Evolution and Impact of Subclonal Mutations in Papillary Thyroid Cancer.

Unlike many cancers, the pattern of tumor evolution in papillary thyroid cancer (PTC) and its potential role in relapse have not been elucidated. In this study, multi-region whole-exome sequencing (WES) was performed on early-stage PTC tumors (n = 257 tumor regions) from 79 individuals, including 17 who had developed relapse, to understand the temporal and spatial framework within which subclonal mutations catalyze tumor evolution and its potential clinical relevance. Paired primary-relapse tumor tissues were also available for a subset of individuals. The resulting catalog of variants was analyzed to explore evolutionary histories, define clonal and subclonal events, and assess the relationship between intra-tumor heterogeneity and relapse-free survival. The multi-region WES approach was key in correctly classifying subclonal mutations, 40% of which would have otherwise been erroneously considered clonal. We observed both linear and branching evolution patterns in our PTC cohort. A higher burden of subclonal mutations was significantly associated with increased risk of relapse. We conclude that relapse in PTC, while generally rare, does not follow a predictable evolutionary path and that subclonal mutation burden may serve as a prognostic factor. Larger studies utilizing multi-region sequencing in relapsed PTC case subjects with matching primary tissues are needed to confirm these observations.

The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

Inferring mutational timing and reconstructing tumour evolutionary histories.

Cancer evolution can be considered within a Darwinian framework. Both micro and macro-evolutionary theories can be applied to understand tumour progression and treatment failure. Owing to cancers' complexity and heterogeneity the rules of tumour evolution, such as the role of selection, remain incompletely understood. The timing of mutational events during tumour evolution presents diagnostic, prognostic and therapeutic opportunities. Here we review the current sampling and computational approaches for inferring mutational timing and the evidence from next generation sequencing-informed data on mutational timing across all tumour types. We discuss how this knowledge can be used to illuminate the genes and pathways that drive cancer initiation and relapse; and to support drug development and clinical trial design.

Clonal evolution and expansion associated with therapy resistance and relapse of colorectal cancer.

Colorectal cancer (CRC) arises by a continuous process of genetic diversification and clonal evolution. Multiple genes and pathways have a role in tumor initiation and progression. The gradual accumulation of genetic and epigenetic processes leads to the establishment of adenoma and cancer. The important 'driver' mutations in tumor suppressor genes (such as TP53, APC, and SMAD4) and oncogenes (such as KRAS, NRAS, MET, and PIK3CA) confer selective growth advantages and cause CRC advancement. Clonal evolution induced by therapeutic pressure, as well as intra-tumoral heterogeneity, has been a great challenge in the treatment of metastatic CRC. Tumors often develop resistance to treatments as a result of intra-tumor heterogeneity, clonal evolution, and selection. Hence, the development of a multidrug personalized approach should be prioritized to pave the way for therapeutics repurposing and combination therapy to arrest tumor progression. This review summarizes how selective drug pressure can impact tumor evolution, resulting in the formation of polyclonal resistance mechanisms, ultimately promoting cancer progression. Current strategies for targeting clonal evolution are described. By understanding sources and consequences of tumor heterogeneity, customized and effective treatment plans to combat drug resistance may be devised.

Lab tests for MPN.

Molecular laboratory investigations for myeloproliferative neoplasm (MPN) can ideally be divided into two distincts groups, those for the detection of the BCR-ABL rearrangement (suspect of chronic myeloid leukemia) and those for the variants determination of the driver genes of the negative Philadelphia forms (MPN Ph neg). The BCR-ABL detection is based on RT-Polymerase Chain Reaction techniques and more recently on droplet digital PCR (ddPCR). For this type of analysis, combined with chromosome banding analysis (CBA) and Fluorescent in situ hybridization (FISH), it is essential to quantify BCR-ABL mutated copies by standard curve method. The investigation on driver genes for MPN Ph neg forms includes activity for erythroid forms such as Polycythemia Vera (test JAK2V617F and JAK2 exon 12), for non-erythroid forms such as essential thrombocythemia and myelofibrosis (test JAK2V617F, CALR exon 9, MPL exon 10), for "atypical" ones such as mastocytosis (cKIT D816V test) and for hypereosinophilic syndrome (FIP1L1-PDGFRalpha test). It's crucial to assign prognosis value through calculating allelic burden of JAK2 V617F variant and determining CALR esone 9 variants (type1/1like, type2/2like and atypical ones). A fundamental innovation for investigating triple negative cases for JAK2, CALR, MPL and for providing prognostic score is the use of Next Generation Sequencing panels containing high molecular risk genes as ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2. This technique allows to detect additional or subclonal mutations which are usually acquired in varying sized sub-clones of hematopoietic progenitors. These additional variants have a prognostic significance and should be indagated to exclude false negative cases.

