Cancer Predisposition Syndrome Research Articles

Update on Cancer Predisposition Syndromes and Surveillance Guidelines for Childhood Brain Tumors.

Tumors of the central nervous system (CNS) comprise the second most common group of neoplasms in childhood. The incidence of germline predisposition among children with brain tumors continues to grow as our knowledge on disease etiology increases. Some children with brain tumors may present with nonmalignant phenotypic features of specific syndromes (e.g., nevoid basal cell carcinoma syndrome, neurofibromatosis type 1 and type 2, DICER1 syndrome, and constitutional mismatch-repair deficiency), while others may present with a strong family history of cancer (e.g., Li-Fraumeni syndrome) or with a rare tumor commonly found in the context of germline predisposition (e.g., rhabdoid tumor predisposition syndrome). Approximately 50% of patients with a brain tumor may be the first in a family identified to have a predisposition. The past decade has witnessed a rapid expansion in our molecular understanding of CNS tumors. A significant proportion of CNS tumors are now well characterized and known to harbor specific genetic changes that can be found in the germline. Additional novel predisposition syndromes are also being described. Identification of these germline syndromes in individual patients has not only enabled cascade testing of family members and early tumor surveillance but also increasingly affected cancer management in those patients. Therefore, the AACR Cancer Predisposition Working Group chose to highlight these advances in CNS tumor predisposition and summarize and/or generate surveillance recommendations for established and more recently emerging pediatric brain tumor predisposition syndromes.

A Validated Highly Sensitive Microsatellite Instability Assay Accurately Identifies Individuals Harboring Biallelic Germline PMS2 Pathogenic Variants in Constitutional Mismatch Repair Deficiency.

Constitutional mismatch repair deficiency (CMMRD) is a rare and extraordinarily penetrant childhood-onset cancer predisposition syndrome. Genetic diagnosis is often hampered by the identification of mismatch repair (MMR) variants of unknown significance and difficulties in PMS2 analysis, the most frequently mutated gene in CMMRD. We present the validation of a robust functional tool for CMMRD diagnosis and the characterization of microsatellite instability (MSI) patterns in blood and tumors. The highly sensitive assessment of MSI (hs-MSI) was tested on a blinded cohort of 66 blood samples and 24 CMMRD tumor samples. Hs-MSI scores were compared with low-pass genomic instability scores (LOGIC/MMRDness). The correlation of hs-MSI scores in blood with age of cancer onset and the distribution of insertion-deletion (indel) variants in microsatellites were analyzed in a series of 169 individuals (n = 68 CMMRD, n = 124 non-CMMRD). Hs-MSI achieved high accuracy in the identification of CMMRD in blood (sensitivity 98.5% and specificity 100%) and detected MSI in CMMRD-associated tumors. Hs-MSI had a strong positive correlation with whole low-pass genomic instability LOGIC scores (r = 0.89, P = 2.2e-15 in blood and r = 0.82, P = 7e-3 in tumors). Indel distribution identified PMS2 pathogenic variant (PV) carriers from other biallelic MMR gene PV carriers with an accuracy of 0.997. Higher hs-MSI scores correlated with younger age at diagnosis of the first tumor (r = -0.43, P = 0.011). Our study confirms the accuracy of the hs-MSI assay as ancillary testing for CMMRD diagnosis, which can also characterize MSI patterns in CMMRD-associated cancers. Hs-MSI is a powerful tool to pinpoint PMS2 as the affected germline gene and thus potentially personalize cancer risk.

Clinical and biological landscape of constitutional mismatch-repair deficiency syndrome: an International Replication Repair Deficiency Consortium cohort study

Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare and aggressive cancer predisposition syndrome. Because a scarcity of data on this condition contributes to management challenges and poor outcomes, we aimed to describe the clinical spectrum, cancer biology, and impact of genetics on patient survival in CMMRD. In this cohort study, we collected cross-sectional and longitudinal data on all patients with CMMRD, with no age limits, registered with the International Replication Repair Deficiency Consortium (IRRDC) across more than 50 countries. Clinical data were extracted from the IRRDC database, medical records, and physician-completed case record forms. The primary objective was to describe the clinical features, cancer spectrum, and biology of the condition. Secondary objectives included estimations of cancer incidence and of the impact of the specific mismatch-repair gene and genotype on cancer onset and survival, including after cancer surveillance and immunotherapy interventions. We analysed data from 201 patients (103 males, 98 females) enrolled between June 5, 2007 and Sept 9, 2022. Median age at diagnosis of CMMRD or a related cancer was 8·9 years (IQR 5·9-12·6), and median follow-up from diagnosis was 7·2 years (3·6-14·8). Endogamy among minorities and closed communities contributed to high homozygosity within countries with low consanguinity. Frequent dermatological manifestations (117 [93%] of 126 patients with complete data) led to a clinical overlap with neurofibromatosis type 1 (35 [28%] of 126). 339 cancers were reported in 194 (97%) of 201 patients. The cumulative cancer incidence by age 18 years was 90% (95% CI 80-99). Median time between cancer diagnoses for patients with more than one cancer was 1·9 years (IQR 0·8-3·9). Neoplasms developed in 15 organs and included early-onset adult cancers. CNS tumours were the most frequent (173 [51%] cancers), followed by gastrointestinal (75 [22%]), haematological (61 [18%]), and other cancer types (30 [9%]). Patients with CNS tumours had the poorest overall survival rates (39% [95% CI 30-52] at 10 years from diagnosis; log-rank p<0·0001 across four cancer types), followed by those with haematological cancers (67% [55-82]), gastrointestinal cancers (89% [81-97]), and other solid tumours (96% [88-100]). All cancers showed high mutation and microsatellite indel burdens, and pathognomonic mutational signatures. MLH1 or MSH2 variants caused earlier cancer onset than PMS2 or MSH6 variants, and inferior survival (overall survival at age 15 years 63% [95% CI 55-73] for PMS2, 49% [35-68] for MSH6, 19% [6-66] for MLH1, and 0% for MSH2; p<0·0001). Frameshift or truncating variants within the same gene caused earlier cancers and inferior outcomes compared with missense variants (p<0·0001). The greater deleterious effects of MLH1 and MSH2 variants as compared with PMS2 and MSH6 variants persisted despite overall improvements in survival after surveillance or immune checkpoint inhibitor interventions. The very high cancer burden and unique genomic landscape of CMMRD highlight the benefit of comprehensive assays in timely diagnosis and precision approaches toward surveillance and immunotherapy. These data will guide the clinical management of children and patients who survive into adulthood with CMMRD. The Canadian Institutes for Health Research, Stand Up to Cancer, Children's Oncology Group National Cancer Institute Community Oncology Research Program, Canadian Cancer Society, Brain Canada, The V Foundation for Cancer Research, BioCanRx, Harry and Agnieszka Hall, Meagan's Walk, BRAINchild Canada, The LivWise Foundation, St Baldrick Foundation, Hold'em for Life, and Garron Family Cancer Center.

Identification of different classes of genome instability suppressor genes through analysis of DNA damage response markers.

Cellular pathways that detect DNA damage are useful for identifying genes that suppress DNA damage, which can cause genome instability and cancer predisposition syndromes when mutated. We identified 199 high-confidence and 530 low-confidence DNA damage-suppressing (DDS) genes in Saccharomyces cerevisiae through a whole-genome screen for mutations inducing Hug1 expression, a focused screen for mutations inducing Ddc2 foci, and data from previous screens for mutations causing Rad52 foci accumulation and Rnr3 induction. We also identified 286 high-confidence and 394 low-confidence diverse genome instability-suppressing (DGIS) genes through a whole-genome screen for mutations resulting in increased gross chromosomal rearrangements and data from previous screens for mutations causing increased genome instability as assessed in a diversity of genome instability assays. Genes that suppress both pathways (DDS+ DGIS+) prevent or repair DNA replication damage and likely include genes preventing collisions between the replication and transcription machineries. DDS+ DGIS- genes, including many transcription-related genes, likely suppress damage that is normally repaired properly or prevent inappropriate signaling, whereas DDS- DGIS+ genes, like PIF1, do not suppress damage but likely promote its proper, nonmutagenic repair. Thus, induction of DNA damage markers is not a reliable indicator of increased genome instability, and the DDS and DGIS categories define mechanistically distinct groups of genes.

Genetic analysis of PALB2 gene WD40 domain in canine mammary tumour patients.

DNA repair mechanisms are essential for tumorigenesis and disruption of HR mechanism is an important predisposing factor of human breast cancers (BC). PALB2 is an important part of the HR. There are similarities between canine mammary tumours (CMT) and BCs. As its human counterpart, PALB2 mutations could be a predisposing factor of CMT. In this study, we aimed to investigate the impacts of PALB2 variants on tumorigenesis and canine mammary tumor (CMT) malignancy. We performed Sanger sequencing to detect germline mutations in the WD40 domain of the canine PALB2 gene in CMT patients. We conducted in silico analysis to investigate the variants, and compared the germline PALB2 mutations in humans that cause breast cancer (BC) with the variants detected in dogs with CMT. We identified an intronic (c.3096+8C>G) variant, two exonic (p.A1050V and p.R1354R) variants, and a 3' UTR variant (c.4071T>C). Of these, p.R1354R and c.4071T>C novel variants were identified for the first time in this study. We found that the p.A1050V mutation had a significant effect. However, we could not determine sufficient similarity due to the differences in nucleotide/amino acid sequences between two species. Nonetheless, possible variants of human sequences in the exact location as their dog counterparts are associated with several cancer types, implying that the variants could be crucial for tumorigenesis in dogs. Our results did not show any effect of the variants on tumor malignancy. The current project is the first study investigating the relationship between the PALB2 gene WD40 domain and CMTs. Our findings will contribute to a better understanding of the pathogenic mechanism of the PALB2 gene in CMTs. In humans, variant positions in canines have been linked to cancer-related phenotypes such as familial BC, endometrial tumor, and hereditary cancer predisposition syndrome. The results of bioinformatics analyses should be investigated through functional tests or case-control studies.

