Clonal Chromosome Aberrations Research Articles

The Importance of Monitoring Non-clonal Chromosome Aberrations (NCCAs) in Cancer Research.

Cytogenetic analysis has traditionally focused on the clonal chromosome aberrations, or CCAs, and considered the large number of diverse non-clonal chromosome aberrations, or NCCAs, as insignificant noise. Our decade-long karyotype evolutionary studies have unexpectedly demonstrated otherwise. Not only the baseline of NCCAs is associated with fuzzy inheritance, but the frequencies of NCCAs can also be used to reliably measure genome or chromosome instability (CIN). According to the Genome Architecture Theory, CIN is the common driver of cancer evolution that can unify diverse molecular mechanisms, and genome chaos, including chromothripsis, chromoanagenesis, and polypoidal giant nuclear and micronuclear clusters, and various sizes of chromosome fragmentations, including extrachromosomal DNA, represent some extreme forms of NCCAs that play a key role in the macroevolutionary transition. In this chapter, the rationale, definition, brief history, and current status of NCCA research in cancer are discussed in the context of two-phased cancer evolution and karyotype-coded system information. Finally, after briefly describing various types of NCCAs, we call for more research on NCCAs in future cytogenetics.

Cytogenetic and molecular analyses of 291 gastrointestinal stromal tumors: site-specific cytogenetic evolution as evidence of pathogenetic heterogeneity.

Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm with variable behavior. An increased understanding of the tumor pathogenesis may improve clinical decision-making. Our aim was to obtain more data about the overall chromosome aberrations and intratumor cytogenetic heterogeneity in GIST. We analyzed 306 GIST samples from 291 patients using G-banding, direct sequencing, and statistics. Clonal chromosome aberrations were found in 81% of samples, with 34% of 226 primary tumors demonstrating extensive cytogenetic heterogeneity. 135 tumors had simple (≤5 changes) and 91 had complex (>5 changes) karyotypes. The karyotypically complex tumors more often were non-gastric (P < 0.001), larger (P < 0.001), more mitotically active (P = 0.009) and had a higher risk of rupture (P < 0.001) and recurrence (P < 0.001). Significant differences between gastric and non-gastric tumors were found also in the frequency of main chromosome losses: of 14q (79% vs. 63%), 22q (38% vs. 67%), 1p (23% vs. 88%), and 15q (18% vs. 77%). Gastric PDGFRA-mutated tumors, compared with gastric KIT-mutated, had a lower incidence of 22q losses (18% vs. 43%) but a higher rate of 1p losses (42% vs. 22%). The present, largest by far karyotypic study of GISTs provides further evidence for the existence of variable pathogenetic pathways operating in these tumors’ development.

Hamartomas of Body: A Revisited Entity - An Experience of a Tertiary Care Hospital

Introduction/Background: Hamartoma is a mass of disorganized tissue indigenous to the particular site. Although traditionally considered as developmental malformations, many hamartomas have clonal chromosomal aberration that is acquired through somatic mutation, thus now considered to be neoplastic. They arise virtually in all organs, either sporadically or in association with autosomal dominant syndrome. Materials and Methods: A retrospective study was carried out in a tertiary care health institute for 3-year period (January 2013 to December 2016), which included all visceral hamartomas from head to toe. Detailed clinicoradiological and pathological examination was done along with ancillary tests for definite diagnosis. Observation: The study included nine cases, categorized site wise as head and neck (one case of cystic lymphangioma), cardiothoracic (one case of chondroid hamartoma), gastrointestinal tract (GIT) (one case of Brunner's gland hamartoma), hepatic (one case of cystic lymphangioma), and genitourinary tract (four cases of angiomyolipoma [AML] and one case of myelolipoma). The age ranged from 1 year to 70 years showing a male predilection. The tumors were variable sized ranging from 0.5 to 20 cm in dimension either solitary or multiple. Some large tumors clinically masqueraded as malignant tumors and presented with complications. The postsurgical course and prognosis of all patients were uneventful. Conclusion: Hamartomas are usually not associated with significant morbidity but for the size and location of the tumor. Despite slow-growing and self-limiting behavior, some predispose to malignancy especially in the epithelioid AML and familial GIT hamartoma or may be seen associated with malignancy in syndromic forms.

