e15604 Background: Microsatellite instability (MSI) and DNA mismatch repair defect (dMMR) are dependable predictive biomarkers of colorectal cancer (CRC) response to checkpoint inhibitors. We explored the correlation between mutations in genes encoding various DNA repair proteins and other genes to PD-L1 expression in CRC. Methods: In this study, 264 CRC tissue samples were collected in a community setting and were evaluated for molecular abnormalities. TP53, KRAS, BRAF, and PTEN mutations were detected with next generation sequencing, PTEN deletions with FISH, PD-L1 expression with IHC, MLH1 promoter methylation and MSI with PCR. Correlations were evaluated using standard statistical Chi Square and Kruskal-Wallis tests. Results: MSI in CRC correlated directly with age (P < 0.0001), MLH1 promoter methylation (P < 0.0001), and BRAF mutation (P < 0.0001), and inversely with PTEN (P < 0.0001), KRAS (P = 0.002), and TP53 (P = 0.005) mutations. PD-L1 expression directly correlated with MSI (P = 0.0047), MLH1 promoter methylation (P = 0.004), and BRAF mutation (P < 0.0001). There was no correlation between PD-L1 expression and TP53 or KRAS mutations. Conclusions: CRC has been shown to express immune checkpoint proteins differently than other cancers. While PD-L1 expression may not be a good predictor of response to checkpoint inhibition, MSI predicts benefit well. The demonstration of a direct correlation between MSI and BRAF and PTEN mutation suggests that therapy targeting these abnormalities may be effective when combined with checkpoint inhibitors. Prevalence and Correlations of Mutations, MSI, and PD-L1 Prevalence Mutant or Expressing MSI Type of correl. PD-L1 Pvalue Pvalue Age N/A 0.04 Positive NS PD-L1 24.9% ( 56/225) 0.005 Positive 0.005 KRAS 52.4% (129/246) 0.002 Negative NS TP53 58.9% (145/246) 0.005 Negative NS BRAF 15.1% ( 31/205) of PD-L1 samples 9.8% ( 21/215) of MSI samples < 0.0001 Positive 0.004 KRAS +/or TP53 81.3% (200/246) < 0.0001 Negative N/A K/RAS 52.4% (129/246) KRAS 0.001 Negative NS MLH1 promoter methylation 20.7% ( 28/135) of PD-L1 samples 23.6% ( 56/237) of MSI samples < 0.0001 Positive 0.01 PTEN Mutation 5.7% ( 14/246) < 0.0001 Positive 0.007

BACKGROUND:The role of MET amplification in lung cancer, particularly in relation to checkpoint inhibition and EGFR WT, has not been fully explored. In this study, we correlated PD-L1 expression with MET amplification and EGFR, KRAS, or TP53 mutation in primary lung cancer.METHODS:In this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for MET amplification by FISH with a MET/centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and TP53, KRAS, and EGFR mutations were tested using next generation sequencing.RESULTS:Our results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with EGFR mutation (P=0.0003), and positively correlated with TP53 mutation (P=0.0001) and MET amplification (P=0.004). Patients with TP53 mutations had significantly higher MET amplification (P=0.007), and were more likely (P=0.0002) to be EGFR wild type. There was no correlation between KRAS mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and KRAS with TP53 co-mutation (P=0.0002). A cut-off for the ratio of MET: centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as MET amplified.CONCLUSIONS:This data suggests that in lung cancer both MET and TP53 play direct roles in regulating PD-L1 opposing EGFR. Moreover, KRAS and TP53 co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification.