Abstract 6376: Establishment of the Consortium for Childhood Cancer Predisposition and the Childhood Cancer Predisposition Study

Abstract Background: Genetic predisposition contributes to a much higher proportion of cancer in children than previously appreciated. This creates a unique opportunity to develop improved methods to identify children at increased risk for cancer, enable early detection or prevention of tumors, and improve cure rates with decreased morbidity. However, several challenges have impeded successful research and systematic approaches to the care of children with cancer predisposition. Methods: We established the Consortium for Childhood Cancer Predisposition (C3P), currently comprised of seven large pediatric institutions in North America. The long-term goal of C3P is to improve outcomes for children with genetic susceptibility to cancer through collaborative research and data sharing. To this end, we have developed the Childhood Cancer Predisposition Study (CCPS), a multicenter registry and biorepository, to achieve three main aims: 1) Establish and maintain the infrastructure for recruitment, participation, collection and sharing of clinical data and biological samples for children with cancer predisposition syndromes (CPS); 2) Define the spectrum of disease in children with CPS and explore the unique characteristics and management approaches of CPS-related tumors compared to their sporadic counterparts; and 3) Evaluate the clinical impact and effectiveness of standard and emerging tumor surveillance strategies. Results: The CCPS opened to enrollment in May, 2021 and is currently recruiting at 3 member sites. To date, 276 primary subjects and 44 family members have been enrolled, 211 in the last year. In total, 37 different cancer predisposition diagnoses are represented among participants, including those that are more prevalent, e.g., Li-Fraumeni syndrome (n=42), DICER1 syndrome (n=27), and PTEN Hamartoma Syndrome (n=21), and those which are very rare, e.g., Rothmund-Thomson syndrome (n=2). There is excellent uptake on obtaining samples, with the CCPS Biorepository at Emory University receiving biospecimens from 261 subjects to date, including constitutional DNA (n=261), frozen peripheral blood mononuclear cells (n=32), and plasma samples for ctDNA analysis (n= 26). Conclusions: C3P has established the infrastructure to facilitate comprehensive collaborative work to improve outcomes for children with cancer predisposition, which represent a wide variety of disorders. Future efforts will include the development of innovative strategies to promote recruitment beyond the primary C3P sites and the support of additional clinical and biological studies of specific CPS. Citation Format: Anita Villani, Garrett M. Brodeur, Lisa R. Diller, Chloe Edgerton, Junne Kamihara, Wendy Kohlmann, Suzanne P. MacFarland, Luke Maese, David Malkin, Kim E. Nichols, Melissa R. Perrino, Sharon E. Plon, Surya Rednam, Christopher C. Porter. Establishment of the Consortium for Childhood Cancer Predisposition and the Childhood Cancer Predisposition Study [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 6376.

Abstract 5463: Germline DICER1 loss promotes rhabdomyosarcoma via innate immune system

Abstract DICER1 syndrome is a cancer predisposition syndrome where affected patients have increased risk of neoplasms including pleuropulmonary blastoma, Sertoli-Leydig cell tumor, and fusion-negative rhabdomyosarcoma (FN-RMS). This syndrome is defined by germline heterozygous loss of function mutations in the gene DICER1, but it remains unclear exactly how these mutations predispose children to develop cancer. One would expect the germline loss of one allele to provide a sensitized background in the tumor cell to acquire a second hit mutation. To gain insight into the role of Dicer1 loss in FN-RMS, we deleted the Dicer1 gene both globally in the germline and conditionally in the tumor cell in our previously established mouse model of FN-RMS. Surprisingly, we observed faster tumor onset and increased penetrance in mice with germline heterozygous Dicer1 loss (Dicer1+/-) but not in mice where Dicer1 loss was restricted to the tumor cells, demonstrating that Dicer1 functions as a non-cell autonomous haploinsufficient tumor suppressor. Single cell RNA sequencing (scRNA-Seq) revealed massive expansion of immature neutrophils in Dicer1+/- tumors, which were enriched for proteases associated with neutrophil extracellular traps (NETs). NETs are webs of chromatin and proteases released when neutrophils undergo NETosis in response to inflammatory stimuli. These NETs are known to cause significant damage to the local extracellular matrix (ECM) and have been implicated in tumor promotion. In addition to the neutrophil enriched immunophenotype, we found that Dicer1+/- tumors show significant ECM remodeling and are enriched for C5a, the potent neutrophil chemoattractant and NET-priming factor. Taken together, these results suggest NETosis is upregulated in Dicer1+/- FN-RMS tumors. Performing iTALK ligand-receptor pair analysis on the scRNA-Seq data revealed a putative interaction between a NET-derived ligand and EGFR and IGF1R on tumor cells, suggesting direct tumor promoting signaling from NET-to-tumor. We validated this novel FN-RMS promotion mechanism in vitro using human FN-RMS cell lines and observed that this NET-derived ligand promoted FN-RMS growth in a dose-dependent manner and could function through both EGFR and IGF1R signaling. Finally, we incorporated a Padi4-/- allele into our tumor model to genetically block NETosis and observed complete rescue of the accelerated tumor onset and increased penetrance observed in Dicer1+/- mice, demonstrating that NETosis promotes the growth of Dicer1+/- FN-RMS tumors. Importantly, these findings may be applicable to many DICER1 syndrome-associated cancers, as neutrophils which get converted to tumor promoters by heterozygous DICER1 loss are present in DICER1 syndrome patients regardless of tumor type. Citation Format: Randolph Larsen, Jason Hanna, Kristin Reed, Hongjian Jin, Wood Kimbrough, Kyna Vuong, Myron Evans, Casey Langdon, Catherine Drummond, Matthew Garcia, David Finkelstein, Patrick Schreiner, Jerold Rehg, Mark Hatley. Germline DICER1 loss promotes rhabdomyosarcoma via innate immune system [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 5463.

