Early Driver Mutations Research Articles

Abstract LB228: Evolutionary trajectories of lung cancer in never smokers

Abstract Purpose of the study Elucidating evolutionary trajectories of cancers allows us to understand the key events, and the order in which they occur, throughout their development. This can help us to find important associations with tumor progression and prognosis. Our aim was to perform de novo identification of the evolutionary trajectories within Sherlock-lung, with a dataset containing the largest collection of lung cancer in never smokers (LCINS) samples ever analyzed. Experimental procedures Our Plackett-Luce ordering model utilized copy number data from Battenberg and mutation cancer cell fraction (CCF) data from DPClust. Frequently-occurring copy number events and driver mutations are ordered within each sample using their copy number states and CCFs. An aggregate ordering is then calculated for a sample set. Mixture model analysis identifies subsets of samples displaying distinct orders of events, uncovering diverse evolutionary trajectories within a tumor set. Dataset The Sherlock-lung whole genome sequencing dataset (n=1217) was filtered to the samples that allowed us to identify subclonal expansions. Samples required at least 10 reads per chromosome copy and a minimum cellularity of 30%. This provided 458 LCINS samples of various histologies. 155 smoker samples were also analyzed for comparison. Results We identified two subsets of LCINS tumors following distinct evolutionary trajectories. The “loss-based” subset commonly saw whole genome duplication (WGD) combined with copy number losses occurring earlier, and at higher prevalence, than gains. Contrastingly, in the “gain-based” subset, WGD was relatively rare but ploidy increased via copy number gains, which were more prevalent than losses. Interestingly, these different trajectories converged on similar overall copy number states. The loss-based subset had a higher mutational burden and a higher proportion of the genome altered, and followed a more smoker-like trajectory than the gain-based subset. Considering these differences alongside the convergence in copy number states, it is intriguing that survival times were similar between the two subsets. Copy number events defined the difference between the two trajectories. However, driver mutations also played important roles in tumor evolution in LCINS. TP53 and EGFR mutations were associated with greater genomic instability. Conversely, KRAS mutations were associated with more stable genomes. Samples with early clonal mutations in TP53, ERBB2, and PIK3CA, as well as those with a copy number gain of ERBB2, exhibited shorter survival times. Conclusions Two distinct evolutionary trajectories of LCINS were identified by de novo Plackett-Luce event ordering analysis. The contrast between the subgroups was defined by different paths of copy number activity, but they ultimately converged on similar overall copy number states and outcomes. Key early driver mutations influenced genomic instability and survival times. Citation Format: Christopher Wirth, Tongwu Zhang, Wei Zhao, Phuc Hoang, Jian Sang, Nathaniel Rothman, Marcos Díaz-Gay, Ruxandra Teslo, Naser Ansari-Pour, Máire Ní Leathlobhair, Iliana Peneva, William Eagles, Lixing Yang, Ludmil Alexandrov, David C. Wedge, Maria Teresa Landi. Evolutionary trajectories of lung cancer in never smokers [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 LB228.

The etiology of clonal mosaicism in human aging and disease

AbstractOur DNA is consistently assaulted by a variety of intrinsic and extrinsic mutational factors. Fortunately, DNA repair provides for protective barriers that limit the full manifestation of DNA damage. Yet, DNA repair represents no panacea as DNA damage continuously slips through these erected defenses and materializes as mutation, which can have undesirable consequences as seen for cancer. Acquisition of early driver mutations can engender mutated stem cells with increased cellular fitness resulting in clonal expansion (CE) and increased risk of malignant disease. Tissue clonal mosaicism as observed in the elderly is therefore the natural outcome of continuous driver mutation acquisition in stem cells and their subsequent clonal outgrowth. Hence, a major emerging theme is that CE is an idiosyncrasy of the aging human tissue. This phenomenon can have diverse health consequences that we here divide into three categories: cancer, non‐cancer morbidity, and disease protection. This review outlines current day knowledge on clonal outgrowth, how it relates to health and aging, and how in the framework of DNA repair deficiencies these subjects are consolidated.

