Molecular Characterization Using Oncoscan Chromosome Microarray in an International Cohort of 51 Patients with Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Abstract Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematological malignancy with multi-organ and frequent skin involvement, and poor clinical outcomes. Based on the limited available data, the estimated incidence is 0.44% of all hematologic malignancies, representing less than 1% of acute leukemias, and 0.7% of cutaneous lymphomas. Due to the rarity of this entity, there have been relatively few studies characterizing the molecular profile of BPDCN. We examined a cohort of 51 patients with BPDCN using OncoScan chromosome microarray, which provides genome-wide copy number abnormality (CNA) analysis. Methods An international cohort of BPDCN cases were collected from centers in Brazil (Laboratorio de Patologia, Botucatu), Swtizerland (University of Zurich), France (Hospital St. Louis, Paris), Peru (Instituto Nacional de Enfermedades Neoplasicas, Lima), Canada (Department of Pathology, University of Montreal), Italy (Derpartment of Pathology, University of Bologna), and US (Department of Pathology - The Ohio State University, Department of Hematopathology - MD Anderson Cancer Center; and Department of Pathology - University of Virginia). A total of 58 tissue blocks from 51 patient samples were retrieved. The diagnosis of BPDCN was done and confirmed by at least three independent hematopathologists or dermatopathologists in accordance with the WHO classification (Lyon 2017). For the purpose of the molecular analysis substratification, cases were classified as 'BPDCN' if they were positive for TCF4, and 'BPDCN-like' if they were negative for TCF4. Immunohistochemistry for CD123, CD4, and CD56 was performed in all cases. Exclusion criteria included expression of MPO, lysozyme, CD3, CD19, CD20, CD22, and/or EBV. DNA was extracted from FFPE samples via standard techniques and processed on OncoScan CNV Plus microarray (ThermoFisher Scientific) according to manufacturer's recommended protocol. Copy number abnormalities and select single nucleotide variants and insertions/deletions (74 mutations in 9 genes) were analyzed on Chromosome Analysis Suite software (ChAS v4.1; ThermoFisher Scientific). Additional analysis was performed using Nexus Copy Number (BioDiscovery, version 10.0). Results To date, we have successfully analyzed 45 cases of BPDCN with Oncoscan, revealing widespread CNA in the vast majority of cases (44/45; 98%). Alterations of chromosome 9 were common in this cohort, particularly CNAs involving CDKN2A/B at 9p21.3. Twenty-five cases (56%) demonstrated CNA including CDKN2A/B, with ten of these cases demonstrating a homozygous loss of CDKN2A/B (22%). Alterations of chromosome 13 were also frequently detected with loss of RB1 (located at 13q14.2) detected in 24 cases (53%). The RUNX1 gene (21q22.12) was a common target of CNAs in this cohort, seen in nine cases (20%). Eight of these cases showed a copy number gain of RUNX1, which is a recurrent finding in a variety of hematological malignancies, particularly myeloid neoplasms. The remaining case with RUNX1 CNA showed a focal, homozygous loss of the gene, demonstrating that dysregulation of RUNX1 through CNA is a common event in BPDCN. We observed frequent deletions of ETV6 (53%), IKZF1 (33%), and TP53(16%) in our cohort. The ARHGAP26 gene (5q31.3), which is associated primarily with juvenile myelomonocytic leukemia, was included in CNA in 13 cases (29%), with both gains and losses observed in this cohort. Oncoscan can detect a limited number of single nucleotide variants in nine genes that are frequently mutated in cancers (BRAF, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, PTEN, and TP53). Mutations were detected in ten cases (22%), with NRAS and TP53 variants detected in three cases each and KRAS and IDH2 variants detected in two cases each. Conclusions Our preliminary data demonstrates complex genomic alterations in BPDCN, with the RB1 locus on chromosome 13, the CDKN2A/B locus on chromosome 9, and the ETV6 locus on chromosome 12 most commonly detected. However, widespread genomic alterations were detected involving a variety of cancer-associated genes further characterizing CNA in BPDCN. Analysis of additional BPDCN cases is progress. Disclosures Khoury: Kiromic: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy. Gru: StemLine: Honoraria, Research Funding, Speakers Bureau; CRISPT Therapeutics: Research Funding; Innate Pharma: Research Funding.

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Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): A Large Single-Center Experience: Analysis of Clinical and Molecular Characteristics and Patient Outcomes

Blood ◽

10.1182/blood.v126.23.3746.3746 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3746-3746 ◽

Cited By ~ 5

Author(s):

Naveen Pemmaraju ◽

Hagop M. Kantarjian ◽

Jorge E. Cortes ◽

Madeleine Duvic ◽

Joseph D Khoury ◽

...

Keyword(s):

