Early Driver Mutations Research Articles

Non-neoplastic Fallopian Tube Epithelium Carrying Gene Mutations of a Novel SOX2 Repressor Region is Soil of High-grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) has a high mortality rate because the disease is asymptomatic in early stages and is resistant to treatments. A novel modality for diagnosis in the early stages of carcinogenesis is needed. In this issue of EBioMedicine, Hellner et al. present novel genetic aspects of small lesions of HGSOC (Hellner et al., 2016). They performed screening by LFR WGS technology for small HGSOC lesions from a patient who underwent treatment and found 750 genomic mutations. Gene ontology analysis revealed that the gene mutations are related to stem cell differentiation, and the authors finally found 6 novel non-coding mutations (BB1 - BB6) in proximity of sex determining region Y-box2 (SOX2), a key driver of stem cell differentiation (Takahashi and Yamanaka, 2006). Deep genetic analysis revealed that the BB5 region is commonly mutated in HGSOC tissues. A reporter assay and genome engineering using CRISPR/Cas9 system revealed that the BB5 region is a novel repressor of the SOX2 gene and that mutation in the BB5 region induces protein expression of SOX2. Mutations in the BB5 region were detected in non-neoplastic fallopian tube epithelium (FTE) of HGSOC cases and also in normal FTE of BRCA1 or BRCA2 mutation carriers who are at high risk for HGSOC. Surprisingly, immunohistochemical analysis of BB5 mutation carriers revealed that SOX2 protein is broadly expressed in even normal FTEs, indicating that non-neoplastic FTEs with BB5 mutation propagated clonally. SOX2 protein is expressed in earlier stages than TP53 protein expression indicating overexpression of SOX2 protein is earlier event than TP53 gene mutation. Interestingly, SOX2 protein expression decreased after the establishment of an ovarian cancer lesion. These results suggest that mutation in the SOX2 BB5 region is an early carcinogenesis driver mutation in HGSOCs and that detection of SOX2 protein is a promising biological marker for prediction of HGSOC.

Premalignant SOX2 overexpression in the fallopian tubes of ovarian cancer patients: Discovery and validation studies

Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p<2−16), which was not found in patients without cancer (n=108). Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n=100), and common in BRCA1-BRCA2 mutation carriers (n=71) who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.

Melanomas Associated With Blue Nevi or Mimicking Cellular Blue Nevi: Clinical, Pathologic, and Molecular Study of 11 Cases Displaying a High Frequency of GNA11 Mutations, BAP1 Expression Loss, and a Predilection for the Scalp.

Melanomas associated with blue nevi (MABN) or mimicking cellular blue nevi (MMCBN) represent exceptional variants of malignant cutaneous melanocytic tumors. Uveal and leptomeningeal melanomas frequently have somatic mutations of GNAQ or GNA11, which are believed to be early driver mutations. In uveal melanomas, monosomy 3, linked to the BAP1 gene, is an adverse prognostic factor. We have studied the clinical, histologic, BAP1 expression profile, and molecular data of 11 cases of MABN/MMCBN and 24 cellular blue nevi. Most of the cases of MABN/MMCBN occurred on the scalps of adult patients and presented as rapidly growing nodules, typically >1 cm, often arising at the site of a preexisting melanocytic lesion. The MABN/MMCBN were composed of dense nests of large dermal atypical melanocytes, in some cases lying adjacent to a blue nevus. Four patients developed metastatic disease, and 2 died from their disease. A GNA11 mutation was found in 8/11 cases and a GNAQ mutation in 1 case. Seven of 11 cases showed loss of nuclear BAP1 immunohistochemical (IHC) expression in the malignant component, sparing the adjacent nevus. Array comparative genomic hybridization revealed recurrent deletions of chromosomes 1p, 3p, 4q, 6q, 8p, 16q, and 17q and recurrent gains of chromosomes 6p, 8q, and 21q. The 24 cases of cellular blue nevi frequently occurred on the sacrum, had GNAQ mutations, and showed normal positive IHC staining for BAP1. These results underscore overlapping features in all blue-like malignant melanocytic tumors. Loss of BAP1 IHC expression was restricted to melanomas, including all metastatic cases.

