scholarly journals The Mutational Profile of Pediatric Therapy-Related Myeloid Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2775-2775
Author(s):  
Jason R Schwartz ◽  
Michael P Walsh ◽  
Jing Ma ◽  
Tamara Lamprecht ◽  
Raul C Ribeiro ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Primary Tumor ◽  
Mapk Pathway ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Mutational Signatures ◽  
Primary Tumors ◽  
Tp53 Mutations ◽  
Myeloid Neoplasms ◽  
Significant Difference

Abstract We and others recently showed that the mutational spectrum of de novo pediatric myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is different than those in adults. MDS and AML also occur in children as a consequence of cytotoxic therapies used to treat childhood malignancies and are collectively referred to as therapy-related myeloid neoplasms (tMN). The incidence of pediatric tMN is ~1% in the pediatric cancer population. These secondary malignancies are usually resistant to conventional chemotherapy and managed with hematopoietic cell transplantation (HCT). These patients have a dismal prognosis. TP53 mutations and somatic alterations in chromatin modifiers predominate in adults with tMN, yet whether children with tMN have a similar constellation of genetic alterations remains unclear since comprehensive genomic profiling has not been completed in a large pediatric tMN cohort. We hypothesize that the mutational profile of pediatric tMN will be different than adult tMN given the patients' younger age and the different spectrum of primary tumor types and chemotherapies. Here we describe the somatic mutational profile of pediatric tMN (including tMDS & tAML) using whole exome (WES) and RNA-sequencing. We evaluated 65 diagnostic bone marrow samples from 61 unique patients, obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1987 & 2018. The cohort contains 26 tMDS and 39 tAML cases; in 4 patients both tMDS and tAML samples were included. Primary tumors included hematological malignancies (n=45), bone and soft tissue solid tumors (n=14), and brain tumors (n=2); acute lymphoblastic leukemia (ALL) was the most common primary tumor (n = 38, 62%). WES was completed for 61 tumor/normal pairs using Nextera Rapid Capture Expanded Exome (Illumina), while WGS was completed on 4 pairs. Normal comparator genomic DNA was obtained from flow-sorted lymphocytes. Median sequencing coverage for the tumor and normal samples were 107x and 95x, respectively. An average of 49 variants/patient (range: 6-217) was observed in the tMN cohort, including coding, non-coding, silent, and splice site variants, which is significantly different than our previously reported 5 variants/patient in pediatric primary MDS (p = 1x10-6). There was not a significant difference in the number of mutations/patient when tMDS was compared to tAML. Mutational signature analysis (https://cancer.sanger.ac.uk/cosmic/signatures) identified 3 major signatures, the most predominant was characterized by a strong bias for C>A mutations (Signature 24), followed by a signature with strong transcriptional strand bias for T>A mutations (Signature 27) and then a smaller subset resembling MDS and AML (Signature 1). Interestingly, patients with Signature 1 had an inferior 2-year overall survival than the other mutational signatures, with a median survival of 0.3 years (p = 0.0005). WES data and conventional karyotyping showed that chromosome 7 deletions (del(7)) were frequent (n=21, 32%), followed by deletions involving chromosome 5 (del(5)) (n=10, 15%). All of the cases with del(5) had complex cytogenetics and 6 of the 10 cases also had del(7). Ras/MAPK pathway mutations were present in 37% of the cases (40 total mutations in 27 cases). Canonical KRAS (n = 14), NF1 (n = 8), and NRAS (n = 7) mutations were the most frequent coding mutations present overall. Only 5 patients had somatic TP53 mutations, all of which had complex karyotypes. RNA sequencing was performed on 55 samples with suitable RNA. KMT2A rearrangements (KMT2Ar) were common (n = 29, 53%), 4 of which were cytogenetically cryptic. KMT2A rearrangements were more common in tAML (n = 25) but were present in tMDS (n = 4). Nearly half of these KMT2Ar cases also harbored an additional Ras/MAPK mutation. Fusions involving NUP98, RUNX1, MECOM, and ETV6 were also detected. In conclusion, we show that the mutational profile of pediatric tMN has fewer TP53 mutations and more KMT2Ar than adults, as well as a unique set of mutational signatures. These differences are likely a reflection of age-specific chemotherapeutic strategies and fewer pre-existing TP53 mutant hematopoietic clones in children. Future studies understanding the clonal evolution of pediatric tMN development will be helpful in describing pediatric tMN further. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comprehensive Genomic Profiling of Pediatric Therapy-Related Myeloid Neoplasms Identifies Mecom Dysregulation to be Associated with Poor Outcome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1394-1394
Author(s):  
Jason R Schwartz ◽  
Jing Ma ◽  
Michael P Walsh ◽  
Xiaolong Chen ◽  
Tamara Lamprecht ◽  
...  
Keyword(s):  
Primary Tumor ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Cells ◽  
Fusion Partner ◽  
Genomic Profile ◽  
Complex Karyotype ◽  
Rna Seq ◽  
Primary Tumors ◽  
Expression Levels ◽  
Myeloid Neoplasms