BRAFV600E mutational status assessment in cutaneous melanocytic neoplasms in a group of the Egyptian population

BackgroundMelanocytic neoplasms range from banal nevi to malignant melanomas. The genetic background has been extensively studied in the Caucasian population. BRAF mutations were reported among the early driver mutations in nevogenesis. Nevertheless, the pathogenesis in the Egyptian population has not been elucidated.Aim and MethodsThe present study was carried out to assess the sensitivity and specificity of immunohistochemistry (IHC) using the RM-08 clone in reference to allele-specific real-time PCR (CAST-PCR) for the detection of the BRAF V600E mutation in 50 formalin-fixed paraffin-embedded blocks of melanocytic neoplasms with prior bleaching using hydrogen peroxide in Tris-HCL and Bovine Serum Albumin respectively.ResultsIHC staining was interpreted using staining reaction (positive versus negative) and staining pattern (negative and heterogeneous versus homogenous). Using the staining pattern, the specificity increased from 73.3 to 88.2%, the negative predictive value increased from 73.3 to 100%, the diagnostic accuracy increased from 71.4 to 90.48% and the overall accuracy increased from 69.9 to 77.3%. The sensitivity and positive predictive value remained unchanged. The K-agreement coefficient increased from 0.364 (fair agreement) to 0.741 (good agreement) and was statistically significant (p = 0.00). Next-generation sequencing was performed in 11 cases, 8 cases with IHC-positive and BRAFwild type in addition to 3 cases that failed PCR analysis and revealed no BRAF V600E. No statistically significant difference was found in the clinicopathological parameters between BRAFV600E and BRAF wild−type melanomas.ConclusionsThese findings suggest that IHC staining homogeneity may be more accurate in predicting BRAFV600E mutational status. However, IHC cannot replace molecular methods.

Thyroid Fine Needle Aspiration, the Bethesda System, and the BRAFV600E Mutation in Papillary Thyroid Carcinoma: Association and Prediction for Biopsy

Introduction: BRAFV600E mutations have been associated with papillary thyroid carcinoma (PTC) histological types including tall-cell and classical, peritumoral infiltration, and nuclear signs, whereas cytological features such as plump cells and sickle nuclei have also been associated with favorable thyroid fine needle aspiration (FNA) results for this tumor. BRAF and RAS are considered early driver mutations that contribute to the development of BRAF-like PTCs and RAS-like PTCs. Our aim was to assess the possible association between all Bethesda System cytological features and thyroid FNAs for PTC and their potential predictive value for future BRAFV600E-related biopsies. Methods: Our study analyzed 63 cases of PTCs operated on at our hospital over a 5-year period between 2005 and 2017 that had previously undergone FNA and had been classified by the Bethesda System. BRAFV600E was identified by pyrosequencing paraffin-embedded tissues and comparing the cytological signs with the Bethesda System. In addition, a statistical and predictive study of the diagnostic factors “non-follicular,” “non-round nuclei,” and “non-clear chromatin” was performed to discriminate BRAF-like signs from other hypothetical RAS-like follicular signs. Results: BRAFV600E was detected in 43/63 cases (68.2%). Histological types were significant (p < 0.001), with the classical variant being the most prevalent 31/63 (49.2%) and independent by multivariate analysis odds ratio of 10.58 [2.67; 41.97]. Follicular cytological signs are negatively associated with BRAFV600E: follicular structure (p < 0.001), round nuclei (p = 0.015), and clear chromatin (p = 0.049), while the diagnostic factors: “non-follicular” (positive predictive value [PPV] 82.9, sensitivity 79.1, negative predictive value [NPV] 59.1, specificity 65.0), “non-round nuclei” (PPV 76.6, sensitivity 83.7, NPV 56.3, specificity 45.0), and “non-clear chromatin” (PPV 75.6, sensitivity 79.1, NPV 50.0, specificity 45.0) have predictive value for the mutation. There was no individual significance for the remaining cytological features. Conclusions: Our study found no association between cytomorphological signs of thyroid FNA and BRAFV600E mutation. Considering the Bethesda System, there is an association (p = 0.045) with numerous cases of mutated PTC in categories V and VI. Our results indicate, however, that the presence of signs referred to as “non-follicular,” “non-round nuclei,” and “non-clear chromatin” in biopsy of papillary thyroid carcinoma is predictive of BRAF type mutation, whereas follicular signs indicate a RAS type PTC, according to published literature. These results need to be confirmed or modified by further research.

