Recurrent Genetic Changes Research Articles

Long-term Multimodal Recording Reveals Epigenetic Adaptation Routes in Dormant Breast Cancer Cells.

This study advances the understanding of therapy-induced dormancy with potential clinical implications for breast cancer. Estrogen receptor-positive breast cancer cells adapt to endocrine treatment by entering a dormant state characterized by strong heterochromatinization with no recurrent genetic changes. Targeting the epigenetic rewiring impairs the adaptation of cancer cells to ETs. See related commentary by Llinas-Bertran et al., p. 704. This article is featured in Selected Articles from This Issue, p. 695.

P445: CRYPTIC TRANSLOCATION T(5;11)(Q35;P15) RESULTING IN NUP98::NSD1 GENE FUSION IN ADULTS WITH DE NOVO ACUTE MYELOID LEUKEMIA (AML)

Background: The NUP98::NSD1 fusion, a product of the cryptic translocation t(5;11)(q35;p15.5), is a recurrent genetic change in cytogenetically normal patients with AML. It occurs most frequently in children (16%) and young (2%) AML patients, very rarely in adult patients. The coexistence of internal tandem duplication of FLT3 gene (FLT3::ITD) found in more than 70% of NUP98::NSD1 positive patients is always associated with a poor prognosis and results in high frequency of induction failure. Aims: The aim of this study was to determine the incidence of the NUP98::NSD1 fusion gene in adults with AML and NUP98 rearrangement. Methods: We examined the bone marrow cells of 268 newly diagnosed AML patients using conventional karyotyping in combination with FISH (Abbott, MetaSystems) and mFISH/mBAND (MetaSystems). We used RT-PCR followed by direct sequencing to detect fusion genes. We processed the PCR product by ExoSAP-IT and directly sequenced on ABI Prism 310 genetic analyzer using the Big Dye Terminator kit v. 3.1. Results: In nine out of 268 cases we identified rearrangement of the 11p13-15 region. We confirmed t(5;11)(q35;p15.5) with NUP98::NSD1 gene fusion in four of them (4/268; 1.5%). Sequence analyses proved the NUP98-NSD1 transcript, arising from fusion of NUP98 exon 12 with exon 6 in NSD1 gene, in all four patients (2M/2F; FAB M4/M5b; age 42, 54, 64 and 64 years). FLT3::ITD mutation was detected in all of them. Specific primers and a probe have been designed to monitor minimal residual disease during therapeutic treatment of the patients. Out of 4 patients, two died (median OS 9 months) and two patients are alive (4 and 1 year after allogenic bone marrow transplantation). Summary/Conclusion: Our study demonstrated the occurrence of t(5;11)(q35;p15.5) with NUP98::NSD1 gene fusion also in adults over 60 years of age. We confirmed the NUP98::NSD1 fusion gene in 1.5% adults AML patients. With respect to the poor prognosis of the patients with NUP98::NSD1 fusion gene, we suggest pre-screening of NUP98 gene rearrangement using FISH in cytogenetically normal AML patients with confirmed FLT3::ITD mutation. Supported by MH CZ-DRO-VFN64165, DRO-UHKT00023736.

P-120: Improving the risk stratification of multiple myeloma with a nucleotide editor/inflammation-based scoring system

<h3>Background</h3> Current clinical prognostication of newly diagnosed MM (NDMM) patients relies on clinical parameters and/or recurrent genetic changes, which mainly reflect early events in the development of MM. However, recent insights into the pathogenesis of MM highlighted genome/transcriptome editing through APOBEC genes as well as inflammation as drivers for the onset and progression of MM. <h3>Aims</h3> To build a superior nucleotide editor/inflammation-based risk scoring system reflecting biological processes that drive the progression of MM. <h3>Method</h3> We hypothesized that a prognostic score reflecting biological processes as well as clinical features is superior to the currently used classification systems for MM patients, such as ISS, R-ISS and the Mayo clinic classification. The Multiple Myeloma Research Foundation CoMMpass study genomics dataset, combining mRNA Seq and clinical data from more than 700 patients, allowed us to evaluate the prognostic value of demographic and clinical parameters, cytogenetics, and gene expression levels of APOBEC family as well as inflammation-modulating cytokines of MM patients. We calculated hazard ratios and Kaplan-Meier survival estimates for all extracted features. Combining clinical variables that were significantly associated with PFS and OS, we then applied machine learning approaches to identify the most accurate classification model to define a new risk score that is easy to compute and able to stratify NDMM patients more accurately than cytogenetics-based classifiers. <h3>Result</h3> Based on our machine learning models, we defined a weighted OS/PFS risk score (Editor-Inflammation score) based on expression of APOBEC2, APOBEC3B, IL11, TGFB1, TGFB3, as well as β-2-microglobulin and LDH serum levels, that achieved the best classification outcome and showed superior performance compared to ISS and R-ISS. Of note, cytogenetic abnormalities did not proof relevant have therefore not been included in the EI score. Besides superior overall risk stratification, the EI score further allowed to identify subgroups of MM patients with very good prognosis who do not significantly benefit from bone marrow transplantation maintenance therapy. <h3>Conclusion</h3> Our findings support the adoption of molecular biomarkers, reflecting dynamic biological processes rather than cytogenetics for a more accurate risk classification of MM. Considering that mRNA-seq is as cost effective as FISH and is being increasingly adopted by diagnostic centers, the EI score is a unique approach to shed light on underlying molecular mechanisms that drive disease progression and the development of true risk-adapted treatment strategies.