Abstract 67: Genomic and mutational profiling of human colon adenomas reveals early driver mutations and a TGF-β-CEA regulated profile

Abstract Introduction: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world with 143,700 newly diagnosed cases in 2012. The rapid development of cancers in small, sessile adenomas could be a contributing factor that may have been overlooked. Identifying high risk patients through genomic analysis of adenomas could potentially lead to an early intervention therapy. Methods: Whole Genome Sequence (WGS) and Whole Exome Sequence (WES) analyses were performed for 4 pairs of normal controls (two from proximal colon and two from the distal colon) and colorectal adenomas (2 sessile serrated (SSA) and 2 tubulovillous adenomas (TVA) less than 1 cm in size). Transcriptome sequence analysis was performed in seven pairs of control-test matched adenomas (6 TVA and 1 SSA). Expression level of CEA and TGF-β pathway members were carried out on 30 non dysplastic adenomas and normal colon tissues by immunohistochemistry. Results: 1. Hyper-mutator profiles were observed in two of the samples (1 SSA and 1 TVA) by WGS with 1709 mutations after normalization with normal paired samples. 2. The samples showed an average mutation frequency of 0.55 mutations per 106 bases. 3. Aberrant mutational profiles was detected in seven of the eleven adenomas, with distinct mutational signatures among the samples, two with high, two intermediate and three low mutational rates. 4. Six of the eleven adenomas (1 SSA and 4 TVAs) showed alteration in the Wnt and p53 pathway. 5. Transitional single nucleotide substitutions of C:T&amp;gt;G:A in the mutational spectrum were observed in 37% of the samples. 6. Subtle localized hyper mutation (kataegis) was observed among two of the samples. 7. Five out of eleven adenomas showed mutations in the TGF-β (transforming growth factor-β) and CEA pathways members, overlapping with Wnt/p53 mutations in four adenomas. 8. Analyses of expression level of CEA and TGF-β pathway members in 30 non dysplastic tissues revealed a marked increase (over 8 fold) in CEA expression in 25% of adenoma samples which was linked to concomitant loss of TGF-β signaling. 9. Further functional studies revealed that CEA associated with TGF-β Type I receptor and disruption of TGF-β tumor suppressor signaling with activation of STAT3. Conclusions: Our studies indicate that small adenomas both TVAs and SSAs can resemble CRCs in genomic profiling and may reflect a high risk population. Genetic and mechanistic analyses reveal that disruption of CEA/TGF-β pathway in early adenomas may reflect a new and early role for these pathways in CRC. This study further supports the biomarker driven targeting of CEA/TGF-β in high risk adenomas and can be used as a prognostic marker for early detection of aggressive adenoma-CRC progression. Citation Format: Vipin K. Menon, Raju S. Gottumukkala, Jian Chen, Xiaoping Su, Nipun Mistry, Avijit Majumdar, Ji-Hyun Shin, Shulin Li, Kirti Shetty, Xifeng Wu, Brian Weston, Ethan Miller, John R. Stroehlein, Marta L. Davila, Mehnaz A. Shafi, Asif Rashid, Bhaskar V. Kallakury, Selvi Thirumurthi, John S. McMurray, Sue-Hwa Lin, Wilma Jogunoori, Lopa Mishra. Genomic and mutational profiling of human colon adenomas reveals early driver mutations and a TGF-β-CEA regulated profile. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 67. doi:10.1158/1538-7445.AM2015-67

The clonal relationships between pre-cancer and cancer revealed by ultra-deep sequencing.

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588.

Sa1914 The Genomic Landscape of Human Colon Adenomas Reveals Early Driver Mutations and a TGF-β-CEA Regulated Profile

Sa1914 The Genomic Landscape of Human Colon Adenomas Reveals Early Driver Mutations and a TGF-β-CEA Regulated Profile

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers.

DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

Lineage-Specific Distribution of CALR EXON 9, JAK2V617F, MPLW515L/K, NPM1 and FLT3 Mutations in Myeloid Disorders