080 Current treatment options for patients with unresectable cutaneous melanoma

Cutaneous Melanoma (CM) remains the leading cause of skin-related mortality in the U.S. The U.S. Surveillance, Epidemiology and End Results database reports 5.6% of all new cancer cases in 2021 attributed to CM, with incidences rising annually since 2009. Several subtypes of CM are thought to arise from early driver mutations in BRAF, RAS, and NF1 genes resulting in unopposed activation of MAPK and PI3K signaling pathways, unregulated growth and malignant transformation of melanocytes. Due to the metastatic potential of CM, emphasis on systemic treatment has been an area of interest when surgical resection is insufficient for curative prognosis.

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.

Anaplastic Thyroid Carcinoma

A thyroidectomy was performed for a rapidly enlarging neck mass in a woman in her 70s. The tumor was diagnosed by pathology as an anaplastic thyroid carcinoma. Which of the following statements regarding anaplastic thyroid carcinoma is most accurate?a.Anaplastic thyroid carcinoma is a highly aggressive malignancy that usually occurs in persons in their second to third decade of life.b.Almost all anaplastic thyroid carcinomas occur in the setting of previous radiation to the neck.c.Anaplastic thyroid carcinomas are highly aggressive malignancies, usually fatal, and affect older persons.d.Anaplastic thyroid carcinomas may be associated with BRAF or RAS mutations but are not associated with TP53 or TERT promoter mutations. Answer: c. Anaplastic thyroid carcinomas are highly aggressive malignancies, usually fatal, and affect older persons. Anaplastic thyroid carcinoma is a highly aggressive thyroid malignancy composed of undifferentiated follicular thyroid cells. These tumors are uncommon, accounting for 1% of thyroid carcinomas in the United States.1Pereira M. Williams V.L. Hallanger Johnson J. Valderrabano P. Thyroid cancer incidence trends in the United States: association with changes in professional guideline recommendations.Thyroid. 2020; 30: 1132-1140Crossref PubMed Scopus (36) Google Scholar Anaplastic thyroid carcinoma usually affects older persons (seventh decade), often presenting as a rapidly enlarging mass that may be associated with compressive symptoms and, in some cases, paraneoplastic manifestations. Anaplastic thyroid carcinoma is generally lethal, and 56% of patients have lymph-node metastases and 38% distant metastases at presentation.2Hvilsom G.B. Londero S.C. Hahn C.H. et al.Anaplastic thyroid carcinoma in Denmark 1996-2012: a national prospective study of 219 patients.Cancer Epidemiol. 2018; 53: 65-71Crossref PubMed Scopus (12) Google Scholar The tumors are highly infiltrative, often have extrathyroidal growth, angiolymphatic invasion, and high-grade nuclear features. A variety of histologic subtypes of anaplastic thyroid carcinoma occur such as epithelioid, spindle cell/sarcomatoid, paucicellular, squamous, giant cell, rhabdoid/rhabdomyoblastic among others (Figures), making the differential diagnosis challenging in some cases. Approximately 10% of these tumors are associated with history of radiation exposure or iodine deficiency, respectively. Anaplastic thyroid carcinomas may occur with a previous or concurrent well-differentiated or poorly differentiated thyroid carcinoma.3Lam K.Y. Lo C.Y. Chan K.W. Wan K.Y. Insular and anaplastic carcinoma of the thyroid: a 45-year comparative study at a single institution and a review of the significance of p53 and p21.Ann Surg. 2000; 231: 329-338Crossref PubMed Scopus (132) Google Scholar,4Xu B. Fuchs T. Dogan S. et al.Dissecting anaplastic thyroid carcinoma: a comprehensive clinical, histologic, immunophenotypic, and molecular study of 360 cases.Thyroid. 2020; 30: 1505-1517Crossref PubMed Scopus (57) Google Scholar Anaplastic thyroid carcinomas are described as having “early” and “late” genetic alterations. Early driver mutations often involve BRAF V600E (especially in tumors associated with papillary thyroid carcinoma) or RAS and late mutations involve TP53 and TERT promoter.5Volante M. Lam A.K. Papotti M. Tallini G. Molecular pathology of poorly differentiated and anaplastic thyroid cancer: what do pathologists need to know?.Endocr Pathol. 2021; 32: 63-76Crossref PubMed Scopus (13) Google ScholarView Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Uveal melanoma pathobiology: Metastasis to the liver.

Uveal melanoma (UM) is a type of intraocular tumor with a propensity to disseminate to the liver. Despite the identification of the early driver mutations during the development of the pathology, the process of UM metastasis is still not fully comprehended. A better understanding of the genetic, molecular, and environmental factors participating to its spread and metastatic outgrowth could provide additional approaches for UM treatment. In this review, we will discuss the advances made towards the understanding of the pathogenesis of metastatic UM, summarize the current and prospective treatments, and introduce some of the ongoing research in this field.