Clinical Trial ◽

Clinical Trials ◽

Dendritic Cell ◽

Board Of Directors ◽

Research Funding ◽

Plasmacytoid Dendritic Cell ◽

Cell Transplant ◽

Advisory Committees ◽

Significant Difference ◽

Cell Neoplasm

Abstract Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy with heterogeneous clinical presentation and no available standard therapy. Little is known about the clinical characteristics, molecular characterization, and outcomes of patients (pts) with BPDCN. Methods: We conducted a retrospective review of pts age ≥18 years with a confirmed pathological diagnosis of BPDCN. Results: 37 pts evaluated at our institution between October 1998-June 2015 were identified. Table 1 shows baseline pt characteristics. Bone marrow (BM) was involved in 23 (62%), skin in 26(70%), lymph nodes in 11(30%), central spinal fluid (CSF) in 3 (8%) and 1 (3%) pt each had disease involving brain, uterus/ovary, elbow/soft tissue, and pleural fluid. Tumor immunophenotype demonstrated: CD4+ (31/32), CD56+ (29/32), TCL-1+ (19/21), CD 123+ (22/23). Additionally, CD22 was expressed in 3/9 pts. Frontline therapies received: 19 (51%) HCVAD; 5 (14%) CHOP, 5 (14%) clinical trials, 2 (5%) bortezomib-based, 1 AML induction with daunorubicin+ARAC, 1 oral MTX, 1 IFN-based therapy, 3 other regimens. 5 (14%) pts received radiation (XRT) as part of their therapy. Median follow-up time was 7 months [1-27 mo]. Median number of chemotherapy regimens was 1 [1-6]. Complete remission (CR1) (by standard AML criteria) was achieved in 19 pts (51%) with a median CR1 duration of 19 mo [1-39 mo]. Median overall survival (OS) was 23 mo [6-45 mo]. 23 (69%) pts died, the most common cause of death being multi-organ failure. Among 14 (38%) pts without BM involvement at diagnosis, all 14 had skin involvement. Comparison of pts with BM involvement versus skin-only showed no difference in outcomes. For pts with BM disease, median OS and median CR1 were 23 mo [1-45 mo] and 21 mo [1-39 mo], respectively. For pts with skin-only disease,median OS and median CR1 were 18 mo [1-31 mo] and 19 mo [1-23 mo], respectively, p =0.43 (OS), p=0.78 (CR1). 10 pts (27%) received stem cell transplant (SCT) [7 allogeneic (including 3 cord blood) and 3 autologous). The median OS for pts receiving SCT (n=10) was 18 mo [8-40 mo] versus 23 mo [1-45] for non-SCT group (n=27), p = 0.98. 19 pts (51%) received HCVAD as part of first-line therapy: median OS was 18 mo [1-45 mo] and median CR1: 21 mo [1-39 mo]. Out of 16 pts evaluable for response, 15 achieved CR1; 1 pt died at day 15 (pneumonia). A clinically validated 28-gene molecular panel (next-generation sequencing for commonly mutated genes in myeloid malignancies) is now being performed prospectively on all new pts with BPDCN seen at our institution (thus far, n=9); notably, all 9 have expressed some form of TET2 mutation [ordered mutations=3(c.1648C>T p.R550; c.3781C>T p.R1261C; c.4365del p.M1456fs*2)], ordered+variant=2,variants=4], confirming our earlier finding of occurrence of TET2 mutations in pts with BPDCN (Alayed K, et al Am J Hematol 2013). Thus far, there has been no statistically significant difference in terms of response rates in pts with known TET2 mutations/variants (n=9) vs all others/not done (n=26). Conclusions: Among patients with BPDCN, we observed an older, male predominance, a high incidence of TET2 mutations and, despite intensive chemotherapy and achievement of CR1 in many pts, most still experience relapse and short survival. Therefore, there is an urgent need for novel therapies. Therapies targeting cell surface CD123 and CD56, are available in 2 separate clinical trials at our institution: SL-401 (DT-IL3), which demonstrated 7/9 (78%) major responses including 5 CR, after a single cycle of therapy, (Frankel et al, Blood 2014) is currently being tested in an ongoing multicenter phase I/II trial (Stemline Therapeutics Inc, ClinicalTrials.gov Identifier: NCT02113982, refer to separate abstract ASH 2015) and Lorvotuzumab Mertansine (ImmunoGen, Inc), an antibody-drug conjugate targeting CD56 (ClinicalTrials.gov Identifier: NCT02420873), is in an ongoing ph II trial in CD56-expressing hematologic malignancies, including BPDCN. Table 1. Baseline characteristics (N = 37) Characteristic N (%) / [range] Median age, years 62[20 - 86] Male 33 (89) Median WBC x 109/L 5.9 [1.7-76.5] Median Hemoglobin g/dL 12.9 [6.8-17.1] Median Platelet x 109/L 130 [22-294] Median BM blast 13[0-95] Cytogenetics (n=27)DiploidComplexDeletion 12p13 17 8 1 Miscellaneous 1 28-gene profile (n=9); includes mutations& variantsTET2ASXL1MPLTP53IDH1IDH2 9 3 2 1 1 1 Disclosures Pemmaraju: Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Off Label Use: No standard of care available. clinical trial drug therapies/investigation/trial only various cytotoxic chemotherapies used in ALL, AML, other blood cancers. Cortes:BMS: Consultancy, Research Funding; BerGenBio AS: Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Duvic:Innate Pharma: Research Funding; Tetralogics SHAPE: Research Funding; Cell Medica Ltd: Consultancy; Array Biopharma: Consultancy; Oncoceutics: Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Spatz Foundation: Research Funding; Therakos: Research Funding, Speakers Bureau; Huya Bioscience Int'l: Consultancy; MiRagen Therapeutics: Consultancy; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Rhizen Pharma: Research Funding; Allos (spectrum): Research Funding; Soligenics: Research Funding; Eisai: Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding. Daver:ImmunoGen: Other: clinical trial, Research Funding. O'Brien:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding. Frankel:Stemline: Consultancy, Patents & Royalties, Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

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Prospective Investigation of Genetic Alterations in Osteolytic Lesions Compared to Paired Random Aspirates

Blood ◽

10.1182/blood.v128.22.4419.4419 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4419-4419

Author(s):

Sandra Sauer ◽

Jens Hillengass ◽

Barbara Wagner ◽

Daniel Spira ◽

Marc Andre Weber ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Iliac Crest ◽