Chromosomal aberrations after induced pluripotent stem cells reprogramming.

Induced pluripotent stem cells (iPSCs) are generated from adult cells that have been reprogrammed to pluripotency. However, in vitro cultivation and genetic reprogramming increase genetic instability, which could result in chromosomal abnormalities. Maintenance of genetic stability after reprogramming is required for possible experimental and clinical applications. The aim of this study was to analyze chromosomal alterations by using the G-banding karyotyping method applied to 97 samples from 38 iPSC cell lines generated from peripheral blood or Wharton’s jelly. Samples from patients with long QT syndrome, Jervell and Lange-Nielsen syndrome and amyotrophic lateral sclerosis and from normal individuals revealed the following chromosomal alterations: acentric fragments, chromosomal fusions, premature centromere divisions, double minutes, radial figures, ring chromosomes, polyploidies, inversions and trisomies. An analysis of two samples generated from Wharton’s jelly before and after reprogramming showed that abnormal clones can emerge or be selected and generate an altered lineage. IPSC lines may show clonal and nonclonal chromosomal aberrations in several passages (from P6 to P34), but these aberrations are more common in later passages. Many important chromosomal aberrations were detected, showing that G-banding is very useful for evaluating genetic instability with important repercussions for the application of iPSC lines.

Cytogenetic complexity and heterogeneity in intravascular lymphoma

AimsTo characterise the karyotypic abnormalities and heterogeneities in intravascular lymphoma (IVL).MethodsG-banded karyotyping was performed on biopsy specimens from a single-centre IVL cohort comprising intravascular large B-cell lymphoma (IVLBCL, n=12) and...

Karyotypic Flexibility of the Complex Cancer Genome and the Role of Polyploidization in Maintenance of Structural Integrity of Cancer Chromosomes.

Ongoing chromosomal instability in neoplasia (CIN) generates intratumor genomic heterogeneity and limits the efficiency of oncotherapeutics. Neoplastic human cells utilizing the alternative lengthening of telomeres (ALT)-pathway, display extensive structural and numerical CIN. To unravel patterns of genome evolution driven by oncogene-replication stress, telomere dysfunction, or genotoxic therapeutic interventions, we examined by comparative genomic hybridization five karyotypically-diverse outcomes of the ALT osteosarcoma cell line U2-OS. These results demonstrate a high tendency of the complex cancer genome to perpetuate specific genomic imbalances despite the karyotypic evolution, indicating an ongoing process of genome dosage maintenance. Molecular karyotyping in four ALT human cell lines showed that mitotic cells with low levels of random structural CIN display frequent evidence of whole genome doubling (WGD), suggesting that WGD may protect clonal chromosome aberrations from hypermutation. We tested this longstanding hypothesis in ALT cells exposed to gamma irradiation or to inducible DNA replication stress under overexpression of p21. Single-cell cytogenomic analyses revealed that although polyploidization promotes genomic heterogeneity, it also protects the complex cancer genome and hence confers genotoxic therapy resistance by generating identical extra copies of driver chromosomal aberrations, which can be spared in the process of tumor evolution if they undergo unstable or unfit rearrangements.