3022 Background: DNA mismatch repair deficiency (dMMR) can be tested by immunohistochemistry (IHC) or microsatellite instability (MSI). While either IHC and MSI is adequate for establishing Lynch syndrome, the relevance of discordant results in selecting patients for immune checkpoint treatment is unknown. We investigated MSI and IHC in detecting dMMR and correlated with PD-L1 expression. Methods: Community-based practice tissue samples were submitted for PD-L1 expression and dMMR by both IHC and MSI. PD-L1 testing was performed by IHC using clone 22C3, dMMR using IHC against four MMR proteins (MHL1, MSH2, MSH6, and PMS2), and MSI using PCR with five Bethesda markers. Results: Of the 396 cases tested for both PD-L1 and dMMR by IHC, 18 (4.5%) were reported dMMR positive. Of the 610 cases tested for both PD-L1 and dMMR by MSI, 27 (4.4%) were dMMR positive. The dMMR positivity was determined as having at least one MMR protein expressed at ≤ 6%. There was no statistically significant correlation between PD-L1 expression and the presence or absence of dMMR as detected by IHC. In contrast, patients with MSI had significantly higher PD-L1 positive cells when PD-L1 expression is considered as a continuous variable (P = 0.04), and at cut-offs of 5% (P = 0.003) and 10% (P = 0.004). When a cut-off point of 6% for IHC is used, 8.9% of positive cases by MSI were negative (FN) by IHC and 2.6% of MSI negative cases were positive (FP) by IHC. If a 20% cut-off for IHC is used, FP was at 4.4% but FP was at 3.6%, and if a 30% IHC cut-off is used, FP was at 3.1% and FP was at 5.7%. This difference between cut-off points was statistically significant (P = 0.0008 for 20% and P = 0.0001 for 30% cut-off). Conclusions: There is significant correlation between PD-L1 expression and dMMR as detected by MSI, but not by IHC testing. Based on this and the established association between tumor mutation burden and MSI, MSI should be considered the gold standard for dMMR testing for checkpoint blockade therapy consideration. dMMR by IHC cut-off MSI % positive % IHC FP % IHC FN Pos Neg Total by MSI by IHC 6% Pos 267 16 283 32.38 31.27 8.9 2.6 Neg 26 596 622 20% Pos 280 22 302 32.38 33.37 4.4 3.6 Neg 13 590 603 30% Pos 284 35 319 32.38 35.25 3.1 5.7 Neg 9 577 586 Total 293 612 905

While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases. Between 2014 and 2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of the patients tested by FISH, 505/3908 (12.9%) also had IHC data. Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. However, 50.5% of equivocal cases were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe: 78% of positive cases by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (p < 0.0001) among the equivocal cases by conventional FISH testing, 44% of IHC negative cases became positive while 36% of the positive IHC cases became negative by alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH. The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine the benefit of anti-HER2 therapy in these patients are urgently needed.

Abstract Introduction: MET gene activation has been reported to be associated with resistance to EGFR inhibitors in lung cancer. Resistance to EGFR inhibitors is also reported to be associated with upregulation of PD-L1. We studied PD-L1 expression levels with MET gene amplification and mutation in EGFR, KRAS, or TP53 in a large number of primary untreated lung cancers. Methods: Tissue samples collected from 397 core biopsies or resections from lung cancers were studied for MET gene amplification by fluorescent in-situ hybridization (FISH) using MET (7q31) probe and centromere 7 as a control. Signals were quantified. PD-L1 expression on the same samples was evaluated using clone SP142 and standard immunohistochemistry (IHC) procedure. Samples were also sequenced using next generation sequencing (NGS) for mutations in TP53, KRAS, and EGFR. Results: PD-L1 expression was detected in 166 patients (42%). Twenty seven (7%) had expression between 1% and 5%, 61 (15%) between 1% and 20%, 92 (23%) between 1% and 50%, and 105 (26%) had PD-L1 > 50% in tumor cells. Ratio of MET: centromere signals was > 1.5 in 16 (4%) of patients. Patients with MET ratio > 1.5% had significantly higher (P=0.004) percentage of PD-L1 as a continuous variable as well as when cut-off points of 5% (P=0.01), 20% (P=0.0006), and 50% (P=0.01) were used. Patients with EGFR mutation had significantly lower levels of PD-L1 expression (P=0.003). When a cut-off point of 50% is used for PD-L1 expression, the EGFR-mutant cases had significantly less number of positive cases (P=0.0003). There was no correlation between the presence of High MET: Ratio and EGFR mutation. There was no correlation between KRAS mutation and overall PD-L1 expression (P=0.4). There was no correlation between MET ratio and KRAS mutation. Patients with TP53 mutation had significantly higher MET ratio when a cut-off at 1.5 is used (P=0.01). Also there was significant correlation between TP53 mutation and overall PD-L1 expression (P=0.0001). This remained significant when cut-off point of 50% or 5% are used (P=0.0004 and P=0.007, respectively). TP53 mutation was significantly more common in EGFR wild-type cases (P=0.0002). Conclusions: Extra copies of MET gene as detected by FISH testing in therapy-naïve lung cancer patients is associated with higher expression of PD-L1, while EGFR-mutant lung cancers had significantly lower expression of PD-L1 when clone SP142 is used and NGS is used for detecting EGFR mutations. Patients with TP53 mutation had strikingly high expression of PD-L1 using SP142 clone and higher copy number of MET gene. This data suggests that in lung cancer, both MET and TP53 genes play a direct role in regulating PD-L1 expression opposing the EGFR gene, which appears to suppress PD-L1 expression. KRAS gene may not be involved in PD-L1 expression in lung cancer. Citation Format: Maher Albitar, Sucha Sudarsanam, Wanlong Ma, Shiping Jiang, Wayne Chen, Vincent Funari, Steven Brodie, Sally Agersborg. Correlation between MET gene amplification and TP53 mutation in upregulating PD-L1 expression in EGFR wild-type lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5618. doi:10.1158/1538-7445.AM2017-5618