Abstract 6372: Germline pathogenic variants are enriched in pediatric central nervous system tumor patients and associate with somatic functional consequences

Abstract The genetic contribution of rare pathogenic germline variation to central nervous system (CNS) tumor formation in pediatric patients without a family history of cancer remains unclear. We sought to characterize the prevalence, spectrum, and clinical significance of pathogenic germline variation in cancer predisposition genes (CPGs) in 838 patients profiled in the Pediatric Brain Tumor Atlas (PBTA) by whole genome sequencing (n=821) or whole exome sequencing (n=17). Rare variants in 214 CPGs were annotated as pathogenic (P) or likely pathogenic (LP) consistent with American College of Medical Genetics criteria using AutoGVP, our open-source automated pathogenicity assessment tool. To assess enrichment of P/LP variants in the PBTA, we compared the frequency of CPG P/LP variants in cases to a tumor-free control cohort from Penn Medicine BioBank (PMBB; n=6,295). Somatic alterations, mutational signatures, and gene set enrichment analyses from matched tumor sequencing were integrated to characterize functional consequences associated with germline pathogenic variation. We observed 215 germline P/LP variants within 76 distinct CPGs in 189 (22.6%) PBTA patients. CPG P/LP variants were most prevalent among patients with a plexiform neurofibroma diagnosis (9/14, 64.3%), and least prevalent among patients with craniopharyngioma (3/39, 7.8%). We detected syndrome-associated P/LP variants in 45/58 (77.6%) patients with a clinically reported cancer predisposition syndrome and incidentally in another 37 patients. CPG P/LP variants were significantly enriched in the PBTA cohort relative to the PMBB control cohort (OR=2.0, p=1.0E-12, CI=1.6-2.4), and NF1 harbored the most significant excess P/LP variant burden (OR=30.7, p=4.57E-13, CI=11.2-105.1). We observed a significant enrichment of mismatch repair (MMR) gene P/LP variants in patients with high-grade gliomas (HGG) and tumors from MMR P/LP carriers exhibited significantly higher MMR-deficiency mutational signature exposures relative to tumors from non-carrier HGG patients (SBS14: W=478, p=1.71E-05). Overall, 90/215 (41.9%) of identified germline P/LP variants co-occurred with at least one corresponding somatic alteration in matched tumors, including oncogenic SNV (n=5), copy number variation (n=28), loss of heterozygosity (n=54), differential gene expression (n=23), and proximal alternative splicing (n=19). In summary, rare germline P/LP variants are enriched in PBTA patients and are associated with somatic events in the same gene, resulting in gene- and pathway-specific functional consequences. Our work emphasizes a role for pathogenic germline variation in pediatric CNS tumor development and frequent under-identification of cancer predisposition syndromes, underscoring the importance of germline sequencing in personalized patient care strategies. Citation Format: Ryan J. Corbett, Rebecca Kaufman, Shelly W. McQuaid, Zalman Vaksman, Saksham Phul, Miguel A. Brown, Jennifer L. Mason, Heena Desai, Ryan Hausler, Ammar S. Naqvi, Antonia Chroni, Zhuangzhuang Geng, Bo Zhang, Chuwei Zhong, Yuankun Zhu, Allison P. Heath, Marilyn Li, Penn Medicine BioBank, Phillip B. Storm, Adam C. Resnick, Kara N. Maxwell, Miriam Bornhorst, Kristina A. Cole, Angela J. Waanders, Suzanne P. MacFarland, Jo Lynne Rokita, Sharon J. Diskin. Germline pathogenic variants are enriched in pediatric central nervous system tumor patients and associate with somatic functional consequences [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 6372.

Abstract 1153: Using spatial transcriptomics to dissect cell to cell cooperation in lung adenocarcinoma

