scholarly journals The Copy Number of Disease-Associated HLA Alleles Predicts the Response to Immunosuppressive Therapy in Acquired Aplastic Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 604-604
Author(s):  
Yoshitaka Zaimoku ◽  
Hiroki Mizumaki ◽  
Tatsuya Imi ◽  
Kohei Hosokawa ◽  
Hiroyuki Maruyama ◽  
...  
Keyword(s):  
Immunosuppressive Therapy ◽  
Odds Ratio ◽  
Copy Number ◽  
Research Funding ◽  
Somatic Mutations ◽  
Response Rate ◽  
Clonal Evolution ◽  
Favorable Alleles ◽  
Hla Alleles ◽  
The U.S

Abstract In immune-mediated acquired aplastic anemia (AA), the presence of an HLA allele, which is highly overrepresented or lost due to somatic mutations, may represent a specific immune pathophysiology and a clinical manifestation. HLA-B*14:02 is one of the most overrepresented class I alleles in AA and is also frequently affected by a somatic loss of expression; the inherited B*14:02 genotype was correlated with high-risk clonal evolution in two independent cohorts in the U.S. (Babushok DV et al. Blood Adv 2017; Zaimoku Y et al. manuscript in preparation). In contrast, HLA-B*14:02 is virtually absent in Japanese, in whom somatic mutations of AA have frequently been detected in HLA-B*40:02, B*54:01, and A*02:06, and occasionally in A*02:01, A*02:07, A*31:01, B*13:01, B*40:01, B*40:03, B*44:03, B*55:02, and B*56:01 (Mizumaki H et al. Haematologica 2021). A class II allele HLA-DRB1*15 is highly overrepresented in AA across various ethnic groups, including those in the U.S. and Japanese. This retrospective study in the Japanese population aimed to explore the clinical significance of disease-associated non-B*14:02 HLA class I and II alleles in AA. A total of 423 enrolled patients with AA (very severe [n = 81], severe [n = 266], transfusion dependent non-severe [n = 76]; median age 60 [range, 1-86] years) had undergone genotyping for HLA-A, HLA-B, HLA-C, and HLA-DRB1 at 2-field resolution. The HLA allele frequencies in these patients were compared to those in a Japanese HLA haplotype dataset (n = 19183; Ikeda N et al. Tissue Antigens 2016). The most overrepresented allele in AA was HLA-DRB1*15:02, followed by DRB1*15:01, B*40:02, and A*02:06 (Table); DRB1*13:02 and B*44:03, which are in linkage disequilibrium, were markedly underrepresented, consistent with a well-known protective role of DRB1*13 against autoimmune diseases. HLA-DRB1*15:02 was also significantly correlated with age and its frequency among patients aged <50 years was below the level of the control group (Figure A). Frequencies of HLA-DRB1*15:01, B*40:02, and A*02:06 were increased in both older and younger groups. HLA-A*31:01 and A*02:01 found to be enriched in young patients instead of DRB1*15:02 (Figure B); B*13:01, B*55:02, and B*56:01 showed a similar tendency for an early onset. HLA-B*40:02 was correlated with disease severity and was especially overrepresented in very severe AA (Figure C). The overall response rate to anti-thymocyte globulin-based immunosuppressive therapy at 6 months was 63% (139 of 220 treated and evaluable patients). A trend for a higher response was observed in patients harboring mutation-related HLA-B alleles (except for minor alleles B*13:01, B*40:03, and B*55:02) and the highly overrepresented or protective HLA-DRB1 alleles, but not in the HLA-A alleles (Figure D). A multivariate logistic regression revealed that the combination of the presence of any favorable alleles in HLA-B (odds ratio 3.6, P < 0.0001) or in HLA-DRB1 (odds ratio 2.3, P = 0.00085) was significantly and independently associated with a hematologic response; the tendencies for a lower or higher response in very severe disease and the presence of paroxysmal nocturnal hemoglobinuria clone did not reach statistical significance. Further, there was likely an additive effect when two favorable alleles coexisted in HLA-B or HLA-DRB1 (Figure E); the copy number of the favorable HLA-B and HLA-DRB1 alleles stratified the response rate to four groups: three or four copies, 95% (19 of 20); two copies, 72% (61 of 85); one copy, 59% (50 of 85); and zero copy, 30% (9 of 30). Only eight patients displayed clonal evolution to monosomy 7, myelodysplastic syndrome, or acute myeloid leukemia after immunosuppression without significant overrepresentation or underrepresentation of the pathogenic HLA alleles. Using a large dataset of homogeneous Japanese population with high-resolution HLA typing, we revealed, for the first time, a strong relationship between disease-associated (overrepresented, inactivated, or protecting) HLA alleles and the responsiveness to immunosuppressive therapy. Figure 1 Figure 1. Disclosures Takamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; SRL: Consultancy; Adaptive Biotechnologies, Eisai: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Yamazaki: Novartis Pharma: Honoraria; Kyowa Kirin: Honoraria; Kyowa Kirin: Research Funding. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding.

Dynamic Changes in MDS Clonal Architecture Following Allogeneic Stem Cell Transplant

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5506-5506
Author(s):  
Meagan A. Jacoby ◽  
Eric J. Duncavage ◽  
Gue Su Chang ◽  
Christopher A. Miller ◽  
Jin Shao ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Copy Number ◽  
Research Funding ◽  
Somatic Mutations ◽  
Stem Cell Transplant ◽  
Clonal Evolution ◽  
Cell Transplant ◽  
Secondary Aml ◽  
Time Points ◽  
Clonal Architecture

