Rare Cancers Prevention and Interception Workshop (Speaker Abstracts)

Brooke Hickey, B.S.; Marisa Ciesielski, B.S.; Henry Mang, B.S.; Emily White, B.S.; Qingbo Lu, B.S.; Steve Angus, Ph.D.; D. Wade Clapp, M.D.

Neurofibromatosis type 1 (NF1) is one of the most common cancer predisposition syndromes, affecting one in 3,000 individuals. Malignant peripheral nerve sheath tumor (MPNST) is a rare yet lethal form of sarcoma that can arise from pre-existing plexiform and atypical neurofibromas in persons with NF1 with little to no early warning. Key genetic events driving the malignant transformation of plexiform and atypical neurofibromas are known to include CDKN2A loss and disruption of components of the PRC2 complex. We have shown that the CDKN2A locus plays a critical role in maintaining plexiform neurofibroma indolence through oncogene-induced senescence. Conditional genetic ablation of Nf1 and Cdkn2a in Schwann cell precursors leads to the development of atypical neurofibromas in mice that progress to MPNST in a gene dosage dependent fashion. Clinically, approximately 50% of patients diagnosed with atypical neurofibroma will develop MPNST by 50 years of age with variable latency. Additionally, nearly one-third of these MPNSTs occurred at locations distinct from the atypical neurofibroma, suggesting that other factors within the tumor microenvironment or epigenetically within the tumorigenic Schwann cells themselves may influence malignant transformation. HMGA2 is a non-histone chromatin-binding protein that binds to AT-rich sites within the minor groove of DNA to regulate gene expression programs through changes in chromatin architecture. We found that HMGA2 is highly overexpressed in MPNSTs from human NF1 patients and in Nf1-Cdkn2a mutant mice – associated with downregulation of inhibitory Let-7 microRNAs, which physiologically suppress HMGA2. Further, we show that HMGA2 is highly expressed in murine Schwann cells during embryogenesis but drops to undetectable levels postnatally. In the developing sciatic nerve, HMGA2 expression is lineage restricted to Schwann cells and is also highly expressed during acute nerve injury. shRNA knockdown of HMGA2 alters gene expression signatures associated with cell cycle control, chromatin assembly, and differentiation in Nf1-Cdkn2a mutant Schwann cell precursors. We further demonstrate that CRISPR-Cas9 knockout of HMGA2 impairs the growth of primary Nf1-Cdkn2a deficient Schwann cell precursors and human MPNST cell lines upon orthotopic implantation into the sciatic nerve. Conversely, Hmga2 conditional knock-in mice overexpressing HMGA2 in conjunction with loss Nf1 in Schwann cell precursors develop massive plexiform neurofibromas. Collectively, these results suggest that oncofetal reprogramming mediated by HMGA2, in conjunction with the loss of key tumor suppressors including Nf1 and Cdkn2a, may contribute to plexiform neurofibroma growth and malignant transformation. Ongoing studies aim to elucidate cellular phenotypes associated with HMGA2-dependence and to characterize the epigenetic mechanisms by which HMGA2 governs embryonic growth programs in PNST mouse models.

Molecular endoscopy is an emerging integrated in vivo imaging technology that combines use of a targeting ligand, such as a fluorescently-labeled antibody or peptide, with wide-field fluorescence endoscopy. The ligand binds specifically to unique cell surface targets expressed by pre-malignant and malignant cells but not normal. This approach offers the advantage of generating an image that reflects the biological behavior of digestive tract neoplasia. The incidence of esophageal (EAC) adenocarcinoma has increased steadily with a poor 5-year survival rate of <15%. When detected early, these patients can have a good clinical outcome following surgery. These observations underscore the importance of early cancer detection. Patients with Barrett’s esophagus (BE) are known to be at increased risk. The overarching goal of this study is to advance new methods of imaging to visualize the effects of spatial distribution of genetic alterations in BE. Novel imaging methods will be used to evaluate tumor heterogeneity with progression toward EAC. A complementary panel of genes that are overexpressed on the cell surface have been identified and were used to develop and validate specific peptide ligands. The targets chosen address important clinical needs, including real-time endoscopic identification of pre-malignant lesions and early stage cancer to guide endoscopic resection; risk stratification of BE patients for timing of endoscopic surveillance; and detection of gastro-esophageal junction adenocarcinomas in patients without endoscopic appearance of BE. Targets were identified and validated using highly specific monomer peptides. Optimized sequences were arranged in a dimer configuration to produce multivalent ligand-target interactions and improve binding performance so that early targets can be detected at low levels of expression. A flexible fiber multi-spectral endoscope was used to detect multiple targets concurrently.