Abstract Lung cancer is the second most common cancer in the United States and the leading cause of cancer related deaths in this country. Unfortunately, efficacious treatments for lung cancer remain suboptimal. Lung cancer is characterized by several distinct subtypes of which lung adenocarcinoma is the most common, comprising about 40% of this malignancy. The hallmark gene mutations of lung adenocarcinoma include TP53, RAS, and STK11. LKB1 is a serine-threonine kinase (coded by the gene STK11) that largely functions as a tumor suppressor, and is mutated in 20-30% of non-small cell lung cancers (NSCLCs). The diversity between and within lung cancer subtypes, as well as within a patient (i.e. metastases vs primary tumor), makes treating this cancer very challenging. Lung adenocarcinoma, in particular, has collective invasion packs of cells adjacent to the primary tumor that correlate with metastatic disease in mouse models. We hypothesize that the transcriptomic profile of the collective invasion packs in lung adenocarcinoma patients varies significantly from the adjacent primary tumor and represents a targetable metastatic sub-population. This work will help to identify specific cell signaling pathways that have the translational potential to develop novel therapeutics for metastatic disease, ultimately improving patient outcomes through precision medicine. Utilizing patient lung adenocarcinoma samples with KRAS and KRAS+LKB1 mutations, we identified regions of interest including bulk tumor and surrounding invasion packs. Then, using GeoMx digital spatial profiling technology (by Nanostring) and next-gen sequencing, we generated transcriptomic profiles from bulk tumor and invasion packs. Preliminary results indicate region specific transcriptomic differences, highlighting the heterogeneity of these cell populations. More specifically, collective invasion packs exhibit upregulation of gene networks involving immune cell differentiation, function, oxidative phosphorylation, tumor invasiveness, and mitochondrial structure. To discern the metastatic potential, transcriptomic profiles and biological functions will be compared between invasion packs, tumor bulk vs invasion packs, and finally, inter-patient differences. Successful completion of this project will characterize transcriptomes of metastatic lung cancer and has the potential to ultimately identify biomarkers of aggressive disease. Lastly, this foundational study will pave the way for future studies on the transcriptomic landscape of metastatic pediatric cancers and cancer predisposition syndromes. Citation Format: Raehannah Jamshidi, Lyra Griffiths, Rich Johnston, Vaunita Parihar, Frank Schneider, Adam Marcus. Using spatial transcriptomics to dissect cell to cell cooperation in 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 1153.

Abstract 6938: Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1

Abstract Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant phenotypes and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was hidden in the bulk tumor, and the extended granularity of NF-1 glioma tumor microenvironment (TME) remained still unexplored. Here, we collected glioma samples from 46 NF-1 patients including 22 high-grade (HGG) and 24 low-grade (LGG) tumors, and we analyzed their gene expression by single nuclei RNA sequencing. A total of 239,044 single cells were classified into tumor and non-tumor components by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, and clustering). We defined the pattern of intra-tumor heterogeneity of NF-1 glioma cells using non-negative matrix factorization and derived 7 malignant meta-programs (MPs) that we respectively defined as Neuronal-like, EMT, Astrocyte-like, Dividing Radial Glia-like, Ependymal-like, Immune, and Glycolytic/Hypoxic-like. These MPs recapitulated normal brain cell subtypes, thereby reflecting broad cell plasticity. The non-tumor cell compartment (121,364 cells, 51%) was dissected for the characterization of the cell types that populate the TME of NF-1 glioma. We identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma ecomodules were highlighted by comparing the relative composition of the TME across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that could mediate a potential anti-tumor response in low-grade NF-1 glioma (LGG immune high). Conversely, regulatory T cell infiltration and effector T cell exhaustion induced immune suppression in a low-grade glioma immune dysfunctional ecomodule. The absence of lymphocytes characterized a large set of cold tumors, mostly including high-grade glioma. Together, the complex interplay of tumor cell states with different TME compartments elucidated the existence of separate ecomodules in NF-1 glioma, with the LGG immune high TME associated with Neuronal-like and the LGG immune dysfunctional with Ependymal-like tumor cells. The Ependymal-like state also exhibits maximal association with brain-specific normal cells, including oligodendrocytes, neurons and astrocytes, whereas the HGG are enriched with Dividing Radial Glia- and Glycolytic/Hypoxic-like tumor cell states. The elucidation of different ecomodules provides novel insights for the application of targeted therapies in NF-1 glioma patients. Citation Format: Luciano Garofano, Fulvio D'Angelo, Michael Oh, Michele Ceccarelli, Franck Bielle, Marc Sanson, Anna Lasorella, Antonio Iavarone. Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1 [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 6938.

Abstract 1533: Characterizing tumor-immune surveillance in Li-Fraumeni syndrome

Abstract Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome associated with germline TP53 mutations and increased lifetime risk of developing multiple cancers. p53, the protein encoded by TP53, plays a role in regulating various aspects of immune cell biology. Accordingly, inactivation of p53 in tumor cells shapes interactions with the immune system and influences the anti-tumor immune response. Mutation or deletion of p53 in immune cells also favors tumor development and progression. However, to date, a comprehensive study of immune cell populations and the anti-tumor immune response has not been undertaken in the context of LFS. We hypothesized that germline TP53 mutation carriers possess dysfunctional immune cells and/or immune responses, supporting LFS-associated tumor progression. Our preliminary data indicate a profound defect in tumor-immune surveillance in an LFS murine model. We observed that MC38 cancer cells develop significantly faster into tumors when injected into LFS mice (harboring a gain-of-function mutation Trp53R172H/WT) compared to wildtype (WT) control mice. Importantly, we identified significant alterations in immune cell compositions and phenotypes within the tumor microenvironment as well as immune organs using flow cytometry. We are currently using ex vivo proliferation assays and cytotoxicity assays to investigate the impact of mutant p53 on the tumor-killing ability of immune effector cells. Altogether, an improved understanding of anti-tumor immune dysfunction in the context of germline TP53 mutation may reveal the potential for innovative therapies to target different facets of the immune system to intercept cancer progression in LFS patients. Citation Format: Camilla Giovino, Nicholas Fischer, Noel Ong, Ashby Kissoondoyal, Ran Kafri, David Malkin. Characterizing tumor-immune surveillance in Li-Fraumeni syndrome [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 1533.

