Recurrent Genetic Abnormalities Research Articles

Cladribine, Cytarabine and Mitoxantrone As Treatment Intensification for Patients with Acute Myeloid Leukemia with the Excess of Bone Marrow Blasts on Day 14 of the First Induction. Prospective, Multicenter Study By the Polish Adult Leukemia Group (PALG)

ObjectivesThe treatment results of acute myeloid leukemia (AML) patients (pts) younger than 60yo are still unsatisfactory. The strategy of double induction in AMLis based on two courses of chemotherapy depends on percentage of bone marrow (BM) blast at 7-14 days of treatment. This therapy intensification may induce higher response rate but toxicity appeared as early death (ED) rates still a medical dilemma. The advantage from addition of cladribine (2CdA) to standard induction and reinduction treatment was previously confirmed in PALG's trials.AimThe aim of this study was an estimation of efficacy and toxicity of treatment intensification based on BM blast at 14 day (D14) by second early induction, CLAM (2CdA + cytarabine + mitoxantron)MethodsWe performed multicenter, prospective, non-randomized trial where pts received DAC induction treatment with subsequent second early induction according as D14 BM biopsy results. When BM examination revealed >10% of blast without hypocellular marrowand pts met all criteria to intensive chemotherapy, second intensive induction with CLAM protocol (2CdA 5mg/m2 iv days 1-5, Cytarabine 2g/m2 iv days 1-5, Mitoxantron 10mg/m2 iv days 1-3) from day 16 was applied. The pts were allocated to risk-adjusted postremission treatment with high-dose cytarabine (HD-AraC) for low-risk group according to European Leukemia Net (ELN) classification and allogeneic stem cell transplantation (allo-SCT) recommended for intermediate and high-risk group.ResultsOne hundred seventy one newly diagnosed AML pts from 8 PALG's centers were enrolled to the study. The median age was 47 yo (range19-60). According to WHO 2008 classification, our group consisted of 9% AML with recurrent genetic abnormalities, 19% AML related to myelodysplasia, 3% therapy-related AML and 69% AML not otherwise classified. Analysis of cytogenetic-molecular risk factors based on ELN classification revealed favorable, intermediate-1, intermediate-2, poor-risk groups in 17%, 44%, 18% and 21% AML pts, respectively. Twenty-six (15%) out of 171 pts had elevated BM infiltration of blasts at D14, with the median 44% (range 11-100%). Overall, 22 of 26 pts were judged as eligible to receive second induction, reasons for exclusion being in all cases active infection.Response evaluation was available in 150 pts, 21 pts were still ongoing in the protocol. Complete remission (CR) rate after single DAC induction course was 71% (n=106), partial remission (PR), non-remission (NR) and ED was 8% (n=12), 18% (n=28) and 3% (n=4), respectively. Response after double induction was evaluated in 21 out of 22 cases; one patient still has not reached time of measurement. CR was observed in 14/21 pts (67%), NR in 5% (n=1), ED 28% (n=6). The CR rate after inductiontreatment applied to first hospitalization (DAC or DAC+ CLAM) was 81% (n=120). Twelve pts who had <10% of blasts at day 14 and achieved PR after first induction were allocated to second DAC induction. Six out of them achieved CR leading to the overall CR rate 85%.In 27 pts who achieved CR allo-SCT was performed till June 2016, however there are pts who are still awaiting allo-SCT.The median duration of hospital stay was 31 (11-73) and 66 (52-76) days respectively for the DAC and the DAC + CLAM induction. During the single DAC induction the median number of days with neutropenia <0.5 G/l was 24 (12-101), and with thrombocytopenia <20 G/l was 12 (0-51). The median number of transfused packed red blood cell (PRBC) was 9 (0-32) and the number of platelet concentrate units was 15 (2-124). During DAC+CLAM double induction the median number of days of the neutropenia <0.5 G/l was 53 (17-73) and thrombocytopenia <20 G/l was 19 (4-57). The median number of PRBC was 18 (10-44) and the number of platelet concentrate units was 30 (5-153).With the median time of follow-up 16 months the median overall survival (OS) was not reached. The probability of 1-year OS was 59% and the probability of 1-year disease free survival was 83% (Figure 1, Panel A and B).ConclusionThe preliminary results of our study suggest that early second induction CLAM is a feasible and effective treatment option in younger AML patients which further improves the CR rate and do not compromise the feasibility of allo-SCT. [Display omitted] DisclosuresGrzybowska-Izydorczyk:BMS: Honoraria; Novartis: Honoraria. Warzocha:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Robak:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Wierzbowska:Novartis: Consultancy, Honoraria; Janssen Cilag: Consultancy.

Expression of Blood Coagulation Factor Xiiia in Lymphoblasts Predicts Favorable Outcome in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction: Previously we identified B-cell lineage leukemic lymphoblasts as a new expression site for subunit A of blood coagulation factor XIII (FXIIIA)1. On the basis of FXIIIA expression, various subgroups of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be identified. Methods. Fifty-five children with BCP-ALL treated within the frame of BFM ALL-IC 2002 clinical trial were studied retrospectively. Bone marrow samples were obtained by aspiration and the expression of FXIIIA was detected by flow cytometry. G-banding and fluorescent in situ hybridization was performed according to standard procedures. Results. The 10-year event-free survival (EFS) and overall survival (OS) rate of FXIIIA-positive and FXIIIA-negative patients showed significant differences (EFS: 84% vs. 61%, respectively; p=0.031; OS: 89% vs. 61%; p=0.008). Of all the parameters examined, the only correlation was between the lack of FXIIIA expression and ’B-other’ genetic subgroup. Further multivariate Cox regression analysis of FXIII-subtype and genetic group or ’B-other’ subgroup identified FXIIIA-negative characteristics as an independent predictor for poor outcome in BCP-ALL. Conclusion. We found an excellent correlation between long-term survival and FXIIIA-positive phenotype of lymphoblasts in de novo childhood BCP-ALL. The results presented seem to be convincing enough to suggest a possible role for FXIIIA expression in the prognostic grouping of childhood BCP-ALL patients. In addition, lack of FXIIIA expression is associated with the ’B-other’ characteristics, therefore, FXIIIA can help to identify those cases that may require further detailed genetic examination by using expensive methods. Acknowledgment. The authors thank Dr. Erzsebet Balogh for performing the cytogenetic analyses and Csaba Antal for his administrative help. The authors are grateful to Dr. Kalman Nagy and their coworkers at the Borsod-Abaúj-Zemplén County Hospital and University Hospital for sending bone marrow samples for flow cytometry. This study was supported by grant OTKA K-108885 (CK). Authors declare no conflict of interest. References. 1. Kiss F, Hevessy Z, Veszpremi A, Katona E, Kiss C, Vereb G, Muszbek L, Kappelmayer JN. Leukemic lymphoblasts, a novel expression site of coagulation factor XIII subunit A. Thromb Haemost. 2006; 96: 176-82. Legend to figures. Figure 1. Prognostic value of FXIII-A expression of lymphoblasts in children with B-cell precursor ALLKaplan Meier plots of event-free (A) and overall survival (B) showed significant difference between the FXIIIA-positive and FXIII-A-negative groups (p=0.031 and p=0.008). Figure 2. Relationship between FXIIIA expression profile and genetic classification The distribution of patients in the various genetic groups differed significantly in terms of FXIII-A profile using Chi square test. Recurrent genetic abnormalities: BCR-ABL1, KMT2A (MLL) gene rearrangements, ETV6-RUNX1 (TEL-AML1), E2A/PBX1 and high hyperdiploidy. DisclosuresNo relevant conflicts of interest to declare.

