Heterogeneity Of Somatic Mutations Research Articles

Abstract PL-01: Breast Cancer Evolution, Immune Evasion and Metastasis Driven by Chromosomal Instability

Abstract Chromosomal instability (CIN) is a poor prognostic feature with high prevalence in breast cancer and is responsible for driving Somatic Copy Number Aberrations and Intratumour Heterogeneity (ITH) within subclones during breast cancer evolution. We have observed that SCNAs are enriched in metastatic samples, including gains in chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. However, there have thus far been no prospective, comprehensive studies of intratumor heterogeneity in breast cancer. We will present data from TRACERx Breast/SCANDARE a prospective, multicentre study of triple-negative breast cancer (TNBC) in which multi-region biopsies have been collected from primary TNBC prior to neoadjuvant treatment, and at longitudinal time points including surgery and relapse. We find prevalent intratumor heterogeneity in somatic mutations and SCNAs in the primary tumor prior to treatment. We have leveraged the rich clinical annotation in this cohort to correlate intratumor heterogeneity scores with key clinical outcomes, including treatment response (pathological complete response to neoadjuvant chemotherapy) and survival. We find that CIN manifests as oncogenic amplification on extrachromosomal DNA (ecDNA), which plays a pivotal role in driving drug resistance and tumor evolution. Through analysis of Whole Genome Sequencing data from 2936 breast tumors from the Genomics England breast cancer cohort, we have identified focal amplifications driven by ecDNA in 46.4% of HER2+ breast cancers. EcDNA were enriched in metastatic tumors and was associated with poor clinical outcomes. In addition to oncogenic amplifications such as HER2 derived from ecDNA during cancer evolution, ecDNAs contained immunomodulatory genes associated with reduced T cell infiltration. Further Immune-modulation by CIN results from loss of heterozygosity of the HLA locus (HLA LOH) and is a prevalent subclonal event in breast cancer. CIN is further drives haploid LOH, resulting in the high prevalence of whole genome doubling and further CIN in TNBC. Tumors are heterogeneous compositions of distinct clones with different compliments of SCNAs and varying levels of fitness, hence measuring the proliferation of individual clones to predict future evolutionary outcomes is likely to be critical. We have developed SPRINTER (Single-cell Proliferation Rate Inference in Non-homogeneous Tumours through Evolutionary Routes), a novel computational method to measure proliferation rates in individual tumour clones using single-cell whole-genome DNA sequencing data. We applied SPRINTER to 42,009 triple negative breast cancer cells, demonstrating significant proliferative heterogeneity between distinct tumor clones. Importantly, we show a correlation between higher proliferation rates and increased rates of somatic variants, suggesting proliferation links to clone evolvability. Taken together, these complimentary analyses provide evidence for the importance of CIN in driving ITH, selection, metastasis and immune evasion and provide a framework to determine recurrent evolutionary patterns in breast cancer evolution. Citation Format: C. Swanton. Breast Cancer Evolution, Immune Evasion and Metastasis Driven by Chromosomal Instability [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PL-01.

Targeted gene next-generation sequencing reveals genomic profile in a cohort of 46 Chinese patients with breast cancer.

