Additional Molecular Aberrations Research Articles

Standards of Genetic Testing in the Diagnosis and Prognostication of Systemic Mastocytosis in 2022: Recommendations of the EU-US Cooperative Group

Mastocytosis comprises rare heterogeneous diseases characterized by an increased accumulation of abnormal mast cells in various organs/tissues. The pathogenesis of mastocytosis is strongly linked to the presence of KIT-activating mutations. In systemic mastocytosis (SM), the most frequent mutation encountered is KIT p.D816V, whose presence constitutes one of the minor diagnostic criteria. Different techniques are used to search and quantify the KIT p.D816V mutant; however, allele-specific quantitative PCR and droplet digital PCR are today the most sensitive. The analysis of the KIT p.D816V allele burden has undeniable interest for diagnostic, prognostic, and therapeutic monitoring. The analysis of non-mast cell hematological compartments in SM is similarly important because KIT p.D816V multilineage involvement is associated with a worse prognosis. In addition, in advanced forms of SM, mutations in genes other than KIT are frequently identified and affect negatively disease outcome and response to therapy. Thus, combined quantitative and sensitive analysis of KIT mutations and next-generation sequencing of other recurrently involved myeloid genes make it possible to better characterize the extent of the affected cellular compartments and additional molecular aberrations, providing a more detailed overview of the complex mutational landscape of SM, in relation with the clinical heterogeneity of the disease. In this article, we report the latest recommendations of the EU-US Cooperative Group presented in September 2020 in Vienna during an international working conference, on the techniques we consider standard to detect and quantify the KIT p.D816V mutant in SM and additional myeloid mutations found in SM subtypes.

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.

How does intestinal-type intraductal papillary mucinous neoplasm emerge? CDX2 plays a critical role in the process of intestinal differentiation and progression.

Intestinal-type intraductal papillary mucinous neoplasm (IPMN) of the pancreas is clinicopathologically distinctive. Our research aimed to elucidate the molecular mechanism of the development and progression of the intestinal-type IPMN. In 60 intestinal-type IPMN specimens, histological transitions from gastric-type epithelia to intestinal-type epithelia were observed in 48 cases (80%). CDX2/MUC2/alcian blue triple staining indicated that CDX2 appeared to precede MUC2 expression and subsequent alcian blue-positive mucin production. Expression of p21 and Ki-67 seemed to be accelerated by CDX2 expression (p = 6.02e-13 and p = 3.1e-09, respectively). p21/Ki-67 double staining revealed that p21 was mostly expressed in differentiated cells in the apex of papillae, while Ki-67 was expressed in proliferative cells in the base of papillae. This clear cellular arrangement seemed to break down with the progression of atypical grade and development of invasion (p = 0.00197). Intestinal-type IPMNs harbored frequent GNAS mutations (100%, 25/25) and RNF43 mutations (57%, 8/14) and shared identical GNAS and KRAS mutations with concurrent gastric-type IPMNs or incipient gastric-type neoplasia (100%, 25/25). RNF43 mutations showed emerging or being selected in intestinal-type neoplasms along with ß-catenin aberration. Activation of protein kinase A and extracellular-regulated kinase was observed in CDX2-positive intestinal-type neoplasm. These results suggest that gastric-type epithelia that acquire GNAS mutations together with induction of intrinsic CDX2 expression may evolve with clonal selection and additional molecular aberrations including RNF43 and ß-catenin into intestinal-type IPMNs, which may further progress with complex villous growth due to disoriented cell cycle regulation, acceleration of atypical grade, and advance to show an invasive phenotype.

