Mutations In Cancer-associated Genes Research Articles

Abstract SY36-01: Somatic evolution, cancer, and our inevitable decline with age: Inextricably linked

Abstract Why do we get cancer? Why is cancer highly associated with old age? Aging is associated with the accumulation of more mutations; some of which can contribute to cancer phenotypes. However, we now understand that carcinogenesis is much more complex than originally appreciated. In particular, there are tissue environmental forces that both impede and promote cancer evolution. Just as organismal evolution is known to be driven by environmental changes, cellular (somatic) evolution in our bodies is similarly driven by changes in tissue environments, whether caused by the normal process of aging, by lifestyle choices, or by extrinsic exposures. Environmental change promotes selection for new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven alterations in tissues drives selection for adaptive mutations, and some of these mutations can confer malignant phenotypes. To better understand the evolutionary forces that control somatic cell evolution, we used mouse models of cancer initiation, mathematical models of cellular evolution, and analyses of human tissue samples. We have shown that aging- and inflammation-dependent changes in stem cells and their tissue environments dramatically dictate whether cancer-causing mutations are advantageous to stem cells in our tissues, starting the cells down the path to cancer and deepening our insight into cancer risk. Recent studies from many labs have shown how as we age our tissues become dominated by clones bearing mutations in known cancer-associated genes. I will present studies from our labs exploring how cigarette smoking and aging can alter mutational landscapes in the non-malignant lung. To observe somatic mutations in lung biopsies from individuals with different smoking histories, we used a rare-mutation detection technique called Duplex Sequencing to analyze somatic variants from over 200 lung samples, including from subjects from the BDRE (Colorado), PEACE, and TRACERx cohorts. Using this method, we have identified large numbers of mutations in each brushing or biopsy, many of which are cataloged in the Catalogue Of Somatic Mutations In Cancer (COSMIC), and most of these are predicted to disrupt protein function. We also observe pervasive positive selection acting on mutations in many but not all of the cancer-associated genes, with different patterns of selection in the lungs of ever-smokers and never-smokers. Ongoing studies are using primary human and mouse lung epithelial cultures and mouse models to study how these mutations interact with smoking and age to alter associated clonal expansions, tissue integrity, and malignant progression. We propose a model whereby aging and microenvironmental changes induced through lifestyle choices like smoking can promote selection for cells with adaptive mutations, which can contribute to not only cancer risk but also tissue aging. Thus, while young tissues impede selection for such adaptive mutations, old age is associated with a feed-forward loop of aging tissue-mediated selection for mutant clones that then increase tissue aging. Thus, understanding the forces controlling clonal selection as we age and due to lifetime exposures could be critical for controlling multiple diseases of old age. [EJE, EL and FM contributed equally.These studies were led by CS and JD.] Citation Format: Edward J. Evans, Emilia Lim, Fabio Marongiu, Shi Biao Chia, William Hill, Clare Weeden, Oriol Pich, Andrew Rowan, Faiz Jabbar, Cristina Naceur-Lombardelli, Raju Veeriah, Moumita Ghosh, Daniel Merrick, York E. Miller, Robert Keith, Mariam Jamal-Hanjani, Charles Swanton, James V. DeGregori. Somatic evolution, cancer, and our inevitable decline with age: Inextricably linked [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY36-01.

Abstract 1470: Advancing cancer research: A novel PanCancer digital PCR tool for simultaneous detection of multiple hallmark mutations in BRAF and EGFR

Abstract The paradigm shift towards precision medicine in oncology emphasizes the importance of identifying specific genetic mutations driving cancer. Some hallmark mutations in cancer-associated genes, such as BRAF and EGFR, are important in analyzing many cancer types. With the emergence of digital PCR (dPCR) technology, high-sensitivity and quantitative mutation analysis can be performed, lending valuable support to cancer research. In this work, we introduce a new and innovative dPCR method, the dPCR PanCancer assays (RUO), designed for concurrently detecting multiple hallmark mutations in BRAF and EGFR. This work includes two novel PanCancer assays for BRAF and EGFR, each tailored to target a spectrum of mutations associated with these genes, facilitating a comprehensive mutation analysis. The dPCR assay for BRAF targets multiple V600 hallmark mutations, and the dPCR assay for EGFR targets various important deletions on exon 19. This also includes a reference gene as a control for PCR efficiency in the duplex reaction. Here, we present our initial data from various sample types, including blood, plasma and FFPE samples. Through a meticulously optimized dPCR setup, we achieved exceptional sensitivity and specificity, enabling the detection of multiple mutations in a single channel at allelic frequencies below 1%. Both dPCR PanCancer Kits (RUO) have potential use in research for pre-screening samples (e.g., prior to next-generation sequencing) or monitoring cancer cells. They simultaneously assess the mutations, reducing time and costs and saving sample material. Additionally, our technology is adaptable to other cancer-associated genes, where similar assays can potentially be developed. Overall, we have demonstrated that our dPCR PanCancer Kits (RUO) provide a robust, fast and efficient technology to identify critical mutations, ultimately enhancing our understanding of BRAF and EGFR-driven cancers. The dPCR PanCancer Kit is for research use only. Not for the diagnosis, prevention, or treatment of a disease. Citation Format: Claudia Kappmeier, Sherina Edward, Corinna Hochstein, Ellen Inga Bruske, Francesca Di Pasquale, Ronny Kellner. Advancing cancer research: A novel PanCancer digital PCR tool for simultaneous detection of multiple hallmark mutations in BRAF and EGFR [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 1470.