The present study aimed to explore the genomic profile in a cohort of Chinese patients with breast cancer (BC), as well as provide potential strategies for clinic treatment in specific subset of BC patients. Paired samples from 46 BC patients were subjected to DNA extraction and 537 gene targeted next-generation sequencing. In total, 742 somatic mutations were detected in these patients, which involved 303 genes. TP53 and PIK3CA were the most frequently mutated genes, with a mutation rate of 45.65% and 26.09%. C>T, T>C and C>A comprised the main single nucleotide base variation for this Chinese cohort. Triple negative breast cancer (TNBC) group had more TP53-mutated patients than the Non-TNBC group (p = 0.0229). In addition, the cohort was also divided into 'Young' and 'Old' groups based on the age of onset. Compared with the 'Young' group, the 'Old' group had more frameshift mutations (p = 0.0190), less missense mutations (p = 0.0269) and more HOXA11-mutated patients (p = 0.0197). Additionally, the HOXA11mt (HOXA11 gene mutated) group had more frameshift mutations than the HOXA11wt (HOXA11 gene without mutation) group (p < 0.0001). In KEGG (i.e. Kyoto Encyclopedia of Genes and Genomes) analysis, the HOXA11wt group had more gene mutations involved in the T cell receptor signaling pathway (p = 0.0197), Jak-STAT signaling pathway (p = 0.0380) and the HIF-1 signaling pathway (p = 0.0489) than the HOXA11mt group. In the present study, the heterogeneity of somatic mutations was revealed between different tumor subgroups, including TNBC/Non-TNBC, age of onset (Young/Old) and HOXA11 mutation (HOXA11mt /HOXA11wt ). The present study revealed the heterogeneity of gene mutation and clinical variables among BC subtypes and might provide guidance for developing a potential target for clinical treatment.

Spatial and temporal intratumour heterogeneity has potential consequences for single biopsy-based neuroblastoma treatment decisions

Intratumour heterogeneity is a major cause of treatment failure in cancer. We present in-depth analyses combining transcriptomic and genomic profiling with ultra-deep targeted sequencing of multiregional biopsies in 10 patients with neuroblastoma, a devastating childhood tumour. We observe high spatial and temporal heterogeneity in somatic mutations and somatic copy-number alterations which are reflected on the transcriptomic level. Mutations in some druggable target genes including ALK and FGFR1 are heterogeneous at diagnosis and/or relapse, raising the issue whether current target prioritization and molecular risk stratification procedures in single biopsies are sufficiently reliable for therapy decisions. The genetic heterogeneity in gene mutations and chromosome aberrations observed in deep analyses from patient courses suggest clonal evolution before treatment and under treatment pressure, and support early emergence of metastatic clones and ongoing chromosomal instability during disease evolution. We report continuous clonal evolution on mutational and copy number levels in neuroblastoma, and detail its implications for therapy selection, risk stratification and therapy resistance.

Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine

BackgroundCancer is a somatic evolutionary disease and adenocarcinomas of the stomach and gastroesophageal junction (GC) may serve as a two-dimensional model of cancer expansion, in which tumor subclones are not evenly mixed during tumor progression but rather spatially separated and diversified. We hypothesize that precision medicine efforts are compromised when clinical decisions are based on a single-sample analysis, which ignores the mechanisms of cancer evolution and resulting intratumoral heterogeneity. Using multiregional whole-exome sequencing, we investigated the effect of somatic evolution on intratumoral heterogeneity aiming to shed light on the evolutionary biology of GC.MethodsThe study comprised a prospective discovery cohort of 9 and a validation cohort of 463 GCs. Multiregional whole-exome sequencing was performed using samples form 45 primary tumors and 3 lymph node metastases (range 3–10 tumor samples/patient) of the discovery cohort.ResultsIn total, the discovery cohort harbored 16,537 non-synonymous mutations. Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. Of the non-synonymous mutations, 53–91% were not present in each patient’s sample; 399 genes harbored 2–4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Multi-sample tree-based analyses provided evidence for branched evolution being most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. We found evidence of parallel evolution and evolutionary trajectories: different mutations of SMAD4 aligned with different subclones and were found only in TP53 mutant GCs.ConclusionsNeutral and non-neutral somatic evolution shape the mutational landscape in GC along its lateral expansions. It leads to complex spatial intratumoral heterogeneity, where lymph node metastases may stem from different areas of the primary tumor, synchronously. Our findings may have profound effects on future patient management. They illustrate the risk of mis-interpreting tumor genetics based on single-sample analysis and open new avenues for an evolutionary classification of GC, i.e., the discovery of distinct evolutionary trajectories which can be utilized for precision medicine.

