Cytogenetic and molecular predictors of response in patients with myeloid malignancies without del[5q] treated with lenalidomide

<p>Abstract</p> <p>Background</p> <p>While lenalidomide (LEN) shows high efficacy in myelodysplastic syndromes (MDS) with del[5q], responses can be also seen in patients presenting without del[5q]. We hypothesized that improved detection of chromosomal abnormalities with new karyotyping tools may better predict response to LEN.</p> <p>Design and methods</p> <p>We have studied clinical, molecular and cytogenetic features of 42 patients with MDS, myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes and secondary acute myeloid leukemia (sAML) without del[5q] by metaphase cytogenetics (MC) who underwent therapy with LEN.</p> <p>Results</p> <p>Fluorescence in situ hybridization (FISH) or single nucleotide polymorphism array (SNP-A)-based karyotyping marginally increased the diagnostic yield over MC, detecting 2/42 (4.8%) additional cases with del[5q], one of whom were responded to LEN. Responses were more often observed in patients with a normal karyotype by MC (60% vs abnormal MC; 17%, <it>p </it>= .08) and those with gain of chromosome 8 material by either of all 3 karyotyping methods (83% vs all other chromosomal abnormalities; 44% <it>p </it>= .11). However, 5 out of those 6 patients received combined LEN/AZA therapy and it may also suggest those with gain of chromosome 8 material respond well to AZA. The addition of FISH or SNP-A did not improve the predictive value of normal cytogenetics by MC. Mutational analysis of <it>TET2, UTX, CBL, EZH2, ASXL1, TP53, RAS, IDH1/2</it>, and <it>DNMT-3A </it>was performed on 21 of 41 patients, and revealed 13 mutations in 11 patients, but did not show any molecular markers of responsiveness to LEN.</p> <p>Conclusions</p> <p>Normal karyotype and gain of chromosome 8 material was predictive of response to LEN in non-del[5q] patients with myeloid malignancies.</p

Recovery index, attentiveness and state of memory after xenon or isoflurane anaesthesia: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Performance of patients immediately after anaesthesia is an area of special interest and so a clinical trial was conducted to compare Xenon with Isoflurane anaesthesia. In order to assess the early cognitive recovery the syndrome short test (SST) according to Erzigkeit (Geromed GmbH) was applied.</p> <p>Methods</p> <p>ASA I and II patients undergoing long and short surgical interventions were randomised to receive either general anaesthesia with Xenon or Isoflurane. The primary endpoint was the validated SST which covering memory disturbances and attentiveness. The test was used on the day prior to intervention, one and three hours post extubation. The secondary endpoint was the recovery index (RI) measured after the end of the inhalation of Xenon or Isoflurane. In addition the Aldrete score was evaluated up to 180 min. On the first post-operative day the patients rated the quality of the anaesthetic using a scoring system from 1-6.</p> <p>Results</p> <p>The demographics of the groups were similar. The sum score of the SST delivered a clear trend one hour post extubation and a statistically significant superiority for Xenon three hours post extubation (p < 0.01). The RI likewise revealed a statistically significant superiority of Xenon 5 minutes post extubation (p < 0.01). The Aldrete score was significantly higher for 45 min. The scoring system results were also better after Xenon anaesthesia (p < 0.001).</p> <p>Conclusions</p> <p>The results show that recovery from anaesthesia and the early return of post-operative cognitive functions are significantly better after Xenon anaesthesia compared to Isoflurane. The results of the RI for Xenon are similar with the previously published results.</p> <p>Trial Registration</p> <p>The trial was registered with the number ISRCTN01110844 <url>http://www.controlled-trials.com/isrctn/pf/01110844</url>.</p

The relationship of TP53 R72P polymorphism to disease outcome and TP53 mutation in myelodysplastic syndromes