Abstract 3934: Smoking's influence on divergent paths to lung cancer

Abstract Lung cancer is the leading cause of cancer mortality in the world. Many studies have highlighted how mutagens from cigarette smoke promote specific mutations such as G12 mutations in KRAS, contributing to clonal evolution that leads to lung cancer. Despite the indisputable correlation between smoking and lung cancer, people who have never smoked account for the 7th leading cause of cancer mortality. Given the differences in predominant exposures, we hypothesize that the clonal evolution between people with and without smoking histories are markedly different. Therefore, we analyzed lung samples from people with different cigarette smoking exposure to elucidate smoking’s effect on the somatic mutational landscape in the progression to lung cancer. To observe somatic mutations subjected to different exposures of smoking, we used a rare-mutation detection technique called DuplexSeq to analyze somatic variants from 200+ lung samples. We acquired DNA from cells from bronchoscopy brushings and lung punches of histologically normal tissue from people with and without lung cancer and who currently, formerly, or never smoked cigarettes. Through collaborations, we characterized mutations using bioinformatic pipelines, various databases, and statistical methods. In samples from people who smoke, we observe an enhancement of mutations with higher variant allele frequencies and pervasive positive selection acting on mutations in many cancer-associated genes. From our bronchoscopy brushings, NOTCH1 mutations show the most definitive increase of protein-altering variants, while NF1 and KRAS show a predominance of driver mutation in lung punches. Unexpectedly, people without lung cancer who never smoked had a notable presence of pathogenic mutations in PTEN and PIK3CA. Furthermore, multiple punches from the same individual highlight how expansions of mutations have stronger similarity within an individual than across individuals regardless of smoking status. This association points to the importance of the inherent lung microenvironment in influencing the mutational landscapes in lung cancer progression. Altogether, these results show how smoking increases the oncogenicity of observable mutations in the lung but does not preclude the presence of oncogenic clones, emphasizing the value of precision diagnoses and prevention. Ongoing research will be centered around functional assays to determine the impact of observed mutations on tissue function and longitudinal studies using mutational landscapes as a proxy for lung cancer risk. Citation Format: Edward James Evans, Fabio Marongiu, Emilia Lim, Oriol Pich, Yonghua Zhuang, Dexiang Gao, William Hill, Cian Murphy, Ryan Turner, Moumita Ghosh, York Miller, Mariam Jamal-Hanjani, Charles Swanton, James DeGregori. Smoking's influence on divergent paths to 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 3934.

Genomic alterations in ovarian endometriosis and subsequently diagnosed ovarian carcinoma.

Can the alleged association between ovarian endometriosis and ovarian carcinoma be substantiated by genetic analysis of endometriosis diagnosed prior to the onset of the carcinoma? The data suggest that ovarian carcinoma does not originate from ovarian endometriosis with a cancer-like genetic profile; however, a common precursor is probable. Endometriosis has been implicated as a precursor of ovarian carcinoma based on epidemiologic studies and the discovery of common driver mutations in synchronous disease at the time of surgery. Endometrioid ovarian carcinoma and clear cell ovarian carcinoma are the most common endometriosis-associated ovarian carcinomas (EAOCs). The pathology biobanks of two university hospitals in Sweden were scrutinized to identify women with surgically removed endometrioma who subsequently developed ovarian carcinoma (1998-2016). Only 45 archival cases with EAOC and previous endometriosis were identified and after a careful pathology review, 25 cases were excluded due to reclassification into non-EAOC (n = 9) or because ovarian endometriosis could not be confirmed (n = 16). Further cases were excluded due to insufficient endometriosis tissue or poor DNA quality in either the endometriosis, carcinoma, or normal tissue (n = 9). Finally 11 cases had satisfactory DNA from all three locations and were eligible for further analysis. Epithelial cells were collected from formalin-fixed and paraffin-embedded (FFPE) sections by laser capture microdissection (endometrioma n = 11) or macrodissection (carcinoma n = 11) and DNA was extracted. Normal tissue from FFPE sections (n = 5) or blood samples collected at cancer diagnosis (n = 6) were used as the germline controls for each included patient. Whole-exome sequencing was performed (n = 33 samples). Somatic variants (single-nucleotide variants, indels, and copy number alterations) were characterized, and mutational signatures and kataegis were assessed. Microsatellite instability and mismatch repair status were confirmed with PCR and immunohistochemistry, respectively. The median age for endometriosis surgery was 42 years, and 54 years for the subsequent ovarian carcinoma diagnosis. The median time between the endometriosis and ovarian carcinoma was 10 (7-30) years. The data showed that all paired samples harbored one or more shared somatic mutations. Non-silent mutations in cancer-associated genes were frequent in endometriosis; however, the same mutations were never observed in subsequent carcinomas. The degree of clonal dominance, demonstrated by variant allele frequency, showed a positive correlation with the time to cancer diagnosis (Spearman's rho 0.853, P < 0.001). Mutations in genes associated with immune escape were the most conserved between paired samples, and regions harboring these genes were frequently affected by copy number alterations in both sample types. Mutational burdens and mutation signatures suggested faulty DNA repair mechanisms in all cases. Datasets are available in the supplementary tables. Even though we located several thousands of surgically removed endometriomas between 1998 and 2016, only 45 paired samples were identified and even fewer, 11 cases, were eligible for sequencing. The observed high level of intra- and inter-heterogeneity in both groups (endometrioma and carcinoma) argues for further studies of the alleged genetic association. The observation of shared somatic mutations in all paired samples supports a common cellular origin for ovarian endometriosis and ovarian carcinoma. However, contradicting previous conclusions, our data suggest that cancer-associated mutations in endometriosis years prior to the carcinoma were not directly associated with the malignant transformation. Rather, a resilient ovarian endometriosis may delay tumorigenesis. Furthermore, the data indicate that genetic alterations affecting the immune response are early and significant events. The present work has been funded by the Sjöberg Foundation (2021-01145 to K.S.; 2022-01-11:4 to A.S.), Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement (965552 to K.S.; 40615 to I.H.; 965065 to A.S.), Swedish Cancer Society (21-1848 to K.S.; 21-1684 to I.H.; 22-2080 to A.S.), BioCARE-A Strategic Research Area at Lund University (I.H. and S.W.-F.), Mrs Berta Kamprad's Cancer Foundation (FBKS-2019-28, I.H.), Cancer and Allergy Foundation (10381, I.H.), Region Västra Götaland (A.S.), Sweden's Innovation Agency (2020-04141, A.S.), Swedish Research Council (2021-01008, A.S.), Roche in collaboration with the Swedish Society of Gynecological Oncology (S.W.-F.), Assar Gabrielsson Foundation (FB19-86, C.M.), and the Lena Wäpplings Foundation (C.M.). A.S. declares stock ownership and is also a board member in Tulebovaasta, SiMSen Diagnostics, and Iscaff Pharma. A.S. has also received travel support from EMBL, Precision Medicine Forum, SLAS, and bioMCC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The relevance of genetic testing in young patients with breast fibroadenomas

