ALBERT

Description: Germ line mutations in growth factor independent-1 (GFI1) have been described in a small subset of patients (pts) with severe congenital neutropenia. Subsequently, the GFI136N polymorphism, present in 3-5% of controls, has been found overrepresented (11%) in pts with primary (p) acute myeloid leukemia (AML), conveying 1.6 fold risk of development of AML. Mutant GFI136S and N variants lack affinity to HOXA9 (overrepresented in corresponding AML cases), shows increased proliferative potential in vitro and accelerates RAS-driven myeloproliferative neoplasm (MPN) disease in mice. Whole exome next generation (WE NGS) technology facilitates comprehensive screens for the presence of both somatic and germ line genetic alteration. In this study, we used NGS to search for germline variants of the GFI1 gene. We screened 140 pts (mean age 66.8 years, range 44-85) with MDS and related disorders (MDS/MPN and secondary (s) AML) for the presence of GFI1 variants. We found non-synonymous variants in 11 cases (8%), including the previously described pathogenic p.S36N (n=8), p.P107A (n=2), or p.L400F (n=1), while the corresponding frequencies for these alterations were .04 and .001, .002 in the general population. This frequency appears comparable or higher to those previously reported for pAML, but our screen of the TCGA AML cohort, perhaps due to very low coverage for this gene, did not reveal any GFI1 polymorphisms. We next focused on the clinical features of altered GFI1 carriers. A significant proportion of GFI1 cases were younger (age

Description: While patients with del(5q) MDS treated with Lenalidomide (LEN) have a response rate as high as 70%, the efficacy of this drug is lower in non-del(5q) cases. Aside from the presence of del(5q), up front identification of potentially responsive patients is difficult, particularly as the mechanistic underpinnings of LEN response have not been elucidated. Although expression signatures of responders were described in 2008, they have not yet been translated into an actionable diagnostic test. Analyses of molecular lesions including somatic mutations and chromosomal defects may predict response to LEN in MDS. We performed deep targeted DNA sequencing on 62 genes in 111 cases of myeloid neoplasms (MDS, MDS/MPN, and MPN) treated with Len for at least 3 months for whom fully annotated clinical outcomes were available. Clinical parameters, FISH, SNP array-based karyotyping and metaphase cytogenetics were also included in our analysis. We assessed response according to IWG 2006 criteria and performed analyses for responses at 3 or 6 months of therapy. Of 111 LEN-treated patients, 77% had lower-risk MDS (IPSS Low /Int-1) and 23% higher-risk disease (IPSS Int-2/High/sAML). Regimens included either LEN alone (52%), or in various combinations (29%) LEN+azacytidine, TLK+LEN (1.8%) or high-dose chemotherapy (7+3)+LEN (0.9%). Any hematologic improvement, cytogenetic response, and complete response (BM) were achieved in 58%, 19% and 18% at 3 months and 84%, 44% and 30% at 6 months, respectively. Responders had better survival, with HR=0.55 (0.32, 0.94; P=.03). The mean age did not differ between responders and non-responders. Using IPSS scoring criteria, there was no difference in proportion of patients with lower-risk disease among responders and non-responders (73% vs. 81%). When IPSS-revised (-R) score was applied, there also was no significant difference between responder and non-responders with very low risk (4% vs. 7%), low risk (30% vs. 41%), intermediate risk (22% vs. 15%), high risk (29% vs. 22%), and very high risk (14% vs.12%). Refractory patients showed significantly lower platelet counts compared to responders (117 vs. 215 K/uL; P=.01). Responders tended to have higher reticulocyte counts prior to therapy compared to non-responders (0.5 vs. 0.3 M/uL; P=.07) and had significantly higher MCVs compared to refractory cases (99 vs. 91fL; P

Description: Key PointsT-cell repertoire deep sequencing clearly identifies the nucleotide and amino acid sequence of the immunodominant clone in T-LGL leukemia patients. Deep-sequencing results suggest that CD8+ T-LGL leukemia is characterized by specific CDR3 clonotypes that are private to the disease.