Abstract Background: MDS is a genetically complex, oligoclonal disease consisting of a founding clone and typically one or more subclones derived from the founding clone. Previously we have shown that in MDS patients treated with chemotherapy, a minor subclone present at diagnosis can expand during disease progression from MDS to secondary AML, highlighting the clinical implications of clonal heterogeneity. Whether a similar pattern of clonal evolution occurs in MDS patients that relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) is not known. Methods: We identified 9 MDS patients who progressed after receiving either a myeloablative (n=3) or reduced-intensity (n=6) alloHSCT (median time to progression 309 days, range 98-881). We performed enhanced exome sequencing (EES) to define the clonal architecture of 23 tumor samples at the following clinical landmarks (with matched skin as a source of normal DNA): diagnosis, <2 months pre-alloHSCT (where available), and relapse post-alloHSCT. Somatic mutations were validated in the 23 discovery samples and genotyped in 35 additional serial banked samples at various time-points post-alloHSCT, including day 30 and 100, using capture probes targeting all putative single-nucleotide variants (SNV) and short insertions and deletions (INDELs) identified by EES. The variant allele fraction (VAF) of all validated somatic mutations was determined. Ultra-deep, error-corrected sequencing (i.e., barcoded sequencing) was performed on 49 tumor samples to provide increased sensitivity to detect low-level mutations post-alloHSCT. Copy number alterations were identified using exome and SNP array data. Results: Averaged sequencing coverage depth was 246x for tumors subjected to EES; 537x for validation sequencing, and 24,150x total and 5,180x unique for the ultra-deep sequencing. In all cases, we observed that mutations found in the diagnostic founding clone were always detected at relapse. However, using SNVs, INDELs, and copy number analysis, we show that the dominant clone at relapse was often derived from a population that was subclonal at presentation. We observed the following, non-mutually exclusive patterns of clonal evolution at relapse: i) A subclone expanded or emerged and became the dominant clone at relapse as compared to presentation (n=6). In 2 of these cases, the subclone contained mutations that were not detected at presentation even via ultra-deep sequencing. ii) A subclone was cleared with alloHSCT (defined as VAF<1% by EES, n=4), confirmed by ultra-deep sequencing when available (n=2). iii) The founding/dominant clone at diagnosis was also the dominant clone at relapse (n=3). However, in 2 of these 3 cases, changes in clonal architecture were observed with evidence of rising or contracting subclones. Although our sample size is relatively small, the intensity of the alloHSCT conditioning regimen did not impact the relapse pattern. No acquired abnormalities were detected in the MHC locus, and no mutations in a particular gene family or cellular pathway were consistently observed in rising or contracting subclones. Finally, we used ultra-deep sequencing to determine if mutations (i.e., tumor cells) could be detected at day 30 post-alloHSCT in 7 of the 8 patients with no evidence of disease, who had available data. Mutations were detected in 6 of 7 patients. The average detectable mutation VAF per patient was 0.37% (ranged from 0.04% to 0.93%)(i.e., 1 mutant cell in 135). Conclusion: Complex clonal dynamics and clonal evolution are observed at relapse post-alloHSCT for MDS. Although minor subclones rise and may become the dominant clone at relapse, mutations present in the dominant (i.e., founding) clone of the diagnostic MDS sample pre-alloHSCT are always detected at relapse. This is similar to the pattern of clonal evolution previously observed for MDS progression to secondary AML following chemotherapy. These observations have implications for targeted therapy and tumor burden monitoring. Ultra-deep sequencing can detect persistent or emerging mutations at early time-points post-alloHSCT that are associated with subsequent relapse. The predictive value of detecting persistent mutations early after post-alloHSCT merits testing in future studies. Disclosures Jacoby: Quintiles: Consultancy; Sunesis: Research Funding; Celgene: Speakers Bureau. DiPersio:Incyte Corporation: Research Funding.

scholarly journals Diverging Clonal Evolution during Sequential Therapy with Chemoimmunotherapy Followed By BTK Inhibitors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 850-850
Author(s):  
Inhye E Ahn ◽  
Yun-Ching Chen ◽  
Chingiz Underbayev ◽  
Erika M Gaglione ◽  
Clare Sun ◽  
...  
Keyword(s):  
Copy Number ◽  
Research Funding ◽  
Clonal Selection ◽  
Clonal Evolution ◽  
Growth Potential ◽  
Driver Genes ◽  
Parent Clone ◽  
Richter’S Transformation ◽  
Richter's Transformation ◽  
Clonal Composition

Background: Treatment confers an evolutionary bottleneck for cancer. Little data exists on the genomic evolution of CLL under different treatment pressure and disease compartments. Methods: We identified 13 patients uniformly treated with chemoimmunotherapy (CIT) as first-line therapy and a BTK inhibitor (BTKi) as second-line. We collected peripheral blood (PB) samples before CIT, at relapse after CIT (which was also before starting a BTKi), and 6 months on a BTKi. Five patients on a long-term BTKi were additionally tested at two years on BTKi. Two patients had samples from bone marrow (BM) and/or lymph node (LN) compartments at 6 months on a BTKi. In total, 48 tumor samples paired with saliva serving as a germline control were tested with whole exome sequencing (WES). We used high confidence variants identified by at least two variant callers for somatic single nucleotide polymorphisms and insertion-deletion variants, and TitanCNA for copy number variations. Clonal composition was reconstructed using PhyloWGS. Results: The median age of the cohort was 60 years at the start of CIT. The median duration of follow-up was 7 years. Median time between the start of CIT and a BTKi was 43 months (range 10-135). To date, eleven patients are alive and remain in response to a BTKi. Two patients died, one due to hepatitis B virus reactivation, and one with Richter's transformation. The median tumor mutational burden (TMB), defined as the number of nonsynonymous coding mutations per Mb, was 1.9 before CIT, 2.1 after CIT, and 1.9 after 6 months on a BTKi, consistent with previous reports in CLL. TMB was loosely associated with time since CIT exposure (R2=0.12, P=0.014). All patients had at least one mutation in known driver genes, most commonly NOTCH1 and SF3B1 (31% each; Figure A). Most of these mutations were subclonal and remained subclonal throughout the treatment course. The number of known driver gene mutations per patient remained relatively stable during the treatment course (median: 2 before CIT, 3 after CIT, 3 at 6 months on a BTKi; all P&gt;0.05). We recomposed clonal structures and estimated the cancer cell fraction (CCF) of each clone. Phylogenetic analysis showed branching evolution in most patients with a median of three child clones originating from a parent clone. In these patients, multiple clonal branches and its descendants were simultaneously being selected during therapy. In addition, we observed linear evolution in three (23%) patients, characterized by a repetitive selection of one parent clone and its leading progeny per generation. Treatment with CIT and a BTKi selected for distinct sets of clones. BTKi therapy effectively downsized clones which grew to dominance after CIT, except in one patient whose subclone with concurrent NRAS and TP53 mutations remained dominant throughout CIT and BTKi therapy. The median CCF decrease of CIT-selected clones was 18% during BTKi therapy (range 1-52). In all patients with SF3B1 mutation, clonal fractions of SF3B1 mutated clones were higher at relapse after CIT and lower during BTKi therapy. There was no consistent pattern of clonal selection for NOTCH1 mutation. We compared patterns of clonal evolution in different compartments in two patients who had PB, LN and/or BM samples available. Each compartment had a distinct clonal composition. Notably, one patient who progressed with Richter's transformation after 6 months on a BTKi showed three main clones shared among all compartments and a set of compartment-restricted clones (Figure B). The LN was preferentially enriched with clones carrying HIST1H1E mutation and complex copy number changes, reflecting transformed clones. The NOTCH1 mutated clone and its progeny, reflecting CLL clones, were dominant in PB and BM, but not in LN. Conclusion: The number and clonality of somatic mutations affecting known driver genes, remained relatively stable over the course of treatment with CIT followed by a BTKi at relapse. Distinct sets of clones evolved under each line of therapy. Clonal composition is shaped by the growth potential of individual clones, the selective pressure of the therapy used, and the tumor microenvironment. Figure Disclosures Wiestner: Pharmayclics: Research Funding; Acerta: Research Funding; Nurix: Research Funding; Merck: Research Funding.