Introduction. Fibroadenomas are the most common benign tumors of the mammary glands in children and adolescents. In some cases, they may be part of hereditary tumor predisposition syndromes associated with a high risk of developing malignant neoplasms throughout life, and therefore genetic testing is relevant.The purpose of the study is to discribe the spectrum of genetic mutations in cancer-associated genes according to the results of next generation sequencing (NGS) in young patients with breast fibroadenomas.Matherials and methods. Sixteen teenage girls with fibroadenomas of the breast who were followed up in Research Institute of Pediatric Oncology and Hematology from 2020 to 2023 were enrolled in this study. Genetic testing by NGS was performed.Results. Pathogenic variants in cancer-associated genes were found in 4 (25 %) patients. In two cases, fibroadenomas were a part of Cowden’s syndrome associated with PTEN inactivation; two patients carried pathogenic variants in the BRCA1 and BRCA2 genes.Conclusion. Genetic testing of young patients with breast fibroadenomas is important to optimize the management strategy in order to reduce cancer risk in high-risk groups of patients.

Molecular and clinical aspects relevant for counseling individuals with clonal hematopoiesis of indeterminate potential.

Clonal hematopoiesis of indeterminate potential (CHIP) has fascinated the medical community for some time. Discovered about a decade ago, this phenomenon links age-related alterations in hematopoiesis not only to the later development of hematological malignancies but also to an increased risk of early-onset cardiovascular disease and some other disorders. CHIP is detected in the blood and is characterized by clonally expanded somatic mutations in cancer-associated genes, predisposing to the development of hematologic neoplasms such as MDS and AML. CHIP-associated mutations often involve DNA damage repair genes and are frequently observed following prior cytotoxic cancer therapy. Genetic predisposition seems to be a contributing factor. It came as a surprise that CHIP significantly elevates the risk of myocardial infarction and stroke, and also contributes to heart failure and pulmonary hypertension. Meanwhile, evidence of mutant clonal macrophages in vessel walls and organ parenchyma helps to explain the pathophysiology. Besides aging, there are some risk factors promoting the appearance of CHIP, such as smoking, chronic inflammation, chronic sleep deprivation, and high birth weight. This article describes fundamental aspects of CHIP and explains its association with hematologic malignancies, cardiovascular disorders, and other medical conditions, while also exploring potential progress in the clinical management of affected individuals. While it is important to diagnose conditions that can lead to adverse, but potentially preventable, effects, it is equally important not to stress patients by confronting them with disconcerting findings that cannot be remedied. Individuals with diagnosed or suspected CHIP should receive counseling in a specialized outpatient clinic, where professionals from relevant medical specialties may help them to avoid the development of CHIP-related health problems. Unfortunately, useful treatments and clinical guidelines for managing CHIP are still largely lacking. However, there are some promising approaches regarding the management of cardiovascular disease risk. In the future, strategies aimed at restoration of gene function or inhibition of inflammatory mediators may become an option.

The Mydrug Platform Trial: Efficacy and Safety of Daratumumab, Ixazomib, Pomalidomide, and Dexamethasone in Patients with Functionally High-Risk Multiple Myeloma

Background: Multiple myeloma (MM) is an incurable malignant neoplasm with a 5-year relative survival rate of 57.9%. Many new agents have improved overall survival; however, some patients rapidly relapse and develop multidrug resistance, which requires the use of new approaches and drug mechanisms to treat. Precision medicine targeting specific genetic abnormalities has the potential to overcome related inherent and/or acquired resistance in patients with MM, but studying this approach has several limitations. Many different mutations occur in MM, and mutation frequency increases with disease progression and relapse, making it difficult to determine dominant genetic abnormalities and assess their relevance to a particular patient. This is a critical factor, as mutations in certain cancer-associated genes (actionable mutations) are targeted by approved drugs for other cancers. Some mutations are found in only a small number of patients. In addition, a substantial subset of patients appear to lack actionable mutations or have low allelic burden, making recruitment to clinical trials studying one specific mutation operationally difficult. To overcome these challenges and understand the role of novel therapies in precision medicine, the MMRF launched MyDRUG, a platform trial of patients with select actionable genetic mutations. Methods: MyDrug (NCT03732703) is an open-label, multicenter, phase 1/2 platform study of adult patients with functionally high-risk MM (defined as early relapse after initial MM treatment), who have received 1-3 prior therapies. Through different subprotocols, patients received targeted agents or immunotherapy/novel agents in combination with a backbone regimen of ixazomib, pomalidomide, and dexamethasone (IPd). Each subprotocol planned to enroll up to 38 patients, and treatment assignment was based on individual genetic sequencing results as determined by Next-Generation Sequencing assay performed in a CLIA-certified Molecular Characterization Laboratory. Approximately 75% of subjects were expected to have actionable mutations (BRAF, NRAS, KRAS, FGFR3, IDH2, CDK activating) or t(11;14) based on historic data, although not all of these would have the necessary allelic burden of ≥30% to qualify for the targeted subprotocols. Patients with no actionable mutation or mutations with allelic burden below 30% were assigned to the Y1 arm and received daratumumab at the standard dose and schedule in combination with IPd. The primary objective of the Y1 sub-protocol was to evaluate the overall response rate (ORR) according to the International Myeloma Working Group consensus criteria. Secondary objectives included assessment of adverse events (AEs). Confidence intervals were computed using the Clopper-Pearson (exact) method. Results: Thirty-eight patients were enrolled to the Y1 daratumumab arm with a median (range) age of 63 (46-79) years, with 16 (42.1%) patients of ages 65-74 and 2 (5.3%) patients ≥75 years. Thirteen (34.2%) patients were female, and 8 (21.1%) patients were Black or African American. The ORR for patients receiving daratumumab plus IPd was 92.1%, and 19 (50%) patients had a very good partial response. Thirty-seven (97.4%) patients experienced treatment-related AEs, 35 (92.1%) of which were related to daratumumab. Among patients receiving treatment, 22 (57.9%) experienced a serious AE; 3 (7.9%) experienced an AE leading to discontinuation; and 2 (5.3%) had fatal AEs. Conclusions: In this platform study of adult patients with treatment refractory, functionally high-risk MM, patients who received daratumumab plus IPd exhibited significant efficacy and a manageable AE profile.

Spatiotemporal dynamics of clonal selection and diversification in normal endometrial epithelium

It has become evident that somatic mutations in cancer-associated genes accumulate in the normal endometrium, but spatiotemporal understanding of the evolution and expansion of mutant clones is limited. To elucidate the timing and mechanism of the clonal expansion of somatic mutations in cancer-associated genes in the normal endometrium, we sequence 1311 endometrial glands from 37 women. By collecting endometrial glands from different parts of the endometrium, we show that multiple glands with the same somatic mutations occupy substantial areas of the endometrium. We demonstrate that “rhizome structures”, in which the basal glands run horizontally along the muscular layer and multiple vertical glands rise from the basal gland, originate from the same ancestral clone. Moreover, mutant clones detected in the vertical glands diversify by acquiring additional mutations. These results suggest that clonal expansions through the rhizome structures are involved in the mechanism by which mutant clones extend their territories. Furthermore, we show clonal expansions and copy neutral loss-of-heterozygosity events occur early in life, suggesting such events can be tolerated many years in the normal endometrium. Our results of the evolutionary dynamics of mutant clones in the human endometrium will lead to a better understanding of the mechanisms of endometrial regeneration during the menstrual cycle and the development of therapies for the prevention and treatment of endometrium-related diseases.

Clonal hematopoiesis and vascular disease.

Somatic mutations in hematopoietic stem cells are common with aging and can result in expansion of clones harboring mutations, termed clonal hematopoiesis. This results in an increased risk of blood cancers but has also been linked with chronic inflammatory disease states. In recent years, clonal hematopoiesis has been established to have a causative role in atherogenesis and cardiovascular disease. Additionally, as the effector cells have been identified to be immune cells, there is ongoing interest in assessing whether dysregulated immune function plays a role in other chronic inflammatory conditions such as rheumatologic disease. Here, we summarize current understanding of clonal hematopoiesis with a focus on cardiovascular disease and inflammation while outlining the potential, yet unexplored, relationship between clonal hematopoiesis and autoimmune disease. Hematopoietic stem cells (HSCs) continually regenerate blood cells. Acquisition of a somatic mutation that provides a selective advantage, a driver mutation, can result in clonal expansion. Clonal hematopoiesis of indeterminate potential, where somatic mutations in certain cancer-associated genes result in clonal expansion in the absence of overt malignancy, can result in atherosclerotic cardiovascular disease in multiple vascular beds, inflammation, and may also contribute to the pathogenesis of autoimmune disease. Many questions remain unanswered regarding the relationship between clonal hematopoiesis and inflammatory disorders.

Life histories of myeloproliferative neoplasms inferred from phylogenies.

Mutations in cancer-associated genes drive tumour outgrowth, but our knowledge of the timing of driver mutations and subsequent clonal dynamics is limited1-3. Here, using whole-genome sequencing of 1,013 clonal haematopoietic colonies from 12 patients with myeloproliferative neoplasms, we identified 580,133 somatic mutations to reconstruct haematopoietic phylogenies and determine clonal histories. Driver mutations were estimated to occur early in life, including the in utero period. JAK2V617F was estimated to have been acquired by 33 weeks of gestation to 10.8 years of age in 5 patients in whom JAK2V617F was the first event. DNMT3A mutations were acquired by 8 weeks of gestation to 7.6 years of age in 4 patients, and a PPM1D mutation was acquired by 5.8 years of age. Additional genomic events occurred before or following JAK2V617F acquisition and as independent clonal expansions. Sequential driver mutation acquisition was separated by decades across life, often outcompeting ancestral clones. The mean latency between JAK2V617F acquisition and diagnosis was 30 years (range 11-54 years). Estimated historical rates of clonal expansion varied substantially (3% to 190% per year), increased with additional driver mutations, and predicted latency to diagnosis. Our study suggests that early driver mutation acquisition and life-long growth and evolution underlie adult myeloproliferative neoplasms, raising opportunities for earlier intervention and a new model for cancer development.

Targeted Deep Sequencing of Mycosis Fungoides Reveals Intracellular Signaling Pathways Associated with Aggressiveness and Large Cell Transformation.

Simple SummaryIn order to decipher the molecular mechanisms of large cell transformation (LCT) in mycosis fungoides (MF), we screened 51 longitudinally obtained skin samples of mycosis fungoides patients (n = 27) with versus without large-cell transformation by the means of targeted deep sequencing in close clinicopathological correlation. The analysis of longitudinally obtained tissue revealed a dynamic mutational profile in the context of an evolutionary selection processes with the example of PLCG1 alterations. In patients with an aggressive clinical course, we detected high mutational heterogeneity revealing the highest frequency of mutations in patients with LCT. The affected genes included members of the JAK/STAT signaling pathway and epigenetic modifiers. The key findings of our analysis included recurrent activating RAS mutations (KRAS and NRAS) being exclusively present in LCT MF; what is of note is that these molecular aberrations were already present in early stages; thus, RAS mutations in MF exhibit a prognostic marker for a higher risk of relapse and progression and restricted prognosis. As RAS mutated tumors are currently in the focus of novel targeted treatment options in several clinical trials, such personalized treatment modalities might offer novel therapeutic options for RAS mutated MF patients.Introduction: Large-cell transformation (LCT) of mycosis fungoides (MF) has been associated with a higher risk of relapse and progression and, consequently, restricted prognosis. Its molecular pathogenesis has not been elucidated yet. Materials and Methods: In order to address molecular mechanisms of LCT, we performed hybrid capture panel-based sequencing of skin biopsies from 10 patients suffering from MF with LCT versus 17 patients without LCT including follow-up biopsies during clinical course, respectively (51 samples in total). The analyzed patients were attributed to three different groups based on the presence of LCT and clinical behavior. Results: While indolent MF cases without LCT did not show pathogenic driver mutations, a high rate of oncogenic alterations was detected in patients with LCT and aggressive clinical courses. Various genes of different oncogenic signaling pathways, including the MAPK and JAK-STAT signaling pathways, as well as epigenetic modifiers were affected. A high inter-individual and distinctive intra-individual mutation diversity was observed. Oncogenic RAS mutations were exclusively detected in patients with LCT. Conclusion: Our data demonstrate that LCT transition of MF is associated with increased frequency of somatic mutations in cancer-associated genes. In particular, the activation of RAS signaling—together with epigenetic dysregulation—may crucially contribute to the molecular pathogenesis of the LCT phenotype, thus conveying its adverse clinical behavior.