Lei Wei, Ph.D.; Shaneen S. Baxter, Ph.D.; Brandon Somerville, M.Sc.; Holli Loomans-Kropp, Ph.D., MPH; Chelsea Sanders, B.Sc.; Ryan N. Baugher, M.Sc.; Stephanie D. Mellott, B.Sc.; Todd B. Young, B.Sc.; Heidi E. Lawhorn, B.Sc.; Teri M. Plona, B.A.; Qiang Hu, Ph.D.; Song Liu, Ph.D.; Alan Hutson, Ph.D.; Simone Difilippantonio, Ph.D.

Monoallelic germline mutations in MMR genes (e.g., MLH1, MSH2, MSH6, and PMS2) predispose individuals to Lynch syndrome (LS) with microsatellite instability (MSI) throughout the genome. Mutations at coding mononucleotide repeats (MNR) usually result in frameshift mutations (FSMs). Recurrent FSMs are thought to play a central role in the increased risk of different types of cancer. Neoantigens produced by FSMs in coding microsatellites have been shown to elicit immune responses, which is the basis for frameshift neoantigen-based vaccines. To develop a prophylactic vaccine and prevention strategy for this high-risk population, we assessed FSMs during tumorigenesis from histologically normal mucosa and fecal DNA of a LS mouse model using targeted next generation sequencing and a panel of FSMs in MNR regions. Msh2LoxP/LoxP;Villin-Cre mice (VCMsh2) started developing detectable tumors at 7-8 months and median survival was 11.5 months with 100% penetrance. Interestingly, FSMs were detectable not only in tumors and mucosa at 7-8 months, but also in young mice (1 month), embryos and pups (before weaning) although FSMs detected and variant allele frequency (VAF) were very low, indicating that FSMs accumulated over time, and FSMs alone may be not sufficient for tumorigenesis since tumors emerge at older age. To determine whether Msh2 was absent in embryos and pups, immunohistochemical (IHC) analysis was performed. Msh2 was lost in almost all intestine epithelial cells in 2-month-old mice but still present in young pups and embryos with very few cells absent of Msh2, indicating that emerging FSMs in these young pups may be due to decreased Msh2 expression because of delayed Cre function. To determine whether low Msh2 expression could lead to the emergence of FSMs, intestine mucosa from 8-, 10-, and 12-month-old Msh2LoxP/+;Villin-Cre heterozygous mice was sequenced. FSMs were detectable with low VAF, although they didn’t develop tumors. IHC staining revealed that Msh2 was absent in only a few intestine epithelial cells in these heterozygous mice, suggesting that loss of heterozygosity was a late event and rare at 12 months. To determine whether MSI emerged in young VCMsh2 pups and heterozygous mice due to haploinsufficiency of Msh2, MSI was assessed using seven markers and fragment size analysis. One marker (mBat67) showed instability in the intestinal mucosa of heterozygous mice and three showed instability in fecal DNA of one month old VCMsh2 mice. Interestingly, mucosal and fecal samples from a time course study in VCMsh2 mice showed progressive increase in MSI with a good correlation between MSI and FSMs during the tumorigenesis process. In addition, FSMs were also highly detectable in organoids and intra-cecal implanted tumors (metastatic) derived from VCMsh2 model. In summary, FSMs emerged at an early stage of tumorigenesis in VCMsh2 mice, which correlated with MSI status. Our data indicates that FSMs and MSI status can be used to monitor the tumor development of LS colorectal cancer. Msh2 deficiency tumor organoids and intra-cecal implantation model can be used for preclinical vaccine and chemo-prevention agents evaluation.
Funded by the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261201500003I