Genomic landscape of platinum resistant and sensitive testicular cancers

While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combine this with published genomic data on an additional 624 TGCTs. We integrate analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised.

Widely metastatic glioblastoma with BRCA1 and ARID1A mutations: a case report

BackgroundGlioblastoma (GBM) is a highly malignant brain neoplasm with poor survival. Despite its aggressive nature, metastatic spread of GBM is identified only rarely. While the molecular alterations associated with GBM and its subtypes are well-described, there remains a gap in understanding which alterations may predispose towards metastasis. In this report, we present a case of GBM with multi-organ metastases and discuss its genomic alterations.Case presentationA 74-year-old woman was diagnosed with left occipital glioblastoma (IDH-wildtype, MGMT-unmethylated), for which she underwent resection, standard chemoradiation, and then stereotactic radiosurgery (SRS) for local recurrence. One month after SRS, work-up for a pathologic hip fracture revealed a left breast mass, lytic lesions involving pelvic bones, and multiple pulmonary and hepatic lesions. Biopsies of the breast and bone lesions both demonstrated metastatic IDH-wildtype GBM. For worsening neurologic symptoms, the patient underwent debulking of a large right temporal lobe recurrence and expired shortly thereafter. Autopsy confirmed metastatic GBM in multiple systemic sites, including bilateral lungs, heart, liver, thyroid, left breast, small bowel, omentum, peritoneal surfaces, visceral surfaces, left pelvic bone, and hilar lymph nodes. Targeted sequencing was performed on tissue samples obtained pre- and postmortem, as well as on cell cultures and an orthotopic mouse xenograft derived from premortem surgical specimens. A BRCA1 mutation (p.I571T) was the only variant found in common among the primary, recurrence, and metastatic specimens, suggesting its likely status as an early driver mutation. Multiple subclonal ARID1A mutations, which promote genomic instability through impairment of DNA mismatch repair, were identified only in the recurrence. Mutational spectrum analysis demonstrated a high percentage of C:G to T:A transitions in the post-treatment samples but not in the primary tumor.ConclusionThis case report examines a rare case of widely metastatic IDH-wildtype GBM with a clonal somatic mutation in BRCA1. Post-treatment recurrent tumor in the brain and in multiple systemic organs exhibited evidence of acquired DNA mismatch repair deficiency, which may be explained by functional loss of ARID1A. We identify a potential role for immune checkpoint and PARP inhibitors in the treatment of metastatic GBM.

Molecular changes during progression from nonmuscle invasive to advanced urothelial carcinoma.

Molecular changes occurring during invasion and clinical progression of cancer are difficult to study longitudinally in patient‐derived material. A unique feature of urothelial bladder cancer (UBC) is that patients frequently develop multiple nonmuscle invasive tumors, some of which may eventually progress to invade the muscle of the bladder wall. Here, we use a cohort of 73 patients that experienced a total of 357 UBC diagnoses to study the stability or change in detected molecular alterations during cancer progression. The tumors were subtyped by gene expression profiling and analyzed for hotspot mutations in FGFR3, PIK3CA and TERT, the most frequent early driver mutations in this tumor type. TP53 alterations, frequent in advanced UBC, were inferred from p53 staining pattern, and potential genomic alterations were inferred by gene expression patterns at regions harboring frequent copy number alterations. We show that early driver mutations were largely preserved in UBC recurrences. Changes in FGFR3, PIK3CA or TERT mutation status were not linked to changes in molecular subtype and aggressive behavior. Instead, changes into a more aggressive molecular subtype seem to be associated with p53 alterations. We analyze changes in gene expression from primary tumors, to recurrences and progression tumors, and identify two modes of progression: Patients for whom progression is preceded by or coincides with a radical subtype shift, and patients who progress without any systematic molecular changes. For the latter group of patients, progression may be either stochastic or depending on factors already present at primary tumor initiation.