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

Examination of clonal evolution in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is one of the most frequent lymphoproliferative diseases. CLL is characterized by unusual heterogeneity, which probably reflects its biological and genetic lack of homogeneity. Clonal chromosome aberrations belong to the most important prognostic and predictive factors in CLL. This research was aimed at observing clonal evolution in CLL at the chromosomal level, and assessing its clinical significance in relation to selected prognostic factors. The study involved 72 untreated patients with CLL. The preliminary investigations using cytogenetic banding analysis (CBA) and FISH were performed at the time of diagnosis, and again after about 24 months to observe clonal changes (clonal evolution). In addition, other parameters were evaluated, i.e., the expression of ZAP-70 kinase, CD38 antigen, and the mutation statuses of IGVH and NOTCH1 genes. Classic prognostic factors, i.e., categorized ZAP70 and CD38 expressions as well as mutations in IGVH and NOTCH1 genes did not influence the course of clonal evolution in the examined group of patients. Clonal evolution was detected in 45.8% of patients by means of CBA, and in 19.4% patients with FISH. Analysis of chromosomal aberrations in the examined group of patients showed that the incidence of 17p deletions and translocations in karyotypes has a negative impact on overall survival. CE was found to be a risk factor for the occurrence of disease progression (OR = 2.22). Our observations indicate that combined CBA and FISH are the most optimal techniques for monitoring clonal evolution in the course of CLL.

Nonclonal Chromosome Aberrations and Genome Chaos in Somatic and Germ Cells from Patients and Survivors of Hodgkin Lymphoma.

Anticancer regimens for Hodgkin lymphoma (HL) patients include highly genotoxic drugs that have been very successful in killing tumor cells and providing a 90% disease-free survival at five years. However, some of these treatments do not have a specific cell target, damaging both cancerous and normal cells. Thus, HL survivors have a high risk of developing new primary cancers, both hematologic and solid tumors, which have been related to treatment. Several studies have shown that after treatment, HL patients and survivors present persistent chromosomal instability, including nonclonal chromosomal aberrations. The frequency and type of chromosomal abnormalities appear to depend on the type of therapy and the cell type examined. For example, MOPP chemotherapy affects hematopoietic and germ stem cells leading to long-term genotoxic effects and azoospermia, while ABVD chemotherapy affects transiently sperm cells, with most of the patients showing recovery of spermatogenesis. Both regimens have long-term effects in somatic cells, presenting nonclonal chromosomal aberrations and genomic chaos in a fraction of noncancerous cells. This is a source of karyotypic heterogeneity that could eventually generate a more stable population acquiring clonal chromosomal aberrations and leading towards the development of a new cancer.

The Evolution of Genetic Aberrations in CML Patients Harboring a Ph-Negative Clone: A 6-Year Follow-up Study of 52 Patients