Founder and subclonal mutations in myelodysplastic syndromes and related myeloid neoplasms.

Somatic mutations constitute key elements of the pathogenesis of myelodysplastic syndromes (MDS), a group of clonal hematologic neoplasms characterized by cytopenias, dysplasia and leukemic evolution. Whole exome sequencing followed by targeted deep sequencing in patients with MDS and related diseases has been performed cross-sectionally and serially. Bioinformatic analysis and confirmatory sequencing led to detection of in 1458 genes affected by somatic alterations, and identification of known and new driver events. For each patient, mutation spectrum as well as clonal hierarchy was determined and for each significantly mutated gene, its role in the clonal succession established. This approach allowed for a dynamic definition of MDS mutatome, including the spectrum of founding mutations and subsequent secondary mutational patterns. We demonstrate that certain founder events determine the mode and speed of disease progression, while secondary mutations may further modulate phenotypic features. Combinations of founder and secondary mutations further contribute to the phenotypic diversity but categorical grouping of cases based on the type of founder mutations may better define molecular subtypes of MDS and correlates with clinical parameters.

A detrimental mutation on USP40 unlocks the tumorigenesis in a rare case of lung cancer.

Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer. Previous research has shown heterogeneity in lung cancer, with the parallel existence of multiple subclones characterized by their own specific mutational landscape. The aim of our study was to gain insight into the evolutionary pattern of lung cancer by investigating the genomic heterogeneity between a nodule and its distant tumor. Luckily, we obtained nodule and tumor samples derived from surgery and a blood sample from a single patient. The samples are very unique, for tissues with the same genetic background from nodules to malignant tumors are rarely available and require precise micro-cutting. In this study, we performed whole-genome sequencing of these two samples, to identify novel candidate driver genes associated with LUAD. The nodule and tumor were found to have common significant ubiquitin-specific protease 40 (USP40) mutations, indicating an important driver role for the gene. Moreover, we also observed the two novel candidate driver genes ASCL5 and CAPNS1 in the LUAD sample. In summary, we pinpoint the predominant mutations in LUAD by WES, highlighting the substantial genetic alterations contributing to LUAD tumorigenesis. This may provide a better understanding of the clonal evolution during tumor development.

Detection and Functional Analysis of TP53 Mutations in CLL.

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

Detecting Rare Mutations and DNA Damage with Sequencing-Based Methods.

There is a great need in biomedical and genetic research to detect DNA damage and de novo mutations, but doing so is inherently challenging because of the rarity of these events. The enormous capacity of current DNA sequencing technologies has opened the door for quantifying sequence variants present at low frequencies in vivo, such as within cancerous tissues. However, these sequencing technologies are error prone, resulting in high noise thresholds. Most DNA sequencing methods are also generally incapable of identifying chemically modified bases arising from DNA damage. In recent years, numerous specialized modifications to sequencing methods have been developed to address these shortcomings. Here, we review this landscape of emerging techniques, highlighting their respective strengths, weaknesses, and target applications.

Detection of driver and subclonal mutations in myelofibrosis: clinical impact on pharmacologic and transplant based treatment strategies.

INTRODUCTION: Myelofibrosis (MF) is the most aggressive form among Philadelphia negative (Ph-) myeloproliferative neoplasms (MPNs). In the last years, the mutational landscape of MF has expanded remarkably by the identification of additional recurrent mutations, called subclonal mutations. Areas covered: Here we describe the available data about the currently identified subclonal mutations and their prognostic value in MF patients. We also review the practical value of including such molecular information in available prognostic models for both outcome prediction and possibly treatment decision with regards to transplant indication. Lastly, we covered the available data on the application of molecular markers for minimal residual disease (MRD) monitoring after transplantation. Expert commentary: The demonstration of the prognostic value of additional mutations suggests to define this molecular profile at diagnosis and when an allogeneic transplant can be advised, particularly in younger patients. The presence of molecular markers might offer the possibility to evaluate the depth of remission and to monitor MRD after transplantation. Prospective clinical studies are needed to validate the use of this molecular data in the routine clinical practice.