Abstract SY25-04: Tumor and host factors regulating immunotherapy responsiveness

Abstract Checkpoint blockade immunotherapy is favored in patients having a T cell-inflamed tumor microenvironment at baseline. Therefore, absence of a T cell-inflamed phenotype represents a first approximation of immunotherapy resistance. Investigation of molecular features of tumor genomics, host genetics, and commensal microbiota that correlate with the degree of spontaneous T-cell infiltration has revealed multiple layers of pathways that impact on relative response versus resistance to immunotherapeutic interventions. The first tumor-intrinsic oncogene pathway demonstrated mechanistically to mediate T-cell exclusion from the tumor microenvironment and lack of checkpoint blockade efficacy is the Wnt/β-catenin pathway. Additional molecular aberrations correlated with deficient immune infiltration in various cancers include loss of function mutation or absence of PTEN, LKB1, or p53 and activation of FGFR3 or PPAR-γ. Mouse modeling has revealed that tumor cell-intrinsic β-catenin pathway activation also mediates downstream resistance to adoptive T-cell therapy, due to defective expression of key chemokines. In one patient case analyzed, transition from a β-catenin-negative to a β-catenin-positive phenotype has been associated with secondary resistance to immunotherapy. TCGA and other large genomic datasets have been an invaluable tool for identifying such molecular correlates, through availability of tumor RNAseq and exome sequencing data as well as germline SNP analysis. Host germline polymorphisms also have been identified that correlate with the degree of T-cell infiltration in the tumor, and mouse models are being utilized to investigate mechanisms. Specific commensal bacteria associated with the degree of T-cell infiltration also have been identified in animal models, and similar analyses are ongoing with human stool samples in patients undergoing checkpoint blockade therapy. A common feature of non-T cell-inflamed tumors is a relative lack of Batf3-dendritic cells and absence of a type I IFN gene signature. Thus, regulation at the level of innate immunity seems to be a key determining factor. Strategies aimed at boosting innate immune activation in the tumor microenvironment represent a major category of therapeutic intervention to convert non-T cell-inflamed tumors to T cell-inflamed, and STING agonists and probiotics are beginning to be investigated clinically. Citation Format: Thomas Frank Gajewski. Tumor and host factors regulating immunotherapy responsiveness [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 SY25-04. doi:10.1158/1538-7445.AM2017-SY25-04

Incidence and Prognostic Impact of Cytogenetics in Combination with Molecular Aberrations in Patients with Systemic Mastocytosis

Systemic mastocytosis (SM) usually arises as a consequence of an acquired mutation in the receptor tyrosine kinase KIT, usually KIT D816V (>80-90% of patients). The presence of additional molecular aberrations, e.g. in SRSF2, ASXL, or RUNX1 (S/A/Rpos), has a strong adverse impact on disease phenotype and prognosis in patients with advanced SM (advSM) (Jawhar et al., Leukemia 30, 2016). However, little is known about the frequency and prognostic impact of cytogenetic aberrations and potential interactions between cytogenetic and molecular aberrations in the context of prognostication. We therefore analyzed clinical, morphological, cytogenetic and molecular characteristics of 109 patients (KIT D816V, n=102, 94%; KIT D816H, n=2, 2%; no KIT mutation, n=5, 5%) with indolent SM (ISM, n=27) and advSM (n=82) with or without an associated hematologic neoplasm (AHN) [SM-AHN, n=60; aggressive SM, n=3; mast cell leukemia (MCL), n=9; MCL-AHN, n=10)]. Next-Generation Deep Amplicon Sequencing (NGS) was performed to investigate 18 candidate genes as previously described (Schwaab et al., Blood 122, 2013). All ISM patients had a normal karyotype. In advSM, an aberrant karyotype was identified in 16/82 (18%) patients. By analogy with other myeloid neoplasms, e.g. MDS or AML, patients were classified according to their karyotype into two groups. The good-risk group (n=73) included patients with a normal karyotype (n=66) plus those with a favorable karyotype (n=7; del(5q), n=4; trisomy 8, n=1; del(1q), n=1; del(12p), n=1), while the poor-risk group (n=9) included patients with a complex karyotype (defined as ≥3 abnormalities; n=8) or monosomy 7 (n=1). Seven of 9 (78%) patients with a poor-risk karyotype and 2/7 (29%) patients with a favorable karyotype progressed to secondary AML (n=7) or MCL (n=2) within a median observation time of 13 months (range, 0-26). Overall, the median overall survival (OS) of patients with a poor-risk karyotype was significantly shorter than in patients with a good-risk karyotype (4 vs. 41 months; hazard ratio, HR, 9.6, 95% CI 3.9-23.2; P<0.0001). Of the 67 cases that were tested for gene mutations 5/9 (56%) with a poor-risk karyotype and 31/58 (53%) patients with a good-risk karyotype were S/A/Rpos. Significant differences regarding OS were observed when comparing the following 3 groups (Figure): good-risk karyotype + S/A/Rneg (n=27) vs. good-risk karyotype + S/A/Rpos (n=31) vs. poor-risk karyotype (n=9). In 6 multi-mutated advSM patients with simultaneous occurrence of cytogenetic [del(5q), n=3; trisomy 8, n=3], we explored the clonal architecture in single-cell-derivedgranulocyte-macrophage colonies (CFU-GM; median number per patient, n=12; range 10-15) using fluorescence in situ hybridization and mutation-specific PCR followed by Sanger sequencing. None of the colonies were positive for both KIT D816V and the cytogenetic aberration, indicating that these abnormalities marked different clones. However, some cytogenetically positive colonies were tested positive for additional mutations such as JAK2 V617F or TP53. In colonies without cytogenetic aberrations, additional mutations, e.g. SRSF2, ASXL1, RUNX1 or TET2, were identified with or without KIT D816V, confirming our previous results that the KIT D816V mutation is not the initial (earliest) event in SM evolution (Jawhar et al., Leukemia 29, 2015). We conclude, that a) molecular and cytogenetic analyses should be routinely performed in patients with advSM in addition to clinical and morphological baseline staging because they may more accurately indicate risk of disease progression/transformation and poor prognosis and b) patients with SM-AHN and an aberrant karyotype frequently harbor two different clones, with one responsible for SM evolution and the other driving AHN evolution independent of KIT D816V. [Display omitted] DisclosuresValent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