▪Background: Clonal chromosome aberrations in Philadelphia chromosome-negative metaphases (CCA/Ph-) occur in a subset of chronic myeloid leukemia (CML) patients. A shorter survival was reported for “non -Y” CCA/Ph- cases (Issa et al. Blood 2017). Besides -Y, the most frequent CCA/Ph- is +8, but a broad spectrum of other abnormalities can be found, including 7q-/-7, which is a typical aberration of myelodysplastic syndromes (MDS).In previous analyses we had shown an increased number of molecular genetic aberrations in CCA/Ph- compared to non-CCA/Ph- patients (Schnittger et al. ASH 2013, 2014). However, the clinical impact and the evolution of the mutation pattern is largely unknown. Here we extended follow-up and genetic characterization of the initial CCA/Ph- cohort.Aim:To determine the pattern of molecular mutations and their evolutionTo determine if mutations are part of the Ph+ or Ph- clonePatients and Methods: We included 52 CCA/Ph- patients (female: 25; male 27), with a median age of 58 [33-81] years, and a median BCR-ABL1/ABL1 ratio of 4.322% [0-58.088%] (N.A. for 4 pts.) at the time of initial CCA/Ph- detection. The following CCA/Ph- were present: trisomy 8 (n=26), other trisomies (n=4), -Y (n=7), del(7q)/-7 (n=4), others (n=7), two CCA/Ph- (n=4). We performed sequencing of myeloid gene panels on follow-up samples (1 to 3 per patient) on Illumina's MiSeq and NextSeq instruments (library preparation: 29-gene panel Thunderstorm RainDance [Lexington, MA] or 28-gene panel TruSeq [Illumina, San Diego, CA]). Data was analyzed with SeqNext (JSI Medical Systems, Kippenheim, Germany). Detected mutations were monitored on additional time points to determine variant allele frequency (VAF: mutated/all reads) development. A reference cohort of 47 patients with no sign of CCA/Ph- after MMR achievement was presented as part of our initial study (Schnittger et al. ASH 2014).Results:Cytogenetic monitoring was available over a median period of 31 [0-126] months for the CCA/Ph- and 26 [12-85] months for the reference cohort. Of the CCA/Ph- patients, 5/52 (10%) acquired additional typical aberrations as CCA/Ph- clone (incl. one -7), while in the reference cohort only one of 47 (2%) patients developed a -Y clone (n.s.).On the molecular level, we conducted a median follow-up of 72 [9-150] months for the CCA/Ph- cohort (mutations and BCR-ABL1/ABL1 ratio). Following the CCA/Ph- detection, somatic mutations were found in 30/52 (58%) patients (up to 4 per patient): ASXL1 (n=13), DNMT3A (n=10), TET2 (n=6), NRAS (n=3), RUNX1 (n=3), non-recurrent (n=8).The VAF of 7 mutations was strongly correlated to the BCR-ABL1 ratio and thus most likely present in the Ph+ clone. Mutations in ASXL1 were present in the Ph+ clone in five patients, of whom four never reached MMR, while 6/8 patients with ASXL1 mutations in Ph-independent clones achieved MMR under first- or second-line TKI therapy.Molecular genetic aberrations in Ph- cells were found in 23/52 (42%) CCA/Ph- patients, but only in 2 of 47 (4%) cases of the non-CCA/Ph- cohort (p<0.001). Importantly, in the reference cohort only TET2 and DNMT3A mutations were identified, which is the typical pattern in age related clonal hematopoiesis (ARCH) and also found in older individuals without a hematological malignancy. In the CCA/Ph- cohort, eight of 23 (35%) showed only TET2 or DNMT3A mutations. However, a highly predictive mutation signature for development of a myeloid malignancy (according to Malcovati et al. Blood 2017) was found in 8/23 (35%). In addition, of three NRAS positive CCA/Ph- cases, one was diagnosed with MDS/MPN overlap (also CALR positive) and two developed s-AML during the follow-up period and the NRAS VAF had increased to ≥35% at our last monitoring time point.Conclusions:ASXL1 mutations can occur in the Ph- as well as in the Ph+ clone and are associated with a poor TKI response, if present in the Ph+ clone.Molecular mutations are significantly more frequent in CCA/Ph- than in non-CCA/Ph-.While the mutation pattern in non-CCA/Ph- resembles ARCH, the spectrum in CCA/Ph- includes a higher risk pattern for development of a myeloid malignancy.Further prospective studies are required to evaluate the clinical impact of mutations acquired during the course of CML in order to determine which mutation pattern is related to a higher incidence of secondary myeloid malignancies. DisclosuresBaer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

3115 - ¬¬Attenuated DNA Damage Responses and Increased Apoptosis Characterize Human Hematopoietic Stem Cells Exposed to Irradiation

Failure to precisely repair DNA damage in self-renewing Hematopoietic Stem and early Progenitor Cells (HSPCs) can disrupt normal hematopoiesis and promote leukemogenesis. Although HSPCs are widely considered a target of ionizing radiation (IR)-induced hematopoietic injury, definitive data regarding cell death, DNA repair, and genomic stability in these rare quiescent cells are scarce. We found that irradiated HSPCs, but not lineage-committed progenitors (CPs), undergo rapid ATM-dependent apoptosis, which is suppressed upon interaction with bone-marrow stroma cells. Using DNA repair reporters to quantify mutagenic Non-Homologous End Joining (NHEJ) processes, we found that HSPCs exhibit reduced NHEJ activities in comparison with CPs. HSPC-stroma interactions did not affect the NHEJ capacity of HSPCs, emphasizing its cell autonomous regulation. We noted diminished expression of multiple double strand break (DSB) repair transcripts along with more persistent 53BP1 foci in irradiated HSPCs in comparison with CPs, which can account for low NHEJ activity and its distinct control in HSPCs. Finally, we documented clonal chromosomal aberrations in 10% of IR-surviving HSPCs. Taken together, our results revealed potential mechanisms contributing to the inherent susceptibility of human HSPC to the cytotoxic and mutagenic effects of DNA damage.