scholarly journals Distinctive Genetic Features of Plasma Cells in POEMS Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4404-4404
Author(s):  
Yuhei Nagao ◽  
Naoya Mimura ◽  
June Takeda ◽  
Motohiko Oshima ◽  
Kenichi Yoshida ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Copy Number ◽  
Monoclonal Gammopathy ◽  
Research Funding ◽  
Somatic Mutations ◽  
Poems Syndrome ◽  
M Protein ◽  
Target Sequencing ◽  
The Mean ◽  
Genetic Profiles

Abstract Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles. Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing. Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman's variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5). WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases). To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case. We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM. Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Longitudinal Genomic Analyses In Chronic Lymphocytic Leukemia (CLL) Patients Reveal Clonal Relationship and Genomic Evolution In Disease Progression and After Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3605-3605
Author(s):  
Esteban Braggio ◽  
Neil E. Kay ◽  
Scott Van Wier ◽  
Stephanie Smoley ◽  
Jeanette Eckel-Passow ◽  
...  
Keyword(s):  
Disease Progression ◽  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Clonal Evolution ◽  
The Other ◽  
Genomic Evolution ◽  
Advisory Committees ◽  
Time Points ◽  
Time Point

Abstract Abstract 3605 CLL is a malignant B-cell disorder characterized by the accumulation of small B lymphocytes with a mature appearance in blood, marrow and lymph nodes. Despite effective treatment options, all patients with CLL will eventually relapse after therapy. This could be due in part to the presence of subclones of the CLL cell population that harbor genetic abnormalities, which confer resistance to treatment. The aims of this study were to investigate the clonal evolution in longitudinal samples of CLL patients and to identify genetic alterations associated with disease progression and resistance to therapy. Sequential analyses were performed in 51 samples from 23 patients who were included in a previously reported clinical trial of pentostatin, cyclophosphamide and rituximab (PCR) given every 3 weeks for 6 cycles in previously untreated CLL (Blood 109:2007). In all cases the first sample analyzed was prior to therapy. In 5 of 23 patients, three time points were analyzed: >6 months prior to entry onto PCR trial (time point A), just before starting with the PCR regimen (time point B), and the time of relapse after PCR trial (time point C). Seven patients were analyzed at time points A and B; 9 at time points B and C and 2 at time points A and C. The median time between points A and B was 17.5 months (range 8–48 months) and between points B and C was 20.5 months (7–60 m). All samples were examined by array-based comparative genomic hybridization (aCGH) using the Agilent Sureprint G3 (1 million probe) array. aCGH findings were confirmed by interphase FISH using probes for D13S319 (MIR16–1/MIR15A), RB1, MDM2, CEP12, CEP6, MYB, TP53, NFKBIA, PERP and FGFR1 loci. Overall, we observed a small increase in the number of copy-number abnormalities (CNA) with disease progression. Twenty-two of the 23 patients with paired samples harbored at least one CNA that persisted in all samples, indicating clonal relationship between the sequential samples. In 15 of the 23 patients the tumor clone was stable and no CNA differences between time points were identified. Conversely, genomic evolution was found in 8 patients. In 3 cases the genetic differences were observed pre treatment (between time points A and B) and in the other 5 cases, the observed changes were found after therapy (between time points A and C or between B and C). One remarkable case with genome evolution exhibited two subclones sharing trisomies 12 and 19, but with several unique CNA confined to each subclone. The first subclone was characterized by deletions of 6q, RB1, MIR16-1/MIR15A and 3 other losses, while the second subclone showed homozygous deletion of MIR16–1/MIR15A and 5 other monoallelic deletions. The first subclone was predominant at time points A and B (60–70% of cells), but was present in only 10–20% of cells at time point C as confirmed by FISH. Conversely, the second subclone was observed in ~20% of cells at time points A and B and became predominant after therapy, found in ~80% of cells at time point C. Another case was characterized by deletion 11q32 (including ATM and others) as the sole abnormality at time point B. Significant genomic complexity was observed at time point C, including deletions of 11q32, 9p21 (CDKN2A), 9q12-q33, 14q13.2 (NFKBIA) and 17p (TP53), and gains of 2p16 (REL) and 9q34. Interestingly, the deletion 11q32 from both time points arose independently at each time point, as they exhibited different chromosomal breakpoints and copy number variants. Moreover, the other CNA found at relapse were not identified at diagnosis (confirmed by aCGH and FISH). For evolution of specific CNA, trisomy 12 was found in 5 cases at the first sample analyzed and was stable with no changes between time points. The frequency of deletions 13q14.3 (MIR16-1/MIR15A) and 17p increased at the later time points. Conversely, –6q decreased in frequency across time points (3 cases in time points A–B and 1 case in time point C). In summary, at least 35% of CLL patients exhibited clonal evolution and at least 9% showed evidence of multiple subclones. This subgroup of CLL patients provides an exceptional framework for comprehensive analysis of genome evolution during disease progression before and after therapy. Our observations also support the hypothesis of a common CLL progenitor cell can give rise to clonally related, but genetically evolving subpopulations of tumor cells. Finally, this study may bring novel information regarding the drug resistance pathways utilized by CLL B cell clones post therapy. Disclosures: 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. Fonseca:Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