e14500 Background: PD-L1 expression as detected by immunohistochemistry (IHC) is significantly lower in colorectal cancers (CRC) when compared with lung cancer or other types of cancer. We explored if mutations in the RAS/RAF gene family, TP53 or PIK3CA can define a subgroup of CRC that express PD-L1. Methods: Tissue samples collected from 107 patients with CRC were studied for the expression of PD-L1 using clone SP142. The same samples were also tested for mutations in NRAS, KRAS, HRAS, BRAF, TP53, and PIK3CA using Next Generation Sequencing (NGS). Results: Of the 107 CRC samples only 15 (14%) showed PD-L1 positive tumor cells (≥1%) and 8 of the 15 (7.5% of total) had PD-L1 in ≤5% of tumor cells. Detected mutations in these samples were as follows: TP53 65%, KRAS 49.5%, PI3KCA 22.5%, NRAS 5%, HRAS 1%, and BRAF 17%. There was no correlation between PD-L1 expression and mutation status in any of the RAS/RAF genes. There was also no correlation between TP53 mutation and PD-L1 expression. This was true irrespective if PD-L1 expression is considered as a continuous variable or when cut-off points of 5%, 20%, or 50% were used. However, patients without any mutation in RAS or TP53 had significantly (P = 0.005) more expression of PD-L1 when cut-off point of 5% is used. This remained true if PD-L1 expression is considered as a continuous variable (P = 0.04). There was no correlation between PIK3CA and PD-L1 expression. Conclusions: PD-L1 expression is significantly more common in CRC that lack mutations in RAS or TP53. PD-L1 expression is detected in 31% of patients with wild-type RAS/TP53 as compared with 12% in patients with RAS/TP53 mutations (P = 0.04). If a cut-off point of 5% is used, 31% of RAS/TP53-wild-type CRC were positive for PD-L1, while only 6% of RAS/TP53- mutant CRC were positive for PD-L1 (P = 0.005). This suggests that in CRC without RAS/TP53 mutation, the PD-L1 may play a more important role in oncogenesis. Exploring immunotherapy in this group of CRC patients might be justified.