Abstract 135: Resolving the tumorigenesis continuum of DICER1 syndrome with novel lineage-trackable genetically engineered mouse model

Abstract Background: DICER1 syndrome is a rare cancer predisposition syndrome associated with germline DICER1 mutations, which develops pulmonary or extra-pulmonary manifestations mostly in pediatric patients. Unlike classic tumor suppressors, the 2nd hit in DICER1 gene is a missense mutation that renders the RNase IIIb domain defective; therefore, this mutant form of DICER1 is the only protein expressed in these cancer cells, leading to a biased 5p-miRNA production deficiency. Many DICER1 cancers are sarcomas; we therefore postulate the cell of origin is mesenchymal. We recently developed a RNase IIIb-mutant mouse strain and embryonic activation of this mutant along with silencing of the other Dicer1 allele in Mullerian mesenchymal progenitor cells led to the development of tumors resembling sarcomas in human patients. To expand the model and enable lineage tracking, we developed a tamoxifen inducible, tdTomato-trackable, Hypermethylated in Cancer 1 (HIC1)-creERT2 driven transgenic mouse strain. Hic1 marks mesenchymal progenitors. With this strain, we created a model that histologically recapitulates the 3 renal tumors in DICER1 syndrome: cystic nephromas, Wilms tumors, and anaplastic sarcomas. Objective: to identify oncogenic events underlying Dicer1 mutation-driven murine kidney tumor development with single cell RNA-sequencing (scRNA-seq). Method: To build the tumorigenesis continuum, we harvested kidneys from Dicer1+/fl-D1693N and Dicer1fl/fl-D1693N mice at 1, 3, 6, months post tamoxifen injection, sorted out tdTomato+/CD45-/Epcam- cells, and performed scRNA-seq. Five endpoint tumors were included for analyses. Single cell transcriptomic profiles were integrated and clustered using Seurat to identify cell populations that emerge and expand along the tumor development continuum. Differential gene expression, pathway analysis were conducted to identify genes/oncogenic pathways that are activated along the course of tumor development. Cell differentiation trajectory analysis will be performed using Monocle. Results: Integration of 5 tumors revealed diverse cell populations: epithelial, mesenchymal, endothelial cells, lymphocytes, and macrophages. We further clustered the mesenchymal population and identified sub-populations such as highly proliferative cells, muscle satellite cells, and terminally differentiated muscle cells. These mesenchymal cells show high expression of blastema markers (Ncam1, Sox11) - a histological component in Wilms tumors. Temporal trajectory of tumorigenesis with time point samples is currently being analyzed. Conclusion: With scRNA-seq, we begin to unravel heterogeneity of these murine tumors and will identify critical oncogenic events driving the development of DICER1 syndrome-associated cancer, enabling us to utilize this model to develop therapeutic approaches to improve patient management. Citation Format: Joyce Zhang, Shary Chen, Yana Moscovitz, Branden Lynch, Maxwell Douglas, Janine Senz, Wilder Scott, Michael Underhill, Yemin Wang, David Huntsman. Resolving the tumorigenesis continuum of DICER1 syndrome with novel lineage-trackable genetically engineered mouse model [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 135.

Abstract 6373: Effectiveness of tumor surveillance across pediatric cancer predisposition syndromes

Abstract Background: Pediatric oncology patients are increasingly recognized as having an underlying cancer predisposing syndrome (CPS). For individuals diagnosed with a CPS, surveillance is often recommended to detect new tumors at their earliest and most curable stages. Screening is heavily based on radiologic imaging, however data on the effectiveness and outcomes of surveillance are limited. Methods: Patients with a clinical and/or molecular CPS diagnosis, including 274 children and young adults with 35 CPS at a single specialty pediatric oncology center, were monitored using standardized surveillance protocols over an average of 3 years (range 1 month - 12 years). Patients with hereditary retinoblastoma were excluded as surveillance was completed by a separate team. The cohort included 139 patients (50.7%) with a prior tumor history while 135 (49.3%) had no prior cancer. This study reviewed tumors diagnosed, method of detection, clinical course, and patient outcomes. Individual reports from 1,533 surveillance tests including diagnostic imaging, colonoscopy, and upper endoscopy, were assessed and classified for accuracy. Sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) were calculated for selected surveillance methods, and themes of common strengths or weaknesses of specific imaging modalities were identified. Results: During the study period from January 1, 2009 - September 31, 2021, 35 asymptomatic tumors were detected in 27 patients through surveillance (9.9% of the cohort), while five symptomatic tumors were detected in five patients (1.8%) outside of surveillance. Ten tumors (28.6%) discovered through surveillance were identified on first surveillance imaging. Among these 35 new tumors, 19 (54.3%) were diagnosed in children with a previously treated cancer, six (17.1%) were diagnosed in patients still being treated for cancer, and 10 (28.6%) were diagnosed in those with no prior cancer history. Malignant solid and brain tumors identified through surveillance were more often localized (20 of 24; 83.3%) than similar tumors detected before CPS diagnosis (71 of 125; 56.8%, p ≤0.0001). Of the 24 tumors identified through surveillance and surgically resected, 17 (70.8%) had completely negative margins. When analyzed across all CPS, the sensitivity (96.4%), specificity (99.6%), PPV (95.7%), and NPV (99.6%) of surveillance testing was high, with few false positive (n=6/1,533, 0.4%) or false negative (n=5/1,533, 0.3%) reports. No invasive interventions were performed due to false positive results. Conclusion: Patients with CPS are at increased risk of primary and secondary tumor development with 10% of our cohort developing a tumor in a short time span. Surveillance effectively enabled early detection of new brain and solid tumors across a wide spectrum of CPS which allowed for complete surgical resection and successful treatment in most cases. Citation Format: Melissa R. Perrino, Alise Blake, Cara E. Morin, Leslie Taylor, Rose B. McGee, Sara Lewis, Stacy Hines-Dowell, Arti Pandey, Paige Turner, Manish Kubal, Yin Su, Li Tang, Laura Howell, Lynn W. Harrison, Zachary Abramson, Ann Schechter, Noah D. Sabin, Kim E. Nichols. Effectiveness of tumor surveillance across pediatric cancer predisposition syndromes [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 6373.