Introduction:Myeloid malignant disorders are clonal diseases arising in hematopoietic stem or progenitor cells. Several somatic mutations involved in these diseases are currently known and routine molecular testing involves screening genes of therapeutic and prognostic significance. Mutational analysis of FLT3 in combination with NPM1 can be used to predict outcome and direct therapy in normal karyotype acute myeloid leukemia (AML). JAK2, MPL and CALR mutation detection complements the molecular diagnostic testing menu for myeloproliferative neoplasm’s (MPN): polycytemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In addition, these molecular markers are utilized for minimal residual disease (MRD) detection, e.g. following stem cell transplants. Little is currently known about the lineage-specific distribution of some of these markers. In this study we aimed to assess the distribution of common genetic mutations in multiple lineages (lymphoid, myeloid, monocyte, multipotent progenitors, myeloblast and erythroid) of MPN and AML utilizing fluorescent activated cell sorting (FACS).Method:Different cell lineage fractions (lymphoid (CD3+), mature (CD16+) and immature (CD16dim) granulocytes, monocyte (CD14+), erythroid(CD36+), multipotent progenitors (34+) and/ or myeloblasts (CD117+) of unseparated bone marrow and peripheral blood specimens of myeloid disorders were sorted on a BD Aria 2. The patient specimens selected were positive for either JAK2V617F, MPLW515L or CALR Exon9 insertion/ deletion (MPN’s) or for NPM1 and/or FLT3 mutations (AML; diagnostic, relapse and minimal residual disease (MRD)). Fractions were subsequently analyzed for the presence of the respective mutation by PCR and/ or bi- directional sequencing.Results:All FACS purified CD34+ progenitors, myeloid and erythroid cell fractions of MPL W515L (3) or CALR exon9 (12) positive MPN specimens demonstrated the presence of mutations, respectively. Interestingly, JAK2V617F was present in the sorted erythroid cell fraction in 5/6 MPN cases tested. However, the granulocyte cell and blast cell fraction of one polycythemia vera specimen tested negative for the presence of Jak2V617F. All lymphoid CD3+ T-cell fractions were negative.The NPM1 exon 12 mutation was uniformly detected in progenitors and all myeloid cell fractions of 3/3 diagnostic, 2/2 relapse and 1/8 MRD AML specimens. For 5/8 MRD cases all lineages tested negative. Surprisingly, for two MRD cases the mutation was observed only in the unseparated and myeloid lineages but not in CD34+ blast fraction. Similar to the above findings, FLT3 mutations were detected in multipotent progenitors, and/ or myeloblasts collections of 4/4 diagnostic specimens. However, the mutation was absent in the granulocyte and monocyte fraction of one case. No detectable signals were observed in the cell fractions of 5 MRD specimens and in the CD3+ lymphoid cell fractions of all AML cases.Conclusion:We conclude that CALR and MPL mutations are uniformly detectable in the unseparated bone marrow specimens of MPN’s as well as separated progenitor, erythroid, granulocyte and monocyte fractions. Interestingly, JAK2 mutations can be exclusively found in the erythroid lineage in PV, whereas it can be absent in the granulocyte and blast compartment. This finding may have implications on specimen processing to ensure that erythroids are retained for clinical Jak2 testing. In addition, our results support the hypothesis that CALR Exon9 mutations are early event driver mutations in comparison to JAK2V167F.Both NPM1 and FLT3 mutations, in AML, were detected in unseparated specimens as well as in the multipotent progenitors or myeloblasts at diagnosis and relapse. However, the NPM1 mutation was observed in unseparated specimens and granulocyte cell fractions of 2 residual disease cases, whereas it was surprisingly absent in the CD34+ cell lineage fractions. Conversely, FLT3-ITD was exclusively found in the progenitor cells and absent in the granulocyte lineage of one case at diagnosis.Our findings reported here may be able to assist assay development efforts for diagnostic and residual disease mutation detection in myeloid disorders. In addition, flow cytometric assessment of monitoring specimens prior to molecular analysis may be beneficial to decide if cell enrichment steps can give additional evidence for the presence of residual disease. DisclosuresBurnworth:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Angela:Hematologics: Employment. Wentzel:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Loken:Hematologics: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.

Proteogenomic strategies for identification of aberrant cancer peptides using large-scale next-generation sequencing data.

Cancer is driven by the acquisition of somatic DNA lesions. Distinguishing the early driver mutations from subsequent passenger mutations is key to molecular subtyping of cancers, understanding cancer progression, and the discovery of novel biomarkers. The advances of genomics technologies (whole-genome exome, and transcript sequencing, collectively referred to as NGS (next-generation sequencing)) have fueled recent studies on somatic mutation discovery. However, the vision is challenged by the complexity, redundancy, and errors in genomic data, and the difficulty of investigating the proteome translated portion of aberrant genes using only genomic approaches. Combination of proteomic and genomic technologies are increasingly being employed. Various strategies have been employed to allow the usage of large-scale NGS data for conventional MS/MS searches. This paper provides a discussion of applying different strategies relating to large database search, and FDR (false discovery rate) -based error control, and their implication to cancer proteogenomics. Moreover, it extends and develops the idea of a unified genomic variant database that can be searched by any MS sample. A total of 879 BAM files downloaded from TCGA repository were used to create a 4.34 GB unified FASTA database that contained 2787062 novel splice junctions, 38,464 deletions, 1,105 insertions, and 182,302 substitutions. Proteomic data from a single ovarian carcinoma sample (439,858 spectra) was searched against the database. By applying the most conservative FDR measure, we have identified 524 novel peptides and 65,578 known peptides at 1% FDR threshold. The novel peptides include interesting examples of doubly mutated peptides, frame-shifts, and nonsample-recruited mutations, which emphasize the strength of our approach.