e21050 Background: BRAF mutation and PD-L1 expression appear independent in melanoma. We have established that PD-L1 clones 22C3 and 28.8 show almost identical results. In this study we correlated between BRAF mutation and PD-L1 expression as detected by 22C3/28.8 and SP142 in melanoma clinical samples. Methods: Melanoma samples were tested for PD-L1 expression and BRAF mutation. IHC testing for PD-L1 22C3 or 28.8 were tested using FDA-approved kits as recommended. Testing with SP142 (Spring Biosciences; LDT) was performed using standard techniques. BRAF testing in combination with SP142 PD-L1 testing was performed using next generation sequencing covering exons 11 and 15. Samples tested with 22C3 and 28.8 clones were tested using FDA-cleared BRAF kits (Cobas, Therascreen) testing V600 only. Results: 68 samples were tested for PD-L1 expression with clone 22C3, 67 with 28.8, and 56 with SP142. There was no overall statistical difference between the three clones in PD-L1 expression (P = 0.41). For combined 22C3 and 28.8, 61 of 135 cases (45%) had PD-L1 expression and 31 (23%) had BRAF mutation detected using FDA kits (V600). There was no statistical correlation between PD-L1 expression and BRAF mutation in this group (P = 0.9) at any cut-off. In cases tested with SP142 clone, BRAF mutation was detected in 31 cases (55%) by NGS, higher than using the FDA kit (P < 0.0001). In V600 mutations by NGS, 14 cases (25%) were positive, similar FDA kits. In SP142 cases, PD-L1 was positive in 45% of cases. There was no correlation between BRAF mutation and PD-L1 using SP142 expression as a continuous variable (P = 0.59) or cut-off points of 5% or 50%. When cut-off of 20% was used, significant inverse correlation with BRAF mutation (P = 0.004) was identified and was maintained if V600 codon only was evaluated. Conclusions: There is no correlation between BRAF mutation and PD-L1 expression as detected using clones 22C3 and 28.8. With SP142, a negative correlation between PD-L1 expression and BRAF mutation is identified with a 20% cut-off. The rate of BRAF mutations practically doubles when NGS is used and exons 11 and 15 are included in the testing, in contrast to FDA approved testing.

e15064 Background: Current recommendations for colorectal cancer testing include KRAS and NRAS for anti-EGFR therapy, and BRAF mutational analysis with microsatellite instability (MSI) testing for prognostic stratification and Lynch syndrome. We developed a multi-modality colorectal cancer profile useful for clinical management. Methods: 133 Colorectal cancer samples were profiled with our platform, which includes 1) 21 gene mutation analysis using deep sequencing (ca. 15K reads), 2) MSI status, 3) MLH1 Promoter Methylation, 4) MET amplification and PTEN deletion by FISH, and 5) PD-L1 expression (30% of samples) by IHC. Results: 99% of tumors contained at least 1 mutation found by NGS. The most commonly observed mutations were TP53(85%), KRAS (49%), PIK3CA(26%), BRAF(19%), EGFR(16.5%), NRAS(8%), FGFR3(6%), HRAS(5%), KIT (5%), SMO(5%), JAK3(5%), and ERBB2(5%). 39 patients had a TP53 mutation allele frequency consistent with germline mutations, raising the possibility of a Li–Fraumeni syndrome. Most tumors (51%) also had at least 1 abnormal non-sequencing result. The most common findings were PTEN deletion (25%), MLH1(17%) methylation, MSI(11%), PDL1 (17.5%) overexpression. PTEN/MET/PDL1 was anticorrelated to MSH/MLH status. (p < 0.05). Our integrated profile (NGS, FISH, MSI, MLH) robustly recapitulated hypermutation profiles that were associated with BRAF+/MLH+/MSH+ profiles using comprehensive WGS in a recent TCGA study. In addition, 25%(10) were KRAS-/MLH-/MSH-/BRAF- of unknown prognosis without PTEN deletion status. The integrated profile also identified ~2 percent (3/131) of patients as candidates for Lynch Syndrome testing (MSH+/MLH-/BRAF- and < 50 years). Conclusions: Multimodality Colorectal Cancer profiling identified patients with potential new targeted therapy. Specifically, we identified a significant number of cases that were PTEN, PI3KCA, HRAS, FGFR3, MET, KIT, ERBB2, and PDL1 positive that change treatment options. In addition, to identifying patients with prognostic status (e.g.BRAF+, MSI+/MLH+), we identified important candidates for Lynch syndrome testing or possibly Li-Fraumeni Syndrome who would benefit from alternative treatments and different management.