Description: Lenalidomide (LEN) is effective in alleviating anemia in patients with MDS. Patients with del5q show particularly good responsiveness to LEN, but this drug has a significant activity in MDS patients without del5q and those with MDS/MPN and MPN. Currently, apart from del5q no routine molecular biomarkers have been identified to better predict response to LEN. Here, based on ability to perform a broad, unbiased mutational screen using generation sequencing (NGS) we hypothesized that somatic mutational patterns may help identify patients with myeloid malignancies who would benefit from LEN. To that end, we identified a cohort of 92 patients with myeloid malignancies who were treated with LEN. Response criteria were by the 2006 International Working Group and only those who received at least 3 months of LEN were selected for outcome analysis. Sequencing was performed using targeted NGS for the 60 most common mutated genes (frequency 〉2%; 87% CI) identified by whole exome sequencing cohort (see another abstract from our group). Our approach focused on previously described and/or confirmed mutations, acknowledging that there may be some false negatives and our spectrum may not be entirely complete. For most of the cases confirmatory targeted Sanger sequencing for the top 19 mutations has been performed, in addition to NGS. For some analyses we also divided mutations into functional gene families; e.g., DNMT family (DNMT1/3A/3B), PRC2 family (EZH2/EED/SUZ12/JARID2/RBBP4/PHF1), IDH family (IDH1/2), RAS family (NRAS/KRAS/HRAS/NF1/RIT1/PTPN11), cohesin, various spliceosomal gene families among others. Median age was 68 years (range, 20-84); according to IPSS there were 66 were lower (Low and Int-1) and 24 higher risk (Int-2; RAEB1/2 and some sAML) patients. According to cytogenetic risk groups there were 60% low, 18% intermediate, 22% poor risk patients; 31% had del5q. Overall response rate was 61%; rate of CR (including marrow/cytogenetic CR) was 19.5%, PR 5%, any HI 37%, SD 4%; 34% of patient were refractory. The cohort was then dichotomized into “responders” and “refractory,” with responders classified as those achieving CR, PR or any HI for the purpose of this analysis. Response rate was higher among del5q compared to non del5q (69% vs. 57%). Within the whole cohort the most frequently mutated genes were SF3B1 (24%), ASXL1 (18%), TET2 (17%), DNMT3A (14%), RUNX1 (12%), and U2AF1 (10%). At least one of the spliceosomal gene mutations was found in 37 (40%) cases and these seemed to be mutually exclusive. We compared the mutations frequencies in responders vs. non responders and conversely response within mutant or wild type (WT) cases and prioritized them according to p value for subsequent analyses. For instance, ASXL1 (29% vs. 13%; p= .052), U2AF1 (20% vs. 4%; p= .025), DNMT3A (22% vs. 10%; p= .1), LUC7L2 (12.5% vs. 0%; p= .02), SETBP1 (12% vs. 2%; p= .071) and RAS family (12% vs. 4%; p= .2) mutations seem overrepresented among refractory cases. Albeit rare, mutations in ASXL1, U2AF1, and LUC7L2 were always associated with refractoriness. Among responders, mutations in RUNX1 (17% vs. 6%; p= .2), EZH2 (7% vs. 3%; p= .6), TP53 (6% vs. 3%; p= 1) were more prevalent. SETBP1 mutant cases were also more likely to be refractory. Interestingly, all 3 DDX41 mutant cases were responders. The results were augmented when mutations were grouped according to pathways showing e.g., that mutations in ASXL1/ASXL2 and SRSF2/ZRSR2/LUC7L2 were significantly associated with refractoriness (32%; p= .025 and 26%; p= .009, respectively). We next adopted the algorithm when multiple genes were combined in “either-or” fashion. Such an analysis revealed that patients with mutations in either/or ASXL1/U2AF1 (39% vs. 14%) were significantly associated with refractoriness (p= .007; OR 1.4). Similarly, patients harboring either/or RUNX1/EZH2 (22% vs. 6%) were significantly associated with response (p= .041, OR 3.8). Multivariate analyses confirmed U2AF1 mutation to be associated with refractoriness to LEN (OR 6.64; CI: 1.18-37.34; p= .032). Of importance is that presence of TP53 (4/5 mutant cases were responders) and RAS gene family mutations (4/6 refractory cases), usually associated to refractoriness to conventional chemotherapy, did not preclude response to LEN. Our results demonstrate that analysis of somatic mutational patterns may be helpful in predicting patients who most likely benefit or those who will be refractory to LEN therapy. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Sekeres:Celgene: Membership on an entity’s Board of Directors or advisory committees; Amgen: Membership on an entity’s Board of Directors or advisory committees. Maciejewski:Celgene: Honoraria, Speakers Bureau.