Temporal dissection of altered pathways during the evolution of cancer

Abstract Background Cancer arise as normal cells acquire insults to the genome through both mutations and gross copy number alterations. Our current understanding of the molecular makeup of cancer is based on a snap-shot, a biopsy taken at a single time point. Large-scale studies have shown an abundance of genomic alterations, but few are shared across samples which makes interpretation of effect difficult, particularly as different processes may be dominant at different time points in the development of cancer. Here, we have performed pathway analysis on timed mutational driver events to investigate if specific pathways are preferentially affected at different time points during the life-history of cancer. Methods We analyzed mutation data and copy number information from bladder cancer acquired from The Cancer Genome Atlas, to time the mutations as early and late relative to copy number events affecting the same genomic location (McGranahan et al., 2015). The mutations were annotated to genes and potential driver mutations were annotated (Jamal-Hanjani et al., 2017). Timed driver events across each cohort were combined into early and late, and subjected to pathway analysis using Reactome (Fabregat et al., 2017). Results Focusing on bladder cancer, 28% of the timed mutations are early and 72% are late. The most frequent early driver mutations are TP53 (12%), MUC4 (8%), and PIK3CA (5%), while in late they are PABPC1 (21%), KMT2C (16%), HLA-A (9%). Pathway analysis on early drivers showed how events that affects cell cycle and proliferation dominates, while late drivers are particularly enriched in pathways associated with the immune system. Conclusions Our results demonstrate a clear time-separated preference for specific events. These indicates that selection in early cancer development may be primarily focused on genomic alterations that increase proliferation. Conversely, late cancer development enriches in events targeting pathways associated with the immune response, consistent with increased immune pressure as the cancer develops. These results show how timing of events may provide novel insights into how cancer develops, and may help determine the evolutionary developmental stage of cancer with potential implications for improved prognostics and differential therapy. Legal entity responsible for the study The authors. Funding The Lundbeck Foundation. Disclosure All authors have declared no conflicts of interest.

ES17.01 Mesothelioma Evolution

Malignant pleural mesothelioma (MPM) is mostly caused by prior exposure to asbestos fibres. It has a long but variable latency period following exposure, with a median of around 40 years but a range between 20-70 years, possibly reflecting the length and level of asbestos exposure as well as other environmental and genetic causes. Upon diagnosis, there are limited treatment options, and median survival time is a year, although, again, this is highly variable. Understanding the genetic events in the mesothelium between asbestos exposure and diagnosis of MPM is important for two reasons. Firstly, it will inform the biology of MPM tumour growth and potentially highlight different environmental and genetic factors that cause the variation in latency. Secondly, it will help identify key early driver mutations in MPM informing biology and candidate changes for developing approaches for early detection of the cancer. Evolutionary genetics is based on inferring past events from current genetic/genomic sequences. It has long been recognised that the development of cancer is an evolutionary process, and the availability of large amounts of DNA sequence data have facilitated an understanding of evolution of tumours using methods mostly borrowed from evolutionary genetics. One powerful approach compares a matched tumour and normal genome, infers the somatic mutations in the tumour, and uses the ratio of mutations that change an amino acid to mutations that don’t change an amino acid (dN/dS ratio) across all genes to identify particular genes that have been positively selected during tumour evolution. This approach can also shed light on overall evolutionary processes that have occurred in the tumour. Genomic sequences from multiple regions of the same tumour not only emphasise the molecular heterogeneity of tumours but allow an explicit phylogenetic tree of the evolution of the tumour for each patient, distinguishing somatic mutations that happened early in the tumour's evolution (and are therefore present throughout the tumour) from those that happened late in the tumour evolution. Here, I report preliminary findings from a British Lung Foundation/Mesothelioma UK-funded project entitled MEDUSA – Mesothelioma Evolution: Deciphering drUgable Somatic Alterations as potential targets for synthetic lethal therapy. This project uses multiregional sampling of MPMs, together with matched whole blood, to infer a phylogenetic tree of MPMs. The preliminary data presented focuses on the first 20 patients, with between 4 and 5 regions of the tumour analysed per patient. Using whole exome sequencing, the project aims to identify truncal changes, that is, mutations that happened early in the tumour and are present throughout the tumour that can be potential targets for drugs, with the aim of developing personalised, effective tumour treatment for each patient. We focus on copy number changes (deletions and duplications of genes) and confirm that MPM is highly heterogeneous with extensive copy number changes in the genome. We focus on truncal copy number changes in ∼20-25% of patients affecting the known mesothelioma tumour suppressor genes BAP1, MTOR, CDKN2A and SETD1. Distinguishing patients that have truncal copy number changes in these genes, in contrast to those patients with copy number changes in the terminal branches of the evolution of the tumour, helps to tailor individualised drug therapies. Our approach emphasises the importance of multiregional sampling of tumours to account for MPM heterogeneity. For example, by sampling the posterior costophenic angle of these MPM from these 20 patients, we would find 10 deletions of CDKN2A, of which only 5 are truncal, with the other five localised to only part of the tumour. Multiregional genomic analysis and evolutionary genetics approaches can illuminate the history of a tumour and have the potential to guide therapy. They also provide the framework for follow-on studies in a patient, such as analysing the origin of metastases and identifying the effects on the tumour of treatment. The extra information provided by multiregional sampling supports the idea that this approach should become routine in tailoring the treatment to the tumour in MPM. evolution, Mesothelioma, Genomics