UNRAVELLING THE HIGH INCIDENCE OF ACUTE MYELOID LEUKEMIA WITH MONOCYTIC BLAST DIFFERENTIATION IN A BRAZILIAN MULTICENTER STUDY

Acute myeloid leukemia with monocytic blast differentiation (AML-MC) is a rare malignancy (5-10% incidence) with a challenging diagnosis, classified as “non otherwise specified” AML (AML NOS), unless recurrent genetic abnormalities, therapy-related or associated myelodysplasia-related changes are reported (WHO 2017). We observed a high incidence of AML-MC and aimed to assess its molecular and immunophenotypic features in a multicenter study in Brazil. 387 AML bone marrow samples (18-84 years old) from 10 Brazilian centers enrolled in International Consortium of Acute Leukemias (ICAL) Study – IC-AML2015, is in accordance Local Ethical Boards committee. From which, 149 were morphologically diagnosed with AML-MC. FLT3 and NPM1 mutations, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype and immunophenotype were assessed. Mononuclear cells were stained with CD45, CD34, CD117, CD11c, CD64 and CD15. Cell acquisition/analysis was performed in FACS Canto II /FACS Diva software (BD Bioscience). Positivity was considered when ≥20% of the leukemic cells. Treatment consisted on 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m 2 and 7 days of cytarabine 200 mg/m 2 ; cycle 2: 6 days of cytarabine 1 g/m2 twice a day) and 1 or 2 consolidation cycles (6 days of cytarabine 1 g/m2 twice a day) and/or BM transplantation. AML-MC corresponded to 38.50% of cases enrolled in the study. Stratifying according to ELN2017 (without TP53 and ASXL1 ), 23% were Favorable, 22% Intermediate and 19% Adverse risk. 35% of patients failed Stratification or had Not Assessed Information. Molecular Assessment showed positive detections for FLT3 (17.48%), RUNX1-RUNX1T1 (2.80%), CBFB-MYH11 (8.51%) and NPM1 (23.77%). According to treatment response evalutation, complete remission CR was achieved by 49,14%, 55,8%, 53,70% and 59,46% of patients on 1 st , 2 nd Inductionof remission cycle of chemotherapy, 1 st and 2sn cycle of Consolidaton of chemotherapy, and 26.37% of patients relapsed. Multiparametric Flow Cytometry (all risks) showed 39% CD34 − , 61% CD34 + ,11% CD34 − /CD117 − , 13% CD117, 87% CD117 + , 95% CD33 + , 89% CD38 + , 47% CD123 + , 8% CD14 + , 21% CD7 + , 44% CD11b + , 19% CD56 + , 29% CD13 − , 2% CD19 + , 27% CD64 + , 74% CD11c + 27%, CD64 + CD11c + , 19% CD15 + /CD133 + and 60% CD15 + cells. Among AML-NOS patients, 26% presented CD15 + and none were stratified as Favorable risk (6% Intermediate, 17% Adverse and 4% Failed Stratification). Since AML-MC is known to be rare, accounting for 5-10% of AML cases worldwide, its high incidence in our study seemed to be odd and raised curiosity in the molecular and phenotypic context. The fact that a great number of these cases are intermediate ELN2017 risk brings up the necessity to look at them closer, since this is a challenging group when it comes to therapy decisions. Although good response to treatment was noticed, immunophenotypically CD15 expression without molecular abnormalities was more frequent in intermediate and adverse risk. These findings may direct that high rates of good treatment response and favorable outcome in these patients are more likely to be due to favorable molecular abnormalities, while expression of CD15 alone may be associated with a more aggressive disease. Knowing that, it should be of value to investigate whether CD15 could be a good prognostic marker.

Recent Advances in Implantation-Based Genetic Modeling of Biliary Carcinogenesis in Mice.