Description: Background: African-American (AA) patients (pts) have a younger age at diagnosis and worse outcomes compared to whites (WTs) across many cancers, including acute myeloid and lymphoblastic leukemias. This difference may be related to disease biology rather than access to medical care or socioeconomic status. The incidence of MDS and age at diagnosis in national cancer registries in AAs is lower than in WTs. Detailed biological and clinical characteristics and outcome of AA pts with MDS compared to WTs have not been defined. Methods: We collected mutational and clinical data on MDS pts diagnosed from 1/2000-1/2012. Next-generation gene-targeted deep sequencing of 62 common gene mutations (selected based on frequencies established in a separate cohort of MDS pts studied by whole exome sequencing) were analyzed as individual mutations and then grouped into several functional pathways which were hypothesized to characterize MDS pathogenesis. International Prognostic Scoring System-Revised (IPSS-R) score was calculated as described previously. Overall survival (OS) was measured from the time of diagnosis to time of death or last follow up. Time-to-event analyses were performed by the Kaplan-Meier method, with curves compared by log rank test. Differences among variables were evaluated by the Fisher’s exact test and Mann-Whitney U test for categorical and continuous variables, respectively. Results: Of 341 pts, 44 (13%) were AA. Comparing WTs to AAs, pts had a similar median age (68 for both), absolute neutrophil count (1.6 vs 2.23) X 109/L, hemoglobin (9.7 vs 9.4) g/dL, platelets (93 vs 91) X 109/L, and bone marrow blasts (2% vs 3%), respectively. IPSS-R risk category distribution for WTs and AA was: very low 15% vs 9%, low 35% vs 30%, intermediate 18% vs 18%, high 16% vs 23%, very high 10% vs 18%, and not applicable 6% vs 2%, respectively. Among AA pts, 25% had very poor risk cytogenetics per IPSS-R criteria (complex 〉3) compared to 10% of WTs (p=.008) which led to 41% of AA pts having high and very high risk IPSS-R scores compared to 26% of WTs (p=.035). Further, WTs were more likely to receive a treatment (86% vs 66%, p

Description: Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation, truncal obesity, gynecomastia, hypogonadism and other dysmorphic features, is known to be caused by germline (GL) mutations of plant homeo domain finger protein 6 (PHF6). PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and blood leukocytes. Human PHF6 is located on chrXq26.2. Recently, rare somatic nonsense mutations and deletions have been detected in patients with T-ALL and AML and found in some T-ALL cell lines. Patients with BFLS with PHF6 mutations have been reported to develop leukemia, suggesting PHF6 mutations may predispose cancer. Although the actual function and molecular pathogenesis is unknown, PHF6 has been suggested to be a tumor suppressor gene involved in the control of myeloid development. In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of marrow, CD3+ cells and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1072 cases were analyzed (728 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 21 cases with PHF6 mutations, 13 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. These results along with ours suggest that PHF6 mutations are common driver events. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, thus, PHF6 mutations occur at a frequency of 2.0% and are most frequently observed among patients with secondary AML (33%, P=.0021). Gender distribution showed a strong male predominance (76%), likely due to the location of PHF6 on chrX and indicating that retention of a single copy of PHF6 may be protective. SNP-array karyotyping showed that deletions of Xq, involving the PHF6 locus (Xq26), were present in about 1.2% of myeloid neoplasms and affect only female patients. As a family, plant homeo domain (PHD) finger genes are affected by mutations associated with various cancers. JARID1A, PHF23, NSD1 and NSD3 were described to serve as fusion partners with the NUP98 in a subset of AML cases. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.08). The most commonly associated somatic mutations were in RUNX1 (N=7; P=.001), U2AF1 (N=5), ASXL1 (N=5), IDH1 (N=4), and DNMT3A (N=4). Interestingly, 6/7 cases with concomitant PHF6 and RUNX1 mutations showed a poor prognosis AML. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene while RUNX1 mutations are acquired as secondary events. Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and an increase of DNA damage. To examine DNA damage and quantify double stranded breaks (DSBs) in primary cells from PHF6-mutants, those with wild-type (WT) PHF6 and normal bone marrow we used a flow cytometric anti-γH2AX assay, following induction of DNA damage with Camptothecin. As judged by greater percentages of anti-γH2AX labeled cells, DSBs were more common in mutant cases consistent with more DNA damage present in PHF6 mutant compared to WT MDS and normal bone marrow cells. In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with genomic instability and thereby provide a basis for a mutator phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