Nonsynonymous TP53 exon 4 single-nucleotide polymorphism (SNP), R72P, is linked to cancer and mutagen susceptibility. R72P associations with specific cancer risk, particularly hematological malignancies, have been conflicting. Myelodysplastic syndrome (MDS) with chromosome 5q deletion is characterized by erythroid hypoplasia arising from lineage-specific p53 accumulation resulting from ribosomal insufficiency. We hypothesized that apoptotically diminished R72P C-allele may influence predisposition to del(5q) MDS. Bone marrow and blood DNA was sequenced from 705 MDS cases (333 del(5q), 372 non-del(5q)) and 157 controls. Genotype distribution did not significantly differ between del(5q) cases (12.6% CC, 38.1% CG, 49.2% GG), non-del(5q) cases (9.7% CC, 44.6% CG, 45.7% GG) and controls (7.6% CC, 37.6% CG, 54.8% GG) (P = 0.13). Allele frequency did not differ between non-del(5q) and del(5q) cases (P = 0.91) but trended towards increased C-allele frequency comparing non-del(5q) (P = 0.08) and del(5q) (P = 0.10) cases with controls. Median lenalidomide response duration increased proportionate to C-allele dosage in del(5q) patients (2.2 (CC), 1.3 (CG) and 0.89 years (GG)). Furthermore, C-allele homozygosity in del(5q) was associated with prolonged overall and progressionfree survival and non-terminal interstitial deletions that excluded 5q34, whereas G-allele homozygozity was associated with inferior outcome and terminal deletions involving 5q34 (P = 0.05). These findings comprise the largest MDS R72P SNP analysis

Cytogenetic Predictors of Response to Lenalidomide In Myeloid Malignancies without Del(5q)

Abstract Abstract 4016 Lenalidomide (LEN) is effective in patients with myelodysplastic syndromes (MDS) and the del(5q) cytogenetic abnormality, and has activity in a proportion of patients without this chromosomal defect. To date, no clinically actionable biomarkers, other than the presence of del(5q) detected by metaphase cytogenetics, have been identified to predict response to LEN. The diagnostic yield of metaphase cytogenetics can be enhanced by application of fluorescence in situ hybridization (FISH) for targeted chromosomal lesions including del(5q), as this technique is more sensitive. Similarly, single nucleotide polymorphism array (SNP-A)-based karyotyping, due to its superb resolution, allows for detection of previously cryptic unbalanced chromosomal defects. Both techniques can be preformed on interphase cells and thereby do not require cell division. We hypothesized that application of these technologies may allow for better identification of putative responders to LEN in patients with MDS without del(5q); we stipulated that i) we may detect previously unrecognized cases of del(5q) and that ii) more precise analysis of the karyotypes may allow for recognition of other chromosomal markers of response or refractoriness to LEN. In patients with MDS (N=82), MDS/myeloproliferative neoplasm (MPN) (N=13), acute myeloid leukemia (AML) (N=23), and MPN (N=4), the detection rate of del(5q) increased only marginally with use of additional techniques, from 24% (metaphase cytogenetics (MC) + FISH), to 25% (MC + SNP-A), 25% (FISH + SNP-A) and 26% (all 3 methods). Within this cohort, we then analyzed by FISH and SNP-A karyotype a subset of 42 patients with myeloid malignancies without del(5q) by MC who received LEN. This cohort included 33 MDS (RA, N=5; RARS, N=12; RARS-T, N=1; RCMD, N=1; RAEB1, N=4; RAEB2, N=6; MDS-U, N=4), 6 MDS/MPN and MPN patients (CMML1, N=1; CMML2, N=3, IMF; N=2) and 3 secondary AML. By MC, 32 (76%) showed normal karyotype, 1 (2.4%) no growth and 9 (21%) abnormal karyotype other than del(5q). The overall response rate (ORR) (2006 International Working Group criteria) was 44%, including 3 CR, 3 PR and 8 HI. Previously cryptic del(5q) was detected in an additional 1/18 patient by both SNP-A and FISH (secondary AML with normal metaphase cytogenetics), but this case was refractory to LEN. Del(5q) was also revealed by FISH in 1 patient with unsuccessful MC, but, due to the small size of the clone (8%), SNP-A did not detect this lesion. This patient had a sustained PR with transfusion independence. In 28 patients who received LEN for more than 3 months, the ORR to LEN in patients with normal metaphase cytogenetics was 62%, and 17% for those with chromosomal aberrations (p=.08); the addition of SNP-A did not improve the predictive value of normal cytogenetics. We also analyzed 10 patients without del(5q) by MC who received combination therapy with azacitidine (AZA) and LEN, for whom the ORR was 80% (7 CR, 1 PR). By metaphase cytogenetics, 7/10 patients had a normal karyogram and a response of 86%, compared to 3 patients with chromosomal lesions, 1 of whom responded. Similar to the results in LEN alone, inclusion of defects detected by SNP-A or FISH did not allow for better separation of responders based on normal cytogenetics by MC. Six out of 38 LEN-treated patients had a gain of chromosome 8 material by FISH or SNP-A. 4 out of 6 patients had CR (all of them received combination therapy of AZA and LEN), 1 out of 6 had HI, and 1 with complex karyotype had NR (ORR was 83%), while ORR in patients with other chromosomal abnormalities by FISH or SNP-A was 18%. In conclusion, FISH and SNP-A, when added to routine metaphase cytogenetics, marginally increased the diagnostic yield leading to detection of only 2/42 additional cases with del(5q). In our cohort, the non-del(5q) patients with normal karyotype and those with trisomy 8 or microduplication on chromosome 8 were associated with a favorable response to LEN. Disclosures: Sekeres: Celgene: Consultancy, Honoraria, Research Funding. List:Celgene: Research Funding. Maciejewski:Celgene: Research Funding