Comparison of the genomic background of MET-altered carcinomas of the lung: biological differences and analogies

Although non-small-cell lung cancer is a leading cause of cancer-related deaths, the molecular characterization and classification of its genetic alterations has drastically changed treatment options and overall survival within the last few decades. In particular, tyrosine kinase inhibitors targeting specific molecular alterations, among other MET, have greatly improved the prognosis of non-small-cell lung cancer patients. Here, we compare the genomic background of a subset of non-small-cell lung cancer cases harboring either a MET high-level amplification (n = 24) or a MET exon 14 skipping mutation (n = 26), using next-generatison sequencing, fluorescence in situ hybridization, immunohistochemistry, and Nanostring nCounter® technology. We demonstrate that the MET-amplified cohort shows a higher genetic instability, compared with the mutant cohort (p < 0.001). Furthermore, MET mutations occur at high allele frequency and in the presence of co-occurring TP53 mutations (n = 7), as well as MDM2 (n = 7), CDK4 (n = 6), and HMGA2 (n = 5) co-amplifications. No other potential driver mutation has been detected. Conversely, in the MET-amplified group, we identify co-occurring pathogenic NRAS and KRAS mutations (n = 5) and a significantly higher number of TP53 mutations, compared with the MET-mutant cohort (p = 0.048). Of note, MET amplifications occur more frequently as subclonal events. Interestingly, despite the significantly (p = 0.00103) older age at diagnosis of stage IIIb/IV of MET-mutant patients (median 77 years), compared with MET high-level amplified patients (median 69 years), MET-mutant patients with advanced-stage tumors showed a significantly better prognosis at 12 months (p = 0.04). In conclusion, the two groups of MET genetic alterations differ, both clinically and genetically: our data strongly suggest that MET exon 14 skipping mutations represent an early driver mutation. In opposition, MET amplifications occur usually in the background of other strong genetic events and therefore MET amplifications should be interpreted in the context of each tumor's genetic background, rather than as an isolated driver event, especially when considering MET-specific treatment options.

Clinical Validation of Coexisting Activating Mutations Within EGFR, Mitogen-Activated Protein Kinase, and Phosphatidylinositol 3-Kinase Pathways in Lung Cancers.

Mutations within the same signature transduction pathway are redundant and, therefore, most are mutually exclusive. Laboratory errors, however, may introduce unexpected coexisting mutations. To validate coexisting mutations within epidermal growth factor receptor (EGFR), mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. In this retrospective study for quality assessment of next-generation sequencing in a clinical diagnostics setting, coexisting mutations within EGFR, KRAS, NRAS, BRAF, AKT1, and PIK3CA genes were examined in 1208 non-small cell lung cancers. EGFR mutations did not coexist with BRAF mutations, neither kinase-activated nor kinase-impaired mutations. There was a low but similar incidence (3.3%-5.1%) of PIK3CA mutations in BRAF-, EGFR-, and KRAS-mutated lung cancers and a rare incidence of coexisting KRAS and EGFR mutations detected in 1 of 1208 lung cancers (0.08%) or 1 of 226 EGFR-mutated lung cancers (0.4%). Coexisting BRAF p.V600E mutation was observed in 3 of 4 AKT1 p.E17K-mutated lung cancers. Mutational profiling of DNA reisolated from subareas with the same or different histomorphology, using an alternative assay, confirmed that coexisting mutations might present within the same (whole or subclonal) population or different populations and clarified that the so-called coexisting activating KRAS and BRAF mutations originally reported in a specimen were indeed present in separate lung nodules submitted in the same block. The results supported that EGFR and BRAF mutations are early driver mutations in lung cancers. Guidelines from official organizations to establish standard operating procedures are warranted to validate unexpected coexisting mutations and, if clinically indicated, to determine their presence in the same or different tumor populations.