Karyotypic and Genetic Abnormalities Associated with Clonal Evolution in Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2371-2371
Author(s):  
Hideki Makishima ◽  
Kenichi Yoshida ◽  
Michael J. Clemente ◽  
Masashi Sanada ◽  
Yasunobu Nagata ◽  
...  
Keyword(s):  
Stem Cell ◽  
Research Funding ◽  
Somatic Mutations ◽  
Chromosomal Abnormalities ◽  
Clonal Evolution ◽  
Clonal Expansion ◽  
Novel Mutations ◽  
Jak2 Mutation ◽  
Myeloid Malignancies ◽  
Whole Exome

Abstract Abstract 2371 PNH is a clonal stem cell disease. While nonmalignant, PNH shows certain similarities to MDS and other neoplasms affecting hematopoietic stem and progenitor cells, including persistence of an aberrant clone, clonal expansion, and phenotypic abnormalities. In a small proportion of patients, subtle chromosomal abnormalities can be found and cases of otherwise classical PNH due to microdeletions involving the PIG-A locus have been described, illustrating similarities to other malignant conditions. PIG-A gene mutations lead to defective biosynthesis of GPI anchors and are responsible for the PNH phenotype. Similarly, phenotypic features of stem cells affected by PIG-A mutations are believed to be responsible for the extrinsic growth advantage and clonal expansion in the context of immune mediated suppression of hematopoiesis. While this scenario is plausible, there are also observations suggesting that intrinsic factors may be also involved. For instance, PNH persists after successful immunosuppression, often for many years, suggesting activation of stem cell maintenance genes. Furthermore, PNH clones can also be encountered (albeit at a very low frequency) in healthy individuals, and PNH can present in a pure form without aplastic anemia. Such extrinsic factors may include additional, secondary genetic events such as somatic mutations. Supporting this theory, clonal rearrangement of chromosome 12, which leads to overexpression of the transcription factor HMGA2 gene, were found in cells with the PIG-A mutation from 2 PNH cases. Also, we recently reported 3 PNH cases with JAK2 V617F mutation, who presented with a MPN phenotype and thrombosis. We theorized that study of clonal architecture in PNH will reveal clues as to the pathogenesis of clonal evolution of the PNH stem cell. We applied next generation whole exome sequencing to detect somatic mutations in PNH cases (N=6). The subsequent validation set included 45 PNH cases. PNH and non-PNH cells were sorted using magnetic beads. DNA from both fractions was analyzed by whole exome sequencing and results of the non-PNH cells were subtracted from the results of the PNH clone. We found biallelic PIG-A mutations in 2 female cases and a single mutation in each male case. In an index female case with thrombosis, a novel somatic heterozygous mutation of NTNG1 (P24S) was detected, while the patient was negative for the JAK2 mutation. Allelic frequency with the NTNG1 mutation (53/160 sequence reads (33%)) was larger than that with a concomitant heterozygous PIG-A mutation (intron 5 splice donor site G<A) (78/333 reads (23%)). In this case, the size of the other heterozygous PIG-A mutation (G68E) was less (31/194 (16%)) than the other PNH clone. These findings suggest that there are 2 different PNH clones in one case and that the NTNG1 mutation might be acquired before PIG-A gene was mutated. Moreover, NTNG1 encodes a GPI-anchored cell membrane protein and the mutation (P24S) was located in the predicted signal peptide. All together, 3 novel mutations were discovered, including MAGEC1 (C747Y) and BRPF1 (N797S) mutations. Of note, BRPF1 mutations have been also reported in AML. Interestingly, BRPF1 encodes a component of MOZ/MORF complex, positively regulating the transcription of RUNX1. To screen pathogenic karyotypic lesions in PNH clonal expansions, we combined metaphase cytogenetics and single nucleotide polymorphism arrays. We detected 14 somatic chromosomal abnormalities in 13 out of 26 PNH cases (50%). Of note is that a microdeletion on 2q13 resulted in the loss of an apoptosis-inducing gene BCL2L11, suggesting a contribution to growth advantage. Somatic UPD lesions strongly suggest the presence of homozygous mutations, for example the SET nuclear oncogene, which is located in UPD9q32qter was observed in another PNH case. Overall, the discovery of these novel mutations, as well the previously described JAK2 mutation, indicates that the pathophysiology of PNH clonal evolution partially overlaps that of other myeloid malignancies. In sum, various novel somatic karyotypic abnormalities and mutations are frequently detected in PNH clones using technology with comprehensive and high resolution. Some of these aberrations play a similar role in the clonal evolution of myeloid malignancies. These results suggest new therapeutic strategies similar to those for other myeloid malignancies should be considered in PNH cases with addition mutations. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

scholarly journals Molecular Epidemiology of AML: Association of Somatic Gene Mutations with Epidemiologic Exposures and Outcomes in the Mayo Clinic AML Epidemiology Cohort

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-36
Author(s):  
Zaid Abdel Rahman ◽  
Yesesri Cherukuri ◽  
Michael G. Heckman ◽  
Laura E. Finn ◽  
Liuyan Jiang ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Board Of Directors ◽  
Mayo Clinic ◽  
Copy Number ◽  
Research Funding ◽  
Somatic Mutations ◽  
Patient Characteristics ◽  
Advisory Committees ◽  
Risk Response ◽  
Response To Chemotherapy