Therapy-related myeloid neoplasms (tMN) occur in children secondary to cytotoxic therapies used to treat pediatric malignancies, are typically resistant to conventional chemotherapy, require hematopoietic cell transplantation as the only curative option, and have a dismal prognosis. The genomic alterations that drive tMN in children have yet to be comprehensively described, and it is unclear if particular genomic lesions hold prognostic value. We have characterized the genomic profile of 62 pediatric tMN cases (tMDS: n=23, tAML: n=39) obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1987 and 2018. These cases arose following treatment for a variety of primary tumors (hematological (74%), bone and soft tissue (23%), and brain (3%)). Acute lymphoblastic leukemia was the most frequent primary tumor (n=39, 63%). Conventional cytogenetics (n=60) showed a complex karyotype (≥3 structural alterations) in 19 (32%) cases, and 7 of these cases contained a deletion involving chromosome 7 (del(7)). Eleven (18%) other cases without complex karyotypes had del(7). Deletions of chromosome 5 were present in 9 (15%) cases, but only in the context of a complex karyotype. We hypothesized that the patients' younger age and the different spectrum of primary tumor types and chemotherapy would give rise to a mutational spectrum distinct from adult tMN. We used whole exome (WES), whole genome (WGS), and RNA sequencing (RNA Seq) to describe the mutational profile of our pediatric tMN cohort. WES was completed for 58 tumor/normal pairs using Nextera Rapid Capture Expanded Exome (Illumina). Fifteen cases were analyzed by WGS (11 also had WES). Normal comparator genomic DNA was obtained from flow-sorted lymphocytes. An average of 21 coding variants/patient (range: 1-131) was observed from the gene-coding region, and these include synonymous, non-synonymous, and splice site variants. Ras/MAPK pathway mutations were present in 44% of the cases (43 mutations in 27 cases). Canonical KRAS (n = 16), NF1 (n = 10), and NRAS (n = 7) mutations were the most frequent coding mutations. Eleven (18%) patients had either heterozygous deletion or a copy neutral loss of heterozygosity event involving chromosome 17p and the TP53 locus; 5 of these cases had concurrent TP53 missense mutations identified at allele frequencies near 100%. Unlike tMN in adults, mutations in PPM1D were not identified. RNA-Seq completed on 56 evaluable cases identified 28 (50%) cases with KMT2A rearrangement (KMT2Ar). MLLT3 was the most common fusion partner (n=13, 46%). In addition to KMT2A rearrangements, RNA-Seq also identified a RUNX1-MECOM fusion. Alterations involving the MECOM locus have been described in some myeloid neoplasms like tMN, and its overexpression is associated with a poor prognosis and some AMLs with KMT2Ar. MECOM expression levels were variable in this cohort (FPKM range: 0.004 - 38.4) with 24 cases (43%) having an FPKM>5 (MECOMHigh). In addition to the RUNX1-MECOM event, these 24 MECOMHigh cases included 18 with KMT2Ar (64% of KMT2Ar group) and 1 with a NUP98 fusion (NUP98-HHEX). The remaining 4 MECOMHigh cases demonstrate allele-specific MECOM expression, suggesting a cis-regulatory element is driving this expression. Two of these 4 cases have WGS and were found to contain a t(2;3)(p21;q26.2) involving MECOM on chromosome 3 and noncoding regions of chromosome 2 adjacent to ZFP36L2, a gene highly expressed in hematopoietic cells. ENCODE data supports that this region of the genome is an active enhancer in hematopoietic cells, suggesting a proximity effect in which this enhancer has been hijacked to drive high levels of MECOM expression. In our cohort, MECOM expression levels are predictive of a worse outcome (overall survival (OS) at 2 years: High=14.6% vs. Low=46.3%; log rank p<0.01). Although KMT2Ar was frequently present in our cohort and enriched in the MECOMHigh group (High=75% (18/24) vs. Low=31% (10/32); p<0.01), high MECOM expression did not confer a significant survival difference within the KMT2Ar group (OS at 2 years: High=16.7% vs Low=40%; log rank p=0.33). Further, the presence of a KMT2Ar or a complex karyotype did not significantly affect the OS in this cohort. In conclusion, we report the genomic profile of a large cohort of pediatric tMN cases and show that high levels of MECOM expression, a portion of which is driven by enhancer hijacking, predicts a worse outcome. Disclosures Gruber: Bristol-Myers Squibb: Consultancy.