The Genomic and Transcriptomic Landscape of Systemic Mastocytosis

Background and AimSystemic mastocytosis (SM) is characterized by a heterogeneous spectrum of disease variants with different clinical features and prognosis. In indolent SM (ISM), the clinical course is usually stable and patients (pts) have a normal life expectancy. In contrast, aggressive SM (ASM) and mast cell leukemia (MCL) are characterized by severe organ damage and are fatal forms with short survival times. All forms of SM share the presence of activating mutations in the KIT tyrosine kinase (most frequently, the D816V mutation), which suggests that cooperating events might be responsible for the profoundly different clinical presentation and outcome of advanced SM (ASM and MCL) as compared to ISM. However, no robust studies using genome-wide approaches to scan for additional molecular aberrations in ASM and MCL have been published to date. This prompted us to use exome and RNA sequencing and single nucleotide polymorphism (SNP)-arrays to search for novel (and potentially druggable) molecular lesions.MethodsSeven pts with MCL, 6 pts with ASM and 2 pts with ISM were studied using whole exome sequencing (WES) and single nucleotide polymorphism (SNP)-arrays. WES (80x) was performed on a Hiseq 2500 (Illumina). SNP-arrays were done using Cytoscan HD Arrays (Affymetrix). Matched normal/mast cell DNA was analyzed in all but 2 archival MCL cases for whom no germline DNA source was available. RNA sequencing was also performed in 3/7 MCL and 1/6 ASM cases for whom high quality RNA was available. Data analysis and integration was performed according to in-house optimized pipelines.ResultsSNP-arrays found arm-level copy number (CN) or copy-neutral loss-of-heterozygosity (CN-LOH) events in 5/7 MCL pts (loss of 7p, 8p, 9q, 12p, 13q and CN-LOH at 4q, 18q and 21q). In contrast, ASM and ISM showed only focal CN and CN-LOH events. Overall, a median of 28 (range, 20-78) and 77 (range, 55-132) submicroscopic CN and CN-LOH events, respectively, were detected. The number of events did not significant differ between ISM, ASM and MCL. A total of 54, 102 and 1835 genes were significantly found to be recurrently involved in loss, gain and CN-LOH events, respectively. The average number of nonsynonymous mutations identified by WES was 15 in ISM and 35 in ASM and MCL. Signature analysis showed that the majority of sequence changes were C>T transitions and C>A transversions. Among genes recurrently mutated in ≥2 patients or concurrently identified by WES and SNP-arrays or known to be associated with cancer, some had already been reported to be mutated in SM: ASXL1, TET2, CBL, RUNX1, NRAS, IDH1, SRSF2, SF3B1. Alterations were also found in genes not previously implicated in SM, including RUNX3, MLL2, MLL3, CDC27, TP53BP1, CCND3, NCOR2, BCORL1, ATM, WRN, ARID1B, ARID3B, KDM1B, ARID4A, SETD1A, SETD1B, PRDM1. Pathway analysis showed enrichment of genetic lesions affecting the following cellular processes: PI3K/Akt and MAPK pathways, calcium signaling, chromatin modification, DNA methylation and DNA damage repair. RNA-seq results, filtered against those of a set of normal bone marrow samples, revealed a high number of RNA chimeras involving two adjacent genes in the same transcription orientation and invariably joining the pre-last exon of the upstream gene to the second exon of the downstream gene - a fusion pattern suggestive of intergenic splicing following transcription read-through events. Deletions at the intergenic region was excluded by SNP-arrays for all the predicted transcription-induced chimeras. In addition, in all the 4 pts analyzed, retention of some or all introns in mature transcripts was observed for some genes, including known tumor suppressors (TS) and other key genes like TP53, BAX, ERCC2, WRN, FANCD2, ATR; U2AF2, U2AF1, SRSF6.Conclusions•SNP-array and WES analysis of SM revealed a heterogeneous landscape of molecular alterations, although some key cellular pathways were found to be recurrently altered.•More interestingly, RNA-seq uncovered perturbation of transcript elongation and splicing in all the advanced SM pts analyzed. Intron retention, in particular, seems to be a novel recurrent mechanism of TS gene inactivation at the posttranslational level in advanced SM. The mechanisms leading to intron retention and their cellular consequences deserve further investigation.Supported by AIRC (project code 16996), progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresSoverini:Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Cavo:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria. Valent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding. Martinelli:MSD: Consultancy; Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; BMS: Speakers Bureau.