Spliceosome GENE MUTATIONS ARE Also PRESENT In the Diverse Mutational Spectrum of CHRONIC Myelomonocytic LEUKEMIA

Abstract Abstract 1402 Chronic myelomonocytic leukemia (CMML) is characterized by monocytic proliferation, cytomorphologic dysplasia and frequent progression to acute myelogeneous leukemia (AML). The molecular basis of CMML is poorly defined, although somatic mutations in a number of genes have recently been identified in a proportion of patients (epigenetic regulatory genes, spliceosomal genes, apoptosis genes, growth signal transducers and others). We performed a comprehensive analysis of molecular lesions, including somatic mutations detected by sequencing and chromosomal abnormalities investigated by metaphase and SNP-array karyotyping. We have selected a cohort of 72 patients (36 CMML1, 16 CMML2 and 20 sAML evolved from CMML). Our mutational screen performed in stages (as new mutations were discovered by our and other groups) and currently reveals mutations in UTX in 8%, DNMT3A in 9%, CBL in 14%, IDH1/2 in 4%, KRAS in 2.7%, NRAS in 4.1%, JAK2 in 1%, TET2 in 48%, ASXL1 in 43%, EZH2 in 5.5%, RUNX1 37%. Based on the discovery of various spliceosomal mutations in myeloid malignancies, novel mutations were also found in CMML, in U2AF1 in 12%, SF3B1 in 14%, SFRS19 in 6 % of cases tested. Chromosomal defects were detected in 60% of patients. In particular, a high frequency of somatic uniparental disomy (sUPD) were identified 71% of patients with abnormal cytogenetics, including UPD1p (N=3), UPD7q (N=8), UPD4q (N=6), UPD2p (N=2), UPD17q (N=2), UPD11q (N=5), UPDX (N=1), UPD21q (N=2). Some of the detected mutations were homozygous through their association with sUPD as for example for 3 EZH2, 1 UTX, 6 TET2, 2 DNMT3A, 5 CBL, 1 NRAS, 1 U2AF1 mutations. Furthermore, UPD17p implies that a P53 mutation is also present in this case as previously LOH17p was shown to be invariably associated with P53 mutations. Similarly, 2 cases of UPD17q imply that homozygous mutation of SRSF2, which is one of the Serine/arginine-rich splicing factor, may be present in this location and the mutation analysis is ongoing. In over 90% of >1 mutation was found but many patients harbored multiple mutations with frequent combinations of TET2/CBL or TET2/ASXL1 as well as RUNX1 and U2AF1 serving as examples. There was an accumulation of mutations from sAML, CMML2 and CMML1 suggesting stepwise accumulation of lesions. In serial studies, some of the mutations were present at the inception (e.g., TET2, ASXL1 and DNMT3A) in some cases originally heterozygous mutations were also while other can occur in the course of disease (e.g. CBL). RAS and DNMT3A mutations were associated with a higher blasts count. In sum, combined analysis of molecular lesions in CMML reveals that similar phenotype may be a result of diverse mutations associated with seemingly unrelated pathways and that clinical phenotype may be a result of a combination of mutations which accumulate as the disease progresses. Survival analyses will require large cohorts to account for various confounding factors including the presence of multiple chromosomal abnormalities and mutations in one patient, however currently EZH2, DNMT3 and CBL mutations appear to convey less favorable prognosis. Disclosures: No relevant conflicts of interest to declare