The Fanconi Cancer Foundation (FCF) stands at the forefront of global efforts dedicated to advancing research, advocacy, and support for individuals impacted by Fanconi anemia (FA) and associated cancers. FA, a rare inherited DNA repair cancer predisposition disorder, manifests with bone marrow failure, leukemia, and an elevated risk of squamous cell tumors. The disease is caused by mutations in up to 22 genes in the FA/BRCA DNA repair pathway, including BRCA1 and BRCA2, and represents a complex medical challenge due to DNA repair-related defects associated with the disease.

Founded in 1989 by FA advocates Lynn and Dave Frohnmayer, FCF has played a pivotal role in enhancing treatments like hematopoietic cell transplant (HCT) that have significantly improved patient outcomes. The Fanconi Cancer Foundation has channeled over $32 million into funding more than 260 research projects worldwide and has catalyzed significant progress in understanding and treating FA. Central to FCF's success is its engagement of the FA community and promotion of collaborative and multidisciplinary research. Caregivers and individuals living with FA serve on the FCF board of directors and a patient leadership council and work cooperatively with FCF scientific advisors and staff to ensure that the voices of FA advocates shape research priorities.

Recent initiatives that highlight FCF's dynamic and collaborative approach to research include strategic partnerships with Stand Up To Cancer and the American Association of Cancer Research. These collaborations aim to elevate the visibility of FA within the cancer research arena and facilitate the funding of innovative and collaborative research projects. Additionally, FCF has spearheaded the creation of the first FA data commons that will host combined data from over 15 global FA registries. The organization recently established three international research consortia focused on gene editing for FA, early detection of FA cancers, and psychosocial aspects of living with the disease. Beyond research, FCF aims to empower the FA community through direct support and educational resources and by providing access to clinical programs such as the FA Cancer Virtual Tumor Board.

Innovative partnerships, dedicated advocacy, and a commitment to community engagement underscore FCF's mission to advance research, provide support, and ultimately find a cure for Fanconi Anemia and associated cancers.

Feasibility of Using Frameshift Mutations in Peripheral Blood as a Biomarker for Surveillance of Lynch Syndrome

Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by germline mutations in DNA mismatch repair (MMR) genes, which lead to high microsatellite instability (MSI-H) and frameshift mutations (FSMs) at coding mononucleotide repeats (cMNRs) in the genome. Recurrent FSMs in these regions are thought to play a central role in the increased risk of various cancers. However, there are no biomarkers currently approved for the surveillance of MSI-H-associated cancers. We have developed a panel approach to detection of FSMs in samples of peripheral blood which can simultaneously detect over 300 variants in mismatch repair genes. Initially we focused on demonstrating that relevant microsatellite regions of the genome could be detected on blood and serve as the basis for amplification and sequencing. Several technologies were evaluated and applied to exosomes, other vesicles and cfDNA. A next generation sequencing technology that incorporates generation of amplified mononucleotide repeat regions was found to be useful. This panel is very sensitive for detection of recurrent FSMs in plasma from patients with MSI-H tumors and has very low background in samples from normal individuals. The FSM panel can be tuned for sensitivity and specificity through selection of specific targets. This panel approach has promise as a tool for improved surveillance of MSI-H/MMRd carriers with the potential to reduce the frequency of invasive screening methods for this high-cancer-risk cohort.

Germline mutations in RUNX1 are responsible for familial platelet disorder with associated myeloid malignancies (FPDMM, or simply FPD), an autosomal dominant disease characterized by defective megakaryocytic development, low platelet counts, prolonged bleeding times, and risk of developing hematological malignancies [1]. Patients with FPD have a 20-50% life-long risk of hematopoietic malignancies, mostly myeloid, but such risk varies sifnificantly between patients, even among affected individuals within the same family who carry the same RUNX1 germline mutation [2, 3]. The fact that many FPD patients do not develop leukemia suggest that RUNX1 germline mutation by itself is not sufficient for leukemogenesis; additional factors, such as genetic, epigenetic, metabolic, or immunologic, may contribute to disease progression.