Genetic Alterations ◽

Advisory Committees ◽

Single Nucleotide ◽

Osteolytic Lesions ◽

Myeloma Cells

Abstract Background: Bone disease is the most frequent clinical manifestation of multiple myeloma. In this prospective study we ask whether osteolytic lesions (OL) are driven by myeloma cells showing a different background of genetic alterations in terms of chromosomal aberrations and expressed single nucleotide variants (SNVs) compared to random aspirates (RA) from diffuse myeloma cell infiltration at the iliac crest (spatial genetic heterogeneity). Material and Methods: Consecutive sample-pairs (n=41) were prospectively obtained by CT-guided biopsies of OLs as well as simultaneous random bone marrow aspirates of the iliac crest, the latter undergoing CD138-purification of myeloma cells, in transplant eligible patients with previously untreated symptomatic multiple myeloma, after written informed consent. Peripheral blood mononuclear cells were used as germline control. Plasma cell infiltration in biopsies was quantified histologically. Samples pairs (n=8) were subjected to RNA-sequencing (Illumina HiSeq2000), gene expression profiling using DNA-microarrays (Affymetrix U133 2.0), whole exome sequencing (Illumina NextSeq 500), and arrayCGH (Affymetrix cytoscan array). Results and Discussion: Expressed single nucleotide variants.The spectrum of mutated genes in our samples comprises two of the most frequently mutated in symptomatic myeloma, i.e. KRAS and FAM46C, alongside those implicated in myeloma pathophysiology, e.g. mutations in IRF4, FGFR3, and CD200. In total, 1-10 clonal expressed non-synonymous SNVs were exclusively found in OL compared to RA, comprising e.g. WHSC1, FAM46C, and ROCK1P1. In 2/8 patients (25%), no expressed clonal differences between RA and OL were present. Single nucleotide variants.In investigated samples, 77-1569 non-synonymous SNVs appear with an allele frequency of ≥10% in OL and RA, clustering in 4-5 groups. The clonal constitution can vary, but subclones are detectable in both. Subclonal complexity is maintained (subclones remain present) in OL compared to RA, and the vast majority of subclonal changes is present in both, especially for expressed non-synonymous SNVs, incompatible with an "osteolytic clonal variant" driving OL in the majority of patients. Copy number alterations and loss of heterozygosity.Subtle differences in copy number between OL and RA are present. However, only 1/8 patients (12.5%) showed further "gained" aberrations in OL compared to RA, i.e. deletions on chromosome 7p, 8p, and 11p as well as 19p gain. Loss of heterozygosity was observed in 3/8 patients (37.5%) with a shared pattern between OL and RA in all of them. Conclusions: In our prospective study, the majority of alterations is shared between RA and OL. Spatial heterogeneity is present, but nature and frequency of alterations detectable exclusively in OL make them unlikely candidates in most myeloma patients for being causative for generation of OL. Disclosures Hillengass: Novartis: Research Funding; Sanofi: Research Funding; BMS: Honoraria; Celgene: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Goldschmidt:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Durie:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

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Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Is Highly BCL-2 Dependent and Sensitive to Venetoclax

Blood ◽

10.1182/blood.v128.22.4045.4045 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4045-4045 ◽

Cited By ~ 1

Author(s):

Joan Montero ◽

Jason Stephansky ◽

Tianyu Cai ◽

Gabriel K. Griffin ◽

Katsuhiro Togami ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cell ◽

Cytochrome C ◽

Board Of Directors ◽

Research Funding ◽

Plasmacytoid Dendritic Cell ◽

Cytochrome C Release ◽

Advisory Committees ◽

Cell Neoplasm ◽

Peptide Stimulation

Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a particularly aggressive hematologic malignancy with median survival of <12 months and no standard therapy. BPDCN involves the skin in nearly all patients, and frequently infiltrates bone marrow and lymph nodes. Its normal counterpart may be the plasmacytoid dendritic cell (pDC), leading to the name BPDCN. Outcomes are poor with cytotoxic chemotherapy. An interleukin 3-diphtheria toxin fusion (SL-401) has activity in BPDCN (Frankel, Blood 2014), but additional novel agents are urgently needed. BPDCN over-expresses the anti-apoptotic protein BCL-2 compared to normal pDCs (Sapienza, Leukemia 2014). We confirmed this by RNA-sequencing 12 BPDCNs and pDCs from 4 normal donors (reads/kb mapped [RPKM] 22.7 vs 1.33, P=0.0005). BPDCN shares some genetic characteristics with myeloid malignancies, and some acute myeloid leukemias (AMLs) are dependent on BCL-2. We performed RNA-seq on 6 BPDCN and 16 AML patient-derived xenografts (PDXs) and found higher BCL-2 expression in BPDCN (RPKM 48.2 vs 11.5, P=0.0005). We analyzed BCL-2 expression by immunohistochemistry in patient skin and bone marrow biopsies and found that all BPDCNs had BCL-2 staining that was equivalent to or stronger than that of any AMLs. We next tested BPDCN cell lines, primary patient samples, and patient-derived xenografts (PDXs) for BCL-2 dependence and sensitivity to the BCL-2 inhibitor venetoclax (previously ABT-199). Using BH3 profiling, we found that BPDCN is markedly dependent on BCL-2 to prevent mitochondrial cytochrome c release in response to apoptotic stimuli. In comparison to AML, BPDCN had significantly more cytochrome c release after BAD peptide stimulation (81.1% vs 11.8%, P<0.0001), suggesting greater dependency on BCL-2 and/or BCL-XL. BPDCN was uniformly sensitive to treatment with venetoclax in vitro, in cell lines and primary cells, as measured by direct cytotoxicity and Annexin V apoptosis assays. We used dynamic BH3 profiling (Montero, Cell 2015) in primary BPDCNs and PDXs (n=7) to measure early apoptotic signals after 4-hr exposure to venetoclax, avoiding the need for prolonged culture. BPDCNs were more likely than AMLs to undergo cytochrome c release in response to BIM peptide stimulation after 4 hours of venetoclax (increase in apoptotic potential, or "delta priming" 63.4% vs 14.5%, P<0.0001). Targeted sequencing of the BPDCNs found various combinations of mutations in TP53, FLT3, JAK2, SRSF2, TET2, ASXL1, IDH2, GNB1, NRAS and/or ZRSR2. All responded equally to venetoclax, suggesting the response was independent of genotype. Next we treated two BPDCN PDXs in vivo in NSG mice with oral venetoclax (100 mg/kg/day x 28 days). PDX genetics were, PDX1: ASXL1 G646fs*, NRAS G13D, JAK2 V617F, TET2 D1017fs*, and TET2 Q1687fs*; PDX2: IDH2 R140Q, TP53 S241F, TP53 C176Y, and ZRSR2 S188*. Venetoclax caused significant reductions in BPDCN burden in peripheral blood, spleen, and bone marrow after 21 days of therapy in both models. Overall survival was improved in venetoclax compared to vehicle treated animals in a leukemia-watch cohort (57 vs 36 days, P=0.0025). On the basis of these findings, we treated a relapsed BPDCN patient with venetoclax. He is an 80 year-old male who had received 3 prior lines of therapy. He had extensive skin disease with multiple cutaneous tumors, lymph node involvement, and >80% bone marrow blasts. His BPDCN carried the mutations ASXL1 Y581fs*, ASXL1 E553fs*, GNB1 K57E, IDH2 R140W, and NRAS G12D, and expressed high levels of BCL-2 protein in bone marrow and skin. BH3 profiling of a skin tumor biopsy revealed marked BCL-2 dependence and dynamic BH3 response to venetoclax (4 hr delta priming 55.6%). We treated him using a regimen recently FDA-approved for chronic lymphocytic leukemia (CLL) consisting of weekly dose escalation (20 -> 50 -> 100 -> 200 mg), to a target dose of 400 mg daily. At the time of this writing, he had reached 200 mg without significant toxicity, including no evidence of tumor lysis syndrome. His skin disease has responded remarkably (Figure), with the first response evident within 10 days. Our data suggests that BPDCN is highly sensitive to BCL-2 inhibition, which could provide an urgently needed new treatment for patients with this disease. We propose that BCL-2 inhibition should undergo expedited clinical evaluation in BPDCN. In addition, this case offers an example of precision cancer medicine by functional rather than genetic means. Figure Figure. Disclosures Davids: Infinity: Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Gilead: Honoraria; Janssen: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria, Research Funding; TG Therapeutics: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria. Stone:ONO: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Celator: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; Juno Therapeutics: Consultancy; Merck: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Letai:AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding. Lane:N-of-1: Consultancy; Stemline Therapeutics: Research Funding.