11557 Background: Expression of PD-L1 is, in general, associated with response to immunotherapy. However, it is believed that additional intrinsic factors play a role in determining the potential of response to immunotherapy. Toward this goal we investigated the relationship between mutation profile and PD-L1 expression in lung and colorectal cancers. Methods: Molecular profiling using a panel of gene 24 genes was performed by next generation sequencing (NGS) on 158 non-small cell lung cancer (NSCLC) and 42 colorectal cancers. The genes studied included ERBB2, FGFR1, FGFR2, FGFR3, SRC, JAK3, ERBB4, ERBB2, and SMAD4. In addition, these tumors were studied for the expression of PD-L1 using immunohistochemistry (IHC). PD-L1 expression was performed using standard IHC approach using SP142 clone (Spring Biosciences). Results: The level of PD-L1 expression was significantly (P = 0.0005) lower in colorectal cancer as compared with NSCLC. The NSCLC cohort had significantly (P = < 0.0001) more cases with 3 or more genes mutated as compared with colorectal cancer. However, there was no significant difference in TP53 mutation frequency between the two tumor types. There was no correlation between PD-L1 expression and the presence or absence of 3 or more gene mutations in either NSCLC or colorectal cancer. However, PD-L1 expression was significantly (P = 0.01) higher in tumors with TP53 mutation in the NSCLC cohort, but not in the colorectal carcinoma cohort (P = 0.5). This was true whether PD-L1 level was used as a continuous variable or if it was dichotomized at 20% (P = 0.02) or 40% (P = 0.03) cutoffs. Conclusions: There is significant difference between NSCLC and colorectal cancers in PD-L1 expression levels. More importantly, TP53 mutation in NSCLC correlates with the expression of PD-L1 protein, but not in colorectal cancer despite similar rate of mutation of the TP53 between the two tumor types. This suggests a possible difference in the mechanism of regulating PD-L1 expression between the two tumor types.

e23146 Background: Companion diagnostic tests are currently recommended for the prescription of multiple therapeutic agents in oncology. Most of these companion tests are FDA-cleared kits. We compared the mutations detected in large number of solid tumors using next generation sequencing (NGS) and confirmation by Sanger sequencing with FDA-cleared testing kits. Methods: Samples from 822 patients were submitted for routine clinical testing for mutations in EGFR, BRAF, and KRAS. This included 442 lung cancers, 168 colorectal, 29 (4%) brain tumors, 33 melanomas, 14 thyroid cancers, and others. All samples were tested by NGS, then confirmed by Sanger sequencing. LNA was used to increase sensitivity of Sanger for EGFR T790M mutation, codons 12/13 for KRAS, and V600 for BRAF mutations. We compared our results with listed detectable mutations in the FDA-approval labeling for EGFR, KRAS and BRAF for cobas tests (Roche, Indianapolis, IN) and Therascreen tests (Qiagen, Germantown, MD). Results: We detected 55 unique EGFR mutations in 99 patients. Of all 99 patients with EGFR mutations, 39 patients (39%) had mutations in codons not covered in the commercial cobas v2 kit and 63 (64%) were not covered by the older cobas v1 kit. Therascreen test also did not cover 64 (65%) mutations detected in EGFR. We detected mutations in BRAF in 114 patients, but only 61 patients had mutations involving codon V600 and 53 (46.5%) patients had mutations involving other codons. These mutations would have been missed if tested using FDA-cleared tests. We detected mutations in KRAS in 320 patients, of which 284 mutations (89%) involved codons 12 and 13. Sixteen (5%) mutations involved codon 61 and 20 (6%) mutations involved other codons. Therefore 6% of patients with KRAS mutation would have been missed if the cobas KRAS test (P = 0.00002) is used and 11% if Therascreen is used. Conclusions: Currently available FDA-cleared kits for testing for mutations in EGFR, KRAS, and BRAF genes are not comprehensive enough and miss significant number of mutations. Genes Detected mutations Missed cases by FDA-Cleared kits Total Unique Cobas V1 Cobas V2 Therascreen EGFR 99 55 63 (64%) 39 (39%) 64 (65%) BRAF 114 42 53 (51%) NA 53 (51%) KRAS 320 30 NA 20 (6%) 36 (11%)

Introduction: Diagnosis of myelodysplastic syndrome (MDS) can be very difficult when blast count in bone marrow is <5%. The demonstration of a mutation in one or more of the MDS-related genes is usually considered an objective confirmation of MDS. However, recent reports suggest that normal individuals may have circulating clonal hematopoietic cells carrying MDS-related mutations. We studied the relevance the mutated allele frequency and number of mutated genes in confirming the diagnosis of MDS in patients with cytopenia as determined using bone marrow samples.