Attenuated DNA damage responses and increased apoptosis characterize human hematopoietic stem cells exposed to irradiation

Failure to precisely repair DNA damage in self-renewing Hematopoietic Stem and early Progenitor Cells (HSPCs) can disrupt normal hematopoiesis and promote leukemogenesis. Although HSPCs are widely considered a target of ionizing radiation (IR)-induced hematopoietic injury, definitive data regarding cell death, DNA repair, and genomic stability in these rare quiescent cells are scarce. We found that irradiated HSPCs, but not lineage-committed progenitors (CPs), undergo rapid ATM-dependent apoptosis, which is suppressed upon interaction with bone-marrow stroma cells. Using DNA repair reporters to quantify mutagenic Non-Homologous End Joining (NHEJ) processes, we found that HSPCs exhibit reduced NHEJ activities in comparison with CPs. HSPC-stroma interactions did not affect the NHEJ capacity of HSPCs, emphasizing its cell autonomous regulation. We noted diminished expression of multiple double strand break (DSB) repair transcripts along with more persistent 53BP1 foci in irradiated HSPCs in comparison with CPs, which can account for low NHEJ activity and its distinct control in HSPCs. Finally, we documented clonal chromosomal aberrations in 10% of IR-surviving HSPCs. Taken together, our results revealed potential mechanisms contributing to the inherent susceptibility of human HSPC to the cytotoxic and mutagenic effects of DNA damage.

Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture

Background aimsSpontaneous mutagenesis often leads to appearance of genetic changes in cells. Although human multipotent mesenchymal stromal cells (hMSC) are considered as genetically stable, there is a risk of genomic and structural chromosome instability and, therefore, side effects of cell therapy associated with long-term effects. In this study, the karyotype, genetic variability and clone formation analyses have been carried out in the long-term culture MSC from human gingival mucosa.MethodsThe immunophenotype of MSC has been examined using flow cytofluorometry and short tandem repeat (STR) analysis has been carried out for authentication. The karyotype has been examined using GTG staining and mFISH, while the assessment of the aneuploidy 8 frequency has been performed using centromere specific chromosome FISH probes in interphase cells.ResultsThe immunophenotype and STR loci combination did not change during the process of cultivation. From passage 23 the proliferative activity of cultured MSCs was significantly reduced. From passage 12 of cultivation, clones of cells with stable chromosome aberrations have been identified and the biggest of these (12%) are tetrasomy of chromosome 8. The random genetic and structural chromosomal aberrations and the spontaneous level of chromosomal aberrations in the hMSC long-term cultures were also described.ConclusionsThe spectrum of spontaneous chromosomal aberrations in MSC long-term cultivation has been described. Clonal chromosomal aberrations have been identified. A clone of cells with tetrasomy 8 has been detected in passage 12 and has reached the maximum size by passage 18 before and decreased along with the reduction of proliferative activity of cell line by passage 26. At later passages, the MSC line exhibited a set of cells with structural variants of the karyotype with a preponderance of normal diploid cells. The results of our study strongly suggest a need for rigorous genetic analyses of the clone formation in cultured MSCs before use in medicine.