Previously there have been no systematic studies of FPDMM, mainly because it is a rare genetic disease. The pathogenic mechanism is largely unknown and there are no specific interventions to delay or prevent development of hematopoietic malignancies. We therefore launched a prospective, longitudinal natural history study of patients with FPDMM at the NIH Clinical Center in 2019 (https://clinicaltrials.gov/study/NCT03854318) to comprehensively characterize the disease through a multiple-disciplinary approach. Through this study we are now actively following the largest cohort of FPDMM patients in the world, and our initial study data have recently been published [4, 5].

An update of our study will be provided at the meeting.

References:

1. Owen, C.J., et al., Five new pedigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy. Blood, 2008. 112(12): p. 4639-45.
2. Godley, L.A., Inherited predisposition to acute myeloid leukemia. Semin Hematol, 2014. 51(4): p. 306-21.
3. Ripperger, T., et al., Clinical utility gene card for: Familial platelet disorder with associated myeloid malignancies. Eur J Hum Genet, 2016. 24(8).
4. Cunningham, L., et al., Natural history study of patients with familial platelet disorder with associated myeloid malignancy. Blood, 2023. 142(25): p. 2146-2158.
5. Yu, K., et al., Genomic landscape of patients with germline RUNX1 variants and familial platelet disorder with myeloid malignancy. Blood Adv, 2024. 8(2): p. 497-511.

Mariam Dixon-Zegeye, MBBS; Kendra Perez-Smith; Louise Izatt, MBBS; Marc Tischkowitz, MBBS, Ph.D; Rachel Harrison, MBBS; Angela George, MBBS, Ph.D; Simon Lord, MBBS, Ph.D; Gareth Evans, MBBS; Emma Woodward, MBBS; Lara Hawkes, MBBS; James Franklin, MBBS

Current standard of care for Li Fraumeni Syndrome (LFS) involves annual surveillance with whole-body and brain MRI (WB-MRI), however there are no licensed chemopreventives. Preclinical studies in LFS murine models show that the anti-diabetic drug metformin is chemopreventive and, in a pilot intervention trial, its short-term use was well-tolerated in adults with LFS. The Metformin in adults with Li-Fraumeni Syndrome (MILI) is a Precision Prevention Phase II open-labelled unblinded randomised clinical trial in which 224 adults aged ≥ 16 years with LFS are randomised 1:1 to oral metformin (up to 2mg daily) plus annual MRI surveillance or annual MRI surveillance alone for up to 5 years. The primary endpoint is to compare 5-year cumulative cancer free survival. Secondary endpoints include comparison of 5-year overall survival and clinical characteristics of emerging cancers between trial arms. Safety, toxicity and acceptability of metformin; impact of metformin on quality of life and impact of baseline lifestyle risk factors on cancer incidence will be assessed. Exploratory end-points will evaluate the mechanism of action of metformin as a cancer preventative, identify biomarkers of response or carcinogenesis and assess WB-MRI performance as a diagnostic tool for detecting cancers in participants with LFS by assessing yield and diagnostic accuracy of WB-MRI. The study is currently opening at 6 UK sites with a parallel MILI study at the National Cancer Institute (NCI). MILI provides a unique opportunity to evaluate the efficacy of metformin as a chemopreventive in LFS alongside exploring its mechanisms of anticancer action and the biology of p53-driven tumourigenesis.

Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant, tumor predisposition syndrome caused by loss-of-function mutations of NF1 gene encoding neurofibromin. The main cause of death among NF1 patients is the malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft tissue sarcoma. NF1 patients have 10-15% lifetime risk of developing this terrible cancer. MPNSTs metastasize early and are often resistant to radiotherapy and chemotherapy. Driven by the urgent need to identify novel therapeutic targets we are leveraging advanced proteomic techniques to explore the cell surface proteome and major histocompatibility complex (MHC) class I immunopeptidome of NF1-MPNSTs. Through comprehensive profiling of cell surface proteins and MHC-bound immunopeptides, we aim to elucidate the molecular landscape of MPNSTs and identify aberrantly expressed cell surface proteins and tumor-associated antigens that could serve as potential therapeutic targets. To isolate HLA bound peptides, we used protocols adapted from Abelin et. al. (2017) and Bassani-Sternberg et. al. (2018). Peptides were isolated from two human MPNST cultured cell lines (STS-26T and S462-TY). This method has garnered over 2,000 unique peptides for each cell line tested. While no neoantigens have yet been identified, tumor associated, immunogenic epitopes from lineage restricted or oncofetal antigens such as MAGEA2, MAGEA3, PRAME, and SOX9 have been identified. To identify cell surface protiens we’ve utilized a well described cell surface biotinylation technique (Elschenbroich et al., 2010). Several are excellent candidates for targeting with antibody drug conjugates (ADC) or chimeric antigen receptor (CAR) transgenic cells such as EPHA2, HEMO, HER2 and EGFR. Moreover, some are strongly essential in one or two MPNST cell lines based on our whole genome CRISPR data including MET, ITGAV, ITGB5, and CDH2. Ultimately, our research seeks to uncover novel therapeutic targets for the development of precision therapies tailored to treat MPNSTs, thereby improving patient outcomes and quality of life.

In this short overview, I will start with a recap of how rare familial tumors were they first identified (linkage, candidate genes, serendipity) and then will talk about how newer techniques such as exome sequencing have made this easier such that some new tumor syndromes have been identified with just one family. I will then discuss how combining germline with tumor sequencing, based on the two-hit framework, might make it easier to identify very rare dominantly inherited disorders. In the final section, I will look to some of the developing themes of importance, for example the need to consider position and lineage when studying the effect of cancer susceptibility genes, and the recent findings in epigenetics. I will finish with a discussion on implementation - better diagnostic tools, rigorous evaluation of surveillance programs and a greater push to "n of 1" trials in rare tumors.



What else is there to discover?

I will discuss the Children’s Oncology Group (COG) approach to studying rare pediatric cancers. This includes efforts to enroll patients with rare tumors on the Project Every Child registry and biospecimen repository and our recent incorporation of the CCDI Molecular Characterization Initiative into this study. I will briefly provide an overview of current and prior COG therapeutic clinical trials for patients with rare tumors, including retinoblastoma, adrenocortical carcinoma, and nasopharyngeal carcinoma, and well as ADVL1823, the first molecularly driven histology agnostic frontline COG study evaluating larotrectinib for NTRK fusion solid tumors. Throughout, I will highlight areas of success and opportunity in the study of rare cancers in children.

Investigating rare tumors in oncology poses significant challenges. With limited cases, obtaining sufficient data for robust analysis is difficult, and the costs involved raise questions about financial feasibility. One promising avenue for research lies in phase one studies and tumor-agnostic trials. For instance, in a phase I trial, Nirogacestat displayed promising efficacy against desmoid tumors. Additionally, Atezolizumab was investigated early in pediatric patients, given that Alveolar Soft Part Sarcoma (ASPS) primarily affects adolescents and young adults. Initiatives like the NCI Precision Medicine Initiative, which includes the tumor-agnostic NCI MATCH study, aim to address rare cancers. Furthermore, the NCI ComboMATCH targets molecular pathways rather than specific histologies, broadening the scope of treatment options. It's worth noting that breakthroughs in rare tumor research can emerge unexpectedly from diverse sources.

This brief presentation will provide a high-level overview of recent successful development programs resulting in approval of drugs and biological products to treat rare cancers. Additionally, regulatory considerations regarding approaches to design and conduct of clinical trials evaluating new products for rare cancers will be provided.

Collaboration among stakeholders and meaningful patient engagement are paramount for driving advancements in rare diseases like RUNX1 familial platelet disorder (RUNX1-FPD; also known as FPDMM and FPD/AML). An autosomal dominant genetic disease caused by over 100 different mutations, RUNX1-FPD confers lifelong health issues for affected families. However, the most significant health concern for patients and their loved ones is their 30-fold increased risk of hematologic malignancies (HM).