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The Genetic Analysis of Hemophilia a: The Application of Next-Generation Sequencing in the Analysis of Causative Variants Deep inside the Intron of the F8 Gene

Blood ◽

10.1182/blood.v128.22.1393.1393 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1393-1393 ◽

Cited By ~ 1

Author(s):

Hiroshi Inaba ◽

Keiko Shinozawa ◽

Kagehiro Amano ◽

Katsuyuki Fukutake

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Hemophilia A ◽

Pcr Amplification ◽

Causative Mutation ◽

Single Nucleotide Variants ◽

Advisory Committees ◽

Single Nucleotide ◽

Long Range Pcr ◽

Generation Sequencing

Abstract Introduction The identification of genetic defects in hemophilia A is essential to understanding the features of a patient's hemophilia. At present, 3 methods are generally applied in the standard analysis of the F8 gene in order to identify genetic defects: 1) direct sequencing is performed after the PCR amplification of the exons and the flanking intronic sequence to detect small defects; 2) long-range PCR amplification is performed to detect inversions; and 3) a multiplex ligation-probe amplification analysis (MLPA) is performed to detect gross rearrangements. However, a causative mutation cannot be found in approximately 2% of patients. Recently, several studies have reported the existence of a causative variant located deep inside an intron. We aimed to develop a whole F8 genetic analysis method using Next-Generation Sequencing (NGS) to investigate deep inside introns. Material & Methods Genomic DNA was extracted from patient peripheral blood cells, and the complete F8 locus was amplified in 14 overlapping regions (5-23 kb) by long-range PCR. In total, approximately 201kb (including the upstream and downstream regions of the F8) was amplified. The PCR fragments were purified using an illustraTM GFXTM PCR DNA and a Gel Band Purification Kit (GE Healthcare UK Ltd.), and were equimolarly mixed. The DNA library was prepared by fragmentation using a Nextera XT DNA sample preparation kit (Illumina Inc.). The paired-end adapter-ligated fragments of the pooled libraries were attached to the flow cell and sequenced using the amplicon sequencing application of the MiSeq software program (Illumina). The nucleotide sequences that were obtained were aligned to the GRCh37/hg19 coordinates of an F8 reference sequence using the Burrows-Wheeler Aligner (BWA). The variants were detected using the Genome Analysis Toolkit (GATK) and were annotated by the VariantStudio software program (Illumina). The Combined Annotation Dependent Depletion (CADD) score, which predicts the deleteriousness of single nucleotide variants as well as insertion/deletions variants in the human genome, was obtained from the CADD (ver. 1.3) website (http://cadd.gs.washington.edu/home). The study was approved by the Ethics Committee of Tokyo Medical University. Written informed consent was obtained from all patients, and studies were carried out in accordance with the principles of the Declaration of Helsinki. Results Forty-seven male hemophilia A patients were investigated, including 32 who had been analyzed previously. Of these, no causative mutation had previously been identified in two patients by standard analysis. Sequencing coverage was sufficiently high (>20 reads) to confirm the sequence, though it varied widely by region and analysis. However, a small part of intron 22, which differed in size (~1-2 kb) according to sample and analysis, showed very low coverage (0-20 reads). Our NGS analysis therefore allowed the identification of genetic variants in about 99% of F8. On average, 140 variants were detected in each patient. In the analysis of samples which had a previously identified causative mutation, single nucleotide variants (such as point mutations) were detected with high levels of accuracy. In contrast, structural variants (such as inversions and large duplications) could not be detected under present conditions. To identify rare and disease causative variants located deep within introns, we ruled out those variants registered in dbSNP, 1000 Genomes, COSMIC, and ClinVar databases. This left a total of 30 variants from 47 patients. The C-score of these variants obtained by CADD analysis ranged from 0.005 to 14.97. One of the two patients with unknown causative mutations carried a variant, c.144-10810T>C, in intron 1 with a C-score of 13.56. This score was sufficiently high and was suggested as the possible etiology of the patient's hemophilia A. Conclusion We established a comprehensive F8 analysis method using NGS. This technique was shown to be very effective for the detection of single nucleotide variants, though improvement was necessary for the detection of structural variants. Our study suggested that the existence of rare variants, which are likely to cause a hemophilia, deep inside the intron of F8, is not uncommon. Disclosures Inaba: Biogen: Honoraria; Bayer: Honoraria. Shinozawa:Pfizer: Honoraria; Bayer: Honoraria; Baxalta: Honoraria, Other: Endowed chair. Amano:Kaketsuken: Honoraria; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees; ViiV: Honoraria; Biogen: Honoraria; Novo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria. Fukutake:EPS: Research Funding; Siemens: Speakers Bureau; simic: Research Funding; Sekisui Medical: Consultancy, Honoraria, Speakers Bureau; Roche Diagnostics: Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbott: Honoraria, Speakers Bureau; Kaketsuken: Honoraria; Japan Blood Products Organization: Honoraria, Research Funding; Torii: Speakers Bureau; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; LSI Medience: Consultancy; SRL Inc: Consultancy; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Baxalta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CSL Behring: Honoraria.