Genetic and immune divergence in pancreatic cancer lesions at the time of metastatic relapse compared to primary tumor.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 4140-4140
Author(s):  
Bereket Gebregziabher ◽  
Derek A. Oldridge ◽  
Emma E. Furth ◽  
Michael D. Feldman ◽  
Andrew J. Rech ◽  
...  
Keyword(s):  
Radiation Therapy ◽  
Adjuvant Chemotherapy ◽  
Rna Sequencing ◽  
Primary Tumor ◽  
Metastatic Tumors ◽  
Kras Mutations ◽  
Primary Tumors ◽  
Metastatic Relapse ◽  
Resected Tumor ◽  
First Relapse

4140 Background: Relapse of pancreatic ductal adenocarcinoma (PDA) is common even after complete resection and adjuvant therapy. Compared to the resected tumor, the biological characteristics of metastatic tumors at the time of first relapse are poorly understood. Methods: Whole-exome sequencing (WES) (250x) and bulk RNA sequencing were performed on samples from 30 patients with PDA. Paired primary tumor samples were obtained after R0 or R1 resection, and metastatic tumor samples were obtained by biopsy at the time of first relapse. 74.1% of patients had received adjuvant chemotherapy and radiation therapy, 7.4% had received adjuvant chemotherapy only, and 3.7% had received adjuvant radiation therapy only. Most common metastatic sites were liver and lung. The cohort was 60% male with a median age at diagnosis of 64 years. The vast majority of patients had stage IIA or IIB disease at diagnosis. Median disease-free survival was 481 days. Analysis used Freebayes (somatic variant calling), Kallisto (transcript quantification), Danaher et al. (cell type deconvolution), and antigen.garnish (neoantigen prediction). Results: High-quality WES and/or RNA sequencing were available for 27/30 patients. Among these were 16 pairs of primary and metastatic samples for WES and 15 paired samples for RNA sequencing. Median tumor purity was 32% (primary) and 42% (metastatic). KRAS mutations were present in 43/48 evaluable samples, with conserved KRAS mutations in 14/16 primary-metastatic pairs. Tumors were otherwise highly variable, with 13/16 patients developing oncogenic mutations in metastatic tumors that were undetected in primary tumors (BRCA1 [3/16], AKT3 [3/16], TP53 [2/16], ROS1 [2/16]). Overall, primary and metastatic tumors had similar tumor mutation burden and neoantigen production rate. However, neoantigens were highly variable at the peptide and gene level, with conservation rates of 2.73% and 11.57%, respectively, across primary-metastatic pairs. PDA transcriptomic subtype also differed across primary-metastatic pairs in all cases. Furthermore, metastatic tumors contained lower immune suppressive signal by transcripts and deconvolution (CTLA4: p = 0.0012, FOXP3: p = 0.0026, PDCD11: p = 0.012, regulatory T cells: p = 0.012), while myeloid cells were higher (CD33: p = 0.0067). Conclusions: With the exception of KRAS, metastatic PDA tumors at relapse contain new oncogenes, distinct neoantigens, and lower immune-suppressive signal compared to primary PDA tumors. These data suggest a potential clinical utility for tumor biopsies at the time of first metastatic relapse and caution against clinical decisions for relapsed, metastatic patients based solely on sequencing of the originally resected tumor.

scholarly journals Landscape of driver mutations and their clinical impacts in pediatric B-cell precursor acute lymphoblastic leukemia

2020 ◽  
Vol 4 (20) ◽  
pp. 5165-5173
Author(s):  
Hiroo Ueno ◽  
Kenichi Yoshida ◽  
Yusuke Shiozawa ◽  
Yasuhito Nannya ◽  
Yuka Iijima-Yamashita ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Tp53 Mutation ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Cell Precursor ◽  
Tp53 Mutations ◽  
Positive Correlations ◽  
Panel Sequencing