Introduction: Population studies have identified genes with germline polymorphisms associated with acute myeloid leukemia (AML) risk and outcome. However, somatic mutations in these genes have not been reported in an AML clinical population and whether they are associated with epidemiologic exposures, clinical AML phenotypes and outcome after therapy. Methods: We systemically interrogated PubMed database (1998-2018), to identify genes with germline polymorphisms associated with AML risk, response to chemotherapy or outcome. To determine the prevalence and relevance of somatic mutations in these genes in an unselected AML population, we performed an analysis using Whole-Exome Sequencing (WES) on remnant diagnostic cytogenetic pellets from 98 patients from the Mayo Clinic AML Epidemiology Cohort, a detailed and highly-annotated cohort of 295 consecutive AML patients treated at Mayo Clinic Florida & Arizona between October, 2000 and December, 2011. Patient characteristics are shown in Table 1. Samples were sequenced at a depth of ~100 million paired-end 100bp reads using Agilent SureSelectXT Human All Exon V5 + UTRs target enrichment kit. Sequencing reads were aligned to human reference genome, and somatic mutations including non-synonymous and truncating single nucleotide variants and small INDELs were identified and filtered using Exome Sequencing Project, 1000 genome, HapMap, & Mayo Clinic internal biobank genetic variants database. Copy number aberrations were identified & filtered using public copy number polymorphism databases. The association analyses were performed at the gene level, with a primary endpoint of whether a given patient harbored a somatic mutation in any genes linked to AML risk or outcome in literature, and to determine the associations of these mutations with epidemiologic exposures, AML phenotype and clinical outcomes. Results: From the literature search, we identified 77 unique genes with known germline polymorphisms associated with AML risk, response to chemotherapy or outcome. Fifty-eight of these were found to be somatically mutated in our WES dataset, with subsequent analysis focusing on the 11 genes (ABCB1, CYP1A1, CYP2B6, EPHX1, ERCC1, ERCC2, ERCC5, JAK2, MEFV, MTRR, and TERT) that had greater than 5 patients with nonsynonymous somatic mutations in the given gene. Significant associations with epidemiologic exposures and outcomes were noted in patients with somatic mutations in ERCC2, CYP1A1 and ERCC5 genes. Table 2 shows a comparison of patient characteristics and associations according to the presence of somatic mutations in these genes. Patients with mutations in CYP1A1 had a significantly younger age at AML diagnosis (Median: 51.7 vs. 71.0 years, P=.02) and significantly shorter OS in age-adjusted analysis (HR=4.45, P=.003). The former is a novel finding, whereas the latter is consistent with previous reports. Patients with mutations in ERCC2 more commonly used statins (66.7% vs. 21.7%, P=.03). Patients with ERCC5 mutations had a lower rate of tobacco use (20.0% vs. 54.5%, P=.049). In unadjusted analysis, there was a significant association between presence of somatic mutations in JAK2 and poorer survival after AML diagnosis (HR=2.83, P=.017), but this attenuated and did not retain significance when adjusting for age at AML diagnosis (HR=2.22, P=.067). Conclusion: Our exploratory study describes a novel association of CYP1A1 somatic nonsynonymous mutations with age of AML onset, as well as novel associations of ERCC2 and ERCC5 mutations with epidemiologic exposures in an unselect cohort of patients with AML. We confirm the association of CYP1A1 with inferior overall survival after AML diagnosis. These findings suggest that some genes associated with AML risk may also harbor somatic mutations that are clinically relevant. These results will guide a planned confirmatory prospective study to determine frequency and impact of both germline and somatic mutations of risk genes in AML patients, and may contribute to a better understanding leukemia risk assessment and potentially to prevention strategies. Disclosures Finn: Jazz Pharmaceuticals: Speakers Bureau; Celgene: Speakers Bureau; Seattle Genetics: Speakers Bureau. Cerhan:BMS/Celgene: Research Funding; NanoString: Research Funding. Foran:Revolution Medicine: Consultancy; Servier: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; H3Biosciences: Research Funding; Xencor: Research Funding; Trillium: Research Funding; Takeda: Research Funding; Kura Oncology: Research Funding; Aptose: Research Funding; Aprea: Research Funding; Actinium: Research Funding; Agios: Honoraria, Research Funding.

scholarly journals Longitudinal Assessment of Circulating Tumor Mutational Burden Using a Next-Generation Sequencing Cancer Gene Panel: A Potential Biomarker of Response to Programmed Cell Death 1 (PD-1) Blockade in Patients with Relapsed/Refractory Classical Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 131-131 ◽  
Author(s):  
Martina Di Trani ◽  
Ettore Rizzo ◽  
Silvia Locatelli ◽  
Fabrizio Marino ◽  
Vanessa Cristaldi ◽  
...  
Keyword(s):  
Research Funding ◽  
Somatic Mutations ◽  
Clonal Evolution ◽  
Advisory Board ◽  
Classical Hodgkin Lymphoma ◽  
Scientific Advisory Board ◽  
Dna Mutations ◽  
Programmed Cell Death 1 ◽  
Scientific Advisory ◽  
Generation Sequencing