Identification of Oncogenic EZH2 Mutations In Myelodysplastic Syndromes and Related Myeloid Malignancies

Abstract Abstract 607 Using single nucleotide polymorphism arrays (SNP-A) as a karyotyping tool led to the recognition that segmental somatic uniparental disomy (UPD) is a common and important defect in myelodysplastic syndromes (MDS) and related conditions. Homozygous mutations associated with UPD have been found. Additionally, SNP-A-detected submicroscopic deletions have proven helpful in narrowing the search for mutant genes to shared regions of loss of heterozygosity (LOH). Mapping of such microdeletions was instrumental in the identification of TET2 and CBL mutations, frequently found in a homozygous constellation within UPD of the corresponding chromosomal regions. UPD7q is one of the most frequent areas of UPD present in MDS, MDS/MPN and sAML. We hypothesized that this region may be associated with the presence of pathogenic mutations in a homozygous configuration. We utilized two strategies: i) because of the large size of the area commonly affected by UPD7, we applied next generation sequencing (NGS) for the identification of affected genes, and ii) we systematically sequenced genes contained in the overlapping deleted regions. In total, 15 patients with UPD7q were identified. Exome libraries were generated from 2 cases with UPD7q and subjected to sequencing. In one, a missense C to T mutation was found at nt148137376 of EZH2 (100% of sequences), resulting in R690H. The somatic origin of this homozygous mutation was confirmed by sequencing of DNA from a skin biopsy. Simultaneously, we identified 2 patients with overlapping microdeletion involving 7q36.1. The minimally affected area involved 2 genes: CUL1 and EZH2. Sequenced all exons of both genes revealed no mutations in CUL1. However, a mutation in EZH2 exon 19 was identified in proximity to the mutation detected through NGS. The mutation involved position Ile715 producing a frame shift. We detected EZH2 mutations in 10/351 patients (7% of MDS/MPN, 2% of MDS and 0.9% of AML). We found 8 different EZH2 mutations present in 4/15 (27%) of cases with UPD7q, 2/30 (7%) patients with deletion 7q and 4/306 (1%) without LOH. Of note is that both hemizygous mutations were found in patients with microdeletions and none of the patients with large del(7q) or monosomy 7 harbored EZH2 mutation. There were 8 missense and 2 frame shift mutations, located in the SET domain of the EZH2 gene. Diagnoses included 3 cases of refractory cytopenia with multilineage dysplasia, while 5 had myelomonocytoid malignancies, and one each with MDS/MPN unclassifiable and atypical chronic myeloid leukemia (aCML). Overall, EZH2 mutations were detected in 4/53 (8%) cases of CMML, consistent with the high prevalence of somatic UPD7 in this disease. Interestingly, no EZH2 mutant case had a chromosome 7 abnormality by metaphase cytogenetics, but SNP-A karyotyping detected cryptic LOH7q in 6/10 patients with EZH2 mutations. EZH2 is a polycomb associated gene encoding a methyltransferase targeting H3K27, thereby producing a repressive mark. Loss of function or hypomorphic mutations of EZH2 would thus be predicted to result in abrogation of inhibitory chromatin marks and chromatin decompaction, conducive to expression of oncogenes. Immunofluorescence and western blot showed that EZH2 mutations in myeloid malignancies lead to decreased methylation of H3K27 and thereby are functionally relevant. Expression analysis of EZH2 did not show significantly decreased expression in MDS or haploinsuffciency in patients with del7/7q. In a patient with aCML and UPD7q, homozygous R690H EZH2 mutation was detected and trimethylated H3K27 was abrogated while H3K9 methylation appeared normal, consistent with functional impairment of the SET domain. Interestingly, an EZH2 mutation was associated with heterozygous TET2 S733fsX21 and heterozygous ASXL1 W538X mutations, suggesting multiple genes associated with epigenetic regulation may be mutated and act synergistically in malignant evolution. These triple mutant cells ultimately formed lethal tumors when injected to NOD SCID gamma mice. In summary, our investigations demonstrate EZH2 mutations in patients with myeloid malignancies frequently associated with UPD7q, or 7q36.1 microdeletion, but not monosomy 7 and del(7q). These findings suggest that an increasing possibility that mutations in the polycomb gene family represent a new class of molecular lesions conveying a clonal epigenetic instability phenotype and can constitute leukemogenic events. Disclosures: No relevant conflicts of interest to declare