ZDOG: zooming in on dominating genes with mutations in cancer pathways

BackgroundInference of cancer-causing genes and their biological functions are crucial but challenging due to the heterogeneity of somatic mutations. The heterogeneity of somatic mutations reveals that only a handful of oncogenes mutate frequently and a number of cancer-causing genes mutate rarely.ResultsWe develop a Cytoscape app, named ZDOG, for visualization of the extent to which mutated genes may affect cancer pathways using the dominating tree model. The dominator tree model allows us to examine conveniently the positional importance of a gene in cancer signalling pathways. This tool facilitates the identification of mutated “master” regulators even with low mutation frequency in deregulated signalling pathways.ConclusionsWe have presented a model for facilitating the examination of the extent to which mutation in a gene may affect downstream components in a signalling pathway through its positional information. The model is implemented in a user-friendly Cytoscape app which will be freely available upon publication.AvailabilityTogether with a user manual, the ZDOG app is freely available at GitHub (https://github.com/rudi2013/ZDOG). It is also available in the Cytoscape app store (http://apps.cytoscape.org/apps/ZDOG) and users can easily install it using the Cytoscape App Manager.

Mitochondrial RNA Expression and Single Nucleotide Variants in Association with Clinical Parameters in Primary Breast Cancers.

The human mitochondrial DNA (mtDNA) encodes 37 genes, including thirteen proteins essential for the respiratory chain, and RNAs functioning in the mitochondrial translation apparatus. The total number of mtDNA molecules per cell (mtDNA content) is variable between tissue types and also between tumors and their normal counterparts. For breast cancer, tumors tend to be depleted in their mtDNA content compared to adjacent normal mammary tissue. Various studies have shown that primary breast tumors harbor somatic mtDNA variants. A decrease in mtDNA content or the presence of somatic variants could indicate a reduced mitochondrial function within breast cancer. In this explorative study we aimed to further understand genomic changes and expression of the mitochondrial genome within breast cancer, by analyzing RNA sequencing data of primary breast tumor specimens of 344 cases. We demonstrate that somatic variants detected at the mtRNA level are representative for somatic variants in the mtDNA. Also, the number of somatic variants within the mitochondrial transcriptome is not associated with mutational processes impacting the nuclear genome, but is positively associated with age at diagnosis. Finally, we observe that mitochondrial expression is related to ER status. We conclude that there is a large heterogeneity in somatic mutations of the mitochondrial genome within primary breast tumors, and differences in mitochondrial expression among breast cancer subtypes. The exact impact on metabolic differences and clinical relevance deserves further study.

Transcriptional landscape of human cancers.

The homogeneity and heterogeneity in somatic mutations, copy number alterations and methylation across different cancer types have been extensively explored. However, the related exploration based on transcriptome data is lacking. In this study we explored gene expression profiles across 33 human cancer types using The Cancer Genome Atlas (TCGA) data. We identified consistently upregulated genes (such as E2F1, EZH2, FOXM1, MYBL2, PLK1, TTK, AURKA/B and BUB1) and consistently downregulated genes (such as SCARA5, MYOM1, NKAPL, PEG3, USP2, SLC5A7 and HMGCLL1) across various cancers. The dysregulation of these genes is likely to be associated with poor clinical outcomes in cancer. The dysregulated pathways commonly in cancers include cell cycle, DNA replication, repair, and recombination, Notch signaling, p53 signaling, Wnt signaling, TGFβ signaling, immune response etc. We also identified genes consistently upregulated or downregulated in highly-advanced cancers compared to lowly-advanced cancers. The highly (low) expressed genes in highly-advanced cancers are likely to have higher (lower) expression levels in cancers than in normal tissue, indicating that common gene expression perturbations drive cancer initiation and cancer progression. In addition, we identified a substantial number of genes exclusively dysregulated in a single cancer type or inconsistently dysregulated in different cancer types, demonstrating the intertumor heterogeneity. More importantly, we found a number of genes commonly dysregulated in various cancers such as PLP1, MYOM1, NKAPL and USP2 which were investigated in few cancer related studies, and thus represent our novel findings. Our study provides comprehensive portraits of transcriptional landscape of human cancers.