Introduction: The programmed cell death 1 (PD-1) monoclonal antibodies (MoAbs) nivolumab and pembrolizumab induce response rates exceeding 70% in relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL). The lack of response to PD-1 MoAbs, and the relapse occurring in most patients who had responded to PD-1 blockade suggest that tools to identify the determinants of response/resistance to PD-1 MoAbs are urgently required. We hypothesized that the characterization of the mutational profile of circulating tumor DNA (ctDNA) could represent a valuable tool to track clonal evolution-driven resistance to checkpoint inhibitors. Patients and Methods: 21 R/R cHL (median age, 32 years; range, 19-51) who had received a median of 5 (range, 3-7) chemotherapy lines, including autologous stem cell transplantation (77%) and brentuximab vedotin (100%), were treated with PD-1 MoAbs. Blood samples were profiled by CAPP-Seq strategy. We analyzed ctDNA and paired DNA from peripheral blood mononuclear cells (PBMCs), as source of germline DNA to filter out polymorphism and sequencing errors. A targeted resequencing panel optimized to include the coding exons and splice sites of 133 genes (320 Kb) that are recurrently mutated in B-cell lymphomas was used. Libraries were prepared from ctDNA and germline gDNA according to the CAPP-seq targeted enrichment strategy (Nimblegen-Roche) and subjected to ultra-deep-next generation sequencing (NGS) using the Nextseq 500 platform (Illumina). The sequencing was performed to obtain a depth of coverage &gt;2000x in &gt;80% of the target region in all samples, which allowed a sensitivity of 3x10-3. A stringent and completely automated bioinformatic pipeline was applied to call non-synonymous somatic mutations, using the somatic function of VarScan2. Results: After a median of 26 (range, 9-63) cycles of PD-1 inhibitors best response was complete remission (CR) for 9 patients (42%), partial remission (PR) for 6 (29%) and progressive disease (PD) for 6 (29%). Patients achieving PR experienced a disease control lasting for 4.5 to 24 months and subsequently underwent PD. Plasma and PBMC samples were collected at baseline, every five cycles of therapy, and end-of-therapy (EOT). At baseline, 18 of 21 patients could be successfully genotyped, whereas three were not. Evaluable patients showed a mean (±SD) number of mutated genes and mutations per patient of 7.3±5.1 (range, 2-22) and 9.9±8.4 (range, 2-37), respectively. Genes recurrently affected by non-synonymous somatic mutations in &gt;20% of R/R cHL included STAT6 (45%), SOCS1 (40%), ITPKB (35%), GNA13 (35%), TP53 (20%), TNFAIP3 (15%). At baseline, no association of distinct DNA mutations with resistance to PD-1 inhibitors could be demonstrated. Signaling pathways targeted by DNA mutations included JAK-STAT, NF-κB, PI3K-AKT, cytokine, NOTCH, immune evasion. The concentration of ctDNA reported as haploid genome equivalent per ml (hGE/ml) was 592.2 (range, 2-2,746), with values of hGE/ml detected in PD patients being significantly higher as compared to CR patients (P=.0437). As compared to cycle 0, the hGE/ml of ctDNA at cycle 5 showed a significant reduction (592.2 vs. 67, P&lt;.0008) which was followed by further hGE/ml decline in CR patients (to 14 P=.05) and further hGE/ml increase in PD patients (to 1,300 P=.1). At cycle 5, all CR/PR patients showed complete disappearance of baseline mutations, which were replaced by completely novel clones. In all CR/PR patients, this pattern of "clonal reshaping" was repeatedly detected over time. In striking contrast, at cycle 5, PD patients showed the persistence of baseline mutations. In all PD patients, this pattern of "clonal persistence", was repeatedly detected over time. In 4 patients, resistance to PD-1 inhibitors was associated with the appearance of a TP53 mutated clone. Although, a formal correlation of circulating DNA mutations with standard FDG-PET imaging was outside the objective of this study, both the "clonal reshaping" and "clonal persistence" patterns could be demonstrated to correlate with the results of FDG-PET. Conclusions: Analysis of ctDNA allows detecting tumor-specific mutations in R/R cHL. The longitudinal tracking of circulating DNA mutations in these patients identifies two different patterns of clonal evolution associated with sensitivity (clonal reshaping) or resistance (clonal persistence) to checkpoint blockade. Disclosures Santoro: Eisai: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Lilly: Speakers Bureau; Sandoz: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Arqule: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; AstraZeneca: Speakers Bureau; Celgene: Speakers Bureau; Servier: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; BMS: Speakers Bureau; Roche: Speakers Bureau; Abb-Vie: Speakers Bureau; Amgen: Speakers Bureau; BMS: Consultancy; Bayer: Consultancy, Speakers Bureau; MSD: Speakers Bureau. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Carlo-Stella:ADC Therapeutics: Consultancy, Other: Travel, accommodations, Research Funding; Sanofi: Consultancy, Research Funding; Celgene: Research Funding; Janssen Oncology: Honoraria; MSD: Honoraria; Servier: Consultancy, Honoraria, Other: Travel, accommodations; Amgen: Honoraria; Boehringer Ingelheim: Consultancy; Novartis: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Other: Travel, accommodations, Research Funding; BMS: Honoraria; Janssen: Other: Travel, accommodations; Takeda: Other: Travel, accommodations; Rhizen Pharmaceuticals: Research Funding; AstraZeneca: Honoraria; Genenta Science srl: Consultancy.

scholarly journals Genomic Profiles of Bone Marrow (BM) Clonal Plasma Cells (PCs) Vs Circulating Tumor Cells (CTCs) and Extramedullary (EM) Plasmacytomas in Multiple Myeloma (MM)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4442-4442
Author(s):  
Gabriel Bretones ◽  
Bruno Paiva ◽  
Rafael Valdes-Mas ◽  
Diego Alignani ◽  
Miguel Garcia ◽  
...  
Keyword(s):  
Protein Function ◽  
Research Funding ◽  
Somatic Mutations ◽  
Stop Codon ◽  
Clonal Evolution ◽  
Dna Amplification ◽  
Individual Patient Level ◽  
Patient Level ◽  
Recurrent Mutations ◽  
The Individual