Integrated genomic analysis identifies recurrent mutations and evolution patterns driving the initiation and progression of follicular lymphoma

Follicular lymphoma (FL) is an incurable malignancy1, with transformation to an aggressive subtype being a critical event during disease progression. Here we performed whole genome or exome sequencing on 10 FL-transformed FL pairs, followed by deep sequencing of 28 genes in an extension cohort and report the key events and evolutionary processes governing initiation and transformation. Tumor evolution occurred through either a ‘rich’ or ‘sparse’ ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histones, JAK-STAT signaling, NF-κB signaling and B-cell development genes. Longitudinal analyses revealed chromatin regulators (CREBBP, EZH2 and MLL2) as early driver genes, whilst mutations in EBF1 and regulators of NF-κB signaling (MYD88 and TNFAIP3) were gained at transformation. Collectively, this study provides novel insights into the genetic basis of follicular lymphoma, the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations within the CPC represents an attractive therapeutic strategy.

Clinical and biological implications of driver mutations in myelodysplastic syndromes

AbstractMyelodysplastic syndromes (MDS) are a heterogeneous group of chronic hematological malignancies characterized by dysplasia, ineffective hematopoiesis and a variable risk of progression to acute myeloid leukemia. Sequencing of MDS genomes has identified mutations in genes implicated in RNA splicing, DNA modification, chromatin regulation, and cell signaling. We sequenced 111 genes across 738 patients with MDS or closely related neoplasms (including chronic myelomonocytic leukemia and MDS–myeloproliferative neoplasms) to explore the role of acquired mutations in MDS biology and clinical phenotype. Seventy-eight percent of patients had 1 or more oncogenic mutations. We identify complex patterns of pairwise association between genes, indicative of epistatic interactions involving components of the spliceosome machinery and epigenetic modifiers. Coupled with inferences on subclonal mutations, these data suggest a hypothesis of genetic “predestination,” in which early driver mutations, typically affecting genes involved in RNA splicing, dictate future trajectories of disease evolution with distinct clinical phenotypes. Driver mutations had equivalent prognostic significance, whether clonal or subclonal, and leukemia-free survival deteriorated steadily as numbers of driver mutations increased. Thus, analysis of oncogenic mutations in large, well-characterized cohorts of patients illustrates the interconnections between the cancer genome and disease biology, with considerable potential for clinical application.Key Points

The utility of diagnostic biopsy specimens for predictive molecular testing in colorectal cancer

If stratified medicine is to be applied in the neoadjuvant setting, predictive testing will have to be undertaken on preoperative diagnostic biopsy specimens. The aim of this study was to evaluate whether a diagnostic biopsy was adequately representative of the main tumour in colorectal cancer. Thirty cases of paired biopsy and subsequent resection specimens were randomly selected. Samples were screened for mutation in KRAS (codons 12/13, 61, and 146), BRAF (codon 600 and exon 11), PIK3CA (exons 1, 9, and 20), TP53 (exons 5-8), and microsatellite instability, using the quick multiplex consensus or standard polymerase chain reaction (PCR) protocols followed by high-resolution melting analysis. A total of 570 paired PCR tests were performed for mutation detection, and identical results were obtained in both biopsy and resection specimens in 569 tests (>99% concordance). Four cases (13%) showed microsatellite instability, and, in all four cases, instability was seen at identical mononucleotide markers in both biopsy and matched resection specimens. This is the first study to show that diagnostic biopsy specimens, even though they are a tiny sample of the tumour, are sufficiently representative for use in predictive testing for early driver mutations in colorectal cancer.

V600E Braf induces gastrointestinal crypt senescence and promotes tumour progression through enhanced CpG methylation of p16 INK4a

The majority of human colorectal cancers (CRCs) are initiated by mutations arising in the adenomatous polyposis coli (APC) tumour suppressor gene. However, a new class of non-APC mutated CRCs has been defined that have a serrated histopathology and carry the V600EBRAF oncogene. Here we have investigated the pathogenesis of serrated CRCs by expressing V600EBraf in the proliferative cells of the mouse gastrointestinal tract. We show that the oncogene drives an initial burst of Mek-dependent proliferation, leading to the formation of hyperplastic crypts. This is associated with β-catenin nuclear localization by a mechanism involving Mapk/Erk kinase (Mek)-dependent, Akt-independent phosphorylation of Gsk3β. However, hyperplastic crypts remain dormant for prolonged periods due to the induction of crypt senescence accompanied by upregulation of senescence-associated β-galactosidase and p16Ink4a. We show that tumour progression is associated with down-regulation of p16Ink4a through enhanced CpG methylation of exon 1 and knockout of Cdkn2a confirms this gene is a barrier to tumour progression. Our studies identify V600EBRAF as an early genetic driver mutation in serrated CRCs and indicate that, unlike APC-mutated cancers, this subtype arises by the bypassing of a V600EBraf driven oncogene-induced senescence programme.