Description: Familial MDS has been rarely reported, with few cases usually being in the context of early-onset disease and germline (GL) mutations. Patients with familial RUNX1 mutations show thrombocytopenia and progression towards MDS/AML. Similarly, germ line-(GL) CEBPA and GATA2 mutations are associated with MDS/AML. Within typical MDS, late presentation makes it difficult to distinguish hereditary factors from aging or environmental exposures. Only in rare cases, a strong family history may suggest a genetic predisposition. Most of the NGS-based projects focused on discovery of somatic mutations, but it can ]also be used to search for disease prone GL-encoded sequence alterations. For the purpose of this study we hypothesized that in a proportion of seemingly sporadic MDS, known or new GL mutations may be present. They may predispose to disease evolution or constitute a first hit with a long latency, making assessment of penetrance difficult. Because of the tremendous amount of data generated through unbiased cross-sectional approaches and validation needed, we have devised various rational prioritization and analytic strategies: i) we stipulated that genes affected by somatic mutations may also be targets of GL alterations; ii) we screened for alterations in a panel of genes known to constitute predisposing factors to cancer and found in a limited WE NGS study (N=106) to be more frequent than expected in MDS. These genes were included in a somatic targeted panel of 81 genes. In total we have analyzed 459 patients with typical MDS and focused on results of the above described analytic strategies. All identified alterations were independently validated to exclude somatic lesions and technical artifacts. Following sequencing, tier-1 lesions were defined as known disease-prone sequence alterations/mutations, new nonsense/frameshift mutations and highly recurrent missense mutations exceedingly rare in the general population. Tier-2 lesions were defined as missense mutations with general population frequency of 1) MDS. Altogether (tier-1/2) we identified 281 non-synonymous GL sequence alterations in 209 cases (45%). Surprisingly, high frequencies of mutations in DNA repair genes (including ATM, ATR or FA genes) were found in heterozygous and even (4 cases) in biallelic configuration. We also identified adult patients with otherwise typical Li-Fraumeni TP53/CHEK2 mutations. In total we found 40 tier-1 FA gene mutations, 7 ATM and 6 telomerase complex mutations and many others. One of FA genes was found to be affected by a novel, recurrent, frameshift GL variant present in 4 cases. Interestingly, the same locus is mutated in 2 non-hematological cell lines and there are proximal somatic mutations reported in that region. Several clinical and molecular associations were found when patients with various tier-1 mutations were studied. For instance, the presence of heterozygous GL mutations in FA/DNA repair genes were associated with somatic deletion of chr.7 or del5q (P

Description: Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic myeloid neoplasms, in which disease progression is quite common, eventually terminating in secondary acute myeloid leukemia (sAML). To elucidate differential roles of mutations in MDS progression and sAML evolution, we investigated clonal dynamics of somatic mutations using targeted sequencing of 699 MDS patients, of which 122 were analyzed for longitudinally collected samples. Combining publicly available data, mutational data in a total of 2,250 MDS cases were assessed for their enrichment in specific disease subtypes. All samples were obtained after informed consent. Genotyping data from samples with low- (n=1,207) and high-risk (n=683) MDS as well as sAML (n=360) were available for most prevalently mutated 25 driver genes. In univariate comparison between low- and high-risk MDS, the majority of differentially mutated genes were enriched in high-risk MDS, except for SF3B1, which was more frequently mutated in low-risk MDS. Multivariate analysis was performed using a least absolute shrinkage and selection operator model. As a result, mutations in 7 genes (FLT3, PTPN11, WT1, IDH1, NPM1, IDH2,and NRAS) designated as 'Type-1' mutations, were significantly enriched in sAML compared to high-risk MDS. When comparison was made between high- and low-risk MDS, mutations in 10 genes, including GATA2, NRAS, KRAS, IDH2, TP53, RUNX1, STAG2, ASXL1, ZRSR2, and TET2, were enriched in high-risk MDS. The latter mutations are designated as 'Type-2' mutations, excluding NRAS and IDH2 mutations, which were already assigned to the Type-1 category. To characterize the chronological behavior of Type-1 and Type-2 mutations, we performed longitudinal analyses of 122 cases, of which 90 progressed to sAML. Overall, driver mutations tended to increase their clone sizes between two time points. In accordance with their significant enrichment in sAML, Type-1 mutations were more likely to be newly acquired at the second time points, compared to Type-2 and other mutations (P=0.0001). By contrast, in patients with high-risk MDS at the second time point, Type-2 mutations were more dominant than Type-1 mutations, and most of the Type-2 mutations (88%) increased their clone sizes at the second sampling. Similarly, Type-2 mutations found in high-risk MDS or sAML evolving from low-risk MDS increased their clone sizes more frequently (30 out of 38 mutations (79%)) than Type-2 mutations in stable low-risk MDS without disease progression over time (4 out of 11 (36%)) (P=0.02). These findings suggest that Type-1 and Type-2 mutations might be associated with progression from high-risk MDS to sAML and low- to high-risk MDS, respectively. To further clarify the effects of the different classes of mutations on progression to sAML, 429 patients with MDS were analyzed for progression free survival (or PFS). Patients with Type-1 mutations (Group-I) had a significantly shorter PFS, compared to those who had Type-2 mutations but lacked Type-1 mutations (Group-II) (HR=1.82, 95% CI:1.08−3.05; P=0.025). Nevertheless, PFS in Group-II cases was still significantly shorter than that in other cases (HR=2.46, 95% CI:1.43−4.23; P=0.001). Of note, some Group-II cases subsequently acquired Type-I mutations during progression to sAML. By contrast, SF3B1-mutated patients tended to show slower progression to sAML, unless they carried either of Type-1 or 2 mutations (Group-III). Finally, the effects of these mutations on overall survival (OS) were assessed in a larger cohort of patients with MDS (n=1,347). Group-I cases were shown to have a significantly shorter OS than Group-II cases (HR=1.50, 95% CI:1.20−1.86; P