Abstract EM disease in MM increases from newly-diagnosed into relapsed patients, and typically predicts for inferior survival. In fact, no treatment seems to be effective in cases with plasma cell (PC) leukemia, which represents the most aggressive form of EM MM. Unfortunately, the mechanisms of extramedullary spread in MM are not well understood, and there is almost no data about the genetic landscape of both forms of EMD (plasmacytomas and peripheral blood CTCs). Here, we performed whole exome sequencing (WES) to analyze the genomic profiles of highly purified FACS sorted BM and EM clonal PCs from 6 patients with relapsed MM. In 5/6 cases we had all three tissue specific clones, whereas in the remaining case no CTCs were detectable. Depending on the amount of genomic DNA from each clone, whole-genome amplification was performed and in such cases, triplicates of 10 ng of DNA were amplified up to 10 µg using the Illustra GenomiPhi HY DNA amplification kit (GE healthcare). Enrichment of exonic sequences was performed for each library using the SureSelectXT Human All Exon V5+UTRs capture kit (Agilent). To identify somatic mutations we used the mutation caller named Varscan. Variants potentially affecting protein function, including non-synonymous variants, frameshifts in the coding sequence, stop codon-introducing (nonsense) or variants potentially affecting splicing, were analyzed. Only those mutations present in 2/3 libraries analyzed per sample were considered positive. Overall, a median of 89 (67 - 474) somatic mutations were detected. EM plasmacytomas showed the highest mutation load, followed by CTCs and BM clonal PCs (85 vs 77 vs 75, respectively; P=.07), supporting a higher genomic instability/evolution of EM clones. Strikingly, all 6 cases showed lack of concordance in the mutation profiles of the three tissue related clones; even 2x2 comparisons between BM clonal PCs vs CTCs or plasmacytomas, or between the two forms of EM MM (ie. plasmacytomas vs CTCs) showed lack of 100% concordance in every single patient. Despite high inter-tissue heterogeneity, it should be noted that whenever present, recurrent and potentially actionable mutations in genes such as KRAS (n=3), KDM4A (n=2), KMT2A (n=2), ARID5B (n=2), TRIO (n=2), BRAF (n=1) or CCND1 (n=1) were detected in all three clones, with the exception of one patient in which CTCs lacked a KRAS mutation. Only one and less frequent actionable mutation in the EPHB2 gene (eg. Herceptin) was exclusively noted in the EM plasmacytoma from one patient. In order to understand the cellular origin of the three tissue related subclones, we then investigated the degree of similarity between each pair of clones at the individual patient level. The pair of clones showing the highest similarity in their mutation profiles were EM plasmacytomas with CTCs (60%, 30% - 94%), followed by BM clonal PCs with CTCs (58%, 35% - 92%), and by BM clonal PCs with EM plasmacytomas (57%, 37% - 91%). Thus, these data suggests that while CTC clones may represent a cellular bridge in between BM and EM plasmacytomas, there is continuous genomic evolution once the different clones have seeded in their respective tissue niches. Accordingly, EM plasmacytomas showed the highest number of specific mutations, followed by BM clonal PCs and CTCs (medians of 9, 4 and 3, respective). We then looked for recurrent mutations specifically present in EM clones, and found that CTCs showed exclusive and recurrent mutations in the CEP152, FSIP2, SYNE1 and TENM1 and ZNF585A genes, whereas EM plasmacytomas showed exclusive and recurrent mutations in the PITRM1 (Metalloprotease 1) gene. Furthermore, ATP7B, MTOR, TBC1D21 and ZNF717 mutations were present in both CTCs and plasmacytomas. In summary, we compared for the first time the genomic profiles of BM vs EM CTCs and plasmacytoma clones, and showed at the individual patient level, the existence of high mutation heterogeneity in between all three tissue related clones. However, actionable mutations in genes such as KRAS or BRAF were typically shared by BM and EM clones. According to the level of similarity between each clone, CTCs would be the most plausible precursor of EM plasmacytomas. Nevertheless, based on the number of specific mutations present in EM clones, it is likely that EM spreading followed by continuous clonal evolution starts much earlier prior to its clinical detection, which emphasizes the need for sensitive techniques to capture the early stages of EM spreading. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Somatic Gene Mutations Serve As Molecular Biomarkers Predictive for Response to Immunosuppressive Therapy (IST) in Myelodysplastic Syndromes (MDS)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1664-1664 ◽  
Author(s):  
Rami S. Komrokji ◽  
Mintallah Haider ◽  
Najla H. Al Ali ◽  
Jeffrey E Lancet ◽  
Qing Zhang ◽  
...  
Keyword(s):  
Immunosuppressive Therapy ◽  
Lower Risk ◽  
Research Funding ◽  
Response Rate ◽  
Gene Mutations ◽  
Cancer Center ◽  
Trisomy 8 ◽  
Significant Difference ◽  
Somatic Gene ◽  
The Impact

Abstract Introduction Immunosuppressive therapy (IST) yields durable hematologic improvement (HI) in a subset of patients (pts) with lower risk MDS. Age, HLA-DR15+, and duration of transfusion dependence are the strongest independent clinical variables predictive for response. We investigated the impact of somatic gene mutations on response to IST in lower risk MDS pts. Methods MDS pts who received ATG +/- CSA were identified at the Moffitt Cancer Center. The National Institutes of Health (NIH) IST response model was calculated for each pt. Next Generation sequencing (NGS) for somatic gene mutations was conducted using DNA extracted from archived BM prior to therapy. All pts underwent mutation analysis by a 49 myeloid gene panel. The library was generated with the ThunderBolt (RainDance Technologies, Billerica, MA) and sequenced on a MiSeq instrument (Illumina, San Diego, CA). Alignment and variant calling was performed with NextGene (Soft Genetics, State College, PA). Results Sixty-six pts treated with ATG +/- CSA were identified. Median age was 61 and the majority of pts had IPSS lower risk disease with favorable risk karyotype. Median time to initiation of IST was 1 year. All pts received ATG (60% rabbit (r-ATG); 32% equine (e-ATG)), and CSA was used in 60% of pts. Overall frequency of HI was 42% with a trend favoring e-ATG vs. r-ATG (52% vs. 39%, p=0.09). Erythroid HI was evaluable in 30 pts with 60% responding, neutrophil improvement was evaluable in 15 pts and 39% responded, while platelet improvement was evaluable in 18 pts with 57% responding. Six of 18 (33%) pts with pancytopenia experienced trilineage response. Mean time from ATG to next therapy was 12 mo (median of 7.7 mo). Neither presence of an LGL clone, hypocellular BM or fibrosis, HLA DR15, trisomy 8, nor age influenced response to IST. Pts classified as IPSS-R Very high or high risk were unresponsive (n= 5), whereas 10 of 19 pts (53%) with intermediate risk responded. Poor risk IPSS karyotype was associated with a trend for lower response rate when compared to intermediate and good risk (25% vs. 41% vs. 44%; p=0.6). The response rate based on the NIH IST model was 38% for low response probability category pts and 45% for high probability category (p=0.5). Response rate to IST was higher if administered within 2 years from diagnosis, with an HI rate of 48% vs. 33% when treated after 2 years (p=0.04). Pts who received ATG as first line treatment or after lenalidomide had a trend for higher response rates than those treated after azacitidine (46%,75%, and 25% respectively). Addition of CSA significantly improved HI rate (51% vs. 27% for ATG alone, p=0.02). Transformation to AML occurred in 10 pts, 7% of responders and 24% of non-responders (p=0.08). Median OS was 67.2 mo with no significant difference based on IST response. Among 40 pts evaluated by NGS, 20 (50%) had at least one demonstrable somatic mutation (SM) and 9 pts (22.5%) had two or more SM. SF3B1 was the most common SM detected (n=9, 22.5%), followed by ASXL-1 (n=7, 17.5%), TET-2 (n= 5, 12.5%), and STAG2, EZH-2 and ZRSR2 (2 pts each, 5%), and 1 pt each with IDH-1, KDM6A, SETBP1, RAD2, GNAS or GATA-2. Absence of a SM was associated with a higher response to IST (70% vs. 40%, p=0.16), whereas number of SM (1 vs. 2+) did not influence response. The presence of an SF3B1 mutation was a significantly associated with IST nonresponse (1/9 SF3B1 SM, 11% vs. 21/31 WT, 68%; p=0.01). All pts with SF3B1 SM had ring sideroblasts >15% (RS) by morphology and the corresponding HI rate was 20% among pts with RS vs 50% for those without RS, p=0.09. Median OS in pts with an SF3B1 SM was 111 mo vs. 54 mo in SF3B1 WT (p=0.016). The two pts with EZH-2 and the single pt with WT-1 S achieved HI. Mean duration of response was 12 mo among pts with no SM vs. 9 mo in those harboring a SM (p=0.09). Rate of AML transformation among pts with a SM other than SF3B1 was higher in pts without SM (4/11 pts, 36%vs. 1/20, 5%; p =0.023, with a corresponding reduced median OS (52 mo vs. 96, p=0.24). Conclusions These findings support an improved response rate to ATG when administered in combination with CSA, and early in the disease course. The presence of an SF3B1 mutation adversely influences response to IST, suggesting a non-immunologic pathogenesis in this molecularly defined subset. The presence of non-SF3B1 somatic mutations adversely affects response duration and probability of AML transformation. SM should be considered in selection of IST in lower risk MDS patients. Disclosures Komrokji: Pharmacylics: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; Incyte: Consultancy. Lancet:Seattle Genetics: Consultancy; Pfizer: Research Funding; Boehringer-Ingelheim: Consultancy; Kalo-Bios: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Research Funding. List:Celgene Corporation: Honoraria, Research Funding. Padron:Incyte: Research Funding; Novartis: Speakers Bureau.