Mutational Complexity Is Related To Disease Severity In Systemic Mastocytosis

Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal accumulation of mast cells in various tissues, predominantly skin, bone marrow and visceral organs. The morphologic phenotype and extent of organ infiltration/dysfunction are basis for the subclassification of SM into indolent SM (ISM), smoldering SM (SSM), SM with associated hematologic non-mast cell disease (SM-AHNMD), aggressive SM (ASM) and mast cell leukemia (MCL). A somatic point mutation in the kinase domain of the receptor tyrosine kinase (TK) KIT at position 816 (KIT D816V) is present in >95% of patients and plays a central role in the pathogenesis and diagnosis of SM. To further explore mechanisms contributing to the clinical diversity of SM, we analyzed 39 KIT D816V mutated patients with different SM subtypes [ISM, n=10; SSM, n=2; ISM-AHNMD, n=5 (CMML, n=2; MDS/MPNu, n=3); ASM, n=1; ASM-AHNMD, n=14 (CMML, n= 5, MDS/MPNu, n=4, HES/CEL, n=2; AML, n=3); MCL, n=3; MCL-AHNMD n=4 (MDS/MPNu, n=2; HES/CEL, n=1; MDS, n=1)] for the presence of additional mutations. We applied next-generation sequencing to investigate ASXL1, CBL, IDH1/2, JAK2, KRAS, MLL-PTD, NPM1, NRAS, TP53, SRSF2, SF3B1, SETBP1, U2AF1 at mutational hotspot regions, and analyzed the complete coding regions of EZH2, ETV6, RUNX1, and TET2. Additional molecular aberrations were identified in 24/27 (89%) patients with advanced SM (SM-AHNMD, 5/5; ASM/MCL, 19/23) while only 3/12 (25%) ISM/SSM patients carried one additional mutation each (U2AF1, SETBP1, CBL) (p<0.001). In TET2, 9 missense, 4 nonsense, 13 frameshift mutations and one in-frame deletion as well as one splice site mutation were found in 15/39 (39%) patients. Ten of 15 (67%) patients carried more than one TET2 mutation. In SRSF2, missense mutations were identified in 14/39 (36%) patients which were clustered at codon 95 (P95H, n=9; P95L, n=2; P95R, n=2), with one additional mutation identified at codon 18 (V18L, n=1). In RUNX1, 10 mutations (9 missense mutations, 1 frameshift) were identified in 9/39 (23%) patients. Ten CBL mutations were identified in 8 patients (8 missense mutations, 1 duplication, 1 splice mutation) and 8 ASXL1 mutations were identified in 8 patients (7 frameshift and 1 nonsense mutation). In 13 patients, two (TET2, n=8; KRAS, n=1), three (CBL, n=2) or four mutations (TET2, n=2) were found in a single gene. Less frequently affected genes were KRAS (n=4), NRAS, JAK2, U2AF1 (n=2, each), EZH2, SETBP1, and ETV6 (n=1, each). No mutations were identified in MLL, IDH1, IDH2, NPM1, SF3B1 and TP53. In advanced SM, 21/27 patients (78%) carried ³3 mutations and 11/27 patients (41%) exhibited ³5 mutations. The median number of mutations was 4. The concurrent presence of KIT-TET2-SRSF2 (10/39, 26%), KIT-SRSF2-RUNX1 (7/39, 18%), KIT-TET2-CBL (5/39, 13%), KIT-SRSF2-ASXL1 (4/39, 10%) and KIT-TET2-ASXL1 (4/39, 10%) was strongly associated with (A)SM/MCL-AHNMD in 16/16 (100%) of patients (CMML, 7/16, 44%; MDS/MPNu, 6/16, 38%; HES/CEL, 3/16, 19%). Clinical follow-up was available for 38 patients. Six of 38 (16%) patients died, all of whom had at least one additional mutation with 5 patients positive for ³3 and 2 patients for ³5 mutations. The median survival of all 26 patients with at least one additional mutation was 12 months. In contrast, none of the 12 cases with KIT D816V mutation only died. This translated into a significant inferior survival (p=0.019) for patients with additional mutations (figure 1). We therefore conclude that, in addition to the multilineage involvement by KIT D816V, the presence of additional molecular aberrations is a new molecular feature that may contribute essentially to the abnormal phenotype and behaviour of neoplastic mast cells in advanced SM and thus to the clinical diversity and prognosis of advanced mast cell disorders. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Comprehensive mutational profiling in advanced systemic mastocytosis