The RUNX1 Research Program (RRP) was founded because there were significant gaps in the understanding of how germline RUNX1 mutations can contribute to such an elevated HM risk. Filling these knowledge gaps requires creative solutions to significant, complex challenges and a fierce commitment to the belief that a patient-engaged collaborative research network can pave the way to delivering treatments quickly and efficiently.
RRP has implemented key strategies for enhancing collaboration by emphasizing open and frequent communication channels, promoting multi stakeholder convenings, and establishing collaborative platforms and tools.

Patient engagement is a cornerstone of RRP’s work, including the education and empowerment of patients and their involvement throughout the research process. Recently, RRP has launched a new educational program to foster greater inclusion of patients from underserved communities.
By embracing these strategies and cultivating a culture of patient-centric collaboration, research efforts are not only scientifically rigorous but also deeply resonant with the needs and perspectives of the patient community.

Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC), with approximately 1 million LS carriers in the US. The lifetime risk of these individuals developing CRC is as high as 70%. Effective cancer prevention strategies are urgently needed to mitigate this risk. LS provides an ideal disease context in which to leverage immune interception strategies, such as vaccines, given that it results in an impaired DNA Mismatch Repair (MMR) system, allowing the generation of frameshift peptides that act as mutated neoantigens capable of inducing an immune response when recognized by specific T-cell receptor (TCR) sequences. Leveraging the largest cohort to-date of pre-cancerous lesions and tumors from LS patients, the Vilar-Sanchez Lab is working to catalog and identify the most frequently recurring neoantigens and corresponding TCRs for development of vaccines to prevent CRC in this unique patient population. Preclinical data from the Vilar-Sanchez Lab suggests that vaccination with a neoantigen-based vaccine will potentially prevent 90% of CRC tumors, 50% of advanced pre-cancers, and 30% of early pre-cancers in LS patients from developing immune tolerance. Within the clinic, the lab is testing two different vaccine strategies in Phase I/II studies of LS patients (NCT05078866 and NCT05419011). Concurrently, the lab is studying how non-steroidal anti-inflammatory agents (NSAIDs) might synergize with vaccines to further augment the immune response. This approach is being tested in a Phase II trial of the effects of aspirin and naproxen on the normal colon in those with LS (NCT5411718). This work has important implications not only for the LS population, but also for the broader field of cancer prevention, as it offers a route toward developing vaccines for sporadic colorectal cancer and other prevalent cancers.

Howard Parnes, MD; Michele Nehrebecky, FNP; Taylor Sundby, MD; Cecilia Higgs, MS; Brigitte Widemann, MD; James Gulley, MD; Philip Castle, PhD

Although cancer incidence and mortality have decreased over the past three decades due to public health measures and improvements in cancer diagnostics and therapeutics, more work is needed to prevent cancer, determine optimum screening modalities, and improve lives of those affected by cancer. The NCI is committed to reducing death and suffering from cancer through effective cancer screening, prevention/interception, and treatment strategies. The Division of Cancer Prevention (DCP) supports numerous extramural preclinical and clinical cancer prevention and interception programs. The Center for Cancer Research’s (CCR), an intramural NCI division, includes basic and clinical research groups aimed at pursuing the most challenging problems in cancer research to prevent, diagnose, and treat cancer. The intramural Division of Cancer Epidemiology and Genetics (DCEG)’s mission is to discover the causes of cancer and inform the means for prevention by conducting transdisciplinary epidemiological and genetic research.
In 2022, DCP, CCR, and DCEG established a trans-NCI Cancer Prevention Clinic (CPC) within the CCR to combine expertise and address next generation approaches to clinical management of high-risk individuals and testing of cancer preventive interventions. The collaboration will also expand opportunities for NCI fellows to conduct clinical cancer prevention research. This talk will describe the virtual CPC goals, study development, and collaborative work, to date.