Simple SummaryBiliary tract cancer (BTC) is often refractory to conventional therapeutics and is difficult to diagnose in the early stages. In addition, the pathogenesis of BTC is not fully understood, despite recent advances in cancer genome analysis. To address these issues, the development of fine disease models is critical for BTC. Although still limited in number, there are various platforms for genetic models of BTC owing to newly emerging technology. Among these, implantation-based models have recently drawn attention for their convenience, flexibility, and scalability. To highlight the relevance of this approach, we comprehensively summarize the advantages and disadvantages of BTC models developed using diverse approaches. Currently available research data on intra- and extrahepatic cholangiocarcinoma and gallbladder carcinoma are presented in this review. This information will likely help in selecting the optimal models for various applications and develop novel innovative models based on these technologies.Epithelial cells in the biliary system can develop refractory types of cancers, which are often associated with inflammation caused by viruses, parasites, stones, and chemicals. Genomic studies have revealed recurrent genetic changes and deregulated signaling pathways in biliary tract cancer (BTC). The causal roles have been at least partly clarified using various genetically engineered mice. Technical advances in Cre-LoxP technology, together with hydrodynamic tail injection, CRISPR/Cas9 technology, in vivo electroporation, and organoid culture have enabled more precise modeling of BTC. Organoid-based genetic modeling, combined with implantation in mice, has recently drawn attention as a means to accelerate the development of BTC models. Although each model may not perfectly mimic the disease, they can complement one another, or two different approaches can be integrated to establish a novel model. In addition, a comparison of the outcomes among these models with the same genotype provides mechanistic insights into the interplay between genetic alterations and the microenvironment in the pathogenesis of BTCs. Here, we review the current status of genetic models of BTCs in mice to provide information that facilitates the wise selection of models and to inform the future development of ideal disease models.

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

SummaryHuman pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

Molecular Diagnostics of Vascular Tumors of the Skin

In this article, the authors have reviewed all the recent news regarding how the discovery of some novel and recurrent molecular and genetic changes has modified the classification of some entities and have addressed to the description of new variants of vascular tumors. And even more important, the authors also reviewed on how these findings, in addition to gain insight into the tumoral biology, portend significant clinical consequences not only regarding to their diagnosis but also to their management and prognosis because some of these mutations are potential targets for treatment. The authors have also highlighted immunohistochemical markers can help us as a surrogate marker of those molecular alterations.

Nonclonal chromosomal alterations and poor survival in cytopenic patients without hematological malignancies

BackgroundClonal chromosomal alterations (CCAs) reflect recurrent genetic changes derived from a single evolving clone, whereas nonclonal chromosomal alterations (NCCAs) comprise a single or nonrecurrent chromosomal abnormality. CCAs and NCCAs in hematopoietic cells have been partially investigated in cytopenic patients without hematological malignancies.MethodsThis single-center retrospective study included 253 consecutive patients who underwent bone marrow aspiration to determine the cause of cytopenia between 2012 and 2015. Patients with hematological malignancies were excluded. CCA was defined as a chromosomal aberration detected in more than two cells, and NCCA was defined as a chromosomal aberration detected in a single cell.ResultsThe median age of the patients was 66 years. There were 135 patients without hematological malignancies (median age, 64 years; 69 females); of these, 27 patients (median age, 69 years; 8 females) harbored chromosomal abnormalities. CCAs were detected in 14 patients; the most common CCA was −Y in eight patients, followed by inv.(9) in three patients and mar1+, inv. (12), and t (19;21) in one patient each. NCCAs were detected in 13 patients; the most frequent NCCA was +Y in four patients, followed by del (20), + 8, inv. (2), − 8, and add (6) in one patient each. Moreover, nonclonal translocation abnormalities, including t (9;14), t (14;16), and t (13;21), were observed in three patients. One patient had a complex karyotype in a single cell. The remaining 106 patients with normal karyotypes comprised the control group (median age, 65 years; range, 1–92 years; 56 females). Further, follow-up analysis revealed that the overall survival of the NCCA group was worse than that of the CCA and the normal karyotype groups (P < 0.0001; log-rank test).The survival of the NCCA-harboring cytopenic patients was worse than that of the CCA-harboring cytopenic patients without hematological malignancies, suggesting that follow-up should be considered for both CCA- and NCCA-harboring cytopenic patients.

Antagonistic Pleiotropy in the Bifunctional Surface Protein FadL (OmpP1) during Adaptation of Haemophilus influenzae to Chronic Lung Infection Associated with Chronic Obstructive Pulmonary Disease.

