Assessing copy number abnormalities and copy-neutral loss-of-heterozygosity across the genome as best practice in diagnostic evaluation of acute myeloid leukemia: An evidence-based review from the cancer genomics consortium (CGC) myeloid neoplasms working group

Structural genomic abnormalities, including balanced chromosomal rearrangements, copy number gains and losses and copy-neutral loss-of-heterozygosity (CN-LOH) represent an important category of diagnostic, prognostic and therapeutic markers in acute myeloid leukemia (AML). Genome-wide evaluation for copy number abnormalities (CNAs) is at present performed by karyotype analysis which has low resolution and is unobtainable in a subset of cases. Furthermore, examination for possible CN-LOH in leukemia cells is at present not routinely performed in the clinical setting. Chromosomal microarray (CMA) analysis is a widely available assay for CNAs and CN-LOH in diagnostic laboratories, but there are currently no guidelines how to best incorporate this technology into clinical testing algorithms for neoplastic diseases including AML. The Cancer Genomics Consortium Working Group for Myeloid Neoplasms performed an extensive review of peer-reviewed publications focused on CMA analysis in AML. Here we summarize evidence regarding clinical utility of CMA analysis in AML extracted from published data, and provide recommendations for optimal utilization of CMA testing in the diagnostic workup. In addition, we provide a list of CNAs and CN-LOH regions which have documented clinical significance in diagnosis, prognosis and treatment decisions in AML

Myeloproliferative neoplasms: from origins to outcomes.

Substantial progress has been made in our understanding of the pathogenetic basis of myeloproliferative neoplasms. The discovery of mutations in JAK2 over a decade ago heralded a new age for patient care as a consequence of improved diagnosis and the development of therapeutic JAK inhibitors. The more recent identification of mutations in calreticulin brought with it a sense of completeness, with most patients with myeloproliferative neoplasm now having a biological basis for their excessive myeloproliferation. We are also beginning to understand the processes that lead to acquisition of somatic mutations and the factors that influence subsequent clonal expansion and emergence of disease. Extended genomic profiling has established a multitude of additional acquired mutations, particularly prevalent in myelofibrosis, where their presence carries prognostic implications. A major goal is to integrate genetic, clinical, and laboratory features to identify patients who share disease biology and clinical outcome, such that therapies, both existing and novel, can be better targeted

Cytogenetic and Molecular Predictors of Outcome in Acute Lymphocytic Leukemia: Recent Developments

During the last decade a tremendous technologic progress based on genome-wide profiling of genetic aberrations, structural DNA alterations, and sequence variations has allowed a better understanding of the molecular basis of pediatric and adult B/T- acute lymphoblastic leukemia (ALL), contributing to a better recognition of the biological heterogeneity of ALL and to a more precise definition of risk factors. Importantly, these advances identified novel potential targets for therapeutic intervention. This review will be focused on the cytogenetic/molecular advances in pediatric and adult ALL based on recently published articles

Molecular and genetic alterations associated with therapy resistance and relapse of acute myeloid leukemia

Background The majority of individuals with acute myeloid leukemia (AML) respond to initial chemotherapy and achieve a complete remission, yet only a minority experience long-term survival because a large proportion of patients eventually relapse with therapy-resistant disease. Relapse therefore represents a central problem in the treatment of AML. Despite this, and in contrast to the extensive knowledge about the molecular events underlying the process of leukemogenesis, information about the mechanisms leading to therapy resistance and relapse is still limited. Purpose and content of review Recently, a number of studies have aimed to fill this gap and provided valuable information about the clonal composition and evolution of leukemic cell populations during the course of disease, and about genetic, epigenetic, and gene expression changes associated with relapse. In this review, these studies are summarized and discussed, and the data reported in them are compiled in order to provide a resource for the identification of molecular aberrations recurrently acquired at, and thus potentially contributing to, disease recurrence and the associated therapy resistance. This survey indeed uncovered genetic aberrations with known associations with therapy resistance that were newly gained at relapse in a subset of patients. Furthermore, the expression of a number of protein coding and microRNA genes was reported to change between diagnosis and relapse in a statistically significant manner. Conclusions Together, these findings foster the expectation that future studies on larger and more homogeneous patient cohorts will uncover pathways that are robustly associated with relapse, thus representing potential targets for rationally designed therapies that may improve the treatment of patients with relapsed AML, or even facilitate the prevention of relapse in the first place.(VLID)484849