Clonal chromosomal aberrations in Philadelphia negative cells such as monosomy 7 and trisomy 8 may persist for years with no impact on the long term outcome in patients with chronic myeloid leukemia

The appearance of clonal chromosomal aberrations in Philadelphia negative cells (CCA/Ph-) during the treatment of chronic myeloid leukemia (CML) was recently confirmed. Importance of these findings has not been clearly defined. We present data on the time of appearance, persistence, size of the CCA/Ph- clone in terms of drugs used and hematological, cytogenetic and molecular response rates. The focus was on the peripheral blood cytopenias and myelodysplastic changes in the bone marrow microscopic evaluation. In 5 out of 155 (3,2%) CML patients, the persistent presence (up to nine years) of CCA/Ph- was found (monosomy 7 and trisomy 8 in unrelated clones in two patients treated with tyrosine kinase inhibitors; trisomy 8 in two patients on imatinib; trisomy 21 in one patient on interferon alfa treatment). Aberrations were present in median 24% Ph- cells in 3-15 subsequent analyses at different cytogenetic and molecular response time points. No evident myelodysplastic changes nor transformation to MDS/AML occurred in patients with CCA/Ph-. All the patients achieved major molecular response (MMR). It seems that CCA/Ph- presence does not affect the long term outcome in patients with chronic myeloid leukemia. Further complex monitoring of the CML patients with CCA/Ph- is still needed.

Prognosis of FLT3, NPM1, DNMT3A and IDH mutations in non-M3 acute myeloid leukemia

Acute myeloid leukemia (AML) is a group of heterogeneous malignant diseases in hematological system, with significant differences in morphology, immunology, genetics, molecular biology and clinical manifestations. Cytogenetics detection has become one of the main bases for the accurate diagnosis, treatment options and prognosis judgement of AML. However, the high heterogeneity at the molecular level leads to low detection rate by conventional cytogenetics detection technology. Clonal chromosome aberrations in 40 %-50 % of patients with AML could not be detected by using standard chromosomal banding, which might be called normal karyotype AML (NK-AML). The common molecular genetic changes in non-M3 AML includes FLT3, NPM1, DNMT3A and IDH mutations. This paper mainly reviews the characteristics of the above four genes, pathogenesis and prognosis of non-M3 AML. Key words: Leukemia, myeloid, acute; Gene, FLT3; Gene, NPM1; Gene, DNMT3A; Gene, IDH

Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.

Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc.

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.

Abstract 01: Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS)

Abstract Background: AML is a clinically and genetically heterogeneous clonal disorder. Approximately 50% are characterized by recurrent clonal chromosome aberrations which have contributed to the classification of disease and are recognized as important prognostic factors. AML patients who lack these recurrent structural abnormalities have been grouped as “intermediate cytogenetic risk” and are being further subcategorized by the sequence alterations that are being identified. A more complete understanding of the genetic changes that are relevant to the pathogenesis of AML will improve the classification of risk and ultimately better selection of therapy. Our hypothesis is that mutational profiling and analysis of patient outcomes will help better define the risk subgroups of patients and predict prognosis in patients with AML. Methods: Archived DNA from 37 patients with various AML diagnoses were obtained with IRB approval (IRB#201502763). A panel of 30 commonly mutated genes in AML were designed using the Ion Ampliseq Designer (Ion Torrent) and targeted for “hotspot” or complete coding sequence (CDS). Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) technology. Library clonal amplification was performed by emulsion PCR using Ion PGM™ Template OT2 Reagents Kit200. Simultaneous sequencing of clonally amplified DNA targets was performed by Ion-Torrent Personalized Genome Machine (IT-PGM) using 318v2 chip. Genomic variants were identified by variant caller3.4 and further filtered and analysed through a custom-made clinically validated pipeline. Novel mutations were confirmed by either single nucleotide primer extension or Sanger sequencing. Results: The assay successfully identified previously identified point mutations in FLT-3 and CEBPA genes. Additional variants were identified in 81% (30/37) of specimens; no mutations were found in 19% (7/37) specimens. The genes most frequently mutated were DNMT3A (23%), TET2 (17%), IDH2 (14%) and TP53 (14%). U2AF1 (11%) and RUNX1 (8%) were exclusively mutated in specimens with non-recurrent cytogenetic abnormalities. The identified variants will be used to risk stratify patients based on clinical outcome. Conclusion: This targeted multi-gene panel successfully identified several variants along with previously known mutations in archived specimens. Citation Format: Anup Tilak, Ramakrishna Sompallae, Aaron Stence, Natalya Guseva, Aaron Bossler. Development of a targeted panel for acute myeloid leukemia (AML) by using next-generation sequencing (NGS). [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 01.