Diagnostic yield and clinical impact of germline sequencing in children with CNS and extracranial solid tumors—a nationwide, prospective Swedish study

Childhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors. gWGS was performed in 309 children at diagnosis of CNS (n=123, 40%) or extracranial (n=186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients. The prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35). Overall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients. The study was supported by the Swedish Childhood Cancer Fund and the Ministry of Health and Social Affairs.

O189: Exploring the polyclonal origins of intestinal tumours

Abstract Introduction Some reports have shown that a proportion of intestinal tumours in the human colorectal cancer (CRC) predisposition syndrome Familial Adenomatous Polyposis (FAP) may have a multi-ancestral structure, challenging the widely-held monoclonal theory of tumour initiation. The nature of these polyclonal tumours and the reasons for their existence have not yet been elucidated. Methods Intestinal tumorigenesis was promoted through chemical mutagenesis in Villin-CreERT2-Apcfl/+ mice containing a multicolour reporter. The clonal status of tumours was determined with confocal microscopy. Targeted cancer driver sequencing was performed to determine if certain driver mutations were associated with monoclonal or polyclonal routes of tumour formation. Finally, transcriptomic analysis was performed to identify differentially expressed pathways. Results Confocal imaging reveals that more than 40% of tumours are heterotypic, and therefore polyclonal. Transcriptomic profiling of clones within these tumours demonstrates a non-cell autonomous interplay between Myc and Ras pathways, suggestive of oncogenic cooperation between adjacent clones. Strikingly, sequencing reveals that the nature of the Apc mutation dictates clonal architecture. Using a large publicly available dataset of sporadic CRCs, these mutational differences are confirmed, suggesting a role for differential Wnt activation in tumour initiation. Conclusions Taken together, these data suggest that a significant proportion of intestinal tumours arise through clonal cooperation, and that early RAS/MAPK pathway activation may obviate the need for this. Clonal cooperation appears to be at play not just in FAP but also in sporadic CRCs. Uncovering this mechanism may pave the way for therapeutics aimed at reducing tumour burden in patients with FAP.

Frameshift mutations in peripheral blood as a biomarker for surveillance of Lynch syndrome.

Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline mutations in DNA mismatch repair genes, which lead to high microsatellite instability and frameshift mutations at coding mononucleotide repeats in the genome. Recurrent frameshift mutations in these regions are thought to play a central role in the increased risk of various cancers, but no biomarkers are currently available for the surveillance of high microsatellite instability-associated cancers. A frameshift mutation-based biomarker panel was developed and validated by targeted next-generation sequencing of supernatant DNA from cultured high microsatellite instability colorectal cancer cells. This panel supported selection of 122 frameshift mutation targets as potential biomarkers. This biomarker panel was then tested using matched tumor, adjacent normal tissue, and buffy coat samples (53 samples) and blood-derived cell-free DNA (cfDNA) (38 samples) obtained from 45 high microsatellite instability and mismatch repair-deficient patients. We also sequenced cfDNA from 84 healthy participants to assess background noise. Recurrent frameshift mutations at coding mononucleotide repeats were detectable not only in tumors but also in cfDNA from high microsatellite instability and mismatch repair-deficient patients, including a Lynch syndrome carrier, with a varying range of target detection (up to 85.2%), whereas they were virtually undetectable in healthy participants. Receiver operating characteristic curve analysis showed high sensitivity and specificity (area under the curve = 0.94) of the investigated panel. We demonstrated that frameshift mutations can be detected in cfDNA from high microsatellite instability and mismatch repair-deficient patients and asymptomatic carriers. The 122-target frameshift mutation panel described here has promise as a tool for improved surveillance of high microsatellite instability and mismatch repair-deficient patients, with the potential to reduce the frequency of invasive screening methods for this high-cancer-risk cohort.

The history of families at-risk for hereditary breast and ovarian cancer: what are the impacts of genetic counseling and testing?