HnRNP K Overexpression Drives AML Progression By Altering Pathways Critical for Myeloid Proliferation and Differentiation

Acute myeloid leukemia (AML) is a disease largely defined by recurrent genetic and chromosomal abnormalities. As such, The Cancer Genome Atlas' recent detailed examination of the most common genetic abnormalities that drive AML uncovered relatively few novel alterations. However, within this list of recurrently mutated genes was heterogeneous nuclear ribonucleoprotein K (HNRNPK). While previously unknown to impact AML, we recently identified HNRNPK as a haploinsufficient tumor suppressor at the 9q21.32 locus. In contrast to its tumor suppressive roles, hnRNP K is most customarily overexpressed, rather than underexpressed, in several solid tumors.In this study, we show through FISH analyses, RNA expression profiling, and reverse phase protein array analyses that the HNRNPK gene is amplified with corresponding mRNA and protein overexpression in a sizeable cohort of patients with AML that do not carry a 9q.21.32 deletion. Together, this indicates that hnRNP K may be an uncharacterized oncogene in this context. Increased hnRNP K correlated with decreased event-free and overall survival, increased WBC, and increased bone marrow and peripheral blood blast percentage. High hnRNP K levels also correlated with elevated expression of histone modifiers (e.g., ASH2L and TRIM24), regulators of ribogenesis (e.g., nucleolin and EBP1), and potent oncogenes, such as c-Myc. Together, these observations indicate that increased hnRNP K expression significantly alters the landscape of myeloid biology and may thus contribute to the etiology of AML.To directly interrogate this possibility in vivo, we generated a Vav-Cre;HnrnpkTg mouse model. In addition to significant differences in hnRNP K expression compared to Vav-Cre mice, Vav-Cre;HnrnpkTg mice exhibited reduced survival, development of transplantable myeloid malignancies, and enhanced HSPC proliferation and differentiation potential. Reflecting data from our patients, Vav-Cre;HnrnpkTg mice also exhibited elevated expression of critical oncogenic pathways such as c-Myc.Currently, we are examining pharmacologic methods to abrogate the effects of aberrant hnRNP K expression on various activated pathways. Transplant models using diseased bone marrow from Vav-Cre;HnrnpkTg mice are being utilized to evaluate the efficacy of specific pathway inhibitors (i.e., bromodomain inhibitors or bortezomib) in prolonging survival and decreasing leukemic burden. These studies will provide the rationale for potential clinical trails that employ these agents for patients with hnRNP K-overexpressing AML. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Stromal Cells May Have Genetic Aberrations and Are Capable to Gain Them in a Culture

Introduction. Stromal microenvironment posses a key role in the regulation of both normal hematopoiesis and its reconstitution after hematopoietic stem cell transplantation (HSCT). Recent data supports the idea that bone marrow stromal cells (BMSC) also have genetic aberrations and may tightly involved in the pathogenesis of HSCT complications. These findings justify the need for more detailed study of genetic aberrations in BMSC.The aim of this study was to evaluate genetic aberrations inBMSC and check the ability to gain them in coculture system.Materials and methods. The interaction of BMSC with hematopoietic tumor cell lines bearing specific genetic aberrations (BCR-ABL fusion transcript for K-562 and JAK2 V617F mutation for Uke-1 cell line) was investigated in stromal cells harvested from 17 patients and 8 healthy donors. We performed cultivation of BMSC monolayer and tumor cells suspension using semipermeable membrane plates inserts with different pore size (0,4 μm and 3,0 μm) in order to exclude direct cell-to-cell contact. We looked also forexisting specific genetic aberrations (point mutations and fusion transcripts) in BMSC of patients with the respective aberration in their leukemic clone. For this purpose we used both karyotyping (7 patients) and RQ-PCR method. BMSC were examined by flow cytometry to evaluate the possible contamination with cells of hematopoietic lineage.Results.We investigated the BMSC karyotype in seven patients and only one case led us to a remarkable finding. The clonal chromosomal rearrangement t(1;7) was detected in 25% of BMSC metaphases. Interestingly, this aberration was never detected in patient's leukemic cells. Moreover, at the moment of this investigation patient had full clinical and hematological remission, full donor chimerism and no signs of minimal residual disease (MRD).We also examined BMSC from leukemia patients bearing recurrent genetic abnormalities and in one case the leukemia-specific marker was detected by RQ-PCR - we observed expression of ETV6-RUNX1 gene (≈0,02%) in BMSC by patient with t(12;21) acute lymphoblastic leukemia. At the moment of BMSC culture initiation ETV6-RUNX1 expression in patient’s bone marrow was detected at high level (ETV6-RUNX1/ABL*100=321%). Before carrying out RNA extraction BMSC were harvested after the second passage and no contamination with CD45+/CD34+ cells by flow cytometry was observed (50,000 events collected from the sample). When BMSCs and Uke-1 cell line were cocultured by using of 3,0 μm pore culture dishes inserts, the BMSC population gained the Jak2V617F mutation (allele burden ≈ 30,39%). We reproduced similar experiments with the K-562 cell line and got similar results - CD45+ cells were also detected in BMSC population (≈ 30%). Moreover we detected CD45+ non-cellular particles by flow cytometry analysis. Implying K-562 cells are not likely to cross the semipermeable membrane (3,0 μm pores versus 20,0 μm cells as measured during microscopy). Besides BCR-ABL gene expression in BMSC was detected by RQ-PCR (BCR-ABL/ABL*100=19%). We repeated same test with 0,4 μm pore inserts and without them in order to check implication of cell-to-cell interaction. We didn’t obtain any similar results with smaller pores, but the fusion transcript was detected in CD45- BMSC population when these two cell populations weren’t devided. Both findings point out at possible horizontal gene transfer mediated by membrane vesicles larger than 0,4 μm and direct whole cell fusion.ConclusionOur data stands for the existence of horizontal gene transfer between leukemic clone and BMSC. This process seems to be mediated by membrane vesicles larger than 0,4 μm in size, though cell fusion can also take place. We also confirmed the fact BMSCs can bear clonal genetic rearrangements which are not specific to tumor cell populations. These findings show tight interaction between tumor and microenvironment cells and can partly explain nature of PCR-based MRD persistence in complete remission. DisclosuresNo relevant conflicts of interest to declare.