Somatic evolutionary timings of driver mutations

BackgroundA unified analysis of DNA sequences from hundreds of tumors concluded that the driver mutations primarily occur in the earliest stages of cancer formation, with relatively few driver mutation events detected in the late-arising subclones. However, emerging evidence from the sequencing of multiple tumors and tumor regions per individual suggests that late-arising subclones with additional driver mutations are underestimated in single-sample analyses.MethodsTo test whether driver mutations generally map to early tumor development, we examined multi-regional tumor sequencing data from 101 individuals reported in 11 published studies. Following previous studies, we annotated mutations as early-arising when all tumors/regions had those mutations (ubiquitous). We then inferred the fraction of mutations occurring early and compared it with late-arising mutations that were found in only single tumors/regions.ResultsWhile a large fraction of driver mutations in tumors occurred relatively early in cancers, later driver mutations occurred at least as frequently as the early drivers in a substantial number of patients. This result was robust to many different approaches to annotate driver mutations. The relative frequency of early and late driver mutations varied among patients of the same cancer type and in different cancer types. We found that previous reports of the preponderance of early driver mutations were primarily informed by analysis of single tumor variant allele profiles, with which it is challenging to clearly distinguish between early and late drivers.ConclusionsThe origin and preponderance of new driver mutations are not limited to early stages of tumor evolution, with different tumors and regions showing distinct driver mutations and, consequently, distinct characteristics. Therefore, tumors with extensive intratumor heterogeneity appear to have many newly acquired drivers.

Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.

Abstract 1573: Detection of late-arising driver mutations using multi-sample tumor sequencing data

Abstract Whole-genome sequencing of tumors has identified genetic mutations that drive the origination, growth, and spread of cancer. Most driver mutations detected from sequencing tumors have been categorized as early-occurring and are believed to play a role in initial tumor formation. However, single-region primary tumor samples represent just one snapshot of the continuously changing mutational landscape of a tumor. Drivers of initial tumor formation, occurring early on in tumor development, are present in more tumor cells and are more likely to be identified from a single tumor sample than drivers of later events like metastasis, which are likely to be found in only one or a few tumor samples. We analyzed 101 multi-region and/or multi-tumor sequencing data sets using driver mutation annotation and phylogenetic inference to determine whether driver mutations occur preferentially early on. We found driver mutations frequently arise late in the course of tumor evolution, even when statistically accounting for small amounts of total drivers and many early total mutations. In addition, the timing of driver mutation occurrence varies widely depending on cancer type as well as among different individuals with a single cancer subtype. Finally, we identify known cancer genes that are enriched in either early or late driver mutations, reflecting different selective pressures at various stages of cancer development. These results illustrate how drivers of primary tumorigenesis must be distinguished from drivers of treatment resistance, metastasis, or recurrence in order to better understand the genetic changes behind cancer progression. Citation Format: Karen Gomez, Sayaka Miura, Sudhir Kumar. Detection of late-arising driver mutations using multi-sample tumor sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1573. doi:10.1158/1538-7445.AM2017-1573

Abstract 4371: Genomic characterization of oropharyngeal squamous cell carcinoma in Korean population