O-066 The naughty genes and 3D structure of the endometrium

Abstract The naughty genes and 3D structure of the endometrium Takayuki Enomoto, Manako Ymaguchi, Kazuaki Suda, Kosuke Yoshihara Department of Obstetrics and Gynecology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. The human endometrium is a highly regenerative tissue and involved in menstruation and implantation of the fertilized egg, giving it a central role in women’s reproductive health. However, the regenerative nature of the endometrial glands can lead to the development and progression of “endometrium-related diseases” such as adenomyosis, endometriosis, endometriosis-associated ovarian cancer, endometrial hyperplasia, and endometrial cancer. To clarify the pathogenesis of endometrium-related diseases and develop effective preventative measures and therapeutic strategies, comprehensive understanding of molecular biological linkage between endometrium and endometrium-related diseases was crucially important. To this end, we focused on genomic alterations of endometrial epithelium which is considered the origin of endometriosis, and sequenced 107 ovarian endometriotic and 82 normal uterine endometrial epithelium samples isolated by laser-microdissection. Intriguingly, several genes recurrently mutated in endometriosis-associated ovarian cancers were frequently mutated in both endometriotic epithelium and normal uterine endometrial glands. In particular, PIK3CA mutation was detected in 41% of endometrial epithelium subjects but none of them had shared PIK3CA mutations across multiple regions collected from the same individuals. Mutation allele frequencies of somatic mutations in uterine endometrial epithelium samples were also significantly lower than those in ovarian endometriotic epithelium samples, suggesting the heterogeneous genomic compositions in uterine endometrium. To interpret this genomic heterogeneity in uterine endometrium, we focused on endometrial gland, the minimum functional unit of uterine endometrium, and conducted 109 single endometrial glands sequencing. As a result, we unveiled that each gland carried distinct somatic mutations in cancer-associated genes, such as PIK3CA, KRAS, and PTEN, with high mutant allele frequencies, suggesting the monoclonality of each gland. The presence of cancer-associated gene mutations in histologically normal endometrial glands provides important clues regarding the pathogenesis of endometrium-related diseases. However, our previous study could not determine the spread of endometrial gland harboring cancer-associated gene mutation because there is a limitation to two-dimensional assessment of the whole shapes of endometrial gland due to its complicatedly winding morphology. Therefore, we tackled with three-dimensional (3D) assessment of human endometrium. To construct a large picture of endometrial gland structure, we performed tissue-clearing-based 3D imaging of full-thickness human uterine endometrial tissue with the use of light-sheet fluorescence microscopy. Our 3D immunohistochemistry discovered some new and unique 3D morphologies of endometrial glands, including plexus network of glands or occluded glands. Notably, computational analysis of 3D layer clarified that the plexus structure of the glands was mainly located in the stratum basalis and expanded along muscular layer horizontally, similar to the so-called “rhizome of grass”. Although previous studies have shown the 3D structure of murine endometrial glands, the bottom of these glands forms a crypt but not a rhizome. This can potentially be explained by the existence of menstruation, which is the crucial difference between the human and murine endometrium. The rhizome structure of endometrial gland in the human endometrium will have a functional advantage over the crypt in terms of the conservation of progenitor/stem cells and regeneration. In addition, some endometrial glands shared the plexus and rose toward the luminal epithelium, suggesting that these glands were the same origin. The rhizome of the endometrium may be a crucial element for understanding the expansion of endometrial glands harboring cancer-associated gene mutations. Integrated analysis of the naughty gene alterations and the 3D structure in human endometrium will lead to a better understanding of the human endometrium in various fields, including histology, pathology, pathophysiology, reproduction, and oncology.

Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes

Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.

Molecular Changes in Retinoblastoma beyond RB1: Findings from Next-Generation Sequencing

This investigation uses hybridization capture-based next-generation sequencing to deepen our understanding of genetics that underlie retinoblastoma. Eighty-three enucleated retinoblastoma specimens were evaluated using a MSK-IMPACT clinical next-generation sequencing panel to evaluate both somatic and germline alterations. Somatic copy number variations (CNVs) were also identified. Genetic profiles were correlated to clinicopathologic characteristics. RB1 inactivation was found in 79 (97.5%) patients. All specimens had additional molecular alterations. The most common non-RB1 gene alteration was BCOR in 19 (22.9%). Five (11.0%) had pathogenic germline mutations in other non-RB1 cancer predisposition genes. Significant clinicopathologic correlations included: vitreous seeds associated with 1q gains and 16q loss of heterozygosity (BH-corrected p-value = 0.008, 0.004; OR = 12.6, 26.7, respectively). BCOR mutations were associated with poor prognosis, specifically metastases-free survival (MFS) (nominal p-value 0.03). Furthermore, retinoblastoma patients can have non-RB1 germline mutations in other cancer-associated genes. No two specimens had the identical genetic profile, emphasizing the individuality of tumors with the same clinical diagnosis.

Driver Mutation Acquisition in Utero and Childhood Followed By Lifelong Clonal Evolution Underlie Myeloproliferative Neoplasms