Tracking bacterial evolution during chronic infection provides insights into how host selection pressures shape bacterial genomes. The human-restricted opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) infects the lower airways of patients suffering chronic obstructive pulmonary disease (COPD) and contributes to disease progression. To identify bacterial genetic variation associated with bacterial adaptation to the COPD lung, we sequenced the genomes of 92 isolates collected from the sputum of 13 COPD patients over 1 to 9 years. Individuals were colonized by distinct clonal types (CTs) over time, but the same CT was often reisolated at a later time or found in different patients. Although genomes from the same CT were nearly identical, intra-CT variation due to mutation and recombination occurred. Recurrent mutations in several genes were likely involved in COPD lung adaptation. Notably, nearly a third of CTs were polymorphic for null alleles of ompP1 (also called fadL), which encodes a bifunctional membrane protein that both binds the human carcinoembryonic antigen-related cell adhesion molecule 1 (hCEACAM1) receptor and imports long-chain fatty acids (LCFAs). Our computational studies provide plausible three-dimensional models for FadL's interaction with hCEACAM1 and LCFA binding. We show that recurrent fadL mutations are likely a case of antagonistic pleiotropy, since loss of FadL reduces NTHi's ability to infect epithelia but also increases its resistance to bactericidal LCFAs enriched within the COPD lung. Supporting this interpretation, truncated fadL alleles are common in publicly available NTHi genomes isolated from the lower airway tract but rare in others. These results shed light on molecular mechanisms of bacterial pathoadaptation and guide future research toward developing novel COPD therapeutics.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in patients with chronic obstructive pulmonary disease (COPD). To elucidate the bacterial pathways undergoing in vivo evolutionary adaptation, we compared bacterial genomes collected over time from 13 COPD patients and identified recurrent genetic changes arising in independent bacterial lineages colonizing different patients. Besides finding changes in phase-variable genes, we found recurrent loss-of-function mutations in the ompP1 (fadL) gene. We show that loss of OmpP1/FadL function reduces this bacterium's ability to infect cells via the hCEACAM1 epithelial receptor but also increases its resistance to bactericidal fatty acids enriched within the COPD lung, suggesting a case of antagonistic pleiotropy that restricts ΔfadL strains' niche. These results show how H. influenzae adapts to host-generated inflammatory mediators in the COPD airways.

The t(1;10)(p22;q24) TGFBR3/MGEA5 Translocation in Pleomorphic Hyalinizing Angiectatic Tumor, Myxoinflammatory Fibroblastic Sarcoma, and Hemosiderotic Fibrolipomatous Tumor.

Pleomorphic hyalinizing angiectatic tumor (PHAT) of soft parts, hemosiderotic fibrolipomatous tumor (HFLT), and myxoinflammatory fibroblastic sarcoma (MIFS) are 3 distinct entities of low-grade spindle cell mesenchymal neoplasm. These tumors have similar clinical presentations and partially overlapping but distinctive pathologic features. A recurrent translocation, t(1;10)(p22;q24), has been detected in a subset of PHAT, HFLT, MIFS, and HFLT/MIFS hybrid cases. Translocation t(1;10)(p22;q24) involves transforming growth factor β-receptor 3 ( TGFBR3) and meningioma-expressed antigen 5 ( MGEA5) genes on chromosomes 1p22 and 10q24, respectively. However, the percentage of translocation in PHAT, HFLT, and MIFS varies significantly among different studies. The relationship among these tumors has been a controversial topic among experts. To discuss the diagnostic and functional significance of translocation t(1;10)(p22;q24) TGFBR3/MGEA5 rearrangement in HFLT, PHAT, and MIFS. PubMed was used for this study. Diagnosis of HFLT, PHAT, and MIFS is challenging because of a lack of unique morphologic, immunophenotypic, molecular, and cytogenetic markers. The recurrent t(1;10)(p22;q24) translocation and/or TGFBR3/MGEA5 rearrangement was reported in 55 patients, with a relatively even distribution among HFLT, PHAT, and MIFS (17 HFLT, 15 MIFS, 13 MIFS/HFLT, and 10 PHAT). This indicates that current morphology-based diagnostic criteria do not identify reliably the subset of soft tissue tumor with t(1;10) translocation. Genetic heterogeneity of these tumors is supported by the recent detection of a mutually exclusive, second recurrent genetic change, t(7;17) TOM1L2-BRAF translocation or BRAF amplification, in a subset of MIFS.

STAT6 is a cargo of exportin 1: Biological relevance in primary mediastinal B-cell lymphoma

Primary mediastinal B-cell lymphoma (PMBL) is a distinct B-cell lymphoma subtype with unique clinicopathological and molecular features. PMBL cells are characterised by several genetic abnormalities that conduct to the constitutive activation of the Janus kinase 2/signal transducer and activator of transcription 6 (JAK2/STAT6) signalling pathway. Among recurrent genetic changes in PMBL, we previously reported that the XPO1 gene encoding exportin 1 that controls the nuclear export of cargo proteins and RNAs, is mutated (p.E571K) in about 25% of PMBL cases. We therefore hypothesized that STAT6 could be a cargo of XPO1 and that STAT6 cytoplasm/nucleus shuttle could be altered in a subset of PMBL cells. Using immunocytochemistry techniques as well as the proximity ligation assay, we showed that STAT6 bound XPO1 in PBML cell lines and in HEK-293 cells genetically engineered to produce STAT6. Moreover, XPO1-mediated export of STAT6 occurs in cells expressing either a wild-type or the E571K mutated XPO1 protein.

RHCG Suppresses Tumorigenicity and Metastasis in Esophageal Squamous Cell Carcinoma via Inhibiting NF-κB Signaling and MMP1 Expression.