The Role of Smc3 in Mouse Embryonic and Adult Hematopoiesis

Acute myeloid leukemia (AML) is a heterogeneous disease, characterized by recurrent genetic mutations. Mutations in the cohesin complex are one of the 8 functional categories of mutations in AML. SMC3 encodes a subunit of the cohesin complex, which has important roles in chromosome segregation, genome instability, and gene expression. In the first chapter of the dissertation, we discuss the genetics of AML, normal functions of the cohesin complex, and the interplay between cohesin mutations and myeloid malignancies. SMC3 is recurrently mutated in AML and other myeloid malignancies. In the second chapter of the dissertation, we compare the consequences of Smc3 deficient and haploinsufficient mouse models to determine whether the heterozygous missense mutations in SMC3 might have dominant-negative effects or phenocopy loss-of-function effects. We found that homozygous deletion of Smc3 during embryogenesis or in adult mice resulted in hematopoietic failure. SMC3 missense mutations are therefore unlikely to be associated with simple dominant negative phenotypes due to incompatibility with hematopoiesis. Smc3 haploinsufficiency, in contrast, was tolerated during embryonic and adult hematopoiesis. Under steady-state conditions, Smc3 haploinsufficiency did not alter colony forming capacity ex vivo and led to modest transcriptional and chromatin accessibility changes in Lin-cKit+ progenitor cells. However, following tamoxifen-induced deletion in competitive transplantations, we observed a significant hematopoietic competitive disadvantage in Smc3 haploinsufficient bone marrow cells across myeloid and lymphoid lineages and within the stem/progenitor compartments. The competitive disadvantage was not affected by different conditions of hematopoietic stresses, but was partially abrogated by concurrent Dnmt3a haploinsufficiency, suggesting that antecedent mutations may be the prerequisites to realize the leukemogenic potential of Smc3 mutations. In the third chapter of the dissertation, we present a case of an older women that initially appeared to be treatment-related AML following non-cytotoxic all-trans retinoic acid (ATRA)/arsenic trioxide (ATO) therapy for acute promyelocytic leukemia (APL), but upon further analysis found to be more consistent with secondary AML. Exome sequencing revealed a TET2-mutated dominant clonal process that preceded the APL diagnosis, persisted, and gave rise to an AML-associated new subclone with a NPM1 mutation. Review of additional cytogenetic abnormalities observed in APL patients showed that cytogenetic abnormalities commonly occur as subclones of the APL clone, although one rare case with del(7) independent of the APL clone was identified. These results demonstrated that APL may emerge within the context of clonal hematopoiesis and caution must be exercised when interpreting the development of tAML after ATRA/ATO therapy, especially in older patients

Measurable residual disease and clonal evolution in acute myeloid leukemia with focus on NPM1-mutations

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, with mutations in the NPM1 gene occurring in almost one third of all cases. The ability to detect residual leukemia below the resolution of conventional microscopy is crucial for evaluation of relapse risk after therapy. In principle, this can be achieved by measuring residual disease (MRD) with two different approaches, both used routinely in everyday hematological practice and in this thesis: multicolor flow cytometry (MFC) and molecular techniques. The latter include methods such as reverse transcription quantitative PCR (RT-qPCR), quantitative PCR (qPCR), droplet digital PCR (ddPCR) and next-generation sequencing (NGS). NPM1 mutations are ideal targets for molecular MRD and the level of NPM1-MRD, as determined by quantification of RNA transcripts, is currently considered the most relevant prognostic factor after first-line treatment. Instead, to explore the clinical relevance of genomic DNA-based molecular MRD methods, this thesis has focused on targeting NPM1 mutations, but also other AML-associated mutations, to decipher patterns of clonal evolution in AML before, during and after treatment.First, a qPCR-based protocol for quantification of the NPM1 type A mutation was refined, validated, and shown to be more sensitive than MFC for determination of MRD. This study was followed by an extensive comparison of DNA- and RNA-based methods for MRD assessment. The DNA-based methods proved highly accurate with respect to RNA thresholds of importance for treatment response. In addition, although RT-qPCR was more sensitive, it failed to detect leukemic transcripts in about 10% of samples with clear-cut NPM1-mutated DNA. Hence, DNA-based MRD techniques can add important information with respect to residual leukemia, of possible clinical relevance for MRD assessment. Next, several mutations in addition to NPM1 were targeted with ddPCR and monitored in follow-up samples after treatment. This strategy revealed several patterns of clonal evolution in relapsing AML. In one pattern, all monitored mutations reappeared at relapse regardless of the number of subclones. In other relapses, a subclone different from the original leukemia was responsible for the recurrence. Finally, in some patients, the leukemia relapsed from persistent clonal hematopoiesis despite complete morphological and immunophenotypical remission. To explore the mutational landscape and clonal evolution in elderly patients, who are often treated outside clinical trials, a cohort of patients older than 75 years with de novo AML with mutated NPM1 was analysed. The results indicate that the mutational pattern may differ between younger and older patients, with more TET2 and SRSF2 mutations but fewer DNMT3A mutations in the elderly.In conclusion, this thesis shows that DNA-based methods are more sensitive than MFC for determination of MRD and that they may complement RT-qPCR, with possible consequences for risk assessment of patients treated for NPM1-mutated AML. Targeting several mutations with ddPCR or other DNA-based techniques may be relevant for accurate and complete MRD assessment in the personalised follow-up of most AML patients. Finally, the mutational landscape seems to differ between younger and elderly AML-patients, with possible implications for risk stratification, and ultimately, treatment