AbstractTo explore mechanisms contributing to the clinical heterogeneity of systemic mastocytosis (SM) and to suboptimal responses to diverse therapies, we analyzed 39 KIT D816V mutated patients with indolent SM (n = 10), smoldering SM (n = 2), SM with associated clonal hematologic nonmast cell lineage disorder (SM-AHNMD, n = 5), and aggressive SM (n = 15) or mast cell leukemia (n = 7) with (n = 18) or without (n = 4) AHNMD for additional molecular aberrations. We applied next-generation sequencing to investigate ASXL1, CBL, IDH1/2, JAK2, KRAS, MLL-PTD, NPM1, NRAS, TP53, SRSF2, SF3B1, SETBP1, U2AF1 at mutational hotspot regions, and analyzed complete coding regions of EZH2, ETV6, RUNX1, and TET2. We identified additional molecular aberrations in 24/27 (89%) patients with advanced SM (SM-AHNMD, 5/5; aggressive SM/mast cell leukemia, 19/22) whereas only 3/12 (25%) indolent SM/smoldering SM patients carried one additional mutation each (U2AF1, SETBP1, CBL) (P < .001). Most frequently affected genes were TET2, SRSF2, ASXL1, CBL, and RUNX1. In advanced SM, 21/27 patients (78%) carried ≥3 mutations, and 11/27 patients (41%) exhibited ≥5 mutations. Overall survival was significantly shorter in patients with additional aberrations as compared to those with KIT D816V only (P = .019). We conclude that biology and prognosis in SM are related to the pattern of mutated genes that are acquired during disease evolution.

Outcomes of patients with advanced cancer and KRAS mutation in phase I clinical trials.

e22086 Background: The prognostic significance of KRAS mutations varies by mutation type. We compared the outcomes of patients with different KRASmutations treated in phase I clinical trials. Methods: Patients with advanced cancer and KRAS mutation who were treated in the phase I program from 9/08 to 12/12 were analyzed. The presence and type of KRAS mutation in the patients’ tumor samples were assessed in a CLIA-certified laboratory using PCR, sequenom analysis or next-generation sequencing. We compared clinical outcomes according to type of mutation, tumor, and therapy. Results: KRAS mutations were identified in 365 patients (codon 12, n=278; codon 13, n = 49; other codon, n = 38). The median age was 59 yrs (range, 18-87); and 46% men. Of 365 patients, 260 had KRAS mutation alone; 105 patients had 130 additional aberrations: PI3KCA, n=45 (39%); p53, n=29 (25.2%); PTEN, n=22 (19%); other, n=34 (29%). Of 260 patients, 159 were treated and evaluable for response. Outcomes in patients with KRAS mutation alone or with KRAS and additional molecular aberrations are shown in the Table. Conclusions: Keeping in mind that the number of patients was small, our results demonstrate heterogeneity in outcomes of patients with KRAS mutations. Combination studies of MEK inhibitors with other drugs may have improved antitumor activity. [Table: see text]