Abstract Recent genetic studies using high-throughput sequencing have disclosed genetic alterations in B-cell precursor acute lymphoblastic leukemia (B-ALL). However, their effects on clinical outcomes have not been fully investigated. To address this, we comprehensively examined genetic alterations and their prognostic impact in a large series of pediatric B-ALL cases. We performed targeted capture sequencing in a total of 1003 pediatric patients with B-ALL from 2 Japanese cohorts. Transcriptome sequencing (n = 116) and/or array-based gene expression analysis (n = 120) were also performed in 203 (84%) of 243 patients who were not categorized into any disease subgroup by panel sequencing or routine reverse transcription polymerase chain reaction analysis for major fusions in B-ALL. Our panel sequencing identified novel recurrent mutations in 2 genes (CCND3 and CIC), and both had positive correlations with ETV6-RUNX1 and hypodiploid ALL, respectively. In addition, positive correlations were also newly reported between TCF3-PBX1 ALL with PHF6 mutations. In multivariate Cox proportional hazards regression models for overall survival, TP53 mutation/deletion, hypodiploid, and MEF2D fusions were selected in both cohorts. For TP53 mutations, the negative effect on overall survival was confirmed in an independent external cohort (n = 466). TP53 mutation was frequently found in IGH-DUX4 (5 of 57 [9%]) ALL, with 4 cases having 17p LOH and negatively affecting overall survival therein, whereas TP53 mutation was not associated with poor outcomes among NCI (National Cancer Institute) standard risk (SR) patients. A conventional treatment approach might be enough, and further treatment intensification might not be necessary, for patients with TP53 mutations if they are categorized into NCI SR.

Genomic characterization of brain metastases (BM) in high-grade serous ovarian cancer (HGSOC).

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13580-e13580
Author(s):  
Renata Duchnowska ◽  
Anna Maria Supernat ◽  
Rafał Pęksa ◽  
Marta Łukasiewicz ◽  
Tomasz Stokowy ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Dna Damage ◽  
Somatic Mutations ◽  
Dna Damage Repair ◽  
Genetic Alterations ◽  
High Grade ◽  
Serous Ovarian Cancer ◽  
Primary Tumors ◽  
Tp53 Mutations ◽  
Damage Repair

e13580 Background: BM are a rare occurrence in ovarian cancer (OC) and their molecular characteristics is virtually unknown. DNA damage repair (DDR) deficiency is prevalent in OC, and co-mutated TP53 and any DDR denotes high tumor mutation burden (TMB). We genetically characterized a unique series of high-grade serous ovarian cancer (HGSOC) patients who developed BM to identify alterations of potential clinical relevance. Methods: Whole-exome sequencing (2x150bp, SureSelectXT Library Prep Kit, Illumina’s NovaSeq platform) was performed in matched BM, primary tumors (PT) and normal tissue. DNA was extracted from FFPE samples using QIAamp DNA FFPE Tissue Kit (Qiagen, Germany). All mutations were checked with Catalogue of Somatic Mutations in Cancer (COSMIC) and Integrative Genomics Viewer (IGV). Results: Study group included 10 HGSOC patients (International Federation of Gynecology and Obstetrics classification (FIGO) II-IV, mean age at diagnosis 48 years, range 35-59). Median time from primary HGSOC diagnosis to BM was 38 months (range, 18 to 149). TP53 somatic mutations were found in both primary tumor (PT) and BM in 8 patients. The other 2 cases harbored TP53 mutations not reported in COSMIC catalogue: p.S60L and intronic TP53 mutation preceding p.I322 (IGV). In 9 cases TP53 mutations coexisted with germline or somatic DNA damage repair deficiency. Four cases contained BRCA1 mutations (all germline), and none harbored germline BRCA2 mutation. Other mutated genes included MLH1 (2 somatic, 2 germline), ATR (4 germline, 1 somatic), AMT (1 somatic), RAD50 (1 somatic), ERCC4 (1 somatic), FANCD2 (1 somatic) and RPA1 (1 germline). Three mutation signatures defined in the COSMIC database were indentified in BM: 6, 20 and 30. In 6 cases these mutations were shared in PT, and in another 4 their presence in PT could not be determined due to technical reasons. Median survival from BM was 31 months (range, 5 to 184). Conclusions: Genomic analysis of BM provides an opportunity to identify potentially clinically informative alterations. Mutational profiles in PT are generally reflected in BM. Detected genetic alterations suggest their potential sensitivity to PARP inhibitors and immunotherapy.