Introduction: Recent data suggest that MDS evolves by accumulating mutations. Early mutations may involve genes that require additional mutations prior to clinical manifestation as MDS. We explored if mutant allele burden and the relative mutation of one gene to another gene could provide information on the interclonal and intraclonal progression of MDS using next generation sequencing (NGS) in patients with early MDS. Methods: NGS data was generated from 96 patients diagnosed with MDS with marrow blast count 10%. A difference of 10% to 20% was considered mild, 20%-30% moderate, and >30% severe. A heat map reflecting these differences in mutant allele frequency was generated. Results: In this group of early MDS patients, 63 patients (66%) had more than one gene mutated and 38 (40%) had a significant (>10%) difference in allele frequency. The median number of genes mutated was 2 (range 1 to 5). Difference in mutant allele frequency was severe in 15 patients (16%), intermediate in 15 patients (16%), and mild in 13 patients (14%). TET2 was the most commonly mutated gene (43 patients, 45%) and was rarely the sole mutation with most cases exhibiting a mutation in a second gene (39 patients, 91%). The mutant allele burden was highest in TET2 in 26 of these 39 patients (67%), reflecting early event in the tumorigenic process. Of the 13 cases with TET2 mutation and allele burden less than the companion gene, 6 had a mutation in SF3B1, 3 had significant cytogenetic abnormalities (monosomy 5, del(7q), and trisomy 8), 2 had a mutation in SRSF2, 1 had a mutation in ZRSR2 and 1 had a mutation in ASXL1, which suggests that these abnormalities might be the initiating event. A second TET mutation (biallelic mutation) was detected in 16 of the 39 patients. SF3B1 was the most common gene having a solitary mutation (10% of all patients), although mutation in SF3B1 was detected in 27 patients (26% of all patients). All solitary SF3B1 mutations were associated with normal karyotypes, except for one patient with del(11q). JAK2 was mutated with SF3B1 in two cases diagnosed as RARS-T (refractory anemia with ring sideroblasts and thrombocytosis). In one case, the JAK2 and SF3B1 mutation allele frequencies were similar, but in the other, the JAK2 mutant allele frequency was 23% higher, suggesting that a myeloproliferative neoplasm was the initiating process. ASXL1 was mutated in 14 cases, 13 of which had additional mutations. DNMT3A gene was mutated in 18 cases, 5 of which were solitary; two of these five showed cytogenetic abnormalities. TP53 was mutated in 13 cases, but except for one case, all had either mutation in another gene or a cytogenetic abnormality. Conclusion: These data suggest that in patients with clinically confirmed early MDS, TET2 mutations are most likely the initiating oncogenic event, but mutations in other genes or cytogenetic abnormalities most likely lead to clinically confirmed MDS. In contrast, patients with SF3B1 mutation can have clinical disease without additional mutations. Our data suggest that SRSF2, ZRSR2, and ASXL1 may initiate mutagenesis in patients with MDS. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is currently distinguished from myelodysplastic syndrome (MDS) based on the presence of 20% blasts in bone marrow, an arbitrary cut-off adopted by the WHO classification and replacing the 30% cut-off required by the older FAB (French, American and British) classification. Patients with t(15;17), t(8;21), or inversion 16 cytogenetic abnormalities are classified as having AML irrespective of the percentage of blasts. We explored the possibility that currently defined molecular abnormalities can distinguish AML from MDS without relying on an arbitrary percentage of blasts in the bone marrow. We compared the molecular profiles obtained by next generation sequencing (NGS) from consecutive patients with a clinical diagnosis of AML or MDS by WHO criteria.