Assessing copy number aberrations and copy neutral loss of heterozygosity across the genome as best practice: An evidence based review of clinical utility from the cancer genomics consortium (CGC) working group for myelodysplastic syndrome, myelodysplastic/myeloproliferative and myeloproliferative neoplasms

Multiple studies have demonstrated the utility of chromosomal microarray (CMA) testing to identify clinically significant copy number alterations (CNAs) and copy-neutral loss-of-heterozygosity (CN-LOH) in myeloid malignancies. However, guidelines for integrating CMA as a standard practice for diagnostic evaluation, assessment of prognosis and predicting treatment response are still lacking. CMA has not been recommended for clinical work-up of myeloid malignancies by the WHO 2016 or the NCCN 2017 guidelines but is a suggested test by the European LeukaemiaNet 2013 for the diagnosis of primary myelodysplastic syndrome (MDS). The Cancer Genomics Consortium (CGC) Working Group for Myeloid Neoplasms systematically reviewed peer-reviewed literature to determine the power of CMA in (1) improving diagnostic yield, (2) refining risk stratification, and (3) providing additional genomic information to guide therapy. In this manuscript, we summarize the evidence base for the clinical utility of array testing in the workup of MDS, myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and myeloproliferative neoplasms (MPN). This review provides a list of recurrent CNAs and CN-LOH noted in this disease spectrum and describes the clinical significance of the aberrations and how they complement gene mutation findings by sequencing. Furthermore, for new or suspected diagnosis of MDS or MPN, we present suggestions for integrating genomic testing methods (CMA and mutation testing by next generation sequencing) into the current standard-of-care clinical laboratory testing (karyotype, FISH, morphology, and flow)

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery.

Since the publication of the Society for Immunotherapy of Cancer\u27s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients

Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia

Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n = 12 of 21), branching (n = 4 of 21), and clonal evolution (n = 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in ∼80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.This work was supported by grants from the Área de Oncología del Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red (CIBER-ONC) (CB16/12/00369, CB16/12/00233, CB16/12/00489, and CB16/12/00284), Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS numbers PI16/01661, PI16/00517, and PI19/01518), and the Plan de Investigación de la Universidad de Navarra (PIUNA 2014-18). This work was supported internationally by the Cancer Research UK, FCAECC, and AIRC under the Accelerator Award Program (EDITOR)

Impact of somatic and germline mutations on the outcome of systemic mastocytosis

[EN]Systemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we investigated the presence and frequency of genetic variants in 34 SM patients with multilineal KIT D816V mutations. Initial screening was performed by targeted sequencing of 410 genes in DNA extracted from purified bone marrow cells and hair from 12 patients with nonadvanced SM and 8 patients with advanced SM, followed by whole-genome sequencing (WGS) in 4 cases. Somatic mutations were further investigated in another 14 patients with advanced SM. Despite the fact that no common mutation other than KIT D816V was found in WGS analyses, targeted next-generation sequencing identified 67 nonsynonymous genetic variants involving 39 genes. Half of the mutations were somatic (mostly multilineal), whereas the other half were germline variants. The presence of ≥1 multilineal somatic mutation involving genes other than KIT D816V, ≥3 germline variants, and ≥1 multilineal mutation in the SRSF2, ASXL1, RUNX1, and/or EZH2 genes (S/A/R/E genes), in addition to skin lesions, splenomegaly, thrombocytopenia, low hemoglobin levels, and increased alkaline phosphatase and β2-microglobulin serum levels, were associated with a poorer patient outcome. However, the presence of ≥1 multilineal mutation, particularly involving S/A/R/E genes, was the only independent predictor for progression-free survival and overall survival in our cohort