Background Recurrent mutations in cancer-associated genes drive tumour outgrowth, however, the timing of driver mutations and the dynamics of clonal expansion remain largely unknown. Philadelphia-negative myeloproliferative neoplasms (MPN) are unique cancers capturing the earliest stages of tumorigenesis through to disease evolution. Most patients harbor JAK2V617F, present as the only driver mutation or occurring in combination with driver mutations in genes such as DNMT3A or TET2. We aimed to identify the timing of driver mutations and clonal dynamics in adult MPN. Method We undertook whole-genome sequencing of individual single-cell derived hematopoietic colonies (n=952) together with targeted resequencing of longitudinal blood samples from 10 patients with MPN who presented with disease between ages 20 and 76 years. We identified 448,553 somatic mutations which were used to reconstruct phylogenetic trees of hematopoiesis, tracing blood cell lineages back to embryogenesis. We timed driver mutation acquisition, characterised the dynamics of tumour evolution and measured clonal expansion rates over the lifetime of patients. Resequencing of bulk blood samples corroborated clonal trajectories and provided population estimates. Results JAK2V617F was acquired in utero or childhood in all patients in whom JAK2V617F was the first or the only driver mutation. Earliest age estimates were within a few weeks post conception, and upper estimates of age of acquisition were between 4.1 months and 11.4 years, despite wide ranging ages of MPN presentation. The mean latency between JAK2V617F acquisition and clinical presentation was 34 years (range 20-54 years). Subsequent driver mutation acquisition, including for JAK2V617F, was separated by decades. Disease latency following acquisition of JAK2V617F as a second driver event was still 12-27 years. DNMT3A mutations, commonly associated with age-related clonal hematopoiesis (CH), occurred as the first driver event, subsequent to mutated-JAK2, and as independent clones representing CH in MPN patients. DNMT3A mutations were also first acquired in utero or childhood, at the earliest 1.2 weeks post conception, and the latest 7.9 weeks of gestation to 7.8 years across 4 patients. A recurrent feature of the clonal landscape in MPN was the observation of similar genetic changes repeatedly occurring in unrelated clones within the same patient. Such 'parallel evolution' was observed for chr9p loss-of-heterozygosity, chr1q+ and mutations in myeloid cancer genes, suggesting that patient-specific factors flavour selective landscapes in MPN. Normal hematopoietic stem cells accumulated ~18 somatic mutations/year, however, mutant clones, particularly those with mutant-JAK2, acquired 1.5-5.5 excess mutations/ year and had shorter telomeres, reflecting increased cell divisions during clonal expansion. We modelled the rates of clonal expansion and found that they varied substantially, both across patients and within individuals. In one patient, an in utero acquired DNMT3A-mutated clone expanded slowly at &amp;lt;10%/year, taking 30 years to reach a clonal fraction of 1%, whilst a clone with mutated-JAK2, -DNMT3A and -TET2 expanded at &amp;gt;200%/year, doubling in size every 7 months. JAK2V617F as a single driver mutation also expanded variably across patients, highlighting that other factors, which may include germline, cytokine or stem cell differences between individuals, also influence selection for driver mutations. JAK2V617F associated clonal expansion rates in MPN were greater than that reported for JAK2-CH. Furthermore, rates of expansion in the cohort predicted time to clinical presentation, more so than age of mutation acquisition or tumour burden at diagnosis. This suggests that JAK2-mutant clonal expansion rates determine both if and when clinical manifestations occur. Driver mutations and rates of clonal expansion would have been detectable in blood one to four decades before clinical presentation. Conclusions MPN originate from driver mutation acquisition very early in life, even before birth, with life-long clonal expansion and evolution, establishing a new paradigm for blood cancer development. Early detection of mutant-JAK2 together with determination of clonal expansion rates could provide opportunities for early interventions aimed at minimising thrombotic risk and targeting the mutant clone in at risk individuals. Disclosures No relevant conflicts of interest to declare.

Acquired genetic changes in human pluripotent stem cells: origins and consequences.

In the 20 years since human embryonic stem cells, and subsequently induced pluripotent stem cells, were first described, it has become apparent that during long-term culture these cells (collectively referred to as 'pluripotent stem cells' (PSCs)) can acquire genetic changes, which commonly include gains or losses of particular chromosomal regions, or mutations in certain cancer-associated genes, especially TP53. Such changes raise concerns for the safety of PSC-derived cellular therapies for regenerative medicine. Although acquired genetic changes may not be present in a cell line at the start of a research programme, the low sensitivity of current detection methods means that mutations may be difficult to detect if they arise but are present in only a small proportion of the cells. In this Review, we discuss the types of mutations acquired by human PSCs and the mechanisms that lead to their accumulation. Recent work suggests that the underlying mutation rate in PSCs is low, although they also seem to be particularly susceptible to genomic damage. This apparent contradiction can be reconciled by the observations that, in contrast to somatic cells, PSCs are programmed to die in response to genomic damage, which may reflect the requirements of early embryogenesis. Thus, the common genetic variants that are observed are probably rare events that give the cells with a selective growth advantage.

The actinic dysplasia syndrome - diagnostic approaches defining a new concept in field carcinogenesis with multiple cSCC.

Usually, SCC lesions are surrounded by a number of clinically visible and non-visible (subclinical) areas of actinically damaged skin containing cells with dysplasia, and thus may be designated actinic dysplasia syndrome. The epithelial damage is caused mainly by UV radiation, inducing mutations in keratinocytes that may confer growth advantages resulting in preneoplastic fields. The development of visible dysplastic lesions (actinic keratosis - AK) and subsequent progression to invasive SCC requires further mutations in cancer-associated genes, like tumour suppressor genes and cell cycle regulators. Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) represent a considerable advantage for the investigation of field cancerization. In addition, imaging allows the non-invasive monitoring of topical treatments for AKs. RCM provides invivo horizontal skin sections with a high, 1-μm lateral resolution (similar to histopathology) but with a limited penetration (about 200μm), which can hamper the visualization of important areas such as the dermal-epidermal junction. Conventional OCT has better penetration (1-2mm) at the expense of a more limited resolution (much lower than histopathology). Line-field confocal OCT (LC-OCT) combines the high precision of RCM and the good penetration of OCT in a single device and therefore appears to be very useful in diagnosing/managing AKs.

Intramuscular fast-flow vascular anomaly contains somatic MAP2K1 and KRAS mutations.