scholarly journals Mutational Type and Configuration of an Individual Gene May Differentially Impact the Clinical and Phenotypic Features

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2992-2992
Author(s):  
Yasunobu Nagata ◽  
Hideki Makishima ◽  
Cassandra M Kerr ◽  
Bhumika J. Patel ◽  
Hassan Awada ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Somatic Mutations ◽  
The Other ◽  
Missense Mutations ◽  
Advisory Committees ◽  
Different Types ◽  
Equity Ownership ◽  
The Impact

Myelodysplastic syndromes (MDS) arise in older adults through the stepwise acquisition of multiple somatic mutations. The genetic heterogeneity that results includes mutations of diverse genes and their combinations, clonal hierarchy, genetic configuration (e.g., bi-allelic or compound heterozygous, hemizygous lesions), specific positions within a gene including canonical hotspots vs. other positions, and types of mutation (truncations vs. missense), all of which could differentially affect pathogenesis. Given the binary status (e.g. mutated vs. wild-type) used in many clinical analyses, the true impact of specific types of mutations may be obscured and their specific roles underestimated. Deep targeted NGS was carried out for a panel of the 36 most frequently mutated genes in 1,809 MDS patients (low-risk MDS (n=839) vs. high-risk MDS (n=607), MDS/MPN (n=212), and sAML n=151). Copy number alterations (CNA) were also evaluated by combining karyotyping, microarray, and digital copy number analysis. With a mean coverage of 862x, after removing SNPs and errors, 3,971 somatic mutations were identified, the most common (>10% of cases) being TET2, SF3B1, ASXL1, del(5q), SRSF2, complex karyotype, and del(7q). For the purpose of this proof of concept analysis we focused on illustrative genes (TP53, RUNX1, TET2, and EZH2) affected by 2 recurrent hits. Bi-allelic TET2 or TP53 mutations were found in 15% (271/1,809) and 4% (72/1,809) of patients, respectively. TET2 and RUNX1 were most likely biallelic, whereas TP53 and EZH2 were most often affected by mutations and somatic deletion. Comparing the distribution of canonical vs. other types of mutations in genes, DNMT3A mutations affected the canonical site (R882) in 17% (35/203) of patients, were truncating in 39% (79/203) and missense in 44% (89/203) have also been found; deletions affecting the DNMT3A locus are rare. Within U2AF1, U2AF1Q157 are more frequent than U2AF1S34 (54% vs. 35%). Next, we checked correlation between these different types of mutations of one gene. 78 significant combinations were found. For instance, U2AF1Q157 mutations more commonly accompanied ASXL1 mutations and del(7q) and less frequently DNMT3A and BCOR mutations, trisomy8 and del(20) when compared to U2AF1S34 mutations [ASXL1 mutations 53% (42/80) in U2AF1Q157 vs. 16% (8/49) in U2AF1S34, P < .0001]. TET2 Bi-allelic mutations were more commonly associated with ZRSR2 and SRSF2 mutations, and less frequently del(5q) when compared to TET2 mono-allelic mutations [SRSF2 mutations 29% (80/276) in TET2-bi vs. 15% (34/227) in TET2-mono, P = .003]. In addition, patients with SRSF2 missense mutations were more likely to have RUNX1 bi-allelic mutations than those with SRSF2 in-frame mutations. We evaluated the impact of different types of mutations and combinations of them on disease phenotypes and survival. We then evaluated the impact of different types of mutations and their combinations on clinical phenotypes including dichotomous morphological (MDS vs. MDS/MPN) features, progressive (low- vs. high risk) subtypes. EZH2 bi-allelic alterations were more commonly associated with myleoproliferative features` compared to EZH2 mono-allelic alteration (q=.016). TET2 bi-allelic alterations and truncating mutations were found more frequently in higher-risk subtypes than TET2 mono-allelic and missense mutations (q<.001). In survival analyses, patients with DNMT3AR882 mutations had a poorer prognosis than those with truncating and the other missense mutations [P = .033, HR 1.86 (1.05-3.3)]. Next, using the PyClone bioanalytic pipeline, we recapitulated for each patient the clonal hierarchy and defined "dominant" vs. "secondary" mutations. DNMT3AR882 mutations were likely to be dominant/founder lesions compared to truncating or the other missense mutations: 77% (27/35) for R882 vs. 51% (40/79) for truncating vs. 45% (47/98) for the other missense, p=.0046. Specific dominant and secondary mutational pairs also differentially affected survival compared to the reverse configuration (q<.1) including EZH2 and RUNX1 or BCOR and U2AF1 or RUNX1 and BCOR. In conclusion, we report a comprehensive analysis of various types and configurations of lesions of individual commonly affected genes. Our results indicate that establishment of clinical or phenotypic correlations requires consideration of the type, rank and configuration of somatic mutations. Disclosures Mukherjee: McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy. Nazha:Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

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