MTOR inhibitor temsirolimus and MEK1/2 inhibitor U0126 promote chromosomal instability and cell type-dependent phenotype changes of glioblastoma cells

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and the RAF/mitogen-activated and extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are frequently deregulated in cancer. Temsirolimus (TEM) and its primary active metabolite rapamycin allosterically block mTOR complex 1 substrate recruitment. The context-/experimental setup-dependent opposite effects of rapamycin on the multiple centrosome formation, aneuploidy, DNA damage/repair, proliferation, and invasion were reported. Similarly, the context-dependent either tumor-promoting or suppressing effects of RAF–MEK-ERK pathway and its inhibitors were demonstrated. Drug treatment-mediated stress may promote chromosomal instability (CIN), accelerating changes in the genomic landscape and phenotype diversity. Here, we characterized the genomic and phenotypic changes of U251 and T98G glioblastoma cell lines long-term treated with TEM or U0126, an inhibitor of MEK1/2. TEM significantly increased clonal and non-clonal chromosome aberrations. Both TEM and U0126 affected copy number alterations (CNAs) pattern. A proliferation rate of U251TEM and U251U0126 cells was lower and higher, respectively, than control cells. Colony formation efficiency of U251TEM significantly decreased, whereas U251U0126 did not change. U251TEM and U251U0126 cells decreased migration. In contrast, T98GTEM and T98GU0126 cells did not change proliferation, colony formation efficiency, and migration. Changes in the sensitivity of inhibitor-treated cells to the reduction of the glucose concentration were observed. Our results suggest that CIN and adaptive reprogramming of signal transduction pathways may be responsible for the cell type-dependent phenotype changes of long-term TEM- or U0126-treated tumor cells.

Importance of FISH combined with Morphology, Immunophenotype and Cytogenetic Analysis of Childhood/ Adult Acute Lymphoblastic Leukemia in Omani Patients.

Genetic changes associated with acute lymphoblastic leukemia (ALL) provide very important diagnostic and prognostic information with a direct impact on patient management. Detection of chromosome abnormalities by conventional cytogenetics combined with fluorescence in situ hybridization (FISH) play a very significant role in assessing risk stratification. Identification of specific chromosome abnormalities has led to the recognition of genetic subgroups based on reciprocal translocations, deletions and modal number in B or T-cell ALL. In the last twelve years 102 newly diagnosed childhood/adult ALL bone marrow samples were analysed for chromosomal abnormalities with conventional G-banding, and FISH (selected cases) using specific probes in our hospital. G-banded karyotype analysis found clonal numerical and/or structural chromosomal aberrations in 74.2% of cases. Patients with pseudodiploidy represented the most frequent group (38.7%) followed by high hyperdiploidy group (12.9%), low hyperdiploidy group (9.7%), hypodiploidy (<46) group (9.7%) and high hypertriploidy group (3.2%). The highest observed numerical chromosomal alteration was high hyperdiploidy (12.9%) with abnormal karyotypes while abnormal 12p (7.5%) was the highest observed structural abnormality followed by t(12;21)(p13.3;q22) resulting in ETV6/RUNX1 fusion (5.4%) and t(9;22)(q34.1;q11.2) resulting in BCR/ABL1 fusion (4.3%). Interestingly, we identified 16 cases with rare and complex structural aberrations. Application of the FISH technique produced major improvements in the sensitivity and accuracy of cytogenetic analysis with ALL patients. In conclusion it confirmed heterogeneity of ALL by identifying various recurrent chromosomal aberrations along with non-specific rearrangements and their association with specific immunophenotypes. This study pool is representative of paediatric/adult ALL patients in Oman.