The term "intramuscular hemangioma capillary type" (IHCT) refers to a fast-flow vascular lesion that is classified as a tumor, although its phenotype overlaps with arteriovenous malformation (AVM). The purpose of this study was to identify somatic mutations in IHCT. Affected tissue specimens were obtained during a clinically indicated procedure. The diagnosis of IHCT was based on history, physical examination, imaging and histopathology. Because somatic mutations in cancer-associated genes can cause vascular malformations, we sequenced exons from 446 cancer-related genes in DNA from 7 IHCT specimens. We then performed mutation-specific droplet digital PCR (ddPCR) to independently test for the presence of a somatic mutation found by sequencing and to screen one additional IHCT sample. We detected somatic mutations in 6 of 8 IHCT specimens. Four specimens had a mutation in MAP2K1 (p.Q58_E62del, p.P105_I107delinsL, p.Q56P) and 2 specimens had mutations in KRAS (p.K5E and p.G12D, p.G12D and p.Q22R). Mutant allele frequencies detected by sequencing and confirmed by ddPCR ranged from 2 to 15%. IHCT lesions are phenotypically similar to AVMs and contain the same somatic MAP2K1 or KRAS mutations, suggesting that IHCT is on the AVM spectrum. We propose calling this lesion "intramuscular fast-flow vascular anomaly."

DNA methylation changes and somatic mutations as tumorigenic events in Lynch syndrome-associated adenomas retaining mismatch repair protein expression.

BackgroundDNA mismatch repair (MMR) defects are a major factor in colorectal tumorigenesis in Lynch syndrome (LS) and 15% of sporadic cases. Some adenomas from carriers of inherited MMR gene mutations have intact MMR protein expression implying other mechanisms accelerating tumorigenesis. We determined roles of DNA methylation changes and somatic mutations in cancer-associated genes as tumorigenic events in LS-associated colorectal adenomas with intact MMR.MethodsWe investigated 122 archival colorectal specimens of normal mucosae, adenomas and carcinomas from 57 LS patients. MMR-deficient (MMR-D, n = 49) and MMR-proficient (MMR-P, n = 18) adenomas were of particular interest and were interrogated by methylation-specific multiplex ligation-dependent probe amplification and Ion Torrent sequencing.FindingsPromoter methylation of CpG island methylator phenotype (CIMP)-associated marker genes and selected colorectal cancer (CRC)-associated tumor suppressor genes (TSGs) increased and LINE-1 methylation decreased from normal mucosa to MMR-P adenomas to MMR-D adenomas. Methylation differences were statistically significant when either adenoma group was compared with normal mucosa, but not between MMR-P and MMR-D adenomas. Significantly increased methylation was found in multiple CIMP marker genes (IGF2, NEUROG1, CRABP1, and CDKN2A) and TSGs (SFRP1 and SFRP2) in MMR-P adenomas already. Furthermore, certain CRC-associated somatic mutations, such as KRAS, were prevalent in MMR-P adenomas.InterpretationWe conclude that DNA methylation changes and somatic mutations of cancer-associated genes might serve as an alternative pathway accelerating LS-associated tumorigenesis in the presence of proficient MMR.FundJane and Aatos Erkko Foundation, Academy of Finland, Cancer Foundation Finland, Sigrid Juselius Foundation, and HiLIFE.

Targeted next-generation sequencing of cancer-related genes in thyroid carcinoma: A single institution's experience.

Thyroid carcinoma (TC) has characteristic genetic alterations, including point mutations in proto-oncogenes and chromosomal rearrangements that vary by histologic subtype. Recent developments in next-generation sequencing (NGS) technology enable simultaneous analysis of cancer-associated genes of interest, thus improving diagnostic accuracy and allowing precise personalized treatment for human cancer. A total of 50 patients who underwent thyroidectomy between 2014 and 2016 at Hokuto Hospital were enrolled. Total DNA was extracted from formalin-fixed, paraffin-embedded tissue sections and quantified. Targeted regions of 24 cancer-associated genes were amplified by PCR, barcoded and sequenced using an Illumina MiSeq platform. Subjects included 30 patients with papillary carcinoma (PC), two with PC tall cell variant (TVPC), two with PC follicular variant (FVPC), eight with follicular carcinoma, seven with poorly differentiated carcinoma (PDC), and one with anaplastic carcinoma (AC). The BRAF V600E mutation was present in 25 of 30 (83%) patients with PC, 2 of 2 (100%) patients with TVPC, 6 of 7 (86%) patients of PDC, and one patient with AC. PIK3CA mutations were present in 3 of 30 (delPV104P, A1046T and C420R; 10%) patients with PC and 1 of 7 (H1047R; 14%) patients with PDC. The TP53 mutation was present in 1 of 30 (R306*; 3.3%) patients with PC and 1 of 7 (Q152*; 14%) patients with PDC. The NRAS mutation was present in 1 of 2 (Q61K, 50%) patients with FVPC. Statistical analysis showed that patients without the BRAF V600E mutation had advanced pathologic T and N stages compared with those with the mutation (P=0.047 and P=0.019, respectively). The BRAF V600E mutation was not correlated with overall and disease-free survival in patients with PC. A patient with PC with a mutation in EGFR (K852Q) and the PIK3CA mutation had an aggressive course with multiple bone and lung metastases. Detection of mutations in cancer-associated genes using NGS could enhance the understanding of the clinical behavior of TC.