Genetic Abnormalities and Prognosis in Pediatric B-Precursor Acute Lymphoblastic Leukemia without Conventional Genetic Abnormalities; Tokyo Children's Cancer Study Group

Introduction; Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease that can be subdivided according to primary recurrent genetic abnormalities that are strongly associated with characteristic biological and clinical features. In approximately two-thirds of pediatric patients with BCP-ALL, well-characterized genetic abnormalities can be detected by standard genetic analyses. In the remaining BCP-ALL patients, major pathogenic or driver cytogenetic abnormalities have yet to be clarified and they are called B-others. Recent studies using advanced analytical approaches have stratified a variety of subgroups harboring novel genetic abnormalities. For example, Ph-like ALL, iAMP21, deletion of ERG and rearrangements of ZNF384 and MEF2D have been identified in B-others. However, a certain portion of B-others still remains as genetically unclassified.Patients and Methods; 291 children with de novo BCP-ALL were enrolled on Tokyo Children Cancer Study Group (TCCSG) ALL L0416 and L0616 trials (between 2004-2007). Conventional genetic abnormalities, including BCR-ABL1, MLL-AF4, MLL-AF9, TCF3-PBX1, ETV6-RUNX1, hyperdiploidy and hypodiploidy were detected in 16 (5.5%), 3 (1.0%), 2 (0.7%), 18 (6.2%), 54 (18.5%), 65 (22.3%) and 3 (1.0%) cases by G-banding, fluorescent in situ hybridization and molecular analyses. 131 cases (44.9%) were classified B-others. We studied 121 (93.1%) out of 131 B-others ALL of the L0416/L0616 cohort. We performed whole transcriptome sequencing (WTS) of 35 pediatric B-others cases. For the confirmation and screening of the fusion transcripts we performed RT-PCR in a total of 121 B-others cases. Alterations of ERG and iAMP21 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) kit P327. We also analyzed the known genetic mutations or copy number alterations associated with these patients using the data from WTS and MLPA kit P335 and P200.Results; A total of 47 candidate in-frame gene rearrangements were identified in 29/35 (82.8%) B-others samples. The fusion genes including ZNF384 (n=12, 4.1%) or MEF2D (n=6, 2.1%) were frequently found in this cohort. All of patients with ZNF384-related fusion genes were found to have a weak or negative expression of CD10. In addition, 7 and 17 patients out of 22 patients with ZNF384-related fusion genes exhibited more than 20% expression of CD13 and CD33. We identified 6 recurrently mutated genes, including NRAS, KRAS, PTPN11, EZH2, MLL2 and ASH1L. Especially, activating mutations in RAS signaling pathway genes, including NRAS, KRAS and PTPN11 were detected in 4 out of 10 ZNF384-translocated patients. Mutations in B-cell developmental genes such as PAX5, BTG1 and IKZF1 were not detected at all in DNA samples with ZNF384-related fusion genes. We identified a total of 6 (1.8%) patients with the fusion genes related with Ph-like ALL, including IGHM-CRLF2, P2RY8-CRLF2, IGH@-EPOR and PAX5-JAK2. Three of 6 patients with Ph-like ALL related fusion gene relapsed and two patients died. We identified iAMP21 in 6 (2.1%) patients and deletion of ERG in 7 (2.4%). One of 6 patients with iAMP21 and 3 of 8 patients with ERG deletion had IKZF1 deletion. No mutations of CRLF2 and JAK2 were identified in patients with iAMP21 and ERG deletion. Among 6 patients with iAMP21, three patients relapsed, and two patients died. No patients with ERG deletion relapsed or died.Conclusions; WTS analysis unmasked a complexity of gene rearrangements in pediatric BCP-ALL without conventional genetic abnormalities. In this study, 10.6% of genetic alterations were revealed in Japanese pediatric B-others cohort. Apart from increasing our understanding of the pathogenesis of pediatric BCP-ALL, this may help improve risk stratification and eventually increase the therapeutic option. DisclosuresNo relevant conflicts of interest to declare.

Langerhans cell histiocytosis is a neoplasm and consequently its recurrence is a relapse: In memory of Bob Arceci.

Langerhans cell histiocytosis (LCH) remains a poorly understood disorder with heterogeneous clinical presentations characterized by focal or disseminated lesions that contain excessive CD1a+ langerin+ cells with dendritic cell features known as "LCH cells." Two of the major questions investigated over the past century have been (i) the origin of LCH cells and (ii) whether LCH is primarily an immune dysregulatory disorder or a neoplasm. Current opinion is that LCH cells are likely to arise from hematopoietic precursor cells, although the stage of derailment and site of transformation remain unclear and may vary in patients with different extent of disease. Over the years, evidence has provided the view that LCH is a neoplasm. The demonstration of clonality of LCH cells, insufficient evidence alone for neoplasia, is now bolstered by finding driver somatic mutations in BRAF in up to 55% of patients with LCH, and activation of the RAS-RAF-MEK-ERK (where MEK and ERK are mitogen-activated protein kinase and extracellular signal-regulated kinase, respectively) pathway in nearly 100% of patients with LCH. Herein, we review the evidence that recurrent genetic abnormalities characterized by activating oncogenic mutations should satisfy prerequisites for LCH to be called a neoplasm. As a consequence, recurrent episodes of LCH should be considered relapsed disease rather than disease reactivation. Mapping the complete genetic landscape of this intriguing disease will provide additional support for the conclusion that LCH is a neoplasm and is likely to provide more potential opportunities for molecularly targeted therapies.

RUNX1 mutations in acute myeloid leukemia are associated with distinct clinico-pathologic and genetic features.

We evaluated the frequency, genetic architecture, clinico-pathologic features and prognostic impact of RUNX1 mutations in 2439 adult patients with newly-diagnosed acute myeloid leukemia (AML). RUNX1 mutations were found in 245 of 2439 (10%) patients; were almost mutually exclusive of AML with recurrent genetic abnormalities; and they co-occurred with a complex pattern of gene mutations, frequently involving mutations in epigenetic modifiers (ASXL1, IDH2, KMT2A, EZH2), components of the spliceosome complex (SRSF2, SF3B1) and STAG2, PHF6, BCOR. RUNX1 mutations were associated with older age (16-59 years: 8.5%; ⩾60 years: 15.1%), male gender, more immature morphology and secondary AML evolving from myelodysplastic syndrome. In univariable analyses, RUNX1 mutations were associated with inferior event-free (EFS, P<0.0001), relapse-free (RFS, P=0.0007) and overall survival (OS, P<0.0001) in all patients, remaining significant when age was considered. In multivariable analysis, RUNX1 mutations predicted for inferior EFS (P=0.01). The effect of co-mutation varied by partner gene, where patients with the secondary genotypes RUNX1mut/ASXL1mut (OS, P=0.004), RUNX1mut/SRSF2mut (OS, P=0.007) and RUNX1mut/PHF6mut (OS, P=0.03) did significantly worse, whereas patients with the genotype RUNX1mut/IDH2mut (OS, P=0.04) had a better outcome. In conclusion, RUNX1-mutated AML is associated with a complex mutation cluster and is correlated with distinct clinico-pathologic features and inferior prognosis.

Characterization of pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia with kinase fusions in Japan.

Recent studies revealed that a substantial proportion of patients with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor fusions involving tyrosine kinase and cytokine receptors, such as ABL1, PDGFRB, JAK2 and CRLF2, which are targeted by tyrosine kinase inhibitors (TKIs). In the present study, transcriptome analysis or multiplex reverse transcriptase–PCR analysis of 373 BCP-ALL patients without recurrent genetic abnormalities identified 29 patients with kinase fusions. Clinically, male predominance (male/female: 22/7), older age at onset (mean age at onset: 8.8 years) and a high white blood cell count at diagnosis (mean: 94 200/μl) reflected the predominance of National Cancer Institute high-risk (NCI-HR) patients (NCI-standard risk/HR: 8/21). Genetic analysis identified three patients with ABL1 rearrangements, eight with PDGFRB rearrangements, two with JAK2 rearrangements, three with IgH-EPOR and one with NCOR1-LYN. Of the 14 patients with CRLF2 rearrangements, two harbored IgH-EPOR and PDGFRB rearrangements. IKZF1 deletion was present in 16 of the 22 patients. The 5-year event-free and overall survival rates were 48.6±9.7% and 73.5±8.6%, respectively. The outcome was not satisfactory without sophisticated minimal residual disease-based stratification. Furthermore, the efficacy of TKIs combined with conventional chemotherapy without allogeneic hematopoietic stem cell transplantation in this cohort should be determined.

Molecular genetics of childhood, adolescent and young adult non-Hodgkin lymphoma.

Molecular genetic abnormalities are ubiquitous in non-Hodgkin lymphoma (NHL), but genetic changes are not yet used to define specific lymphoma subtypes. Certain recurrent molecular genetic abnormalities in NHL underlie molecular pathogenesis and/or are associated with prognosis or represent potential therapeutic targets. Most molecular genetic studies of B- and T-NHL have been performed on adult patient samples, and the relevance of many of these findings for childhood, adolescent and young adult NHL remains to be demonstrated. In this review, we focus on NHL subtypes that are most common in young patients and emphasize features actually studied in younger NHL patients. This approach highlights what is known about NHL genetics in young patients but also points to gaps that remain, which will require cooperative efforts to collect and share biological specimens for genomic and genetic analyses in order to help predict outcomes and guide therapy in the future.

A Series of 116 Therapy-Related Myeloid Neoplasms (t-MN) Patients from 2 French Comprehensive Cancer Centers

Background: Cancer patients (pts) who receive chemotherapy and/or radiation therapy for their primary tumor are at risk for developing therapy-related myeloid neoplasms (t-MN). Estimates of t-MN prevalence are mainly derived from retrospective series from hematology departments: they account for 10 to 20% of all AML cases. However this condition is frequently diagnosed in daily practice in comprehensive cancer centers (CCC). This suspected higher frequency might be due to optimized follow-up protocols and improved survival outcomes after tumor remission. The aim of this study was to assess clinical factors associated with t-MN in 2 French CCC focusing on delay of onset, survival, cytogenetically defined prognosis, treatment received for primary tumor and myeloid disease presentation.Methods: We conducted a retrospective study of cancer pts who developed t-MN seen at Institut Gustave Roussy, Villejuif, France and Institut Curie, Saint-Cloud, France between 2004 and November 2014. As defined in the current 2008 WHO classification, t-MN includes therapy-related acute myeloid leukemia (t-AML), myelodysplastic syndrome (t-MDS) and myelodysplastic/myeloproliferative neoplasms (t-MDS/MPN). The study inclusion criteria were development of t-MN after chemotherapy and/or radiation therapy for any primary tumor.Results: 116 patients were retrieved from our records. 86 patients were women (74%) and the median age at t-MN diagnosis was 65 years (range, 25-91 years). The distribution of primary malignancies by site was as follows: breast cancer 46 pts, hematologic malignancy (lymphoma and myeloma) 24 pts, gynecologic cancer 14 pts, head and neck cancer 8 pts, urologic cancer 6 pts, gastrointestinal cancer 4 pts, miscellaneous (sarcoma, neurooncology, neuroendocrinology cancer) 14 pts. The median interval between primary cancer and t-MN was 60 months (range, 12-324). At the time of t-MN diagnosis, 69 (59%) had t-AML, 46 (40%) had t-MDS and 1 (1%) had t-MDS/MPN. Of note, among t-AML pts 15 (13%) had AML with recurrent genetic abnormalities and 14 had AML (12%) with MDS-related changes. Median survival from the diagnosis of t-MN was 6.5 months (5y OS was below 20%). Adverse risk karyotypes were prominent (63 pts, 59%) followed by intermediate risk (32 pts, 30%). Of note 12 pts (10%) had favorable risk karyotype. The impact of karyotype on survival was crucial: median survival was 60 months for favorable risk pts, 14 months for intermediate risk pts and only 6 months for adverse risk karyotypes. 26 pts (22%) were treated by radiation therapy alone. Treatment modality (radiation therapy alone, chemotherapy alone or combined treatment) did not affect survival or delay of onset. Of note, when we isolated pts treated by alkylating agents without topoisomerase inhibitors from those treated by topoisomerase inhibitors without alkylating agents we found that the disease presentation did not differ (same proportion of t-AML vs. t-MDS in each group), an observation in contrast with the classical description of post-alkylating agent t-MDS vs. post- topoisomerase inhibitor t-AML.Conclusion: We report a heterogeneous cohort of 116 pts with numerous primary tumors and treatment protocols. Prominent adverse risk karyotypes are in line with a dismal survival yet favorable risk karyotypes are not uncommon and remain associated with a relatively good prognosis. DisclosuresNo relevant conflicts of interest to declare.

Recurrent Genetic Abnormalities Are Reflected in Phenotype at Diagnosis in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group Protocol AAML0531

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with high genomic and phenotypic complexity. Although a relationship between phenotype and t(15;17) is recognized, the effect of other genotypic abnormalities on phenotype remains to be elucidated.Objective: To explore the relationships between genotypic and phenotypic abnormalities in diagnostic specimens in pediatric AML.Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 769 satisfied three criteria for this study: (1) submission of a blood or bone marrow sample for multidimensional flow cytometry (MDF) at diagnosis, (2) consent to blood bank specimen testing and (3) leukemia comprising >10% of non-erythroid cells by MDF. The diagnostic AML tumor population was identified by gating on CD45 vs log-SSC. A fifteen-dimensional immunophenotypic expression profile (IEP) was defined by computing mean fluorescent intensities, antigen intensity coefficient of variations, and light scatter characteristics of each leukemia without analyst imposed cut-offs.Unsupervised hierarchical clustering was performed to mathematically cluster patients with similar IEPs. The underlying karyotypic and mutational profile of each patient was compared to the IEPs to identify clusters of patients with relationships between phenotype and identifiable genetic abnormalities. Supervised bagged tree-based models were used to quantify the importance of each cell-surface antigen in identifying these clusters. Levene's test was used to assess the homogeneity of mean cell-surface antigen expression for important markers.Results: Analysis of hierarchical clustering results revealed 7 immunophenotypic clusters (A-G) of patients with relationships between IEP and 4 common genetic abnormalities (Figure 1).Cluster A and Cluster B were both strongly associated with the t(8;21) karyotype (Figure 1). 54/62 patients in Cluster A (87%) and 24/26 patients in Cluster B (92%) were positive for t(8;21). The most important cell-surface antigens in identifying patients with the Cluster A associated phenotype were dim CD33, expression of CD56, expression of CD34 and expression of HLA-DR. The mean antigen expression of these markers was significantly more homogenous for patients in Cluster A compared to all other patients for CD33 (p = 0.02), CD34 (p < 0.01) and HLA-DR (p<0.01). Mean expression of CD56 was notably more heterogeneous (p = 0.40) but positive in 91% of patients in this cluster. The most important cell-surface antigens in identifying the Cluster B associated phenotype were dim CD13, expression of CD34, expression of HLA-DR and dim CD33. The mean expression of these markers was significantly more homogeneous for patients in Cluster B compared to all other patients (all p values <0.01). In contrast to Cluster A, CD56 MFIs were negative for patients in Cluster B. Collectively, 72% of all patients with t(8;21) segregated into Cluster A (50%) or Cluster B (22%).Cluster C was strongly associated with chromosome 16 abnormalities (Figure 1). Of patients in this cluster, 50/55 (91%) were positive for inv(16)/t(16;16). The mean high-expression of CD13 was the most important antigen in identifying patients with this phenotype, and patients in Cluster C had more consistent mean CD13 expression compared to all other patients (p<0.01). 53% of all patients with chromosome 16 abnormalities comprised this cluster.Cluster D was strongly associated with 11q23 abnormalities (Figure 1). Of patients in this cluster, 105/135 (78%) were positive for 11q23 translocations. The most discriminating markers in identifying these patients were dim-to-negative CD34 and dim-to-negative CD117. 69% of all patients with 11q23 abnormalities comprised this cluster.Clusters E, F and G were associated with FLT3 ITD mutations (Figure 1). Of patients in these clusters, 44%, 49% and 53% were positive for FLT3 ITD mutations. Cumulatively, 63% of all patients with FLT3 ITD mutations were grouped into one of these three clusters.Conclusion: Hierarchical clustering algorithms identify IEPs associated with common genetic abnormalities in pediatric AML, providing insight into the consistent dysregulation of cell-surface gene product expression in patients with common genetic abnormalities. Further analysis may reveal that IEPs can identify patients otherwise classified as good or standard risk that will have a poor outcome. [Display omitted] DisclosuresVoigt:Hematologics Inc.: Employment. Brodersen:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Equity Ownership.

Frequency, Characteristics and Prognostic Significance of RUNX1 mutations in Patients with Acute Myeloid Leukemia, Not Otherwise Specified

Background: Somatic mutations in RUNX1 gene have been identified in a substantial proportion of patients with de novo acute myeloid leukemia (AML). It is suggested as a new candidate molecular marker and, therefore, is suggested to be routinely performed at the diagnostic stage of AML. Despite its clinical importance, however, previous cohorts have been heterogeneous in terms of cytogenetic and molecular subtypes of AML. Here, the aim of this study was to evaluate the frequency, biologic characteristics, and prognostic significance of RUNX1 mutations focusing on patients with AML, not otherwise specified (NOS).Methods: Diagnostic samples from 202 patients with AML were analyzed for RUNX1 mutations. We excluded AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, and therapy-related AML because these entities have prognostic relevances of their own. RUNX1 mutations were detected using standard PCR techniques and direct sequencing.Results:RUNX1 mutations were found in 27 (13.4%) patients. The mutations were clustered in Runt homology domain (13, 48.1%) and transactivation domain (9, 33.3%). Frameshift mutations were most common (52.9%), followed by missense mutations (35.3%) and nonsense mutations (11.8%). As shown in Table 1, patients with RUNX1 mutations had a lower platelet count (P = 0.03), a higher rate of trisomy 8 (P = 0.02) and trisomy 13 (P = 0.039), and a trend toward older age (P = 0.063) than patients without mutations. Presence of RUNX1 mutations and NPM1 or CEBPA mutations were mutually exclusive. At the median follow-up of 12.1 months, RUNX1 mutations predicted for shorter overall survival (OS; P = 0.007) and relapse-free survival (RFS; P = 0.003). In the multivariate analysis, RUNX1 mutation was a significant marker for inferior OS (hazard ratio, 3.037; P = 0.014) and RFS (hazard ratio, 5.699; P = 0.001).Conclusion: The findings of our study further strengthen the previous data about RUNX1 mutations in AML. Furthermore, AML NOS with RUNX1 mutations is characterized by distinct biology and is associated with adverse clinical outcome. Our study supports the notion that RUNX1 mutational status would be integrated into diagnostic workup of AML, particularly for AML, NOS subgroup.Table 1Clinical and biologic features of the cohort by RUNX1 mutationsRUNX1 mutationsP -valueMutated, n (%)Wild type, n (%)Number27 (13.4)175 (86.6)Male sex17 (63.0)94 (53.7)0.489Median age, years (range)63 (14 - 80)55 (1 - 83)0.063WBC count, ¡¿109/L (median, range)7.9 (1.1 - 133.3)14.0 (0.8 - 231.3)0.636Hemoglobin, g/dL (median, range)8.6 (5.0 - 10.6)8.8 (4.1 - 17.3)0.376Platelet count, ¡¿109/L (median, range)35 (14 - 230)59 (9 - 900)0.03Blood blasts, % (median, range)29 (0 - 94)38.5 (0 - 93)0.312FAB subtypesM03 (11.1)11 (6.3)0.609M1, M221 (77.8)129 (73.7)0.831M4, M53 (11.1)27 (15.4)0.767M6, M708 (4.6)0.546Cytogenetic abnormalitiesNormal karyotype (%)11 (40.7)104 (59.4)0.106Trisomy 8 (%)5 (18.5)8 (4.6)0.02Trisomy 11 (%)1 (3.7)4 (2.3)0.823Trisomy 13 (%)3 (11.1)3 (1.7)0.039Trisomy 21 (%)1 (3.7)2 (1.1)0.866Distribution of other mutationsFLT3 -ITD5 (18.5)50 (28.6)0.39FLT3 -TKD1 (3.7)4 (2.3)0.823NPM1055 (31.4)0.002CEBPA017 (9.7)0.187MLL -PTD1 (3.7)14 (8.0)0.691 DisclosuresNo relevant conflicts of interest to declare.

Molecular Characterization of Relapsed Core-Binding Factor (CBF) Acute Myeloid Leukemia (AML)

Background: CBF-AML is defined by recurrent genetic abnormalities which encompass t(8;21)(q22;q22), inv(16)(p13.1q22) or less frequently t(16;16)(p13.1;q22). Most frequent secondary chromosome aberrations in t(8;21) AML are del(9q) or loss of a sex chromosome, and in inv(16)/t(16;16) AML trisomy 22 or trisomy 8. At the molecular level mutations involving KIT, FLT3, or NRAS were identified as recurrent lesions in CBF-AML. However, the underlying genetic alterations which might trigger relapse in CBF-AML are not well delineated. Thus, the aim of our study was to characterize the clonal architecture of relapsed CBF-AML.Methods: We performed mutational profiling (KIT, FLT3-ITD, FLT3-TKD, NRAS, ASXL1) in paired samples obtained at diagnosis and at relapse from 66 adults with CBF-AML [inv(16), n=43; t(8;21), n=23] who all were treated within the AMLSG studies.Results: In inv(16) AML, the following mutation pattern was identified at diagnosis: KIT 13/40 (33%; exon 8, n=6; exon 17, n=5; exon 8+17, n=1; exon 11, n=1; missing data, n=3), NRAS 18/43 (42%), FLT3-TKD 4/43 (9%); none of the pts harboured FLT3-ITD or ASXL1 mutations. At the time of relapse, there was a shift in the mutation pattern in 26 pts (60%): KIT mutations (exon 8, n=5; exon 17, n=2; exon 8+17, n=1) were lost in 8 pts and 1 pt acquired an exon 17 KIT mutation; similarly, 15 pts lost and 1 pt gained NRAS mutation, respectively. Of note, all FLT3-TKD mutations were lost at the time of relapse, and only one pt gained a FLT3-ITD mutation. Based on these findings we calculated the stability in inv(16) AML for KIT, NRAS and FLT3-TKD mutations as 38%, 17%, and 0%, respectively.AML with t(8;21) presented a different diagnostic mutation profile: KIT 9/23 (39%; exon 17, n=8; exon 11, n=1), FLT3 -ITD 3/23 (13%), NRAS 2/23 (9%), and ASXL1 1/23 (4%); there were no FLT3-TKD mutations. At the time of relapse, the mutation pattern changed in 9 pts (39%); KIT mutations were lost in 4 pts (exon 17, n=3; exon 11, n=1), but acquired in 2 pts with both of them located in exon 17; only 1 pt lost the NRAS mutation. FLT3-ITD was lost in 2 and gained in 3 pts. There was no change in the ASXL1 mutation status. Thus, the stability for KIT, NRAS, FLT3-ITD and ASXL1 mutations in t(8;21) AML was calculated as 56%, 50%, 33% and 100%, respectively.Of note, mutations affecting the KIT and NRAS gene were almost mutually exclusive; there were only 3 pts with concurrent KIT and NRAS mutations at diagnosis [inv(16), n=2; t(8;21), n=1].Conclusion: CBF-AML cases display a high degree of molecular heterogeneity with shift of the mutation pattern at relapse in both CBF-AML subtypes. The frequent loss of KIT and NRAS mutations at relapse suggests that there might be other important secondary lesions driving relapse. Ongoing high-resolution genome-wide profiling will further unravel the clonal hierarchy and genomic landscape in CBF-AML. DisclosuresGötze:Novartis: Honoraria; Celgene Corp.: Honoraria. Greil:Celgene: Consultancy; Ratiopharm: Research Funding; Sanofi Aventis: Honoraria; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Astra-Zeneca: Honoraria; GSK: Research Funding; Novartis: Honoraria; Genentech: Honoraria, Research Funding; Janssen-Cilag: Honoraria; Merck: Honoraria; Mundipharma: Honoraria, Research Funding; Eisai: Honoraria; Amgen: Honoraria, Research Funding; Cephalon: Consultancy, Honoraria, Research Funding; Bristol-Myers-Squibb: Consultancy, Honoraria; AOP Orphan: Research Funding; Roche, Celgene: Honoraria, Research Funding. Schlenk:Boehringer-Ingelheim: Honoraria; Teva: Honoraria, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Arog: Honoraria, Research Funding.

Mutations Modifying Risk in NPM1+ AML

Introduction:Favorable risk acute myeloid leukemia (AML) is defined by recurrent genetic abnormalities including core binding factor rearrangements such as inv(16) and t(8,21), normal cytogenetics with isolated mutations of NPM1, or bi-allelic mutations of CEBPA. Approximately 60% of patients are cured with standard 7+3 induction and cytarabine consolidation, which is comparable to survival rates of patients who receive allogeneic stem cell transplant (SCT). However, 20-30% of patients treated with chemotherapy still relapse, and relapsed disease remains the leading cause of death in this group. Even if relapsed patients achieve CR2 with salvage chemotherapy, their survival with allogeneic SCT is reduced compared to patients transplanted in CR1. Thus, predicting which favorable risk AML patients are more likely to relapse after chemotherapy would help guide therapy and improve patient outcomes. Recent publications have proposed that additional mutations in genes such as IDH1, IDH2, or DMT3A may impact relapse risk, but reports are conflicting. In addition, studies using minimal residual disease to evaluate disease burden after induction and consolidation has also been shown to predict relapse. We have used high throughput next generation sequencing (NGS) as diagnostic panel for AML at our institution for the past 2 years. This panel looks for mutations in 42 different genes known to be associated with acute leukemias and can quantitatively evaluate genetic MRD at a sensitivity of about 0.1%. We are analyzing favorable risk AML patients in an effort to identify additional mutations that predict relapse and in the process of evaluating the ability of this panel to evaluate MRD.Methods:Clinical samples were obtained with informed consent and with the approval of our institutional IRB. A targeted NGS panel was designed using multiplexed Ion AmpliSeq Designer (Life Technologies) software to amplify and sequence 42 genes relevant to hematopoietic malignancies. 20ng of DNA from bone marrow or blood was used to generate amplicon-based libraries that were sequenced using an Ion Torrent PGM. Bioinformatics analysis was performed using the Torrent Suite v.3.2 pipeline. Open source programs and lab-developed algorithms were used for variant annotation and mutation prediction.Patients with favorable risk AML diagnosed within the past two years had sequencing data available in their medical records. For patients diagnosed >2 years ago, archival samples were obtained and DNA extracted from isolated mononuclear cells or formalin-fixed paraffin-embedded tissue according to standard protocols.Results:We identified 57 patients with favorable risk AML diagnosed at OHSU over the past five years. 48 had enough biopsy material for genetic analysis. Of the 48, 17% had t (8;21), 29% had inv(16), 46% were NPM1+, and 8% had CEBPA mutations. Seven patients received transplant in CR1 for either residual disease or physician discretion. Of the remaining 41 patients, 11 patients relapsed (26%) and 8 of the 11 were NPM1+. Of the 8 relapsed NPM1+ patients, all had additional DNMT3A R882 mutations, IDH1/2 mutations, or both. 13 NPM1+ non-relapsed patients have been evaluated by sequencing to date and only 5 of 13 in the non-relapsed group had additional mutations in DNMT3A and/or IDH1/2(P=0.0185), however only 2 of the 5 had the R882 DNMT3A mutation. No DNMT3A, IDH1 or IDH2 mutations were identified in patients with t(8;21), inv (16) or biallelic CEBPA AML.Conclusions:Although our numbers are small, the presence of the R882 DNMT3A mutation appears to increase the risk of relapse in NPM1+ patients (Figure 1). Other DNMT3A point mutations do not seem to impact the risk of relapse and should be considered separately for relapse risk. Complete analysis and evaluation of MRD is underway. [Display omitted] DisclosuresDunlap:Oregon Health & Sciences University: Employment. Druker:Bristol-Myers Squibb: Research Funding; McGraw Hill: Patents & Royalties; Oncotide Pharmaceuticals: Research Funding; Millipore: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Aptose Therapeutics Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CTI Biosciences, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Henry Stewart Talks: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharamceuticals: Research Funding; Molecular MD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health and Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sage Bionetworks: Research Funding.

Abstract IA09: Therapy of lymphoma inspired by functional and structural genomics

Abstract Diffuse large B cell lymphoma (DLBCL) is a heterogeneous diagnostic category that is comprised of two prominent molecular subtypes, termed activated B cell-like (ABC) and germinal center B cell-like (GCB). These DLBCL subtypes are now viewed as molecularly distinct diseases since they arise from distinct stages of normal B cell development, require distinct recurrent genetic abnormalities to become malignant, have distinct cure rates with current chemotherapy regimens, and respond differentially to targeting agents. We defined a chronic active form of B cell receptor (BCR) signaling that activates NF-kB in ABC DLBCLs. Such ABC DLBCLs are killed by knockdown of BCR signaling components, such as the kinase BTK or components of the BCR itself. Over one fifth of ABC DLBCLs have mutations affecting the CD79B or CD79A subunits of the BCR that augment BCR signaling. To attack chronic active BCR signaling therapeutically, we initiated clinical trials in relapsed/refractory DLBCL of ibrutinib, an irreversible and highly selective inhibitor of BTK. Ibrutinib monotherapy induced a high rate of complete and partial responses in ABC DLBCL, while GCB DLBCL tumors rarely responded. Genetic analysis of responding tumors demonstrated an enrichment for those with CD79B mutations, but most responses were observed in tumors with wild type BCR subunits. This observation allowed us to define a non-genetic mechanism of BCR activation that depends on reactivity of the lymphoma BCR to self antigens. In addition to chronic active BCR signaling, the signaling adapter MyD88 is activated by oncogenic mutations in 40% of ABC DLBCL cases. Since these mutant MyD88 isoforms act in parallel to BCR signaling to activate the NF-kB pathway in ABC DLBCL, they might have been predicted to mitigate the clinical effects of ibrutinib. On the contrary, ABC DLBCL tumors with both CD79B and MYD88 mutations were highly responsive to ibrutinib, leading us to demonstrate that the BCR and MyD88 pathways positively regulate one another. To extend the efficacy of ibrutinib, we have identified additional therapeutic targets in the oncogenic signaling pathways in ABC DLBCL including: 1) ubiquitin ligases, such as LUBAC, that are required to assemble active signaling complexes downstream of the BCR; 2) the kinase IRAK4, which mediates oncogenic MyD88 signaling; 3) the PI3 kinase pathway, which is activated by chronic active BCR signaling; 4) BCL2, which is expressed highly in most ABC DLBCL tumors and is the target of recurrent amplifications. Several synergistic, mechanism-based drug combinations that exploit these redundant survival pathways will be presented. Citation Format: Louis M. Staudt. Therapy of lymphoma inspired by functional and structural genomics. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr IA09.

Targeted exome sequencing profiles genetic alterations in leiomyosarcoma.

Leiomyosarcoma (LMS) belongs to the class of genetically complex sarcomas and shows numerous, often non-recurrent chromosomal imbalances and aberrations. We investigated a group of LMS using NGS platform to identify recurrent genetic abnormalities and possible therapeutic targets. Targeted exome sequencing of 230 cancer-associated genes was performed on 35 primary soft tissue and visceral (extra-uterine) LMS. Sequence data were analyzed to identify single nucleotide variants, small insertions/deletions (indels), and copy number alterations. Key alterations were further investigated using FISH assay. The study group included patients with median age of 64 years and median tumor size of 7 cm. The primary sites included retroperitoneal/intra-abdominal, extremity, truncal, and visceral. Thirty-one tumors were high grade LMS, while four were low grade. Losses of chromosomal regions involving key tumor suppressor genes PTEN (10q), RB1 (13q), CDH1 (16q), and TP53 (17p) were the most frequent genetic events. Gains mainly involved chromosome regions 17p11.2 (MYOCD) and 15q25-26 (IGF1R). The most frequent mutations were identified in the TP53 gene in 13 of 35 (37%) cases. FISH analysis showed amplification of the myocardin (MYOCD) gene in 5 of 25 (20%) cases analyzed. None of the four low grade LMS showed losses or mutations of PTEN or TP53 genes. Genetic complexity is the hallmark of LMS with losses of important tumor suppressor genes being a common feature. MYOCD, a key gene associated with smooth muscle differentiation, is amplified in a subset of both retroperitoneal and extremity LMS. Further studies are necessary to investigate the significance of gains/amplifications in the development of these tumors.

A122: Correlation between expression of antigens on tumor cells and recurrent genetic abnormalities in chronic lymphocytic leukemia

Background Tumor cells in chronic lymphocytic leukemia (CLL) usually have the “classic” immunophenotype CD19+, CD5+, CD23+, SmIgdim, CD20dim, FMC7-, CD79b-/dim, CD22-/dim. However, sometimes some deviations in antigenic profile CLL cells are observed such as enhanced expression of CD79b, CD22, SmIg and CD20. The most frequent cytogenetic abnormalities in CLL are trisomy 12 and deletions of 13q14, 13q34, 6q, 11q, and 17p. It is noted that immunophenotypic deviations in CLL often correlate with some of these cytogenetic anomalies. The investigation of the links between the antigens expression and caryotype of CLL cells leads to differential diagnostics and prognosis in CLL. The goal of the investigation is search for the links between immunophenotypic characteristics of CLL cells and their karyotype. Material and methods Bone marrow and blood cells from 35 CLL patients are investigated. Immunophenotyping was made using flow cytometer “Cytomics F500” (Beckman Coulter) with the use of monoclonal antibodies CD3/CD19/CD45, SmIg κ SmIg λ /CD19, CD5/CD23/CD19, FMC7/CD38/CD19, CD22/CD79b/CD19, CD10/CD20/CD19. For karyotype determination the standard cytogenetic methods and FISH were used. Results The most frequent deletion in CLL patients was del(13)(q14.3), it was sometimes observed together with (13)(q34) and was found in18 patients (51% of all examined patients). 56% (10/18) of patients in this group had CLL cells phenotype CD19+, CD5+, CD23+, SmIgdim, CD20dim, FMC7-, CD79b-, CD22- and CD38-. Weak (dim) expression of CD79b antigen (in 24–92% of cells) was found in 39% (7/18) of patients with del(13)(q14.3), and expression of CD38 (in 22–86% of cells) – in 17% (3/18). The same phenotype was observed in 2 patients (6%) having only del(13)(q34), in one patient (3%) with (13)(q34) trisomy and in 6 patients (17%) with normal karyotype 46, XY. Trisomy 12 was found in 14% (5/35) of explored CLL patients. The immunophenotype of CLL cells in this group was very different with the previous group. All patients (100%) with trisomy 12 had CLL cells with expression of CD79b (94.2% of cells), CD22 (54–94.6% of cells), CD38 (31.1–99.3% of cells). Additionally 2 of 5 patients (40%) had CLL cells with more bright (dim to mod) CD20 expression and 1 patient (20%) had cells with bright expression of CD79b (in 94.2% of cells). The same phenotype was registered in one patient with del(6q23), comprising CD78b+ (in 97.8% of cells), CD22+ (in 62.6% of cells) and CD38+ (in 32.5% of cells). In 2 patients (6%) with del (11)(q23) expression of CD79b (in 47.3–44.8% of cells) and CD38 (in 35.3–64.4% of cells). Conclusion Expression of CD79b, CD22, CD38 is the main feature of cells with recurrent genetic injuries. Immunophenotype without expression of CD79b, CD22, CD38 in most cases differentiates CLL patients with del(13)(q14.3), del (13)(q34), (13q)(34) trisomy and with normal karyotype. Coexpression in the most of the cells of the three antigens CD79b, CD22, CD38 and more bright expression of CD20 proposes trisomy 12 or del(6q). Partial expression of CD79b and CD38 may correlate with del(11)(q23).

Mutually exclusive mutations in NOTCH1 and PIK3CA associated with clinical prognosis and chemotherapy responses of esophageal squamous cell carcinoma in China.

BackgroundRecurrent genetic abnormalities that correlate with clinical features could be used to determine patients' prognosis, select treatments and predict responses to therapy. Esophageal squamous cell carcinoma (ESCC) contains genomic alterations of undefined clinical significance. We aimed to identify mutually exclusive mutations that are frequently detected in ESCCs and characterized their associations with clinical variables.MethodsWe analyzed next-generation-sequencing data from 104 ESCCs from Taihang Mountain region of China; 96 pairs were selected for deep target-capture-based validation and analysis of clinical and pathology data. We used model proposed by Szczurek to identify exclusive mutations and to associate these with pathology findings. Univariate and multivariate analyses with Cox proportional hazards model were used to examine the association between mutations and overall survival and response to chemotherapy. Findings were validated in an analysis of samples from 89 patients with ESCC from Taihang Mountain.ResultsWe identified statistically significant mutual exclusivity between mutations in NOTCH1 and PIK3CA in ESCC samples. Mutations in NOTCH1 were associated with well-differentiated, early-stage malignancy and less metastasis to regional lymph nodes. Nonetheless, patients with NOTCH1 mutations had shorter survival times than patients without NOTCH1 mutations, and failed to respond to chemotherapy. In contrast, patients with mutations in PIK3CA had better responses to chemotherapy and longer survival times than patients without PIK3CA mutations.ConclusionsIn a genetic analysis of ESCCs from patients in China, we identified mutually exclusive mutations in NOTCH1 and PIK3CA. These findings might increase our understanding of ESCC development and be used as prognostic factors.

Update on Recurrent Genetic Aberrations in Acute Myeloid Leukemia

Recurrent chromosomal aberrations have long been recognized to influence prognosis in acute myeloid leukemia (AML), however, 50% of AML patients have a normal karyotype. The new millennium ushered in discoveries of gene mutations at the molecular level that predict outcome in patients with normal karyotype. Some recurrent mutations are already used in routine practice for AML risk stratification. With the development of high-throughput sequencing technologies, there has been a storm of new data, uncovering a complex genetic landscape in AML. In this review, we describe the significant progress in characterizing recurrent genetic abnormalities in AML in the last 5 years, focusing on prognostic significance and therapeutic implications.

Long-term remission of therapy-related acute myeloid leukemia with a new t(11;18)(q23;q21.2) translocation and KMT2A-ME2 (MLL-ME2) fusion gene

We describe a unique case of a woman with acute myeloid leukemia with a new, previously undescribed translocation, t(11;18)(q23;q21.2), affecting the KMT2A (MLL) gene and resulting in an KMT2A(MLL)-ME2 fusion. This disease occurred secondarily following chemotherapy for a different acute myeloid leukemia with the recurrent genetic abnormality inv(16)(p13.1;q22). The secondary leukemia was treated with intensive chemotherapy without allogeneic hematopoietic cell transplantation. Complete remission lasting more than 10 years has been achieved with concurrent and sustained remission of the primary leukemia.