Cancer Genetic Counseling (CGC) and genetic testing (GT) assume a paramount role for hereditary cancer predisposition syndrome families. We assessed the effects of CGC and GT on women affected by cancer who are at risk for hereditary breast and ovarian cancer predisposition syndrome (HBOC). This study encompasses four time points: before the CGC session, after the CGC session when blood is drawn for GT, after disclosure of GT results, and six months following disclosure of GT results. The impacts of CGC and GT were assessed using psychosocial questionnaires. Additionally, a pedigree, genogram, and ecomap were constructed through a semistructured interview. A total of sixty women were included in the study. Most participants considered their perception of cancer risk to be equivalent to that of the general population, even among those with pathogenic variants. An increased perception of breast and ovarian cancer risks was associated with a heightened inclination toward religious engagement as a coping mechanism. Patients carrying variants of uncertain significance expressed greater concerns about developing another cancer compared to those who had BRCA1 and BRCA2 wild type or pathogenic variants. Qualitative analysis of the genograms and ecomaps demonstrated that the CGC/GT processes facilitate communication within families. The genogram analyses revealed the impact of CGC and GT processes on families at risk for hereditary cancer. Changes in some family relationships were observed, and an improvement in communication was noted following the GT process. These findings can assist healthcare professionals considering a personalized approaches in clinical practice.

A tale of two pathways: Review of immune checkpoint inhibitors in DNA mismatch repair-deficient and microsatellite instability-high endometrial cancers.

The DNA mismatch repair (MMR) pathway is critical for correcting DNA mismatches generated during DNA replication. MMR-deficiency (MMR-D) leads to microsatellite instability (MSI) associated with an increased mutation rate, driving cancer development. This is particularly relevant in endometrial cancer (EC) as 25%-30% of tumors are of MMR-D/MSI-high (MSI-H) phenotype. Comprehensive assessment using immunohistochemistry (IHC) and sequencing-based techniques are necessary to fully evaluate ECs given the importance of molecular subtyping in staging and prognosis. This also influences treatment selection as clinical trials have demonstrated survival benefits for immune checkpoint inhibitors (ICIs) alone and in combination with chemotherapy for MMR-D/MSI-H EC patients in various treatment settings. As a portion of MMR-D/MSI-H ECs are driven by Lynch syndrome, an inherited cancer predisposition syndrome that is also associated with colorectal cancer, this molecular subtype also prompts germline assessment that can affect at-risk family members. Additionally, heterogeneity in the tumor immune microenvironment and tumor mutation burden (TMB) have been described by MMR mechanism, meaning MLH1 promoter hypermethylation versus germline/somatic MMR gene mutation, and this may affect response to ICI therapies. Variations by ancestry in prevalence and mechanism of MMR-D/MSI-H tumors have also been reported and may influence health disparities given observed differences in tumors of Black compared to White patients which may affect ICI eligibility. These observations highlight the need for additional prospective studies to evaluate the nuances regarding MMR-D heterogeneity as well as markers of resistance to inform future trials of combination therapies to further improve outcomes for patients with EC.

The evolutionary impact of childhood cancer on the human gene pool

Germline pathogenic variants associated with increased childhood mortality must be subject to natural selection. Here, we analyze publicly available germline genetic metadata from 4,574 children with cancer [11 studies; 1,083 whole exome sequences (WES), 1,950 whole genome sequences (WGS), and 1,541 gene panel] and 141,456 adults [125,748 WES and 15,708 WGS]. We find that pediatric cancer predisposition syndrome (pCPS) genes [n = 85] are highly constrained, harboring only a quarter of the loss-of-function variants that would be expected. This strong indication of selective pressure on pCPS genes is found across multiple lines of germline genomics data from both pediatric and adult cohorts. For six genes [ELP1, GPR161, VHL and SDHA/B/C], a clear lack of mutational constraint calls the pediatric penetrance and/or severity of associated cancers into question. Conversely, out of 23 known pCPS genes associated with biallelic risk, two [9%, DIS3L2 and MSH2] show significant constraint, indicating that they may monoallelically increase childhood cancer risk. In summary, we show that population genetic data provide empirical evidence that heritable childhood cancer leads to natural selection powerful enough to have significantly impacted the present-day gene pool.

Adult-Onset Cancer Predisposition Syndromes in Children and Adolescents-To Test or not to Test?

With the increasing use of comprehensive germline genetic testing of children and adolescents with cancer, it has become evident that pathogenic variants (PV) in adult-onset cancer predisposition genes (aoCPG) underlying adult-onset cancer predisposition syndromes, such as Lynch syndrome or hereditary breast and ovarian cancer, are enriched and reported in 1% to 2% of children and adolescents with cancer. However, the causal relationship between PVs in aoCPGs and childhood cancer is still under investigation. The best-studied examples include heterozygous PVs in mismatch repair genes associated with Lynch syndrome in children with mismatch repair deficient high-grade glioma, heterozygous PVs in BARD1 in childhood neuroblastoma, and heterozygous PVs in BRCA2 in children with rhabdomyosarcoma. The low penetrance for pediatric cancers is considered to result from a combination of the low baseline risk of cancer in childhood and the report of only a modest relative risk of disease in childhood. Therefore, we do not advise that healthy children empirically be tested for PVs in an aoCPG before adulthood outside a research study. However, germline panel testing is increasingly being performed in children and adolescents with cancer, and exome and genome sequencing may be offered more commonly in this population in the future. The precise pediatric cancer risks and spectra associated with PVs in aoCPGs, underlying cellular mechanisms and somatic mutational signatures, as well as treatment response, second neoplasm risks, and psycho-oncological aspects require further research.