Background and Aims: Esophageal squamous cell carcinoma (ESCC), a major histologic subtype of esophageal cancer, is increasing in incidence, but the genetic underpinnings of this disease remain unexplored. The aim of this study is to identify the recurrent genetic changes, elucidate their roles and discover new biomarkers for improving clinical management of ESCC.Methods: Western blotting and immunohistochemistry were performed to detect the expression level of RHCG. Bisulfite genomic sequencing (BGS) and methylation-specific PCR (MSP) were used to study the methylation status in the promoter region of RHCG. The tumor-suppressive effect of RHCG was determined by both in-vitro and in-vivo assays. Affymetrix cDNA microarray was used to identify the underlying molecular mechanism.Results: RHCG was frequently downregulated in ESCCs, which was significantly correlated with poor differentiation (P = 0.001), invasion (P = 0.003), lymph node metastasis (P = 0.038) and poorer prognosis (P < 0.001). Demethylation treatment and bisulfite genomic sequencing analyses revealed that the downregulation of RHCG in both ESCC cell lines and clinical samples was associated with its promoter hypermethylation. Functional assays demonstrated that RHCG could inhibit clonogenicity, cell motility, tumor formation and metastasis in mice. Further study revealed that RHCG could stabilize IκB by decreasing its phosphorylation, and subsequently inhibit NF-κB/p65 activation by blocking the nuclear translocation of p65, where it acted as a transcription regulator for the upregulation of MMP1 expression.Conclusions: Our results support the notion that RHCG is a novel tumor suppressor gene that plays an important role in the development and progression of ESCC.

Expanded molecular profiling of myxofibrosarcoma reveals potentially actionable targets

Myxofibrosarcomas are morphologically heterogeneous soft tissue sarcomas lacking a specific immunohistochemical expression profile and recurrent genetic changes. The study was designed to gain further insights into the molecular landscape of myxofibrosarcomas by targeted re-sequencing of known cancer driver hotspot mutations and the analysis of genomewide somatic copy number alterations. A well-defined group of myxofibrosarcomas, including myxofibrosarcomas G1 (n=6), myxofibrosarcomas G3 (n=7), myxofibrosarcomas with morphologically heterogeneous and independently selectable G1 and G3 areas within a tumor (n=8), and myxofibrosarcomas G3 with subsequent tumor recurrence (n=1) or metastatic disease (n=3) were evaluated. Mutational analysis demonstrated mutations in TP53, PTEN, FGFR3, CDKN2A, and RB1. TP53 mutations were seen in 11 (44%) of patients and detected in myxofibrosarcomas G1, G3, with heterogeneous morphology and G3 with subsequent metastases in 1 patient (16%), 3 patients (42%), 2 patients (62.5%), and 3 patients (75%), respectively. Additional mutations were detected in 2 patients, intratumoral mutational heterogeneity in 1 patient. We observed a variety of copy number alterations typical for myxofibrosarcomas, with higher numbers in G3 compared with G1 myxofibrosarcomas. Cluster analysis revealed distinctive features especially in metastatic and recurrent disease. Focal alterations affected CDKN2A, CCND1, CCNE1, EGFR, EPHA3, EPHB1, FGFR1, JUN, NF1, RB1, RET, TP53, and additional novel amplifications in CCNE1, KIT, EGFR, RET, BRAF, NTRK2 were seen in G3 compared with the G1 tumor areas. The total number of focal events in G1 versus G3 tumors differed significantly (P=0.0014). TRIO and RICTOR co-amplification was seen in 8 (44%) G3 and 1 (10%) G1 myxofibrosarcomas and RICTOR amplification alone in 4 (40%) G1 myxofibrosarcomas. TRIO amplification was significantly (P=0.0218) higher in G3 myxofibrosarcomas indicating a late genetic event. These findings support the use of expanded molecular profiling in myxofibrosarcomas to detect drug-able targets to allow patients to participate in basket trials.

Recurrent DNA copy number alterations in intestinal-type sinonasal adenocarcinoma

Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumour related to occupational wood dust exposure. Few studies have described recurrent genetic changes on a genome-wide scale. The aim of this study was to obtain a high resolution map of recurrent genetic alterations in ITAC. Copy number alterations were evaluated by microarray CGH and MLPA in 37 primary tumours. The results were correlated with pathological characteristics and clinical outcome. Microarray CGH identified the following recurrent aberrations, in descending order: gains at 5p15 (22 cases, 60%), 8q24 (21 cases, 57%), 20q13 (20 cases, 54%), 20q11, and 8q21 (19 cases, 51%), 20p13, and 7p11 (16 cases, 43%), and losses at 5q11-qter, 8p12-pter, and 18q12-23 (15 cases, 40%), and 17p13, and 19p13 (13 cases, 35%). MLPA analysis confirmed this global pattern of gains and losses. Chromosomal loss at 4q32-ter and gains at 1q22, 6p22 and 3q29, as well as deletion of TIMP2 and CRK correlated with unfavourable clinical outcome. ITACs have a unique pattern of chromosomal abnormalities. The four different histological subtypes of ITAC appeared genetically similar. Four chromosomal gains and losses and two specific genes showed prognostic value and may be involved in tumour progression.