Panitumumab: A Summary of Clinical Development in Colorectal Cancer and Future Directions

Panitumumab is a fully human, monoclonal antibody targeting the EGF receptor with proven clinical activity in KRAS wild-type metastatic colorectal carcinoma. Treatment with panitumumab has been shown to significantly improve response rate and progression-free survival in this subgroup of patients, with a manageable toxicity profile. Panitumumab's first worldwide indication was as a single agent in chemorefractory patients. Recently, the EMA approved its use as part of a chemotherapy regimen in first- and second-line settings, following the encouraging results of large randomized Phase III trials. In order to identify patients with higher chances of benefiting from the treatment, additional molecular aberrations in the EGF receptor signaling pathway are being investigated as predictive biomarkers. In this article we review 10 years of drug development, focusing on the clinical evidence for panitumumab's indication in metastatic colorectal cancer and future strategies of investigation.

A pilot monocentric analysis of efficacy and safety of Fludarabine‐Campath combination (Flucam) as first line treatment in elderly patients with chronic lymphocytic leukaemia and Tp53 disfunction

A pilot monocentric analysis of efficacy and safety of Fludarabine‐Campath combination (Flucam) as first line treatment in elderly patients with chronic lymphocytic leukaemia and Tp53 disfunction

Characterization of CEBPA Mutations and Promoter Hypermethylation in Pediatric Acute Myeloid Leukemia.

Abstract 1605▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-631CCAAT/enhancer binding protein alpha (C/EBPá) function is frequently disrupted in acute myeloid leukemia (AML). This can be caused by different mechanisms, including mutations in CEBPA, the gene encoding for C/EBPá. Recently, promoter hypermethylation resulting in CEBPA silencing has been described. CEBPA mutations are associated with a favorable outcome in adult AML. Recent studies however suggested that the favorable outcome is uniquely associated with CEBPA double-mutated AML (CEBPAdoublemut) (presence of >1 CEBPA mutation), and not with CEBPA single-mutated AML (CEBPAsinglemut). In pediatric AML, data on outcome of CEBPA-mutated AML is limited to one study, showing favorable outcome for CEBPAdoublemut as well as CEBPAsinglemut, which is in contrast with the adult data. So far, data on CEBPA hypermethylation in pediatric AML is lacking.We therefore studied a large pediatric AML cohort (n=252) to characterize CEBPA mutations by sequencing the entire coding region, and CEBPA promoter hypermethylation by methylation-specific PCR (MSP). Survival analyses were performed in 185 patients with de novo AML (excluding t(15;17) and secondary AML) treated on uniform DCOG and BFM protocols. Furthermore, we generated gene expression profiles using the Affymetrix HGU133 plus 2.0 microarrays to compare subgroups with CEBPA aberrations.Thirty four CEBPA mutations were identified in 20/252 diagnostic samples (7.9%). In 14 cases double mutations were present, which combined an N-terminal frame shift mutation with an in-frame mutation in the bZIP region (n=13) or with a frame shift-causing insertion before bZIP (n=1). In 6 cases a single mutation was present; i.e. in-frame bZIP mutation (n=4), or frame shift mutation respectively in the TAD2 domain (n=1) or before the bZIP domain (n=1). CEBPAdoublemut were only present in children above 3 years of age and in FAB M1/2 subtypes, in contrast to CEBPAsinglemut, which presented also in children <3 years of age (1/6) and in other FAB subtypes (3/6). CEBPAdoublemut and CEBPAsinglemut were both exclusively found in cases with a normal karyotype (57% and 33%, respectively) and in cases with ‘other' karyotypes (defined as 'other than t(8;21), inv(16), t(15;17) and MLL-rearrangements'), in 36% and 50%, respectively. However, in both subgroups additional molecular aberrations, i.e. in RAS, FLT3/ITD and WT1, were equally distributed. CEBPAdoublemut patients (n=10) had a significantly better overall survival compared with CEBPAsinglemut (n=5) (5-years pOS 79±13% vs. 25±22%, p=0.04; pEFs 58±16% vs. 30±24%, p=0.16). Furthermore, they showed a trend for favorable outcome compared with CEBPA wild-type AML patients, after excluding CBF-AML cases (n=120; pOS 79±13% vs. 47±5%, p=0.07; pEFS 58±16% vs. 34±4%; p=0.06). Their survival was comparable to the CBF-AML subgroup (n=50) (pOS 91±4%, pEFS 61±8%). Multivariate analysis, including age, WBC, CBF-AML, NPM1 mutations and FLT3/ITD, showed that CEBPAdoublemut were an independent favorable prognostic factor for pOS (HR 0.23; p=0.04) and pEFS (HR 0.32; p=0.03). CEBPA promoter hypermethylation was detected in 3/237 cases, which resulted in CEBPA silencing. Using an unsupervised clustering analysis as previously published by Valk et al. (NEJM 2004) of our de novo AML cases (n=237), CEBPA-mutated cases predominantly aggregated in 1 cluster with the CEBPAsil cases, revealing a common underlying gene expression profile. Two additional cases with silenced CEBPA (CEBPAsil) were identified in this cluster, resulting in 5/237 (2.1%) CEBPAsil cases. All CEBPAsil showed T-lymphoid characteristics, (e.g. high expression of CD7 and high LCK expression). However, NOTCH1 mutations were not found. Three of 5 patients relapsed within 1 year of diagnosis, but the other 2 are in continuous complete remission for 4.8 and 8.5 yrs.In conclusion, CEBPAdoublemut were identified as an independent predictor of good clinical outcome. Hence, if these results could be confirmed in a prospective serie, CEBPAdoublemut may be used for further refinement of risk-group stratification. Of interest, the other cases with CEBPA aberrations, i.e. CEBPAsinglemut and CEBPAsil, seem to predict for poor outcome, in line with data presented in adults. The subgroup with CEBPAsil due to hypermethylation may potentially benefit from the use of demethylating agents. DisclosuresNo relevant conflicts of interest to declare.