Abstract Objective: Oropharyngeal squamous cell carcinoma (OPSCC) is a subtype of head and neck squamous cell carcinoma (HNSCC). Environmental risk factors such as tobacco, alcohol, and especially, human papillomavirus (HPV) are known to be associated with HNSCC. Also, large-scale sequencing studies identified several HNSCC-associated genes such as TP53, EGFR and PIK3CA. However, still the initiation and progression of OPSCC and mutational profiles of OPSCC in Korean population are unclear. Methods: Frozen non-tumor mucosa, tumor tissues and blood were collected from nine OPSCC patients; 6 HPV positive and 3 negative. Adjacent non-tumor tissues were obtained about 1 cm apart from the tumor tissue and were histologically confirmed as non-neoplastic. We then performed whole exome sequencing with Agilent SureSelect Human All Exon Kit v4+UTR, yielding average on-target coverage of 100X. Results: First, we compared somatic alterations of adjacent non-tumor and tumor tissue to understand the genomic status of non-tumor mucosa. Comparison of genome-wide copy number alterations (CNA) between non-tumor and OPSCCs showed that majority of somatic CNA (SCNA) was occurred after tumor progression. Also, non-tumor mucosa rarely harbored genomic changes, showing average nonsilent mutations rate of 0.07 per Mb. In contrast, OPSCC showed 1.0 mutations per Mb with few shared mutations with non-tumor. These results indicate occurrence of new distinct subclones after tumor progression. Second, somatic mutations and CNA analysis of 9 OPSCC identified two distinct groups divided by the HPV status of tumor. HPV(+) tumors showed lower mutation burden than HPV(-) tumors (0.63 and 1.73 mutations per Mb, respectively). And TP53 and CDKN2A mutations were detected exclusively in HPV(-) group, accounting for 66.6% of HPV(-) tumors. Furthermore, HPV(-) tumors showed greater degree of chromosomal alterations compared to HPV(+) tumors. Conclusions: Although there are many studies to understand the relationships between premalignant and malignant tissue, there is a lack of understanding for genetic changes further from non-neoplastic mucosa. Our data show that genetic profile of normal crypt mucosa rarely changes and tumor develops in distinct subclones. This would provide a glimpse into the fundamental cellular changes of non-tumor cells of tonsillar crypts and oral cavity. Also, we provide a mutational landscape in HNSCC of Korean population. Although our OPSCC showed recurrent mutations and copy number alterations of genes related to RTK/PI(3)K- and differentiation-signaling pathways, similar as western, mutation burden in tumors was relatively lower, and somatic mutations on well-known HNSCC-associated genes such as PIK3CA, FAT1 and HRAS weren’t detected. Further investigation and molecular experiments are required to understand the mechanism of carcinogenesis and to discover early driver mutations of HNSCC. Citation Format: Jeesoo Chae, Weon Seo Park, Dongwan Hong, Jong-Il Kim, Yuh-Seog Jung. Genomic characterization of oropharyngeal squamous cell carcinoma in Korean population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4371. doi:10.1158/1538-7445.AM2017-4371

Genomic Discovery, Prognosis, and Target Therapy Development

For over three decades now, the t(14;18)(q32.3;q21.3) has been synonymous with the indolent B cell malignancy, follicular lymphoma (FL). Through the widespread uptake of next generation sequencing, we recognize that these tumors acquire many additional genetic aberrations, targeting several distinct signaling pathways (HVEM, mTORC1, NOTCH, NF-κB and JAK-STAT) all of which fall onto a backbone of epimutations that arise in nearly all FL tumors and preferentially target the histone methyl (KMT2D) and acetyl (CREBBP) transferase machinery. We are still in the early stages of understanding how these mutations impact on the biology of FL, their weight of contribution to FL initiation, progression and transformation and indeed whether we can harness this new knowledge to improve on existing treatment. At the very least, we now realize that the acquisition of these mutations and onset of FL may occur several years earlier from the study of rare examples of clonally-related tumors developing post bone marrow transplantation in both donor and recipient. This has helped formulate the notion of an uncharacterized common progenitor pool of B cells (CPC) that are capable of seeding each new genetically distinct episode of disease and providing an explanation for the relapsing remitting nature of FL. We can also now infer the nature of the apparent early driver mutations that seemingly propagate the disease in all FL clones, including the CPC, by using a gene’s mutation load or clonal composition as a surrogate marker. It is apparent that many epimutations represent early events and appear to alter normal B cell programming by favoring sets of genes that promote proliferation and survival while switching off anti-tumor mechanisms, with CREBBP-mutated FLs for example showing a decreased antigen presentation gene signature. The prospect of following a precision medicine approach informed by a patient’s mutation composition should be encouraged given the frequency of activating EZH2 mutations, an underappreciated role of the NF-κB pathway and mutations in the mTORC1 pathway, that are unique to FL, together with the availability of targeted therapeutic agents. There is of course a need to focus efforts on the 20% of patients that succumb to early disease relapse, and the emergence of a new clinical-genetic risk model (m7-FLIPI), as an outcome predictor offers a promising first step in developing a robust risk stratification approach. We do however need to embrace these new opportunities and embed biomarker discovery and validation studies as part of all FL trial programs. Indeed, a case can be made for utilizing material from specific retrospective studies, where updated mutation analysis may demonstrate a potential novel predictive and therapeutic biomarker (e.g. RRAGC mutations/mTOR inhibitors). Altogether, while the genetic portrait of FL is still far from complete, we are now much closer to delivering a final canvas and with it the prospect of changing the landscape of future FL treatment. DisclosuresFitzgibbon:Epizyme: Research Funding; Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria.