Additional Aberrations Research Articles

Genomic Copy Number Variants in CML Patients With the Philadelphia Chromosome (Ph+): An Update.

BackgroundSubmicroscopic segmental imbalances detected by array-comparative genomic hybridization (array-CGH) were discovered to be common in chronic myeloid leukemia (CML) patients with t(9;22) as the sole chromosomal anomaly. To confirm the findings of the previous study and expand the investigation, additional CML patients with t(9;22) as the sole chromosomal anomaly were recruited and copy number variants (CNVs) were searched for.MethodsKaryotyping tests were performed on 106 CML patients during January 2010–September 2019 in our Genetics Laboratory. Eighty-four (79.2%) patients had the Philadelphia (Ph) chromosome as the sole chromosomal anomaly. Only 49(58.3%) of these 84 patients had sufficient marrow or leukemia blood materials to additionally be included in the array-CGH analysis. Fluorescence in situ hybridization (FISH) was carried out to confirm the genes covered by the deleted or duplicated regions of the CNVs.Results11(22.4%) out of the 49 patients were found to have one to three somatic segmental somatic segmental (CNVs), including fourteen deletions and three duplications. The common region associated with deletions was on 9q33.3-34.12. Identified in five (45.5%) of the 11 positive patients with segmental CNVs, the deletions ranged from 106 kb to 4.1 Mb in size. Two (18.2%) cases had a deletion in the ABL1-BCR fusion gene on der (9), while three (27.3%) cases had a deletion in the ASS1 gene. The remaining CNVs were randomly distributed on different autosomes.ConclusionSubtle genomic CNVs are relatively common in CML patients without cytogenetically visible additional chromosomal aberrations (ACAs). Long-term studies investigating the potential impact on patient prognosis and treatment outcome is underway.

Cytogenetic influence on prognosis in acute promyelocytic leukaemia: A cohort study in Vietnam

Objective/BackgroundTo analyse the influence of chromosomal aberrations in addition to t(15;17)(q22;q21) in acute promyelocytic leukaemia (APL) on clinical characteristics and treatment outcomes. MethodsFifty-seven patients with new APL diagnoses underwent conventional cytogenetic analysis; fluorescence in situ hybridization for t(15;17)(q22;q21) and reverse transcriptase–polymerase chain reaction detected PML/RARα in two forms: L (length) and S (short) and accepted treatment with all-trans retinoic acid and chemotherapy. Patients with additional chromosome aberrations were designated as the complex karyotype group and were compared with patients with only t(15;17), who were designated as the simple karyotype group. ResultsAdditional chromosome aberrations was observed in 18/57 patients (31.6%) at initial diagnosis. Outcome was significantly different between the simple karyotype group and the complex karyotype group for complete remission (92.3% vs. 66.7% respectively, p = .025), overall survival at 3 years (92.3% vs. 65.0%, respectively, p = .017), and progression-free survival at 3 years (81.4% vs. 44.4%, respectively, p = .024). ConclusionsAdditional chromosome aberrations had adverse effects on the prognosis in APL.

Characterization of RB1 in Pediatric TCF3-PBX1+ acute Lymphoblastic Leukemia

Introduction. Genomic alterations are frequently associated with acute lymphoblastic leukemia (ALL) prognosis. TCF3-PBX1 fusion is one of these alterations associated with B-cell precursor ALL (BCP-ALL) subtype. Additional genomic aberrations, observed in most pre-leukemic clones, are crucial in both BCP-ALL leukaemogenesis and treatment management. Aims. To explore the role of deletions in genes involved in lymphoid differentiation, cell cycle regulation and cytokine receptors on BCP-ALL TCF3-PBX1 + patients; To evaluate the difference in the gene expression profile of genes involved in cell cycle regulation when comparing patients with and without RB1 deletions (RB1del); To correlate the alterations found with clinical-laboratorial characteristics and with risk stratification variables. Methods. BCP-ALL patients younger than 18 years-old and without previous leukemic treatment were included in the present study. Submicroscopic deletions were identified by multiplex ligation-dependent probe amplification (MLPA) using SALSA MLPA P335 A4/B1 kit. RB1delwill be confirmed by FISH using in-house probes targeting the regions of interest in RB1 . Gene expression of genes involved in cell cycle regulation was assessed using the TaqMan Array 96-well plate - Human cyclins & Cell Cycle Regulation. Univariable analyses of characteristics were performed using the Fisher's exact test or χ2 test for qualitative variables using GraphPad Prism software. Results. Fifty cases harboring the TCF3-PBX1 (6.9% of the overall BCP-ALL series) were identified in the present study. They were predominantly male, aged 1-10 years-old, with WBC count ≤50x109/L and classified as pre-B ALL. Additional alterations in those TCF3-PBX1 + cases were identified by MLPA, with the most frequent alterations affecting genes involved in cell cycle regulation and lymphoid differentiation: CDKN2A/Bdel (40.0%) and PAX5del (37.7%), followed by RB1del (31.1%) and BTG1del (17.7%). The frequency of RB1del in TCF3-PBX1 +patients was markedly different from those with other cytogenetic subgroups of BCP-ALL (P <0.05). RB1del cases were mainly female (68.7% vs 31.3%) and molecular characterized by 9p21 deletions (37.5% CDKN2A/Bdel plus PAX5del). FISH analyses revealed heterogeneity of nuclei harbouring RB1del, varying from 12% to 97% of the interphase nucleus evaluated. Regarding the expression profile, we observed that the presence of RB1del is associated with significantly increased expression of CCND2 (P =0.032) , while the expression of CDKN2D was reduced compared to cases without RB1del (P =0.043). Additionally, we observed that the complete deletion of RB1 (involving all exons) was significantly associated with increased expression of CCNA2, CCNB1 , CDC2 and E2F3 , as a consequence of the loss of critical phosphorylation sites that regulate pRB interactions (P <0.05). On the other hand, RB1 partial deletion (involving exons 17 to 26) was associated with the reduction of CDKN2D expression. Conclusions.RB1 deletions were frequently found as an additional aberration particularly in TCF3-PBX1 + patients. Deletions in 9p21 locus were also frequently found in this cytogenetic subgroup. The loss of critical RB1 phosphorylation sites, as a consequence of RB1del affecting all exons, deregulate the expression of E2F3 , an important transcript factor that interacts directly with pRB and regulate the expression of other genes involved in the cell cycle, such as cyclins A2 and B1 , and CDC2 , essentials for G1/S and G2/M phase transitions. DisclosuresNo relevant conflicts of interest to declare.

Characteristics and Outcomes of Pediatric Patients with t(16;16)-AML: Results of a Case Series By the International BFM Study Group

IntroductionCore binding factor (CBF) acute myeloid leukemia (AML) inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFβ-MYH11 [inv(16)/t(16;16)] is associated with a favorable outcome in children. Aberrations inv(16) and t(16;16) result in the same fusion of the CBFβ subunit at 16q22 and the myosin-11 heavy chain gene at 16p13, and are thought to share common biological features and outcomes. However, CBFβ-MYH11 fusion transcripts are heterogeneous and may vary in terms of biological activity. Therefore, in this cohort study by the International Berlin-Frankfurt-Munster (I-BFM) study group, we evaluated clinical characteristics, prognostic features and outcomes in pediatric patients with t(16;16)-AML.MethodsPatients (age 0‒19 years) with cytogenetically or molecularly confirmed t(16;16)-AML, diagnosed by members of the I-BFM study group between 1990 and 2015, were included in the study. Patients with previous malignancies, chemotherapy, or radiotherapy were excluded. Data on baseline characteristics, including cytogenetic and molecular information, fusion type, treatment details and follow up were collected using standardized case report forms. If data on the CBFβ fusion type were missing, real-time quantitative PCR for fusion types A, D, and E was performed on available biobank material. The molecular characterization part of the study is ongoing. Herein, we present descriptive statistics and estimated outcomes related to achieving complete remission (CR) and probabilities of event-free survival (pEFS) and overall survival (pOS).ResultsThe study included 46 patients with t(16;16)-AML. Median age was 9.8 [interquartile range (IQR) 1.7‒11.8] years, median white blood cell (WBC) count at diagnosis was 74 [IQR 35‒80] ×109/L and the predominant FAB-type was M4Eo (49%). Eleven patients (24%) had central nervous system involvement at diagnosis. Information on common additional molecular aberrations (e.g. cKIT and RAS mutations) was unavailable for more than 50% of patients and therefore not considered. Information on fusion types was available for 13 patients (28%). Biobank material was used to determine CBFβ fusion type in 2 patients for whom it had previously been unknown. Another 9 samples from international groups are available and pending analysis. Most patients for whom information was available on CBFβ fusion type had type A (n=7, 54%), followed by type D (n=3, 23%) and type E (n=3, 23%). No other fusion types were identified. Patients with type non-A (n=6, 46%) were predominantly male (67%) and had FAB-type M4Eo (83%). They were older than patients with type A (11.9 vs 8.8 years, respectively) and had a higher WBC count (69 vs 48 ×109/L, respectively), but these differences were not significant. Additional cytogenetic aberrations occurred in 38% of patients. Gain of chromosome 8, 21 or 22 were the most common additional chromosomal aberrations. Distribution of additional cytogenetic aberrations was comparable between patient with fusion type A and other types.Patient outcome was excellent with a 100% CR rate and a 5-year pEFS and pOS of 80% [standard error (SE), 6.4%] and 87% [SE, 5.6%], respectively (Figure 1). Five patients suffered a relapse. Of them, 4 had WBCs >50 ×109/L at diagnosis. None of the 5 patients had additional cytogenetic aberrations. Two patients had fusion type A and type was unknown for the other 3 patients. Of 46 patients, 2 died after relapse and 3 died from toxic complications in first CR. These complications included failure to recover bone marrow hypocellularity and subsequent multi-organ failure (n=1), and sepsis after stem cell transplantation in CR (n=2).ConclusionThis is the first report of a relatively large cohort of pediatric t(16;16)-AML patients. Patient outcomes were very good, especially compared with those reported for patients with non-CBF leukemias. However, these patients were at risk of toxic death in CR. Clinical characteristics and outcomes were comparable with those reported previously for patients with inv(16). Although this case series was large, patient numbers remained too small to identify significant prognostic factors associated with outcome. Prospective trials are needed to determine other biological features that may explain differences in outcomes among patients with either t(16;16) or inv(16)-AML. [Display omitted] DisclosuresKaspers:Janssen-Cilag: Research Funding.

High Grade B Cell Lymphoma with MYC and BCL2 and/or BCL6 Rearrangements Depict a High Complexity on the Cytogenetic, but Not on the Molecular Genetic Level and Show MYC Mutations As Prognostic Marker

Background: In the revised WHO classification of 2016, cases of large B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements were categorized as “HGBL (high grade B cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements)”, except for cases that fulfill the criteria for a follicular or lymphoblastic lymphoma. A detailed molecular analysis of these cases and comparison of accompanying aberrations has not been available to date.Aims: Evaluation of associated genetic alterations and prognosis in HGBL cases with rearrangement of (i) MYC + BCL2 , (ii) MYC + BCL6 or (iii) MYC + BCL2+BCL6.Patient cohorts and methods: The total cohort comprised 70 cases with MYC and BCL2 and/or BCL6 rearrangements (39 male, 31 female). Cases were selected by detection of the rearrangements by FISH (fluorescence in situ hybridization), cases diagnosed as CLL, follicular or lymphoblastic lymphoma were excluded. Median age was 67 years (range: 44-87 years). All patients were investigated using chromosome banding analysis (CBA) and FISH using probes for MYC , IGH-BCL2 and BCL6 . Additionally, mutation analyses of ATM , BCL2, BIRC3, BRAF, BTK, CBL, CSF3R, CSNK1A1, CXCR4, EGR2, ETNK1, FBXW7, FLT3, ID3, IDH1, IDH2, KIT, KLF2, KRAS, MAP2K1, MYC, MYD88, NFKBIE, NOTCH2, PHF6, PLCG2, POT1, PTPN11, SAMHD1, SETBP1, SF3B1, SRSF2, STAT3, STAT5B, TET2, TP53, U2AF1, UBR5, WT1, XPO1 and ZRSR2 was performed. Variants of unknown significance were excluded from statistical analysis.Results: We identified 33/70 cases with rearrangement of MYC + BCL2 , 10/70 cases with MYC + BCL6 and 27/70 cases with MYC + BCL2 + BCL6 (47%, 14% and 39%, respectively). In the total cohort, CBA revealed a complex karyotype (>3 abnormalities) in 59/70 (84%) of patients, 10/70 (14%) harbored an aberrant but non-complex karyotype, whereas in 2/70 (3%) of cases, CBA analyses failed due to insufficient cell proliferation. Regarding the subgroups, a complex karyotype was observed in 82%, 80% and 89% of cases, respectively (for MYC + BCL2 , MYC + BCL6 and MYC + BCL2 + BCL6 rearrangement). If only aberrations additional to MYC / BCL2 / BCL6 rearrangements were calculated, >3 aberrations were detected in 64% of cases (67%, 70% and 60%, respectively). The most frequent additional aberrations included 13q deletions, +12, gain of 1q and 6q deletions. Median number of additional cytogenetic aberrations was n=8 (range: 0-36). Despite the high cytogenetic complexity, the frequency of TP53 deletions was relatively low (6% in the total cohort), three of them harboured a concomitant TP53 mutation in the other allele. Mutation analyses of 40 genes revealed a median number of n=1 mutations in the total cohort with no differences between the three subgroups. The range of the number of molecular mutations was 0-3 (0 mutations: 31%; 1 mutation: 44%; 2 mutations: 20%; 3 mutations: 5%). Interestingly, out of the 40 genes analysed, mutations were almost exclusively detected in BCL2 (40%), MYC (21%) and TP53 (21%) in the total cohort. Other recurrent mutations were observed for ID3 and PHF6 only (3%, respectively). Cases with MYC+BCL6 rearrangement showed a higher - although not statistically significant - frequency of MYC mutations compared to the other cases (40% vs. 18%), while BCL2 mutations were rarely detected in this subgroup (10% vs. 45%, p=0.033). Of note, mutations in TP53 were found to show a trend towards a lower frequency in cases with a “triple hit” (MYC+BCL2+BCL6 ) in comparison to the “double hit” cases (MYC+BCL2 and MYC+BCL6 ) (11% vs. 28%).Median overall survival (OS) in the total cohort was 61 months. Cases with MYC + BCL2 showed a shorter OS compared to cases with MYC+BCL2+BCL6 rearrangements, although this was not statistically significant (37 vs.61 months). Unfortunately, OS could not be determined for cases with MYC+BCL6 due to the low number of cases. Mutations in MYC, but not in BCL2 or TP53 , showed a negative impact on OS in the total cohort (16 vs. 61 months, p=0.046).Conclusion: 1) Cases with rearrangements in MYC , BCL2 and/or BCL6 depict a high complexity on the cytogenetic, but not on the molecular genetic level. 2) Molecular mutations are almost exclusively detected in BCL2 , MYC and TP53. 3) Cases with MYC+BCL6 rearrangements differ from the other subgroups by showing a higher frequency of MYC mutations and a lower frequency of mutations in BCL2 . 4) Mutations in MYC negatively impact on OS. 5) Thus, our data support the current WHO classification and complement it on a molecular level. DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Drilling high aspect ratio holes by femtosecond laser filament with aberrations.

A near-infrared femtosecond laser is focused by a 100 mm-focal-length plano-convex lens to form a laser filament, which is employed to drill holes on copper targets. By shifting or rotating the focusing lens, additional aberration is imposed on the focused laser beam, and significant influence is produced on the aspect ratio and cross-sectional shape of the micro-holes. Experimental results show that when proper aberration is introduced, the copper plate with a thickness of 3 mm can be drilled through with an aspect ratio of 30, while no through-holes can be drilled on 3-mm-thickness copper plates by femtosecond laser with minimized aberration. In addition, when femtosecond laser filament with large astigmatism is used, micro-holes that had a length to width ratio up to 3.3 on the cross-section are obtained. Therefore, the method proposed here can be used to fabricate long oval holes with high aspect ratios.

Chromosome Aberrations Detected By SNP Array Technique Indicating a High Risk of MDS Transformation in Patients with Icus and Poor Prognosis of Patients with MDS

AbstractPurpose: To evaluate the value of single nucleotide polymorphism array (SNP-A) analysis on the diagnosis and prognosis of Chinese patients with myelodysplastic syndromes (MDS).Methods: 416 cases were recruited from two medical centers in China; of which 200 cases were diagnosed with MDS, 25 with myeloproliferative neoplasm (MPN), 63 with primary acute myeloid leukemia(AML), 40 with aplastic anemia (AA), 62 with idiopathic cytopenia of undetermined significance (ICUS), and 26 were healthy individuals. We analyzed chromosome abnormalities in all the cases with both SNP-A and metaphase cytogenetics (MC) methods, and evaluated their clinical significance as well. Clinical data analyzed included age, sex, blood counts, marrow morphology, blast counts, progression free survival (PFS) and overall survival(OS).Results: 1. SNP-Aanalysis leaded to a significantly higher detection yield of chromosome abnormalities than MC examination. The abnormal detection rate for MDS, MPN and AML by MC was 42%, 48% and 36.5%, but by SNP-A significantly increased to 73.5%,72% and 69.8%, respectively. Moreover, SNP-A analysis revealed complex types and genomic distribution patterns of chromosome aberrations. 2. Notably, 20 of the 62 ICUS patients had abnormal detections by SNP-A, and interestingly 17 of these 20 ICUS patients with abnormal SNP-A detections transformed to typical MDS with a median follow-up time at 11months(Table 1). 3. Prognosis of the MDS patients with abnormal SNP-A detections was significantly worse than that of those patients without abnormal SNP-A observations in OS (24 months vs not reached (NR); P=0.004) and PFS (15 vs 40 months; P=0.002). Prognosis of the patients identified with abnormal SNP-A detections in addition to a normal or a good IPSS-R prognostic MC finding in a patient was significantly inferior than that of those patients with no such additional SNP-A aberrations in OS (25 months vs NR; P=0.004) and PFS (17 months vs NR; P=0.002). For the patients stratified as high-risk and very-high-risk MDS by the revised International Prognostic Scoring System(IPSS-R), there were no differences between the patients with and without SNP-A abnormalities in OS (13 vs 12 months; P =0.537) and PFS (14 vs 10 months; P =0.368); However, in the group of patients with very-low-risk, low-risk, and intermediate-risk MDS, the patients with SNP-A abnormalities showed significantly inferior survival than those without abnormal SNP-A detections in OS (NR vs NR; P=0.001) and PFS (25 months vs NR; P=0.008). 4. By multivariable analysis the presence of SNP-A aberrations additional to MC analyses in a patient and a higher number of SNP-A abnormalities (≥3 per patient) were independent predictors of poor prognosis for patients with MDS (HR=2.33, P=0.003; HR=2.40, P=0.002).Conclusion:SNP-A analysis can help significantly improve the diagnosis and better evaluate the prognosis of patients with MDS, and help better assess the risk of MDS transformation in patients with ICUS. SNP-A analysis is of great practical value in clinical management. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RIZ1 Gene Promoter Methylation Increases with Progression of Chronic Myeloid Leukaemia to Advanced Phases

Back ground:Chronic myeloid leukaemia CML is a clonal disorder with Philadelphia Ph chromosome as the sole genetic abnormality. The heterogeneity of CML is due to additional genetic aberrations which may include SNPs, mutations, epigenetic alterations of various tumor suppressor or cell cycle regulatory genes and even their abarent expression pattern. Epigenetic changes like histone DNA methylation, histone acetylation, microRNA expression have an important role in disease prognosis and drug resistance in CML. Tumor suppressor genes are the key targets of promoter hypermethylation in cancer. The role of DNA methylation on progression of CML and response to drug therapy has not been elucidated completely. We studied 100 CML patients in different clinical phases, 50 CP, 25 AP and 25 BC, and 100 healthy controls to elucidate the role of RIZ1 gene in chronic myeloid leukaemia CML.Methodology:We characterized RIZ1 gene promoter methylation and expression in 100 CML patients, 50 Chronic phase, 25 Accelerated phase and 25 Blast crisis, recruited from Lok Nayak Hospital, Maulana Azad Medical College, New Delhi, with respect 100 age and sex matched healthy controls. RIZ1 promoter methylation was studied by methylation specific PCR (MS-PCR). RIZ1 gene expression study was performed using SYBR green based qRT-PCR and the results were expressed as mean fold change.Results:In this study, it was found that RIZ1 gene promoter methylation significantly p 0.009 increases with CML disease progression to advanced phases. Also, RIZ1 promoter methylation was also found to be significantly associated with haematological p less than 0.01 and molecular (p less than 0.003) response of patients to Imatinib. Patients with RIZ1 promoter methylation showed a higher survival (12.66 plus minus 2.14 months) in comparison to patients without RIZ1 promoter methylation (40.75 plus minus 2.11 months) with a statistically significant p value of 0.0001.The RIZ1 gene expression was found to decrease progressively during the progression of CML disease with 0.16, 0.6 and 1.5 fold decreased expression in CP, AP and BC phases respectively. Moreover, patients with RIZ1 promoter methylation had a lower RIZ1 gene expression pattern compared to patients without RIZ1 promoter methylation p 0.0008.Conclusion:Thus, it may be concluded that RIZ1 gene promoter methylation increases and its expression decreases progressively during CML disease advancement. Hence, this may be cause among other for poor drug response of some CML patients to Imatinib therapy. DisclosuresNo relevant conflicts of interest to declare.

Comparison of the Outcome between Newly Diagnosed Patients in the Accelerated Phase and Chronic Phase with Chronic Myeloid Leukemia Treated with Frontline Tyrosine Kinase Inhibitors

Background: Accelerated phase (AP) chronic myeloid leukemia (CML) has an aggressive clinical course. Some patients with CML present as AP at the time of diagnosis. Although many studies have confirmed that tyrosine kinase inhibitors (TKIs) significantly improved survival in patients with CML in the chronic phase (CP), there is limited data on the outcome of newly diagnosed CML-AP patients in the era of TKIs.Aims: To compare progression-free survival and overall survival in CML-AP patients with those in CML-CP patients treated with frontline TKIs and identify factors associated with the outcome.Methods: According to the definition of European LeukemiaNet 2013, data of newly diagnosed CML-AP and CML-CP patients receiving TKIs at least 3 months from 2002 to 2016 were retrospectively analyzed. Kaplan-Meier curves were used to compare the probabilities of PFS and OS. Uni- and multivariate Cox regression models were built to identify independent variables (demographics; co-morbidity; disease phase and risk, CBC, and additional cytogenetic aberrations in Ph-positive cells at diagnosis; interval from diagnosis to start TKI-therapy; initial TKI used and early treatment-response) associated with the outcome.Results: In total, 101 CML-AP patients [with excess blasts (blasts ≥15%), n=13; with excess basophils (basophils ≥20%), n=88] and 656 CML-CP patients (Sokal low-risk, n=291; intermediate-risk, n=194; high-risk, n=111; unknown, n=60) were included. 463 (61%) were male. Median age was 39 (14-87) years. Patients received initial therapy with imatinib (n=660), nilotinib (n=85), or dasatinib (n=11) within 6 months from diagnosis. At 3 months, CML-AP patients with excess blasts (62%, 15% and 23%) and excess basophils (65%, 27% and 8%) achieved similar response (optimal, warning and failure) rates to CML-CP patients in Sokal intermediate-risk (66%, 20% and 14%) and high-risk (58%, 26% and 16%) (p=0.338), but significantly lower than those in Sokal low-risk (83%, 13% and 4%) and unknown (77%, 17% and 7%) (p<0.001). With a median follow-up of 39 months (range, 3-184 months), probabilities of PFS (p=0.014) and OS (p=0.152) at 5 years were the lowest in the CML-AP patients with excess blasts (78% and 92%), followed by CML-CP patients in Sokal high-risk (87% and 91%), intermediate-risk (92% and 95%), low-risk (95% and 97%), CML-AP patients with excess basophils (95% and 97%), and those with unknown-risk (96% and 98%). In multivariate analyses, while analyzing baseline and initial therapy variables, interval from diagnosis to starting TKI-therapy >2 months [HR 2.4 (1.1-4.9), p=0.020 and HR 2.6 (1.1-6.5), p=0.037) was associated with inferior PFS and OS; in addition, CML-CP patients in Sokal high-risk [HR 3.6 (1.1-6.5), p=0.038] was associated with inferior PFS, >60 years [HR 3.9 (1.2-13.3), p=0.027], inferior OS. While involving early therapy response, interval from diagnosis to starting TKI-therapy >2 months [HR 3.1 (1.4-6.6), p=0.009 and HR 3.2 (1.2-8.4), p=0.016] and achieving a response of “failure” at 3 months [HR 7.6 (3.7-15.6), p<0.001 and HR 8.1 (3.1-21.3), p<0.001] were variables associated with inferior PFS and OS; in addition, >60 years [HR 4.8 (1.4-16.1), p=0.010], inferior OS. There was no impact of initial TKI used (imatinib or 2nd generation TKI) on the outcome.Conclusions: Newly diagnosed CML-AP patients have comparable outcome to CML-CP patients treated with frontline TKIs, especially CML-AP patients with excess basophils. However, delayed therapy and poor early response to TKIs are significantly associated with unfavorable prognosis in either AP or CP patients with CML. Our data indicates timely and proper TKI-therapy and close monitoring will be a reasonable option for the treatment strategy in CML-AP patients in the era of TKIs. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetic Alterations Are Differently Associated with Disease Progression Depending on t(14;18) in Follicular Lymphoma

[Introduction] Follicular Lymphoma (FL) is characterized by indolent clinical behavior and subsequent histological transformation which links to aggressive clinical course and poor outcome. The hallmark of cytogenetic abnormality in FL is the translocation t(14;18)(q32;q21), which is found in more than 75% of cases. Several other cytogenetic abnormalities, which might contribute to disease progression, have been found. However, the prognostic and biological significance of cytogenetic or genomic features of FL has not been fully revealed. Last year we have shown clear evidence that cytogenetic abnormalities, +21 and 3q27, were independent prognostic factor in the rituximab era. Next we evaluated the pattern of chromosomal abnormalities in FL and the relation between cytogenetic and biological feature in this study.[Patients and Methods] Cells from lymph nodes or other sites of disease at diagnosis from 201 patients with FL admitted to our hospital and affiliated hospitals between 2001 and 2013 were cytogenetically analyzed using standard methods of G-banding. Ninety nine (49.3%) men and 102 (50.7%) women with a median age of 59 years (range, 28 - 83 years) were included in the analysis. The median follow up period was 48.3 months. Forty three patients (21.4%) were Stage I or II; 156 patients (77.6%) were Stage III or IV; and 2 patients (1%) were unknown. Eighty patients (39.8%) were follicular lymphoma international prognostic index (FLIPI) low, 55 patients (27.4%) were intermediate, and 43 patients (21.4%) were high; and 23 patients (11.4%) were unknown. The distribution of FL pathological subgroups was as follows: FL Grade 1 - 2, 142 patients (70.6%); FL Grade 3a, 30 (15.0%); and unknown, 29 (14.4%). One hundred and fifty seven patients (78.1%) received Rituximab-containing chemotherapy as an initial treatment.[Result] t(14;18)(q32;q21) was the most common abnormality observed in 119 patients (59.2%). Other numerical or structural abnormalities that were identified in more than 5% of the patients were as follows: +X (17.9%), del(6)(q) / −6 (16.9%), +7 (14.4%), abnormality of 1q12-21 / 1q (12.9%), del(13)(q) / -13 (11.9%), abnormality of 3q27 (10.4%), abnormality of 10q22-24 (10.0%), +12 / dup(12)(q) (10.0%), abnormality of 1p21-22 / 1p(9.0%), +18 (9.0%), del(17)(p) / −17 (5.0%), and the number of cytogenetic aberration higher than 3 (54.7%). Patients with the number of cytogenetics aberration higher than 3 significantly correlated with higher FLIPI grade (p= 0.0028). As is the case with much higher number of aberration (higher than 4, p= 0.0019 and higher than 5, p= 0.0024). The higher number of cytogenetic aberration also showed a trend of good correlation with advanced stage of Ann Arbor system. These suggest that additional chromosomal aberration closely related to disease progression in FL. To investigate the relationship between the disease progression and each cytogenetic abnormality logistic regression analysis was performed. In all cohort +X (Oods Ratio (OR) 2.604, 95%CI 1.184 - 5.751, p= 0.0175) and +12 / dup(12)(q) (OR 3.955, 95%CI 1.397 - 12.22, p= 0.0095) were significant abnormalities for higher FLIPI grade. To evaluate significance of t(14;18) multivariate analysis was performed for both FLs with and without t(14;18) again. In FLs with t(14;18) the same cytogenetic abnormalities, +X (OR 3.293, 95%CI 1.212 - 9.459, p= 0.0216,) and +12 / dup(12)(q) (OR 6.748, 95%CI 1.779 - 33.998, p= 0.0042), were detected as significant factor for disease progression. On the contrary in FLs without t(14;18) quite different cytogenetic abnormalities, such as +5 (OR 4.01e7, 95%CI 2.180 - , p= 0.0144) and +21 (OR 11.065, 95%CI 1.318 - 232.1, p= 0.0267), were chosen. However, these cytogenetic abnormalities did not influence patients' survival except for +21.[Conclusion] In patients with FL disease progression was closely associated with cytogenetic abnormalities, +X and +12 / dup(12)(q) in those with t(14;18). However, in patients without t(14;18) disease progression developed in distinct manner with respect of cytogenetic alteration. Important cytogenetic alterations for disease progression may be different from those for survival in Rituximab era. DisclosuresMurakami:Ono: Honoraria; BMS: Honoraria; Fujimoto: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Celgene: Honoraria.

Comprehensive Molecular Characterization of Myeloid Malignancies with 9q Deletion

Background and aims: Deletions in the long arm of chromosome 9, del(9q), are recurrent cytogenetic aberrations in myeloid neoplasms. While relatively common in patients with acute myeloid leukemia (AML), del(9q) occurs less frequently in other myeloid malignancies such as myelodysplastic syndromes (MDS) or myeloproliferative neoplasms (MPN). To present, cases with del(9q) in MDS and MPN are poorly characterized on a molecular level. We set out to define the size and minimally deleted region (MDR) of del(9q) as well as the spectrum of accompanying gene mutations in patients with MDS or MPN. Furthermore, the prognostic impact of del(9q) in MDS is still under debate. According to the revised international prognostic scoring system (IPSS-R), MDS patients with isolated del(9q) fall into the cytogenetic intermediate risk group (median overall survival, OS, of 32 months) based on the presence of a single aberration (Greenberg et al, Blood 2012). We therefore aim to evaluate if this risk stratification is appropriate for our patient cohort.Patients and methods: A total of 55 (26 female and 29 male) patients with MDS (n=40), MPN (n=8) and MDS/MPN overlap (n=7) were included in this study. The median age was 75 years (range: 23-94 years). Conventional cytogenetic analysis was performed for all patients. Cases (n=38) with sufficient clone size were investigated by array CGH (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). Mutation status was examined using an amplicon-based NGS panel targeting 40 genes commonly affected in myeloid neoplasia.Results: In 36/55 (65%) of patients, del(9q) was the sole cytogenetic aberration. Additional recurrent cytogenetic abnormalities included unbalanced rearrangements, n=8; del(5q), n=5; del(11q), del(20q), +8, +21 (n=2, each). Clinical follow-up data was available for 16 MDS patients with isolated del(9q), median OS was 35 months (range, 1- 91 months). In cases with array CGH data, size of the 9q deletion varied between 2.2 and 70.5 Mb (median, 33.9 Mb). We identified a MDR of 3.6 Mb ranging from genomic position 85,716,785 to 89,340,559 (UCSC hg19). A total of 21 genes are located in the MDR, including FRMD3, UBQLN1, KIF27, HNRNPK, SLC28A3 and NTRK2, which have previously been described in the MDR of del(9q) AML patients. Driver gene mutations were identified in 43/55 (78%) of patients. The number of mutated genes ranged from 1-5 (median: n=2). We did not observe significant differences in the number of mutated genes between younger or older patients (cut-off: 70 years), and patients with isolated del(9q) or additional cytogenetic aberrations. However, patients without evidence for a gene mutation (n=12) were significantly younger than patients with respective mutations (median age 70 vs. 76 years, p=0.014). Recurrently mutated genes included TET2 (39%), ASXL1 (31%), JAK2 (25%), U2AF1 (17%), RUNX1 (11%), EZH2 (8%), NRAS (7%), BCOR, KRAS, TP53 (6% each) and SRSF2 (5%). ASXL1 and TET2 were frequently co-mutated, as 12/20 (60%) of TET2 mutated cases also harbored ASXL1 mutations (p< 0.001). Overall, we identified a high prevalence of TET2 mutations in patients with del(9q). This association was even more pronounced when we limited our analyses to patients with isolated del(9q), where we observed a TET2 mutation frequency of 45%. In 7/31 (23%) of patients without TET2 mutation, other genes associated with epigenetic regulation, namely DNMT3A, ASXL1, EZH2, WT1, IDH1 and IDH2, were affected, underlining a high mutation rate (49% of total cohort) in this functional class. Mutations linked to the spliceosome machinery, SF3B1, SRSF2 and U2AF1, were mutated in 13/55 (24%) of patients in the total cohort and in 9/40 (23%) patients with MDS.Conclusions: Deletions in 9q are rare aberrations in MDS and MPN. Our analyses provide novel insights into the molecular profile of this cytogenetic subgroup. 1) Our patient cohort with MDS or MPN and del(9q) demonstrated a minimal deleted region in concordance with previous reports on the MDR of del(9q) AML patients, indicating a general pathogenic mechanism. 2) We identified gene mutations in 78% of our patient cohort, TET2 and other epigenetic regulators were mutated in 49% of patients with del(9q), suggesting cooperative pathogenic potential. 3) We observed a median OS of 35 months in patients with MDS and isolated del(9q), confirming the current stratification according to IPSS-R. [Display omitted] DisclosuresHartmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Zieschang:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Mutation Screening at Diagnosis Reveals a High Frequency of ASXL1 Mutations in CML Patients Who Fail to Achieve Molecular Remission Criteria after One Year of TKI Treatment

Background: Tyrosine kinase inhibitors (TKI) revolutionized treatment of chronic myeloid leukemia (CML). While response rates are high, a subset of patients does not show the expected decrease or eradication of BCR-ABL1 . According to ELN guidelines (Baccarani et al. 2013) BCR-ABL1/ABL1 ratio>1% after one year of TKI is considered failure. Mechanisms of failure include the acquisition of additional genetic changes, e.g. cytogenetic aberrations and mutations in the ABL1 kinase domain but also in other genes. Here, we ask the question, if specific mutations are not only acquired but may already be present at initial diagnosis of patients with slow or no TKI response (primary failure according to ELN). Aim: To screen CML patients at diagnosis, who failed ELN remission criteria after one year, for mutations in genes, which are frequently mutated in myeloid malignancies. Patients and Methods: In a backtracking approach, we selected diagnostic samples of 48 CML patients (25 males, 23 females) with a median age of 64 (range: 23-84) years, who failed first year molecular remission criteria. Median BCR-ABL1/ABL1 ratio was 3.603 [1.158-58.318] after 12 (11-16) months of imatinib (n=42), second generation TKI (n=5) treatment or both (n=1). Patients had a p210 BCR-ABL1 fusion (e13a2 [n=24], e14a2 [n=18], both [n=6]) and no additional cytogenetic aberrations at diagnosis. We sequenced DNA of bone marrow (n=28) or peripheral blood (n=20) samples drawn at diagnosis on NextSeq or MiSeq sequencers after TruSeq library preparation (Illumina, San Diego, CA). We used SeqNext 4.3 for data analysis (JSI, Kippenheim, Germany). The panel covered 48 genes including the epigenetic regulators: ASXL1, ASXL2, BCOR, DNMT3A, EZH2, IDH1, IDH2 and TET2 . Results: We identified mutations in 13/48 samples at initial diagnosis (27%). Only one patient had two mutations (total, n=14 mutations). There was no correlation of mutations with types of transcript, age, gender, hemoglobin level, WBC count, percentage of Ph+ cells by FISH, BCR-ABL1/ABL1 ratio at diagnosis or with BCR-ABL1/ABL1 ratio after one year of TKI. Twelve of the 14 (86%) mutations were found in genes involved in chromatin modification or DNA methylation. We screened four splicing machinery genes (SF3B1, SRSF2, U2AF1, ZRSR2), which are frequently mutated in other myeloid malignancies, but no mutation was found. In 8/48 (17%) patients, we found an ASXL1 mutation (57% of all mutations). Other mutated genes were ASXL2, BCOR, CALR, NRAS (n=1 each) and DNMT3A (n=2). The comparison of ASXL1 mutation types in this CML cohort to other myeloid malignancies revealed a significant shift in mutation types: e.g. in myelodysplastic syndrome (Haferlach et al. 2014) and chronic myelomonocytic leukemia (Meggendorfer et al . 2012), c.1934dup and c.1900_1922del (ENS0375687) constitute the majority of mutations (64 [42%] and 23 [15%] of 152, respectively), whereas there was only one c.1900_1922del and no c.1934dup in our CML cohort (p=0.023). Further, we used follow-up samples after one year of TKI to evaluate if mutations are derived from the CML clones. In all patients with available data for ASXL1 (n=6), mutation burdens showed a quantitative course which paralleled BCR-ABL1 ratio indicating that ASXL1 mutations are part of the CML clone present at diagnosis. In the patient with a CALR mutation, the CALR burden remained unchanged despite BCR-ABL1 reduction, indicating a BCR-ABL1 gain in a preexisting CALR mutated myeloproliferative neoplasm. In comparison to ASXL1 mutation rates (4/74 [5%]) in a published cohort with optimal responders (Kim et. al. 2017), we found a significantly higher number of ASXL1 mutations (8/48 [17%]) at diagnosis of our CML patients, who failed first year molecular remission criteria (p=0.041). Conclusions: (1) At diagnosis of CML, mutations were detected (esp. in ASXL1) in 27% of patients with TKI failure according to ELN criteria after one year. Compared to other myeloid malignancies, patients showed a different pattern of ASXL1 mutations. (2) No mutations in spliceosome genes were found. (3) Stringent BCR-ABL1 monitoring allows early adjustment of therapy in case of suboptimal response, but analysis of additional mutation patterns could help understanding the biology of failure and would allow up-front adjusted therapy decisions or different monitoring strategies. (4) Mutations such as ASXL1 at diagnosis may contribute to suboptimal TKI response. Disclosures Baer: 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.

Impact of 9q Deletions on the Classification in AML

Background: In the 2016 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia the deletion of the long arm of chromosome 9 (del(9q)) was excluded as a defining cytogenetic abnormality for AML with myelodysplasia-related changes due to its frequent association with NPM1 and biallelic CEBPA mutations.Aim: Investigation of the frequency of del(9q) in AML and its accompanying molecular and cytogenetic abnormalities to assess whether or not del(9q) is associated with a myelodysplasia-related mutation profile.Patients and Methods: We evaluated 9762 AML patients (pts) diagnosed between 2005 and 2017 for del(9q) using chromosome banding analysis (CBA). Pts with del(9q) were further analyzed for mutations in NPM1 , CEBPA and RUNX1 with amplicon sequencing to categorize them according to the WHO classification. Pts without a class defining aberration or a complex aberrant karyotype were screened for AML- or MDS-related mutations (ASXL1, BCOR, EZH2, FLT3- ITD , FLT3- TKD , SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1 and ZRSR2 ). Variants of unknown significance were excluded from statistical analysis. The size of the deleted region on chromosome 9 was evaluated by array CGH (Agilent Technologies, Santa Clara, CA) and NUP98 -rearrangements were investigated by FISH(MetaSystems, Altlussheim, Germany).Results: In 114/9762 pts (1 %) a del(9q) was detected by CBA. This cohort with del(9q) comprised 60 male and 54 female pts, median age was 63 years (range: 16 - 90 years). Del(9q) was accompanied by the following rearrangements: RUNX1-RUNX1T1 rearrangement in 20/114 pts (18 %), PML-RARA in 5/114 pts (4 %), KMT2A in 2/114 pts (2 %) and MECOM in 1/114 pts (1 %). In 20/114 pts (18 %) a NPM1 mutation and in 7/114 pts (6 %) a biallelic CEBPA mutation was observed. Furthermore, RUNX1 mutations were found in 3/114 pts (3 %). The remaining 56 pts (49 %) were divided into the following three groups: pts with del(9q) as a sole cytogenetic abnormality (del(9q) sole, 8/114 pts, 7 %), pts with one or two additional cytogenetic aberrations (del(9q) other, 19/114 pts, 17 %) and pts with complex aberrant karyotype (complex, 29/114 pts, 20 %) harboring 4 to 29 cytogenetic aberrations (median: 8). Cases with del(9q) sole and other (n = 27) were further characterized. NUP98 -FISH analysis revealed a NUP98 -rearrangement in 5/27 pts (19 %) in the del(9q) sole and other group. Moreover, two pts with del(9q) showed AML specific rearrangements (t(8;16)(p11;p13) and t(3;5)(q25;q35)). The most frequently mutated genes in del(9q) sole and other were: FLT3 -ITD (8/23 pts, 35 %), TET2 (7/27 pts, 26 %), FLT3 -TKD (4/21 pts, 19 %) and WT1 (3/26 pts, 11 %). A very high frequency of FLT3 -ITD and FLT3 -TKD were found in pts with del(9q) and balanced rearrangements (5/7 pts, 71 %).To identify whether pts with del(9q) sole and other rather resemble a molecular mutation profile typical for secondary AML arising from MDS (s-AML), the presence of gene mutations related to s-AML (according to Lindsley, et al., 2015, Blood) was evaluated. 9 pts (33 %) had a mutation in at least one of these genes, 14 pts (52 %) showed no such mutation and 4 pts (15 %) could not be fully analyzed.Array CGH showed that in pts with del(9q) sole the deletion size ranged from 14 - 42 Mb (median: 28 Mb), in pts with del(9q) other from 19 - 70 Mb (median: 32 Mb). A minimal deleted region for pts with del(9q) sole and other could not be determined for all pts due to the strong variation between the respective deleted regions.Follow up data was available for 75 pts with a median follow-up of 27 months. The median overall survival (OS) for pts with RUNX1-RUNX1T1 , NPM1 and bialleic CEBPA mutation was not reached. It was 73 month for pts with del(9q) sole and other and 16 month for pts with complex karyotype, RUNX1 mutations or MECOM rearrangements. The OS of first two groups did not differ significantly, but was significantly distinct from the last group (p = 0.03 and 0.03, respectively).Conclusion: In 1 % of AML pts a del(9q) was present. Del(9) frequently co-occured with RUNX1-RUNX1T1 , biallelic CEBPA and NPM1 mutations, NUP98 -rearrangements and other AML-typical translocations. A mutation signature typical for s-AML was infrequent. Thus, it seems reasonable that the del(9q) is no longer regarded as a defining cytogenetic abnormality for AML with myelodysplasia-related changes, in particular as prognosis in del(9q) cases with non-complex karyotype is favorable. DisclosuresBalk:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan: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.

PPM1D Mutations in MDS Patients with Deletion of Chromosome 5q

Background: Patients with myelodysplastic syndrome with deletion of chromosome 5q (MDS del(5q)) have a favorable prognosis, but TP53 mutations occur in 13-18% of these patients and are known as unfavorable prognostic marker. Recently, Protein Phosphatase Magnesium-Dependent 1 (PPM1D) mutations have been described in clonal hematopoiesis and were found more commonly in patients with therapy-related MDS than in patients with primary MDS (15% vs. 3%) (Lindsley et al. 2017). PPM1D encodes a serine-threonine protein phosphatase that inhibits the p53 tumor suppressor pathway. It is therefore of great interest to evaluate the frequency and potential co-occurrence of PPM1D mutations in patients with MDS del(5q).Aim: To determine the frequency and potential clinical impact of PPM1D mutations in a large cohort of MDS del(5q) patients.Methods: 219 patients with MDS or AML following MDS (sAML) and deletion of chromosome 5q were included in the analysis. Patients were classified following the 2008 WHO classification. Patients with MDS (n=196) or sAML (n=23) were cytogenetically characterized by chromosome banding analysis and molecularly analyzed for mutations in all exons (1 to 6) of PPM1D by Sanger or next-generation sequencing (NGS). Patients were also analyzed for mutations in TP53 and CSNK1A1 .Results: A total of 11 PPM1D mutations were found in 10 (4.6%) MDS patients with del(5q). The mutation frequency was similar in patients with MDS with isolated del5q (8 of 167, 4.8%) compared to patients with more advanced MDS or secondary AML (2 of 52, 3.8%, P=.78). Four patients had nonsense mutations, 3 patients had frameshift mutations (one patient with 2 frameshift mutations), and 3 patients had missense mutations. Nine mutations were found in the previously described hotspot region of PPM1D between amino acids 427 and 542, which is located downstream of the phosphatase domain of PPM1D. Three of the 10 PPM1D mutated patients had additional cytogenetic aberrations besides del(5q) (trisomy 8; complex karyotype in two patients). TP53 was also mutated in the two patients with complex karyotype and in one additional patient with a nonsense mutation of PPM1D . None of the PPM1D mutated patients had a mutation in CSNK1A1 .Patients with mutated PPM1D were slightly older than PPM1D wildtype patients (median age 77.7 vs 71 years, not significant). Eight of 10 PPM1D mutated patients had a diagnosis of MDS del(5q) and 2 patients had MDS RAEB2 with del(5q) accompanied by a complex karyotype. There were no significant differences in the distribution of sex, WHO subtypes, cytogenetic risk according to the international prognostic scoring system (IPSS), WBC count, hemoglobin, platelet count and IPSS risk score between PPM1D wildtype and mutated patients. Two of the 10 patients were transfusion dependent and none of 7 patients with available information transformed to AML.Survival information was available for 156 patients with MDS del(5q) (excluding other WHO subtypes of MDS/sAML) including 8 patients with mutated PPM1D and 15 patients with mutated TP53 . Median follow-up from the time of sample harvest was 2.5 years. The probability of survival at 2 years was 100% for PPM1D mutated and TP53 mutated patients, respectively, and 84% for PPM1D/TP53 wildtype patients (P=.18, log-rank test, Figure 1A). None of 7 PPM1D mutated patients and 1 of 13 TP53 mutated patients with available information underwent allogeneic hematopoietic cell transplantation.Survival information was available for 45 patients with del(5q) and more advanced MDS or secondary AML. The 14 patients with TP53 mutation (including 2 patients with additional PPM1D mutation) had a significantly shorter OS than TP53 wildtype patients (P<.001, 2-year OS 0% vs 51%, Figure 1B). One PPM1D mutated patient responded well to lenalidomide treatment for the duration of 3.2 years.Conclusion:PPM1D mutations are recurrently mutated in MDS patients with del(5q) with a frequency of 4.6% and may co-occur with TP53 mutations. PPM1D mutated patients were not specifically associated with patient characteristics or outcome. Interestingly, in our cohort of patients with MDS with isolated del(5q), TP53 mutations were not associated with unfavorable outcome.VP and MM contributed equally as first authors. TH and MH contributed equally as last authors. [Display omitted] DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Platzbecker:Novartis: Consultancy, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Thiede:Novartis: Consultancy, Speakers Bureau; Roche: Consultancy; Agendix: Employment; Bayer: Consultancy, Speakers Bureau. Germing:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Schroeder:Celgene: Consultancy, Honoraria, Other: travel support. Kobbe:Celgene: Other: Advisory Board, Research Funding. Haase:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Dohner:Novartis: Honoraria, Research Funding. Sperr:Novartis: Other: Register; Phadia: Research Funding; Meda: Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria. Valent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Incyte: Honoraria; Blueprint: Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Pfizer: Honoraria; Deciphera: Honoraria, Research Funding; Teva: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Whole Exome Sequencing Reveals High Genetic Diversity in Relapsed Multiple Myeloma

Background: Treatments like lenalidomide and proteasome inhibitors alone or in combination with chemotherapy have dramatically improved the outcome in multiple myeloma in the past years. Although the majority of patients achieves a remission, almost all patients eventually relapse. Previous studies have found recurrent mutations in TP53 and FAM46C (Weinhold et al., Blood 2016) in relapsed multiple myeloma as well as CRBN mutations (Kortüm et al., Blood 2016) specifically in lenalidomide-treated patients.Methods: In order to identify additional genetic aberrations that are associated with therapy resistance, we analyzed CD138-selected multiple myeloma cells in bone marrow samples obtained pretreatment (n=12) and at different time points at progression during treatment (n=17) by fluorescence in situ hybridization (FISH) and whole exome sequencing (WES). Peripheral blood was used as a germline control. The median number of therapy lines between pretreatment and progression samples was 1 (range, 1 - 4). Progression samples were obtained after treatment with lenalidomide (n=16), bortezomib (n=14), or high-dose melphalan/autologous stem cell transplantation (n=9) alone or in combination or as sequential treatment. WES was performed by paired-end sequencing (read length 100 bp) on an Illumina HiSeq platform. The mean coverage was 117x. A variant allele frequency (VAF) of 5% was set as a cut-off for mutation calling.Results: In the pretreatment samples, the median number of cytogenetic aberrations was 2 (range, 1 - 4) and the median number of somatic variants was 39 (range, 24 - 63) per sample. There was no significant change in these numbers in posttreatment samples: median number of cytogenetic aberrations 2 (range, 0 - 4), median number of somatic variants 52 (range, 11 - 107).The most recurrent cytogenetic alterations at diagnosis were +1q21 (n=6), +9q34.2 (n=4), and del13q14 (n=3). At relapse, 13 of the 18 cytogenetic abnormalities detected at diagnosis were still present, 5 were lost and 5 were newly acquired. The most frequently lost aberration was +9q34.2 (n=2) and the most commonly acquired aberrations were +1q21 (n=2) and del13q14 (n=2). In pretreatment samples we found recurrent mutations in NRAS (n=4), KRAS (n=3), DIS3 (n=3), and IGLL5 (n=3). The median stability of mutations between pre- and posttreatment samples was 62% (range, 13 - 81%). NRAS mutations were lost in 2 progression samples and DIS3 in 1 progression sample while KRAS mutations remained stable. We found 430 relapse-specific mutations in the 17 posttreatment samples. Mutations in TP53 (n=4) were the most frequently newly acquired genetic events in progression samples, which is consistent with previous studies. Of the newly acquired gene mutations, only eight genes were affected in more than one progression sample, all other gene mutations were non-recurrent. Among genes involved in the mechanism of lenalidomide we found a DDB1 mutation in one progression sample after lenalidomide treatment. Another patient had an IKZF3 mutation at relapse. However, the mutation was located outside the lenalidomide degron sequence and the VAF was only 5%, suggesting that it was not responsible for lenalidomide resistance in the major clone. No relapse-specific mutations were found in other genes encoding proteins involved in lenalidomide or proteasome inhibitor activity like IKZF1, IRF4, CRBN, CUL4A, CUL4B or proteasome subunits. Pathway analyses revealed that mutations in genes involved in WNT/beta catenin and KRAS signaling were enriched in the progression samples.Conclusions: In conclusion, we found a high degree of genetic heterogeneity in relapsed multiple myeloma. There were no obvious mutations in genes involved in lenalidomide or proteasome activity that might drive drug resistance. TP53 mutations and +1q21 were the most recurrent gene aberrations identified at relapse, however, these events may cause a general robustness of multiple myeloma cells to multiple treatment modalities and might not be treatment specific. These results suggest that the genetic basis for drug resistance is heterogeneous and warrants genetic characterization of further patients by DNA and RNA sequencing as well as functional analyses of candidate genes and pathways. DisclosuresKronke:Celgene: Honoraria, Other: Travel Support; Takeda: Consultancy, Other: Travel Support.

Genomic Complexity in Chronic Lymphocytic Leukemia Defined By Array-Based Analysis: Definitions, Associations with Other Biomarkers and Clinical Impact

Background: The advent of efficacious but also very expensive novel treatment regimens for high-risk chronic lymphocytic leukemia (CLL), has highlighted the need for accurate risk-stratification. Published evidence indicates that classic chromosome banding analysis (CBA) offers valuable complementary information to the routinely performed FISH analysis in CLL, potentially improving risk assessment. Indeed, recent studies identified complex karyotype (CK; defined as ≥3 abberations) as an independent predictive marker for refractoriness to ibrutinib and venetoclax, although for CLL, a precise evidence-based definition of genomic complexity (GC) is yet to be made. Array-based (CGH/SNP) analysis is capable of globally assessing the CLL genome, does not require in vitro mitogens, offers increased detection depth, and allows for easier standardization.Aims: 1. Determine a prognostic relevant definition of GC in CLL by array-based analysis. 2. Head-to-head comparison of the sensitivity of CBA versus array-based analysis.Methods: 1911 CLL and monoclonal B-cell lymphocytosis (MBL) patients (CLL=1895, 99% and MBL=16, 1%, respectively) were analyzed with CGH or SNP arrays mostly performed within the first year from diagnosis and before treatment administration (66% and 84%, respectively). For a subgroup (N=206) both CBA and array results were available (<1 year in between analyses). Main features of the studied cohort: median age: 60.7 years, males: 1252 (68%), Binet: A/B/C:623/324/141 (57%/30%/13%), IGHV-mutated CLL (M-CLL): 536/1082 (50%), del(13q): 1020/1910 (53%), del(11q): 353/1911 (19%), TP53 aberrations (TP53 abs, deletions and/or mutations of the TP53 gene): 205/1911 (11%). Recurrent chromosomal aberrations i.e. del(13)(q14), del(11)(q22.3) and del(17)(p13.1) defined by array were included in all analyses irrespective of size, while the cut-off for other aberrations are indicated.Results: First, analysis was performed using a size cut-off ≥5Mb according to Schoumans et al (Genes Chrom Cancer 2016). Applying the criteria ≥3 structural and/or numerical aberrations to define GC, 451/1911 cases (24%) displayed GC analyzed with CGH/SNP arrays. GC was significantly related (p<0.001) with advanced clinical stage (Binet B/C), IGHV-unmutated CLL (U-CLL), del(11q), del(13q) and TP53 abs. On univariate analysis, GC was associated with inferior OS (median: 10.0 vs 12.2 (p<0.001) years). GC cases were stratified into those with 3 (‘low-GC’, N=174, 39%), 4 (‘intermediate-GC’, N=103, 23%) and ≥5 (‘high-GC’, N=174, 39%) aberrations. High-GC cases differed significantly (p<0.05) from the other two subgroups, being enriched for U-CLL and TP53 abs (84% and 45%, respectively). The median OS was 5.8 years for the high-GC vs 10 years for the low- and 8.1 years for the intermediate-GC group (p<0.01). Only ‘high-GC’ remained significant (p<0.01) on multivariate analysis along with TP53 abs and IGHV status (see table).Lowering the array cut-off to 1Mb (N=353) resulted in detection of a higher number of aberrations (3.04 vs 2.34) compared to 5Mb cut-off (95% CI paired differences 0.475-0.924). Although a lower array cut-off resulted in upstaging of high risk GC (22 vs 12%), the median OS in the ‘high risk’ group was increased as compared to the cut-off of 5Mb (8.4 vs 5.5 years).Array-analysis (5Mb cut-off) showed a higher number of total detected aberrations as compared to CBA (2.35 vs 1.84, 95% CI paired differences 0.221-0.798). With array more patients were defined as GC with higher discriminatory power regarding survival (concordance index: 58 vs 55%) than with CBA. Prognostic impact of array vs CBA and of specific aberrations is currently being analyzed.Conclusion: According to this largest performed array-based analyses, current definitions for CK defined by the presence of ≥3 structural or numerical aberrations based on classical cytogenetic approaches may be suboptimal when applied for cases analyzed by array techniques. Instead, ≥5 aberrations detected by arrays were linked to high-risk disease, independent of clinical stage, IGHV and TP53 status. Since a lower array cut-off resulted in the detection of additional aberrations, without however been accompanied by the same dismal outcome as the 5Mb cut-off, further studies are warranted in order to define the actual prognostic relevance of smaller aberrations. [Display omitted] DisclosuresEldering:Roche: Research Funding; Gilead: Research Funding; Celgene: Research Funding. Ghia:Pharmacyclics LLC, an AbbVie Company: Consultancy; Janssen: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Adaptive: Consultancy; AbbVie: Consultancy; Roche: Consultancy; Novartis: Research Funding. Stamatopoulos:Janssen Pharmaceuticals: Honoraria, Research Funding; Novartis SA: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Strefford:Roche: Research Funding. Kater:Celgene: Consultancy, Research Funding; Johnson & Johnson: Research Funding; Abbvie: Research Funding.

Chromosomal complexity as a biomarker to de-escalate adjuvant imatinib treatment in high-risk gastrointestinal stromal tumor.

11535 Background: Gastrointestinal stromal tumors (GISTs) are characterized molecularly by oncogenic KIT or platelet-derived growth factor alpha ( PDGFRA) mutations. Malignant progression of primary GISTs occurs through stepwise accumulation of additional chromosomal aberrations, such as losses of chromosome arms 14q, 22q, 1p, 15q and Xp. After surgical resection of primary GIST, three years of adjuvant imatinib treatment is recommended for patients with an estimated high risk of recurrence. Still, nearly half of high-risk patients are cured by surgery alone, indicating that selection of patients could be improved. We hypothesized that high-risk GISTs with few chromosomal aberrations had a favorable outcome, and might not benefit from adjuvant therapy. The aim of the study was to investigate if chromosomal complexity could be used as a biomarker in de-escalation of adjuvant imatinib treatment. Methods: GIST patients undergoing surgical resection of their primary tumor between 1998 and 2020 were identified in the sarcoma database at Oslo University Hospital. All samples with available karyotype analysis made on fresh tumor tissue were included. Karyotypes were categorized as simple if they had ≤5 chromosomal changes, and complex if there were &gt; 5 chromosomal aberrations. Results: Chromosomal aberrations were detected in 226 tumors, of which 181 (80.1 %) were gastric. The most frequent resulting imbalances were loss of 14q (75.9 %), 22q (43.5 %), 1p (36.6 %), and 15q (29.6 %). One-hundred and thirty-six tumors (60.2 %) had simple karyotypes whereas 90 (39.8 %) were complex. Cytogenetically complex tumors were larger ( P&lt; 0.001), had a higher mitotic count ( P= 0.009), and were more often non-gastric ( P&lt; 0.001). There was a strong association between chromosomal complexity and risk classification according to the modified NIH criteria ( P&lt; 0.001). Thirty-eight of 58 (65.5 %) high-risk tumors were karyotypically complex compared to 37 of 144 (25.7 %) tumors that were not high-risk. In the high-risk group, 17 patients experienced disease recurrence, of whom one had a simple and 16 had a complex tumor karyotype. Estimated 5-year recurrence-free survival (RFS) for patients with simple tumor karyotypes was 94 % compared to 51 % for patients with cytogenetically complex tumors ( P= 0.004). Adjuvant and/or neoadjuvant imatinib treatment was administered to 40 high-risk patients with a median treatment duration of 33 months (range 2-60 months). A complex karyotype was associated with poor RFS both in patients with ( P= 0.016) and without ( P= 0.046) adjuvant imatinib. Conclusions: Chromosomal complexity was strongly associated with poor RFS in localized, high-risk GIST. Recurrences were infrequent for tumors with simple karyotypes, indicating that de-escalation of adjuvant imatinib treatment should be further explored in patients with cytogenetically simple GISTs.

Secondary cytogenetic abnormalities in core-binding factor AML harboring inv(16) vs t(8;21)

Patients with core-binding factor (CBF) acute myeloid leukemia (AML), caused by either t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22), have higher complete remission rates and longer survival than patients with other subtypes of AML. However, ∼40% of patients relapse, and the literature suggests that patients with inv(16) fare differently from those with t(8;21). We retrospectively analyzed 537 patients with CBF-AML, focusing on additional cytogenetic aberrations to examine their impact on clinical outcomes. Trisomies of chromosomes 8, 21, or 22 were significantly more common in patients with inv(16)/t(16;16): 16% vs 7%, 6% vs 0%, and 17% vs 0%, respectively. In contrast, del(9q) and loss of a sex chromosome were more frequent in patients with t(8;21): 15% vs 0.4% for del(9q), 37% vs 0% for loss of X in females, and 44% vs 5% for loss of Y in males. Hyperdiploidy was more frequent in patients with inv(16) (25% vs 9%, whereas hypodiploidy was more frequent in patients with t(8;21) (37% vs 3%. In multivariable analyses (adjusted for age, white blood counts at diagnosis, and KIT mutation status), trisomy 8 was associated with improved overall survival (OS) in inv(16), whereas the presence of other chromosomal abnormalities (not trisomy 8) was associated with decreased OS. In patients with t(8;21), hypodiploidy was associated with improved disease-free survival; hyperdiploidy and del(9q) were associated with improved OS. KIT mutation (either positive or not tested, compared with negative) conferred poor prognoses in univariate analysis only in patients with t(8;21).

Evidence of Cooperation between Hippo Pathway and RAS Mutation in Thyroid Carcinomas.

Simple SummaryRAS mutations have been reported in a wide range of thyroid cancer histological types, from benign to aggressive phenotypes. The presence of RAS mutations in benign lesions suggests that the mutation alone is unlikely to lead to a malignant transformation per se, and thus, additional aberrations are necessary for this transformation. In this study, we initially screened a cohort of 120 thyroid carcinomas with a panel of known driver mutations and identified 11 RAS-mutated samples. An RNA-Seq analysis of those 11 RAS-positive samples identified that the Hippo pathway was both mutated and differentially expressed in the RAS-positive tumors. The gene expression analysis of 60 RAS-positive The Cancer Genome Atlas (TCGA) papillary thyroid carcinomas (PTC) samples supported our findings. Thyroid cancer incidences have been steadily increasing worldwide and are projected to become the fourth leading cancer diagnosis by 2030. Improved diagnosis and prognosis predictions for this type of cancer depend on understanding its genetic bases and disease biology. RAS mutations have been found in a wide range of thyroid tumors, from benign to aggressive thyroid carcinomas. Based on that and in vivo studies, it has been suggested that RAS cooperates with other driver mutations to induce tumorigenesis. This study aims to identify genetic alterations or pathways that cooperate with the RAS mutation in the pathogenesis of thyroid cancer. From a cohort of 120 thyroid carcinomas, 11 RAS-mutated samples were identified. The samples were subjected to RNA-Sequencing analyses. The mutation analysis in our eleven RAS-positive cases uncovered that four genes that belong to the Hippo pathway were mutated. The gene expression analysis revealed that this pathway was dysregulated in the RAS-positive samples. We additionally explored the mutational status and expression profiling of 60 RAS-positive papillary thyroid carcinomas (PTC) from The Cancer Genome Atlas (TCGA) cohort. Altogether, the mutational landscape and pathway enrichment analysis (gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genome (KEGG)) detected the Hippo pathway as dysregulated in RAS-positive thyroid carcinomas. Finally, we suggest a crosstalk between the Hippo and other signaling pathways, such as Wnt and BMP.

Aberrations in (3+1)-dimensional Bragg diffraction using pulsed Laguerre-Gaussian laser beams

We analyze the transfer function of a three-dimensional atomic Bragg beamsplitter formed by two counterpropagating pulsed Gaussian laser beams. Even for ultracold atomic ensembles, the transfer efficiency depends significantly on the residual velocity of the particles as well as on losses into higher diffraction orders. Additional aberrations are caused by the spatial intensity variation and wavefront curvature of the Gaussian beam envelope, studied with (3+1)D numerical simulations. The temporal pulse shape also affects the transfer efficiency significantly. Thus, we consider the practically important rectangular-, Gaussian-, Blackman- and hyperbolic secant pulses. For the latter, we can describe the time-dependent response analytically with the Demkov-Kunike method. The experimentally observed stretching of the $\pi$-pulse time is explained from a renormalization of the simple Pendell\"osung frequency. Finally, we compare the analytical predictions for the velocity-dependent transfer function with effective (1+1)D numerical simulations for pulsed Gaussian beams, as well as experimental data and find very good agreement, considering a mixture of Bose-Einstein condensate and thermal cloud.