Integrated analysis of somatic mutations and immune microenvironment in malignant pleural mesothelioma

ABSTRACTTo investigate the link between the genomic landscape of cancer cells and immune microenvironment in tumor tissues, we characterized somatic mutations and tumor-infiltrating lymphocytes (TILs) in malignant pleural mesothelioma (MPM), including mutation/neoantigen load, spatial heterogeneity of somatic mutations of cancer cells and TILs (T-cell receptor β (TCRβ) repertoire), and expression profiles of immune-related genes using specimens of three different tumor sites (anterior, posterior, and diaphragm) obtained from six MPM patients. Integrated analysis identified the distinct patterns of somatic mutations and the immune microenvironment signatures both intratumorally and interindividually. MPM cases showed intratumoral heterogeneity in somatic mutations with unique TCRβ clonotypes of TILs that were restricted to each tumor site, suggesting the presence of a neoantigen-related immune response. Correlation analyses showed that higher neoantigen load was significantly correlated with stronger clonal expansion of TILs (p = 0.048) and a higher expression level of an immune-associated cytolytic factor (PRF1 (p = 0.0041) in tumor tissues), suggesting that high neoantigen loads in tumor cells might promote expansion of functional tumor-specific T cells in the tumor bed. Our results collectively indicate that MPM tumors constitute a diverse heterogeneity in both the genomic landscape and immune microenvironment, and that mutation/neoantigen load may affect the immune microenvironment in MPM tissues.

Abstract P6-07-09: Somatic mutations in A kinase anchoring proteins (AKAPs) in metastatic tumors - A potential characteristic of breast cancer metastasis

Abstract Introduction Genomic heterogeneity in primary solid tumors has been extensively studied using deep sequencing technologies during the last decade. The heterogeneity of cancer tumors is today a well-established concept partly reflected in the low number of genes being recurrently mutated in over 10% of the tumors. However, most available data relates to the primary breast cancer tumors and little has been described about the mutational profiles of the metastatic lesions and their relation to its original malignant cell population. Prospective and retrospective studies have demonstrated that altered receptor status in the metastatic lesion occurs at high rates during cancer progression and is additionally affected by adjuvant therapy with major implications for management of the metastatic disease. Here, we report the exome sequences of paired primary and metastatic lesions from ten breast cancer patients. Results We found a marked heterogeneity of somatic mutations as well as chromosomal aberrations in the metastatic lesions. A number of mutated genes were enriched in the metastases including, significantly, members of the A-kinase anchoring protein family (AKAPs), p &amp;lt; 0.02; Fisher's exact test. The enrichment of AKAP mutations in metastatic lesions was confirmed in an independent cohort containing 20 patients with paired primary and metastatic lesions, which showed the same mutational pattern. In total, 14 nonsynonymous mutations were found in ten of the fourteen AKAP family members. Out of the totally 30 patients examined, ten (30%) carried one or more mutations in AKAP genes either in primary tumor, metastasis, or both. In seven of these ten patients, the AKAP mutation was found uniquely in the metastatic lesion. Several copy number variations (CNV), mostly deletions in regions containing AKAP genes were detected. For example, the down-regulation of AKAP12 is often associated with promoter hypermethylation or loss of its locus 6q24-25.2 and has been associated with tumor progression and metastasis. In our data deletion of the AKAP12 locus is present in six out of twenty patients. Discussion AKAPs are members of a protein family acting as anchors for Protein Kinase A (PKA) by specifically associate PKA regulatory subunits to cellular organelles and direct its active signal transduction spatially and temporally. Several of the AKAP members have been associated to cancer development and metastatic spread, mostly based on differential expression and effects on migration in in vitro assays but both polymorphisms and somatic mutations have been reported in human tumors. Our findings indicate that in metastatic lesions, the primary tumor genome is extensively transformed, with enrichment of mutations in a distinct set of genes. Together, these findings suggest the involvement of AKAPs in the metastatic process and provide a potential avenue for targeted therapy directed at metastatic breast cancer. Molecular and genetic characterization of the metastatic lesions is not only important in the clinical setting but should also provide the means to reveal genetic patterns specific for the disseminated malignancy. Citation Format: Kjällquist U, Erlandsson R, Alkodsi A, Tobin N, Karlsson E, Hatschek T, Hartman J, Linnarsson S, Bergh J. Somatic mutations in A kinase anchoring proteins (AKAPs) in metastatic tumors - A potential characteristic of breast cancer metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-09.