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Education on Blastic Plasmacytoid Dendritic Cell Neoplasm Significantly Impacts the Interdisciplinary Physician Team

Blood ◽

10.1182/blood-2021-145378 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3021-3021

Author(s):

Lauren Willis ◽

Anthony S. Stein ◽

Kendra Sweet ◽

Joan Guitart ◽

Naveen Pemmaraju ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Plasmacytoid Dendritic Cell ◽

Learning Objective ◽

Advisory Committees ◽

Cutaneous Manifestations ◽

Cell Neoplasm ◽

Rare Malignancy ◽

Additional Education ◽

Physician Team

Abstract Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive malignancy that originates from precursors of plasmacytoid dendritic cells. BPDCN is a difficult disease to diagnose and manage and it is often misdiagnosed or underreported. The literature widely supports the need for an interdisciplinary team of physicians with specialized expertise to care for patients with BPDCN, such as dermatologists, pathologists, hematologists/oncologists (hem/oncs), stem cell transplant physicians and others. Aim: The objective of this study was to determine if online education could improve the knowledge of the interdisciplinary physician team members about BPDCN as well as their skills and confidence diagnosing this rare malignancy. Methods: Dermatologists, pathologists, and hem/oncs participated in a series of 6 live continuing medical education (CME)-certified activities, after which the recorded content was posted online as a single online enduring CME-certified activity. Content for the CME activities was developed by a multidisciplinary group of BPDCN experts and was delivered through an approximately 1-hour lecture. Data presented here is for the online enduring activity only. Educational effect was assessed using a repeated-pair design with pre-/post-assessment. Three multiple choice questions assessed knowledge/skills, and 1 question rated on a Likert-type scale assessed confidence. A paired samples t-test was conducted for significance testing on overall average number of correct responses and for confidence rating, and a McNemar's test was conducted at the question and learning objective level (5% significance level, P <.05). Data were collected from December 10, 2020 to May 3, 2021. Results: There were 246 dermatologists, 302 pathologists, and 316 hem/oncs included in this analysis, for overall n=864. PRACTICE SETTING: Dermatologists: 57% community, 15% academic, 13% government, 15% other; Pathologists: 37% community, 37% other, 23% academic, 4% government; Hem/Oncs: 48% community, 31% academic, 14% government, 7% other.OVERALL RESULTS: Overall 46% of dermatologists, 42% of pathologists, and 48% of hem/oncs improved their knowledge/skills related to BPDCN (P <.001 for all), showing a relative increase in responses correct from pre- to post-CME of 67% for dermatologists, 38% for pathologists, and 45% for hem/oncs.CONFIDENCE: 50% of dermatologists, 50% of pathologists, and 49% of hem/oncs had a measurable increase in confidence (P <.001 for all), resulting in 30% of dermatologists, 31% of pathologists, and 36% of hem/oncs who were mostly or very confident diagnosing BPDCN post-CME (9%, 14%, 17% pre-CME, respectively).The Table shows the mean percentage of correct responses by learning objective and the question used to test each learning objective. 20%/54% of dermatologists, 22%/57% of pathologists, and 20%/55% of hem/oncs improved/reinforced their knowledge of the most common cutaneous manifestations of BPDCN and 26%, 22%, 25% need additional education, respectively. CME improved skills ordering tests to diagnose BPDCN, however 57% of dermatologists, 58% of pathologists, and 45% of hem/oncs demonstrate a need for additional education about stains that can aid in diagnosing BPDCN. Conclusions: This online CME-certified educational activity led to statistically significant improvements in the knowledge and skills of dermatologists, pathologists, and hem/oncs about BPDCN as well as their skills and confidence diagnosing this rare malignancy. The results indicate that unique educational methodologies which are available on-demand can be effective tools for advancing clinical decision making. Additional education is recommended on the topics of cutaneous manifestations of BPDCN and case-based education to improve skills diagnosing BPDCN. Acknowledgements: This CME activity was supported by an independent educational grant from Stemline Therapeutics, Inc. Reference: https://www.medscape.org/viewarticle/942245 Figure 1 Figure 1. Disclosures Stein: Amgen: Consultancy, Speakers Bureau; Celgene: Speakers Bureau; Stemline: Speakers Bureau. Sweet: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Guitart: Miragen, Kyowa Kirin: Consultancy; Galderma: Consultancy, Research Funding; Solygenix, Elorac, Nanostring: Research Funding. Pemmaraju: LFB Biotechnologies: Consultancy; Aptitude Health: Consultancy; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Incyte: Consultancy; Daiichi Sankyo, Inc.: Other, Research Funding; Plexxicon: Other, Research Funding; Springer Science + Business Media: Other; Cellectis S.A. ADR: Other, Research Funding; CareDx, Inc.: Consultancy; Affymetrix: Consultancy, Research Funding; Roche Diagnostics: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Blueprint Medicines: Consultancy; Celgene Corporation: Consultancy; DAVA Oncology: Consultancy; Sager Strong Foundation: Other; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; MustangBio: Consultancy, Other; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; Bristol-Myers Squibb Co.: Consultancy; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Poligone: Stemline, Helsinn, Kyowa Kirin: Consultancy; Soligenix, Miragen, Helsinn, Bioniz: Research Funding; Stemline, Therakos, Regeneron: Speakers Bureau.

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Whole Genome Copy Number Variation Analysis of Chronic Lymphocytic Leukemia (CLL) Cells From Early-Intermediate Stage, High Risk CLL Patients Prior to First Treatment Reveals New Loss of Heterozygosity and Duplication Events in the CLL Genome.

Blood ◽

10.1182/blood.v114.22.1265.1265 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1265-1265

Author(s):

Steven A. Schichman ◽

Annjanette Stone ◽

Maria Winters ◽

Weleetka Carter ◽

Lori Frederick ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Homozygous Deletion ◽