Description: MDS and related disorders, including MDS/MPN and sAML that evolved from these conditions constitute disease continuum characterized by a wide spectrum of molecular lesions which often overlap. Here, we defined general mutational spectrum and clonal architecture in a large cohort (n=718) of MDS studied by whole exome sequencing (WES) and target deep sequencing. Within this cohort 97 cases were studied at multiple time points to clarify the clinical impact of clonal dynamics on phenotype commitment or outcomes. All samples were obtained after informed consent, according to protocols approved by the respective ethics boards of the participating institutions. When mean and maximum variant allele frequency (VAF) for whole mutations were at one time-point evaluated in disease phenotypes, significantly higher averaged values suggested their larger clones in sAML and CMML compared to MDS. Clustering analysis of multiple mutational events by Pyclone software discriminated the cases with multiple mutational clones (positive heterogeneity) and those with a single expansion of MDS clone (no heterogeneity detected). Over 80% of low-risk MDS and all the sAML harbored multiple clusters of mutations. These results suggest that intra-tumor heterogeneity of MDS is most likely due to various sizes of clonal and subclonal mutations, likely impacting clinical behavior. To delineate clonal dynamics in MDS, we assessed mutational burden and their temporal changes in serially collected samples (n=97). Among these, Pyclone analysis was applied to exome sequencing at two time points (n=11 pairs). All cases showed various mutational clusters with individual expansions and declines, including initially present, newly acquired or disappearing during clinical course. Initial subclones were identified at disease presentation in 55% of cases, of which in 86% the subclones expanded to occupy whole MDS population with clonal sweep. New subclones acquired during clinical course were identified in 91%, in which 60% cases harbored clonal sweep. Disappearing clones were observed in 55% of cases. Next, we applied clustering analysis on clonal size of driver mutations evaluated at multiple time points (n=97 cases) to categorize the most frequently mutated genes into 3 subtypes. Mutational burden of PTPN11 most frequently increased and were associated with leukemic evolution (an example of type I gene). Similarly, CBL, NRAS, STAG2, RUNX1, and IDH1 were categorized into the type I genes, demonstrating increased clonal size resulting in the evolutions into high-risk phenotypes. Although JAK2 mutations were related to the stable clinical course when the mutational burden decreased, cases with highly expanded JAK2 mutations resulted in leukemic evolution (occasional evolution or expansions; type II gene). DNMT3A, SRSF2, TP53, U2AF1, and ASXL1 mutations were also categorized into such type II consequences with occasional progression. The last category (type III) included clonal/founder genes EZH2, TET2, SF3B1 and PRPF8, demonstrating random shifts of clonal size and lack of association with leukemic evolution. The proposed hierarchical categorization correlates with clinical parameters. Cases with the increasing burden of type I gene mutations showed most significant increases in myeloblasts. Overall survival measured from second sampling time points in the cases with increasing type I mutations was significantly shorter in the whole cohort (HR=2.05, 95%CI; 1.14-3.79, P=0.016) and in the cases solely with IPSS INT-1 (HR=2.37, 95%CI; 1.01-5.97, P=0.048). Subcohorts classified according to the presence or absence of increasing type I mutations did not differ with regard to the IPSS categories. In contrast, increased mutational burden of type II and III genes did not correlated with any of the clinical parameters examined, even though some gene mutations including TP53, EZH2, and U2AF1 represented poor prognostic factors at disease presentation. In conclusion, this work demonstrates that detailed understanding of clonal dynamics allows for new insights into clinical significance of somatic mutations, made possible only by serial sample sequencing at multiple time points. Increasing clonal burden of extracted genes associated with predictive prognostic impact should be prospectively validated in more uniform and larger cohort of MDS. Disclosures Sekeres: TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Shih:Novartis: Research Funding.