Deep sequencing reveals a high rate of temporal heterogeneity of somatic mutations in paired primary and recurrent ovarian carcinomas

Deep sequencing reveals a high rate of temporal heterogeneity of somatic mutations in paired primary and recurrent ovarian carcinomas

Chromosomal Instability Causes Genetic and Clonal Heterogeneity in Myeloproliferative Neoplasms and Is Not Restricted to JAK2-V617F Positive Cells

A number of oncogenic mutations have been identified in myeloproliferative neoplasms (MPN) in the past few years. Among these JAK2-V617F is most frequent, followed by mutations of the thrombopoietin receptor MPL and JAK2 exon 12. In addition, cytogenetic lesions occur frequently in MPN detected either at diagnosis or later in the course of the disease. To explore the genetic aberrations present in MPN patients, we performed microarray genotyping using Affymetrix SNP 6.0 arrays in a series of 71 MPN patients with variable presence of JAK2 and MPL mutations. More than half of the analyzed patients exhibited loss of heterozygosity (LOH) in at least one chromosomal region. Complex karyotypes with two and more regions with LOH were detected in 18 patients. Uniparental disomy (UPD) on chromosomes 9p, 1p, 11q, 14q and 17q represented the largest proportion of LOH detected followed by deletions on chromosome 13q, 20q, and 12p. All patients with UPD on chromosome 1p were homozygous for the MPL-W515L mutation. We observed frequent aberrations of chromosome 7 including monosomy, deletions on 7p and 7q, and UPD of 7q. Using microsatellite PCR, we validated the microarray findings and further determined the frequency of these aberrations in a total of 367 MPN patients. Multiple occurrences of individual chromosomal lesions allowed us to define the minimal genomic regions involved in deletions or UPDs. The sizes of the common deleted regions (CDRs) were variable ranging from 9 mega base pairs (Mb) to 0.5 Mb. The CDR on chromosome 7p included only the IKZF1 and FIGNL1 genes previously shown to associate with leukemic transformation. To determine the clonal composition of the hematopoietic progenitor pool of patients with complex karyotypes we genotyped individual BFU-E and CFU-GM colonies in a series of 27 patients. We observed a remarkable clonal heterogeneity at the progenitor cell level. Using four clonal markers we defined 9 different types of clonal structures. In a set of patients, JAK2-V617F or MPL-W515L mutations occurred before the acquisition of chromosomal deletions. Other patients acquired deletions before the acquisition of JAK2-V617F. In summary, our results show that somatic mutations in MPN are not acquired in a predetermined order as seen in other malignancies, but occur randomly. The chromosomal instability in MPN is not caused by JAK2-V617F exclusively, since many patients show aberrations outside of the JAK2-V617F positive clone. Heterogeneity of somatic mutations in MPN leads to high clonal variability within the progenitor pool potentially affecting therapeutic outcome. Thus, targeting JAK2-V617F alone may not lead to restoration of polyclonal hematopoiesis. An individualized therapeutic approach and/or combination therapy might be necessary to achieve clonal remission in MPN.