Chromosome 12 ◽

Whole Genome ◽

Advisory Committees ◽

Hemizygous Deletion ◽

Number Variation ◽

Trisomy 12

Abstract Abstract 1265 Poster Board I-287 Introduction Fluorescence in situ hybridization (FISH), in combination with other markers, is used as a prognostic tool for CLL patients at diagnosis. The presence or absence of trisomy 12 and deletions at 13q, 11q, and 17p helps to predict disease progression and to stratify patients for therapeutic decisions. We hypothesized that whole genome single nucleotide polymorphism (SNP)-based copy number variation (CNV) analysis would capture all of the information in current CLL FISH panels and would reveal new CNV features in the CLL genome. Patients and Methods Nineteen early-intermediate clinical stage, untreated CLL patients aged 29 to 77 were determined to be at high risk for disease progression by FISH, IgVH mutation status, ZAP-70, and CD38 prognostic markers. CLL cells and normal cells were separated by magnetic bead selection from patient peripheral blood samples with absolute lymphocyte counts that ranged from 7.4 to 162 × 109/L. CNV analysis was performed on purified genomic DNA from the CLL cells and from normal cells for each patient in order to distinguish acquired CNVs in malignant cells from polymorphic CNVs in the human genome. We used the Illumina human660w-quad beadchip, a SNP-based microarray for whole-genome genotyping and CNV analysis that contains more than 550,000 tag SNPs and approximately 100,000 additional markers that target regions of common CNV. CNV data was analyzed using CNV partition (Illumina Genome Studio software) and PennCNV. Results 100% concordance is found between del(13q), del(11q), and del(17p) FISH abnormalities and loss of heterozygosity (LOH) at 13q, 11q, and 17p by CNV analysis. All three patients with trisomy 12 by FISH show copy number(CN)=3 of chromosome 12 by CNV analysis. Of 15 patients with del(13q) by FISH, 12 out of 15 have regions of hemizygous deletion on 13q that vary from ∼830 Kb to ∼38 Mb. The smallest region of LOH is located within 13q14.3. Three out of 15 patients show homozygous deletion within 13q14.3. One of these 3 patients has copy-neutral LOH of the entire 13q arm with an embedded 835 Kb segment of homozygous deletion at 13q14.3. Two patients have large discontinuous segments of LOH on 13q, indicating complex interstitial deletion events. Two out of 5 patients with del(13q) as a sole FISH abnormality show additional CNV events in the CLL genome. One of these patients has copy neutral LOH at 2q33.1-telomere(tel). One other patient with sole del(13q) FISH shows LOH events at 10q23.31-23.33 and at 15q15.1. Five out of six patients with del(11q) by FISH have either 13q LOH (n=4) or chromosome 12 CN=3 (n=1) without any other CNV events detected in the CLL genome. One patient with trisomy 12 and del(11q) by FISH has three additional acquired CNV abnormalities in the CLL genome: LOH at 7p15.2-tel, LOH at 11p13, and CN=3 at 3q24-tel. In contrast to patients with del(11q), del(13q), and trisomy 12, patients with del(17p) by FISH have numerous acquired CNV abnormalities in the CLL genome. These include LOH events at 1p34.3-p34.2, 2q34-q36.3, 3p21.31-tel, 4p13, 4p15.1-tel, 15q11.2-q14 and 15q14-q15.3, 16p13.3-tel, 16p13.11, 16p13.2, 18p11.21-tel, 20p11.21-tel, and 20q13.2-q13.31. CN=3 at 2p12-tel is detected in 2 out of 5 patients with 17p hemizygous deletion. One out of 5 patients with 17p hemizygous deletion shows CN=3 at 10q22.2-tel. One other patient also with 17p hemizygous deletion shows CN=3 at 22q12.2-tel. Conclusions Whole genome CNV analysis by SNP-based microarrays greatly expands our ability to detect acquired genomic events in CLL cells. These events include hemizygous deletion, homozygous deletion, copy-neutral LOH, and CN=3 duplication. Detection of copy-neutral LOH is not possible by FISH or array comparative genomic hybridization technology. The current study reveals a high number of acquired CNV events in earlier stage, untreated CLL patients with 17p hemizygous deletion. This observation, indicative of genomic instability, is consistent with the known poor prognosis of del(17p) patients. The new somatic CNV abnormalities detected in CLL cells may help to discover additional genes or signaling pathways involved in CLL initiation and progression. In addition, the new CNV markers may be used in larger clinical studies to improve CLL prognosis and patient stratification for therapy. Disclosures Shanafelt: Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Zent:Genentech, Bayer, Genzyme, Novartis: Research Funding.

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Progressive but Previously Untreated CLL Patients with Greater Array CGH Complexity Exhibit A Less Durable Response to Chemoimmunotherapy

Blood ◽

10.1182/blood.v116.21.2406.2406 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2406-2406

Author(s):

Neil E. Kay ◽

Jeanette Eckel Passow ◽

Esteban Braggio ◽

Scott Van Wier ◽

Tait Shanafelt ◽

...

Keyword(s):

Clinical Outcome ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Progression Free Survival ◽

Advisory Committees ◽

Free Survival ◽

Genomic Complexity ◽

Duration Of Response ◽

Gains And Losses

Abstract Abstract 2406 The outcome for a given CLL patient is difficult to predict. While there are promising models, they require collation of multiple clinical and laboratory parameters, and it remains to be seen whether they will apply to typical CLL patients in the community. To further dissect out explanations for this dramatic clinical heterogeneity, we sought to understand genomic complexity of clonal B-cells as a possible explanation of clinical variability with specific application to genomic complexity as a predictor of therapeutic response and clinical outcome in CLL. Thus we wished to identified global gains and losses of genetic material in order to define copy-number abnormalities (CNA) in 48 clinically progressive CLL patients who were about to be treated on a chemoimmunotherapy protocol. This protocol was previously reported by us (Blood. 109:2007) and had an induction phase with pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and then responding patients were followed ever three months until relapse. In order to estimate CNA, we employed array-based comparative genomic hybridization (aCGH) using a one-million oligonucleotide probe array format on the leukemic B-cells from the 48 patients entering this trial. In those same patients, the aCGH data were compared to a) FISH detecxtable data using a panel for the common recurring genetic defects seen in CLL and b) to their clinical outcome on this trial. With aCGH we found that 288 CNA were identified (median of 4 per patient; range 0–32) of which 215 were deletions and 73 were gains. The aCGH method identified most of the FISH detected abnormalities with a complete concordance for 17p13.1- deletion (17p-) between aCGH and FISH. We also identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. We found that CLL patients with ≥15 CNA had a significantly worse progression free survival (PFS) than patients with <15 CNA (p=0.004)(figure). Patients with ≥15 CNA also had a shorter duration of response than those with <15 CNA (p=0.0726). Of interest, more complex genomic features were found both in patients with a 17p13.1 deletion and in more favorable genetic subtypes such as 13q14.1. Thus, for 5 patients with >15 CNAs the following FISH patterns were seen: +12/13q14.1-x1/13q14.1 -x2, 13q14.1 ×1 (n=2), and 17p13.1 (n=2). In addition, a 17p- by FISH was positively associated with the number of CNA and total deletion size. The odds of having an overall response decreased by 28% (95% CI: 5–55%; p=0.015) with each additional CNA for the 17p13.1- patients. In addition to defining genomic complexity as the total number of CNA for each patient, we also defined complexity as the sum of the lengths of all interstitial chromosomal gains and losses. When defined as the total size of chromosomal gains or losses, genomic complexity was significantly associated with 17p13.1 and worse overall clinical response. In summary, this analysis utilized the global assessment of copy number abnormalities using a high-resolution aCGH platform for clinically progressive CLL patients prior to initiation of their treatment. One outcome was that we found higher genomic complexity was associated with shorter progression-free survival, reduced duration of response and predicted a poor response to treatment. In addition since we did find genomic complexity in more traditionally favorable FISH categories, such as 13q14.1 type defects, this may explain why some of the latter patients do not fare as well as might be expected even with aggressive chemoimmunotherapy approaches. This study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. Disclosures: Off Label Use: Pentostatin. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