A Zebrafish Model to Study Co-Operating Mutations in CEBPA-Mutated AML

The current model for the pathogenesis of AML involves a normal haematopoietic stem or progenitor cell (HSPC) acquiring successive genetic abnormalities leading to clonal expansion and malignant transformation. Next generation sequencing (NGS) has facilitated the identification of more than 250 recurrent genetic changes in AML, with each case possessing between 5-20. Only a fraction of these have been validated as driver events, and the epistatic relationships and cellular consequences of combined mutational genotypes remain largely unexplored. A unique insight into this is afforded by a subset of patients who have inherited a mutation that predisposes to the development of AML, so-called familial leukaemia with predisposition to AML (FPD-AML) since these mutations are unequivocally the initiating driver mutations. One such mutation occurs in the N-terminal domain of the transcription factor CEBPA. To further understand the role of N-terminal CEBPA mutations and co-operating secondary driver mutations we have used a TALEN pair to generate a zebrafish model carrying an analogous N-terminal frame-shift mutation in Cebpa (cebpaNterm).Initial analysis shows cebpaNterm/Nterm mutants have severe defects in developmental myelopoeisis. By 5 days post fertilisation (dpf), homozygous cebpaNterm/Nterm mutants show a complete absence of Sudan Black (SB) staining myeloid cells. cebpaNterm/+siblings display a minor delay in myeloid development, but no significant difference in numbers of SB staining cells at 5 dpf (Figure 1A,B). cebpaNterm/Nterm mutants are morphologically indistinguishable from their siblings during the first 5 days of life, however, they show diminished growth and survival thereafter. At 4 weeks, remaining juvenile cebpaNterm/Nterm mutants show markedly reduced length and weight compared to age matched controls (cebpa+/+and cebpaNterm/+) and are not viable beyond 8 weeks (Figure 1C).To facilitate assessment of HSPCs/thrombocytes and myeloid cells, we crossed cebpaNterm/+mutants to transgenic reporter lines expressing eGFP from the CD41 promoter (Tg(cd41:eGFP)) and mCherry (mCh) from the lysozyme C promoter (Tg(LyzC:mCherry)). Tg(LyzC:mCherry);cebpaNterm/Nterm fry can be distinguished from their siblings in vivo by the absence of mCherry expressing myeloid cells from 3.5dpf. Flow cytometric analysis in surviving juveniles at 32dpf demonstrated a continued absence of mCh expressing myeloid cells in both peripheral blood and whole kidney marrow. However, evaluation of cebpaNterm/Nterm mutant juveniles at 6 weeks showed evidence of increased numbers of mCh positive cells, compared to wild type and heterozygous sibling controls. Blood smears in homozygous mutants at this stage also revealed cells morphologically suggestive of primitive myeloid origin. Finally, epifluorescent microscopy of 4-6 week old fish demonstrated a widespread GFP dim fluorescence in the cebpaNterm/Nterm mutants. In contrast siblings displayed only GFP bright thrombocytes, suggesting an expansion of CD41-GFPdimexpressing HSPC-like cells in mutant animals.The occurrence of biallelic N-terminal CEBPA-mutations in human AML is however extremely rare. By contrast the most frequent "second hit" mutation occurs in the C-terminal DNA binding domain of the other allele of CEBPA. These mutations are universally in frame deletions or insertions, suggesting retained dimerization capability but abrogation of DNA binding. We have generated a knock-in C-terminal model of the most commonly observed human mutation in the orthologous zebrafish sequence using TALENs and plasmid mediated homology directed repair. This donor plasmid adds 3bp encoding an additional leucine within the C-terminal zipper domain. MISEQ analysis of F0 putative founder eggs and sperm has shown a 2-5% germline transmission rate of the knockin allele. In addition to CEBPA C-terminal mutations, a number of other recurrently mutated genes have been found in CEBPA mutated AML. Our ongoing work will assess the effects of combined N and C terminal cebpa mutation, along with CRISPR-targeting of other common "second hits" including cohesion proteins, WT1, GATA2 and TET2 to define their role in leukaemogenesis in CEBPA-mutated AML. Our ongoing experiments are also defining the self-renewal capacity of myeloid and HSPCs derived from 4-6 week old cebpaNterm/Nterm mutants using rag2E450fsknockout transplantation assays. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recurrent DNA copy number alterations in intestinal-type sinonasal adenocarcinoma.

Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumour related to occupational wood dust exposure. Few studies have described recurrent genetic changes on a genome-wide scale. The aim of this study was to obtain a high resolution map of recurrent genetic alterations in ITAC. Copy number alterations were evaluated by microarray CGH and MLPA in 37 primary tumours. The results were correlated with pathological characteristics and clinical outcome. Microarray CGH identified the following recurrent aberrations, in descending order: gains at 5p15 (22 cases, 60%), 8q24 (21 cases, 57%), 20q13 (20 cases, 54%), 20q11, and 8q21 (19 cases, 51%), 20p13, and 7p11 (16 cases, 43%), and losses at 5q11-qter, 8p12-pter, and 18q12-23 (15 cases, 40%), and 17p13, and 19p13 (13 cases, 35%). MLPA analysis confirmed this global pattern of gains and losses. Chromosomal loss at 4q32-ter and gains at 1q22, 6p22 and 3q29, as well as deletion of TIMP2 and CRK correlated with unfavourable clinical outcome. ITACs have a unique pattern of chromosomal abnormalities. The four different histological subtypes of ITAC appeared genetically similar. Four chromosomal gains and losses and two specific genes showed prognostic value and may be involved in tumour progression.

Myeloid malignancies in the real-world: Occurrence, progression and survival in the UK’s population-based Haematological Malignancy Research Network 2004–15

BackgroundPopulation-based information on cancer incidence, prevalence and outcome are required to inform clinical practice and research; but contemporary data are lacking for many myeloid malignancy subtypes. MethodsSet within a socio-demographically representative UK population of ∼4 million, myeloid malignancy data (N=5231 diagnoses) are from an established patient cohort. Information on incidence, survival (relative & overall), transformation/progression, and prevalence is presented for >20 subtypes. ResultsThe median diagnostic age was 72.4years (InterQuartile Range 61.6–80.2), but there was considerable subtype heterogeneity, particularly among the acute myeloid leukaemias (AML) where medians ranged from 20.3 (IQR 13.9–43.8) for AML 11q23 through to 73.7 (IQR 57.3–79.1) for AML with no recurrent genetic changes. Five-year Relative Survival (RS) estimates varied hugely; from <5% for aggressive entities like therapy-related AML (2.6%, 95% Confidence Interval 0.4–9.0) to >85% for indolent/treatable conditions like chronic myeloid leukaemia (89.8%, 95% CI 84.0–93.6). With a couple of notable exceptions, males experienced higher rates and worse survival than females: the age-standardized incidence rates of several conditions was 2–4 higher in males than females, and the 5-year RS for all subtypes combined was 48.8% (95% CI 46.5–51.2) and 60.4% (95% CI 57.7–62.9) for males and females respectively. During follow-up (potential minimum 2 years and maximum 11years) myelodysplastic syndrome (MDS) progression to AML ranged from 25% for refractory anaemia with excess blasts through to 5% for refractory anaemia with ring sideroblasts: the median interval between MDS and AML diagnosis was 9.0 months (IQR 4.8–17.4months). ConclusionsThe marked incidence and outcome variations seen by subtype, sex and age, confirm the requirement for “real-world” longitudinal data to inform aetiological hypotheses, healthcare planning, and future monitoring of therapeutic change. Several challenges for routine cancer registration were identified, including the need to link more effectively to diagnostic and clinical data sources, and to review policies on the recording of progressions and transformations.

Why it is crucial to analyze non clonal chromosome aberrations or NCCAs?

Current cytogenetics has largely focused its efforts on the identification of recurrent karyotypic alterations, also known as clonal chromosomal aberrations (CCAs). The rationale of doing so seems simple: recurrent genetic changes are relevant for diseases or specific physiological conditions, while non clonal chromosome aberrations (NCCAs) are insignificant genetic background or noise. However, in reality, the vast majority of chromosomal alterations are NCCAs, and it is challenging to identify commonly shared CCAs in most solid tumors. Furthermore, the karyotype, rather than genes, represents the system inheritance, or blueprint, and each NCCA represents an altered genome system. These realizations underscore the importance of the re-evaluation of NCCAs in cytogenetic analyses. In this concept article, we briefly review the definition of NCCAs, some historical misconceptions about them, and why NCCAs are not insignificant “noise,” but rather a highly significant feature of the cellular population for providing genome heterogeneity and complexity, representing one important form of fuzzy inheritance. The frequencies of NCCAs also represent an index to measure both internally- and environmentally-induced genome instability. Additionally, the NCCA/CCA cycle is associated with macro- and micro-cellular evolution. Lastly, elevated NCCAs are observed in many disease/illness conditions. Considering all of these factors, we call for the immediate action of studying and reporting NCCAs. Specifically, effort is needed to characterize and compare different types of NCCAs, to define their baseline in various tissues, to develop methods to access mitotic cells, to re-examine/interpret the NCCAs data, and to develop an NCCA database.

The role of inflammation in brain cancer.