Integrated Genomic Profiling Identifies Preferential Expression of the Insulin Receptor in CLL with Deletion 11q.

Chronic lymphocytic leukemia (CLL) is associated with characteristic genomic aberrations as defined by FISH and SNP arrays. CLL with del11q is characterized by a relatively aggressive clinical course, and about one third of cases carry mutations in ATM on the retained allele. Little information is available on additional molecular aberrations associated with del11q in CLL. We have analyzed del11q in a subset of 178 CLL cases using DNA from FACS-sorted CD19+ cells compared with buccal DNA using the Affymetrix 50k SNP array platform. Genomic copy number analysis using dChipSNP identified two non-overlapping minimal deleted regions on 11q, one of which spans ATM. We proceeded to use comparative expression array profiling of RNA derived from sorted CD19+ cells from 10 CLL cases with and nine cases without del11q using the Affymetrix U133 2.0 Plus platform. Analysis of normalized data identified ~85 probe sets with differential expression (del11q versus reference) that displayed a false discovery rate of <0.1. Approximately 70% of these genes were under-expressed and mapped to 11q22–23, thus providing confidence that genes lost through del11q displayed reduced expression under our assay conditions. Three probe sets identified the insulin receptor (INSR) to be expressed at substantially higher levels (log2 scale 2.5–3.1 fold) in del11q CLL as opposed to the reference cases that were used in the expression array-based analysis. Validation of differential INSR expression in CLL was accomplished by Q-PCR using cDNA synthesized from RNA purified from sorted CD19+ cells from 178 CLL cases. Using normalized Ct values for INSR expression (delta Ct INSR-GAPD) and a delta Ct cut-off of <10, 65% of CLL with del11q expressed the INSR as opposed to 15% (del13q14), 6% (del17p), 11% (normal FISH) and 23% (trisomy 12) and ~20% of all CLL cases, independent of genetic subtype. Finally, immunoblot analysis on cellular lysates derived from negatively enriched and >90% pure CLL cells from 10 cases with del11q and with high INSR expression versus cases without del11q and absent expression confirmed the accuracy of Q-PCR-based estimates of INSR expression. Work is in progress to measure effects of INSR expression on CLL survival and insulin-induced signaling in CLL in an effort to dissect functional consequences of INSR expression in CLL.