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Stem Cell Transplantation Can Provide Durable Disease Control in Blastic Plasmacytoid Dendritic Cell Neoplasia (BPDC): A Retrospective Study From the European Group for Blood and Marrow Transplantation (EBMT)

Blood ◽

10.1182/blood.v118.21.3077.3077 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3077-3077

Author(s):

Sascha Dietrich ◽

Damien Roos-Weil ◽

Ariane Boumendil ◽

Emanuelle Polge ◽

Jian-Jian Luan ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Board Of Directors ◽

Cell Transplantation ◽

Research Funding ◽

Nk Cell ◽

Plasmacytoid Dendritic Cell ◽

Advisory Committees

Abstract Abstract 3077 Blastic plasmacytoid dendritic cell neoplasm (BPDC), formerly known as blastic NK cell lymphoma, is a rare hematopoietic malignancy preferentially involving the skin, bone marrow and lymph nodes. The overall prognosis of BPDC is dismal. Most patients show an initial response to acute leukemia-like chemotherapy, but relapses with subsequent drug resistance occur in virtually all patients resulting in a median overall survival of only 9–13 months. However, anecdotal long-term remissions have been reported in young patients who received early myeloablative allogeneic stem cell transplantation (alloSCT). We therefore performed a retrospective analysis of patients identified in the EBMT registry in order to evaluate the outcome of autologous stem cell transplantation (autoSCT) or alloSCT for BPDC. Eligible were all patients who had been registered with a diagnosis of BPDC or Blastic NK cell lymphoma and had received autologous stem cell transplantation (autoSCT) or alloSCT in 2000–2009. Centres were contacted to provide a written histopathology and immunophenotyping report and information about treatment and follow-up details. Patients who did not have a diagnostic score ≥ 2 as proposed by Garnache-Ottou et al. (BJH 2009) were excluded. RESULTS: Overall, 139 patients could be identified in the database who fulfilled the inclusion criteria (alloSCT 100, autoSCT 39). Of 74 patients for whom the requested additional information could be obtained, central review confirmed the diagnosis of BPDC in 39 patients (34 alloSCT, 5 autoSCT). The 34 patients who had undergone alloSCT had a median age of 41 years (range: 10–70 years), were transplanted from a related (n=11) or unrelated donor (n=23); received peripheral blood stem cells (n=9), bone marrow stem cells (n=19) or cord blood (n=6); and had been treated with a reduced intensity conditioning regimen (RIC, n=9) or myeloablative conditioning (MAC, n=25). Nineteen of 34 patients were transplanted in CR1. After a median follow up time of 28 months (range: 4–77+ months), 11 patients relapsed (median time to relapse: 8 months, range: 2–27 months) of whom 8 died due to disease progression. 9 patients died in the absence of relapse. No relapse occurred later than 27 months after transplant. Median disease free survival (DFS) was 15 months (range: 4–77+ months) and median overall survival (OS) was 22 months (range: 8–77+ months; Figure 1a). However, long-term remissions of up to 77 months after alloSCT could be observed. Patients allografted in CR1 tended to have a superior DFS (p=0.119) and OS (p=0.057; Figure 1b). MAC was associated with a better OS (p=0.001) which was attributable to the significantly higher non-relapse mortality (NRM) rate of patients after RIC (p=0.014), who had been significantly older (age RIC: 56 years, age MAC: 36 years, p=0.0014). The relapse rate was not different in patients after RIC and MAC, respectively. However, there was no survivor after RIC. Median age in the autoSCT group was 47 years (range: 14–62 years). Three of 5 patients were transplanted in CR1 of whom 1 patient relapsed after 8 months, 1 patient experienced treatment related mortality and 1 patient remained in CR for 28 months. The 2 remaining patients had more advanced disease at autoSCT and relapsed 4 and 8 months thereafter. CONCLUSION: AlloSCT is effective in BPDC and might provide curative potential in this otherwise incurable disease, especially when performed in CR1. However, it remains to be shown by prospective studies if the potential benefit of alloSCT in BPDC is largely due to conditioning intensity, or if there is a relevant contribution of graft-versus-leukemia activity. Disclosures: Tilly: Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau, Travel/accommodations/meeting expenses; Genentech: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; Janssen Cilag: Speakers Bureau.

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Absence Of Mutation In Cereblon (CRBN) and DNA Damage Binding Protein 1 (DDB1) Genes In Myeloma Cells and Patients and Its Clinical Significance

Blood ◽

10.1182/blood.v122.21.3139.3139 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3139-3139

Author(s):

Anjan Thakurta ◽

Anita K Gandhi ◽

Michelle Waldman ◽

Chad C. Bjorklund ◽

Suzanne Lentzsch ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Heterozygous Mutation ◽