Malignant brain tumors are among the most lethal of human tumors, with limited treatment options currently available. A complex array of recurrent genetic and epigenetic changes has been observed in gliomas that collectively result in derangements of common cell signaling pathways controlling cell survival, proliferation, and invasion. One important determinant of gene expression is DNA methylation status, and emerging studies have revealed the importance of a recently identified demethylation pathway involving 5-hydroxymethylcytosine (5hmC). Diminished levels of the modified base 5hmC is a uniform finding in glioma cell lines and patient samples, suggesting a common defect in epigenetic reprogramming. Within the tumor microenvironment, infiltrating immune cells increase oxidative DNA damage, likely promoting both genetic and epigenetic changes that occur during glioma evolution. In this environment, glioma cells are selected that utilize multiple metabolic changes, including changes in the metabolism of the amino acids glutamate, tryptophan, and arginine. Whereas altered metabolism can promote the destruction of normal tissues, glioma cells exploit these changes to promote tumor cell survival and to suppress adaptive immune responses. Further understanding of these metabolic changes could reveal new strategies that would selectively disadvantage tumor cells and redirect host antitumor responses toward eradication of these lethal tumors.

DNA Methylation Changes In Aging Human CD34+ Cells Coincide With Hotspots Of Disordered Methylation In AML At Imprinted and Allelically Methylated Regions

Aging of the hematopoietic system in humans is associated with increased incidence of myeloid malignancies. Epigenetic machinery such as DNA methylation is known to change with age, and is disproportionately impacted by recurrent genetic mutations in acute myeloid leukemia (AML). We hypothesized that epigenetic changes in CD34+ hematopoietic stem and progenitor cells (HSPCs) may precede recurrent genetic changes in AML, and might be detected in normal aging HSPCs with increasing frequency. We also hypothesized that areas with increased variability in methylation may be hot-spots for the emergence of epigenetically distinct HSPC clones.In order to characterize these changes, we performed methylome-wide profiles of human HSPCs from different age groups (20-25 years (10), 40-45 years (10) and >60 years (9)).Adult HSPCs were obtained from Leukocyte Reduction System cones from healthy platelet donors. CD34+ cells were then isolated by Fluorescence Assisted Cell Sorting. 200 ng of DNA extracted from the CD34+ cells was processed using the Infinium Methylation 450k Beadchip (Illumina). Differentially methylated regions (DMRs) were identified using the bump hunting procedure of Jaffe (2011) to pool information across CpG loci into regions of consistent change and to quantify statistical significance.893 differentially methylated regions (DMRs) varied linearly with age in HSPCs; a set of 31 such regions yielded an accurate predictor of age in lineage-sorted cells (N=48, Reinius et al., 2012) and whole blood (N=656, Hannum et al., 2011), with a root-mean-squared error of 5.3 years. While age-related lymphopenia has previously been reported, DNA methylation marks for lineage commitment (Houseman et al., 2012) were nearly uniform within our subjects' CD34+ cells, and exhibited no relationship with age. However, regional summaries of methylation provided more accurate age predictions than specific CpG loci. We reasoned that differential variability at individual loci might be the cause.We thus investigated regions where methylation variability increased with age. Known imprinted clusters and allelically methylated regions (AMRs) identified by Fang (2012, PNAS) were disproportionately represented among these; 27% of known imprinting regions and 33.3% of allelically methylated regions in the genome coincided with at least one such region, while comprising only 0.3% of the genome and 0.7% of loci assayed. Among these, the H19 imprinting control region has been shown to crucially regulate long-term HSPC homeostasis in mice via IGF2, and the allelically methylated WT1/WT1-AS region on chromosome 11p is a highly recurrent hotspot for disordered methylation in AML, as well as sequential epigenetic defects in Wilms' tumor. The allelically methylated vault RNA VTRNA2-1 (recently shown to predict survival in AML) on chromosome 5q, and the monoallelically expressed TP73 and DIRAS3 genes on chromosome 1p, were also sites of greater methylation variability with age in normal HSPCs. Wu et al. (1997) showed that loss of imprinting at H19/IGF2 is common in AML, seemingly conferring a selective metabolic advantage, and global loss of imprinting in mice leads to widespread tumorigenesis (Holm et al., 2005). Recurrent methylation aberrations in induced pluripotent stem cells favor imprinted clusters (Nazor, 2012), and epigenetic polymorphisms arise in these regions over time in cultured cells (Tanay et al, 2012). However, to our knowledge, ours is the first report of this type of heterogeneity with age in normal human adult HSPCs.Clonal hematopoiesis has previously been documented in healthy elderly adults (Levine 2012), and the majority of patients in the Cancer Genome Atlas (TCGA) AML study exhibited mutations in one or more genes regulating epigenetic machinery. We propose that increased epigenetic heterogeneity in aging HSPCs, particularly at regions with allele-specific methylation (such as H19/IGF2), may precede malignant evolution in some leukemias, and warrants further investigation. Disclosures:No relevant conflicts of interest to declare.