Spliceosomal gene mutations are frequent events in the diverse mutational spectrum of chronic myelomonocytic leukemia but largely absent in juvenile myelomonocytic leukemia

Chronic myelomonocytic leukemia is a heterogeneous disease with multifactorial molecular pathogenesis. Various recurrent somatic mutations have been detected alone or in combination in chronic myelomonocytic leukemia. Recently, recurrent mutations in spliceosomal genes have been discovered. We investigated the contribution of U2AF1, SRSF2 and SF3B1 mutations in the pathogenesis of chronic myelomonocytic leukemia and closely related diseases. We genotyped a cohort of patients with chronic myelomonocytic leukemia, secondary acute myeloid leukemia derived from chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia for somatic mutations in U2AF1, SRSF2, SF3B1 and in the other 12 most frequently affected genes in these conditions. Chromosomal abnormalities were assessed by nucleotide polymorphism array-based karyotyping. The presence of molecular lesions was correlated with clinical endpoints. Mutations in SRSF2, U2AF1 and SF3B1 were found in 32%, 13% and 6% of cases of chronic myelomonocytic leukemia, secondary acute myeloid leukemia derived from chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, respectively. Spliceosomal genes were affected in various combinations with other mutations, including TET2, ASXL1, CBL, EZH2, RAS, IDH1/2, DNMT3A, TP53, UTX and RUNX1. Worse overall survival was associated with mutations in U2AF1 (P=0.047) and DNMT3A (P=0.015). RAS mutations had an impact on overall survival in secondary acute myeloid leukemia (P=0.0456). By comparison, our screening of juvenile myelomonocytic leukemia cases showed mutations in ASXL1 (4%), CBL (10%), and RAS (6%) but not in IDH1/2, TET2, EZH2, DNMT3A or the three spliceosomal genes. SRSF2 and U2AF1 along with TET2 (48%) and ASXL1 (38%) are frequently affected by somatic mutations in chronic myelomonocytic leukemia, quite distinctly from the profile seen in juvenile myelomonocytic leukemia. Our data also suggest that spliceosomal mutations are of ancestral origin. (C) 2013 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2012.064048981107113National Institutes of Health (Bethesda, MDNIH) [RO1HL-082983, U54 RR019391, K24 HL-077522]AA & MDS International Foundation (Rockville, MD, USA)Robert Duggan Charitable Fund (Cleveland, OH, USA)NIH) [RO1HL-082983, U54 RR019391, K24 HL-077522