences in overall survival were observed among groups. Despite similar EORs, Black patients had worse clinical outcomes following meningioma resection, characterized by a higher prevalence of somatic hedgehog mutations, increased recurrence rates, and shorter PFS. Meanwhile, Hispanic patients had less aggressive meningiomas, a predisposition for TRAF7 mutations, and no difference in PFS. These findings could inform the care and treatment strategies for meningiomas, and they establish the foundation for future studies focusing on the genomic origins of these observed differences.

Atypical meningioma: Histopathological, genetic, and epigenetic features to predict recurrence risk.

Grading assessed according to World Health Organization (WHO) criteria is a major prognostic factor for determining the risk of recurrence in patients with meningiomas and establishing the most appropriate therapeutic strategy after surgery. However, the main issue is to predict the recurrence risk of WHO grade 2 meningioma and, more specifically, of the atypical subtype. Indeed, owing to a reported recurrence rate of 50%, either radiotherapy or observation is currently considered an option after gross total surgical resection of atypical meningiomas. These heterogeneous clinical outcomes are likely related to the broad histopathological diagnostic criteria for this subtype, and whether meningiomas only present as brain invasion should be classified as atypical remains controversial. Over the last few years, several studies have shown that DNA methylation profiling, next-generation sequencing, and transcriptomics can better stratify meningiomas for their recurrence risk than histology. The main limitations to the widespread use of these approaches to classify meningiomas are their high cost and the need for sophisticated technologies. However, all studies concurred that atypical meningiomas without chromosome 1p deletion display a low recurrence risk, suggesting that the assessment of this cytogenetic alteration could represent an easy and quick method to determine which patients could benefit from adjuvant treatment after surgery. In addition, prognostically unfavorable molecular groups can be distinguished using specific immunostainings, although further validation is required.

A custom next-generation sequencing panel for 1p/19q codeletion and mutational analysis in gliomas.

The World Health Organization has updated their classification system for the diagnosis of gliomas, combining histological features with molecular data including isocitrate dehydrogenase 1 and codeletion of chromosomal arms 1p and 19q. 1p/19q codeletion analysis is commonly performed by fluorescence in situ hybridization (FISH). In this study, we developed a 57-gene targeted next-generation sequencing (NGS) panel including 1p/19q codeletion detection mainly to assess diagnosis and potential treatment response in melanoma, gastrointestinal stromal tumor, and glioma patients. Loss of heterozygosity analysis was performed using the NGS method on 37 formalin-fixed paraffin-embedded glioma tissues that showed 1p and/or 19q loss determined by FISH. Conventional methods were applied for the validation of some glioma-related gene mutations. In 81.1% (30 of 37) and 94.6% (35 of 37) of cases, 1p and 19q were found to be in agreement whereas concordance for 1p/19q codeletion and no 1p/19q codeletion was found in 94.7% (18 of 19) and 94.4% (17 of 18) of cases, respectively. Overall, comparing NGS results with those of conventional methods showed high concordance. In conclusion, the NGS panel allows reliable analysis of 1p/19q codeletion and mutation at the same time.

Bilateral Peters' anomaly, aniridia and Wilms tumour (WAGR syndrome) in monozygotic twins.

This study reports the bilateral association of Peters' anomaly and congenital aniridia in monozygotic twins subsequently diagnosed with Wilms tumour (WAGR syndrome). Two monozygotic female twins were referred at age 2 months with bilateral corneal opacity. A diagnosis of Peters' anomaly associated to aniridia was made in both eyes of both twins. Physical examination and ultrasonography were carried out at 12 months of age to explore the possibility of WAGR-related anomalies, specifically Wilms tumour. DNA were isolated and subjected to whole exome sequencing. Peters' anomaly associated to aniridia in both eyes as well as bilateral Wilms tumour in both children were diagnosed. Exome analyses showed a large heterozygous deletion encompassing 6 648 473 bp in chromosome 11p13, using Integrative Genomics Viewer and AnnotSV software. WAGR syndrome is a rare contiguous gene deletion syndrome with a greater risk of developing Wilms tumour associated with Peters' anomaly and congenital aniridia. However, co-occurrence of both anomalies was rarely reported in twins, and never in both eyes of monozygotic twins. Here, we report the bilateral association of Peters' anomaly and congenital aniridia in monozygotic twins with WAGR syndrome.