Employment Equity ◽

Advisory Committees ◽

Single Nucleotide ◽

Truncating Mutation ◽

Equity Ownership

Abstract Background CRBN, a target of thalidomide and IMiDs® immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM), is a component of the E3 ubiquitin cullin 4 ring ligase (CRL4) complex that also includes DDB1, Roc1, and Cul4. Two CRBN mutations have been reported in multiple myeloma (MM) patients: truncating mutation (Q99) and point mutation (R283K). One copy of the CRBN gene was shown to be deleted in the MM1S and MM1S.R cell lines. No DDB1 mutation has been described previously. Results We investigated the incidence of CRBN and DDB1 mutations by next-generation sequencing in 20 MM cell lines and MM subjects. Of 90 MM patients, 24 were newly diagnosed and 66 were relapsed and refractory of which 36 patients were LEN resistant. Out of the cell lines tested, 1 heterozygous CRBN mutation (D249Y) was found in the LEN-resistant ANBL6R cells, which is located in the putative DDB1 binding domain, and 2 single silent mutations were identified in the KMS-12-BM (rs17027638) and OPM-2 cells. One DDB1 heterozygous mutation (E303D) was identified in ANBL6 cells. In the cohort of patients assessed, no CRBN mutation was detected; however, 5 single nucleotide variations (SNV) were identified. Three of the 5 SNVs were at position 735 (Y245Y) and 1 each at position 219 (H73H) and 939 (C313C), respectively. The first 2 SNVs (rs17027638 and rs1045309) are described but not the last. We found a single SNV (P51P; rs2230356) in DDB1 gene the patient samples. Conclusion Mutations within the coding sequences of CRBN and DDB1 are rare in MM patients and cell lines. Most intrinsically LEN-resistant cells and cell lines made resistant to LEN or POM do not have CRBN or DDB1 mutations, suggesting the potential role of other sources, such as genetic or epigenetic pathways in developing resistance to IMiD drug–based therapy. Disclosures: Thakurta: Celgene: Employment, Equity Ownership. Gandhi:Celgene: Employment, Equity Ownership. Waldman:Celgene: Employment, Equity Ownership. Bjorklund:Celgene: Employment, Equity Ownership. Lentzsch:Celgene: Research Funding. Schey:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; NAPP: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees. Madan:Covance Genomics Lab: Employment. Ning:Celgene: Employment, Equity Ownership. Mendy:Celgene: Employment, Equity Ownership. Lopez-Girona:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Avet-Loiseau:Celgene: Research Funding. Chopra:Celgene: Employment, Equity Ownership.

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Identification of Initiating Trunk Mutations and Distinct Molecular Subtypes: An Interim Analysis of the Mmrf Commpass Study

Blood ◽

10.1182/blood.v126.23.722.722 ◽

2015 ◽

Vol 126 (23) ◽

pp. 722-722 ◽

Cited By ~ 1

Author(s):

Jonathan J Keats ◽

Gil Speyer ◽

Legendre Christophe ◽

Christofferson Austin ◽

Kristi Stephenson ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Copy Number ◽

Interim Analysis ◽

Research Funding ◽

Molecular Subtypes ◽

Bayesian Clustering ◽

Clustering Method ◽

Advisory Committees

Abstract The Multiple Myeloma Research Foundation (MMRF) CoMMpass trial (NCT145429) is a longitudinal study of 1000 patients with newly-diagnosed multiple myeloma from clinical sites in the United States, Canada, Spain, and Italy. Each patient receives a treatment regimen containing a proteasome inhibitor, immunumodulatory agent, or both. Clinical parameters are collected at study enrollment and every three months through the five-year observation period. To identify molecular determinants of clinical outcome each baseline and progression tumor specimen is characterized using Whole Genome Sequencing, Exome Sequencing, and RNA sequencing. This will be the first public presentation of the interim analysis seven cohort with 760 enrolled patients of whom 565 are molecularly characterized. This cohort of patients includes 14 patients with baseline and secondary samples along with 7 patients with characterized tumor samples from the bone marrow and peripheral blood. Although the median follow-up time for the cohort is only 260 days the patients on proteasome and IMiD based combinations are currently showing a PFS and OS benefit compared to those receiving combinations with each agent alone. From the raw mutational analysis we identified 24 significant genes that are recurrently mutated and the mutated allele is detectably expressed in all but one, DNAH5. Suggesting these mutations are likely contributing to myelomagenesis through an unconventional mechanism. Interestingly, DIS3 mutations are independent of KRAS, NRAS, and BRAF indicating a potential mechanistic link while PRKD2 mutations are associated with t(4;14). To identify events driving the initiation of myeloma we performed a detailed clonality analysis using a bayesian clustering method that corrects for copy number abnormalities and tumor purity to assign mutations into distinct clonal branches versus the initiating trunk mutations. On average 63.8% of mutations are trunk mutations and in 86.7% of patients at least one trunk mutation is associated with somatic hypermutation of an immunoglobulin gene as expected in a late stage B-cell malignancy. This identified many expressed trunk mutations that did not come out in the classic significance analysis like ATM, EGR1, and CCND1. To identify molecular subtypes we performed unsupervised clustering using a consensus clustering approach on independent discovery and validation cohorts, which identified 12 distinct subtypes, using a combination of silhouette score and cumulative distribution of consensus scores. This analysis identified two distinct groups associated with t(4;14) with mutations in FGFR3 and DIS3 being exclusive to one subgroup. In addition, this analysis separates patients with cyclin D translocations into three different groups, with one group having the second lowest PFS proportion. Three patients without CCND1 or CCND3 translocations were found to have IgH translocations targeting CCND2. The MAF subgroup was associated with the lowest OS and PFS proportion, and the three MAF/MAFB translocation negative patients in the subgroup all had MAFA translocations. The remaining 6 subgroups are associated with hyperdiploid copy number profiles and harbor the majority of the IgH-MYC translocation events. Two of the hyperdiploid groups are associated with a low level of NFKB activation compared to the remaining four, one of these is defined by the highest proliferation index but paradoxically the other has the second worst OS proportion. Another group is enriched with FAM46C and NRAS mutations. The genomic profiles of the paired tumors isolated from the peripheral blood and bone marrow are highly similar indicating these are not genetically distinct tumor compartments, at least in this subset of seven patients. Applying our bayesian clustering method to the serial samples resolved additional clonal clusters as mutations with similar cancer cell fractions at diagnosis clearly diverged at later timepoints. These analyses have identified tumor initiating mutations and new subtypes of myeloma, which are associated with distinct molecular events and clinical outcomes. Disclosures Jagannath: Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Merck: Honoraria; Janssen: Honoraria. Siegel:Celgene Corporation: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Merck: Speakers Bureau. Vij:Takeda, Onyx: Research Funding; Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy. Zimmerman:Amgen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria. Niesvizky:Celgene: Consultancy, Speakers Bureau. Rifkin:Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lonial:Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding.

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