Comprehensive genomic profiling (CGP) in patients with relapsed/refractory germ cell tumors (GCT).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e17053-e17053
Author(s):  
Reem Akel ◽  
Bilal Anouti ◽  
Sean Kern ◽  
Clint Cary ◽  
Timothy A. Masterson ◽  
...  
Keyword(s):  
Primary Tumor ◽  
Germ Cell Tumors ◽  
Genetic Alterations ◽  
Immune Checkpoint Blockade ◽  
High Status ◽  
First Line ◽  
Genomic Alterations ◽  
Primary Tumors ◽  
Entire Cohort ◽  
Pure Seminoma

e17053 Background: Understanding the genetic alterations in patients with relapsed/refractory GCT (rrGCT) could delineate the pathogenesis of cisplatin resistance. Our study uses CGP to characterize genomic alterations (GA) in refractory GCT and correlate with clinical outcomes. Methods: 432 patients with rrGCT were seen at Indiana University between Jan 2016 to Sep 2019 of whom 52 patients underwent CGP using a hybrid-capture based commercial assay to evaluate all classes of GA. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and reported as mutations/Mb and microsatellite instability (MSI) was determined on 114 loci. PDL1 expression was determined by IHC (DAKO 22C3 antibody). Results: All patients relapsed after first-line cisplatin-based combination chemotherapy. Median age at diagnosis was 33 (range 15-68). Primary site of GCT was testicular in 85% and mediastinum in 8%. 6 patients had pure seminoma and 46 had non-seminoma. Platinum refractory disease, defined as serologic or radiographic progression within 4 weeks of first-line chemotherapy comprised 23% of patients. The primary tumor was used for sequencing in 6 cases (12%) and non-primary tumor metastatic site (lymph node, lung, liver, brain, omentum) in 46 cases (88%). The most common GA in the entire cohort were FGF6 (27%), FGF23 (27%), KDM5A (27%), CCND2 (27%), KRAS (18%), TP53 (14%), KIT (8%), APC (8%), ZNF217 (6%), MUTYH (6%), AURKA (6%), NRAS (6%), EGFR (6%), CTNNB1 (6%), GNAS (6%). Most common alterations for testicular primary tumors were FGF6, FGF23, KDM5A, CCND2, KRAS, TP53, KIT. For non-testicular primary GCT, most common GA were APC, TP53, EGFR. Most common GA for non-seminoma were FGF6, FGF23, KDM5A, CCND2, KRAS, TP53, APC. Most common GA for pure seminoma was KIT. Potentially targetable genomic alterations were found in 17 patients (33%). 10 of 17 patients (59%) tested had PDL1 score ≥1% and 3 patients had PDL1 ≥50%. Median TMB was 3.5 mutations/MB. There were 4 patients (8%) with TMB ≥ 10 mutations/Mb and 2 patients (4%) with TMB ≥ 20 mutations/Mb. 1 of 48 patients (2%) evaluated for MSI had MSI-High status. Isochromosome 12p was detected in the majority of samples where it was tested. Outcomes with GA-directed therapy will be presented at the conference. Conclusions: CGP can reveal potential therapeutic targets in patients with rrGCT including EGFR, ERBB3, KIT, and MET. Consistent with reported clinical trials in rrGCT, biomarkers predicting response to immune checkpoint blockade are uncommon with most patients having low TMB, absence of MSI-H status, and low expression of PDL1.

scholarly journals A Fast and Accurate Diagnostic Method for Ph-like ALL Using the Ncounter System

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 375-375
Author(s):  
Kensuke Sasaki ◽  
Kohta Miyawaki ◽  
Yuichiro Semba ◽  
Koji Kato ◽  
Jumpei Nogami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Sequencing ◽  
Clinical Outcomes ◽  
Induction Therapy ◽  
Diagnostic Method ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Pattern ◽  
Genetic Alterations ◽  
Rna Seq ◽  
Significant Difference

Ph-like acute lymphoblastic leukemia (also known as BCR-ABL1-like ALL) is a new disease entity of B-cell ALL (B-ALL) that exhibits a mRNA expression profile similar to that of Philadelphia chromosome-positive ALL (Ph+ ALL). Ph-like signature is presumably driven by kinase-activating gene alterations. Thus, both gene expression pattern and DNA mutational status should be assessed to make a definitive diagnosis for Ph-like ALL. A variety of approaches combining multiple methods, including RNA sequencing (RNA-seq), Taqman low-density array (LDA), fluorescence in situ hybridization (FISH) and targeted DNA sequencing, are being tested; however, such multi-omics approaches are available only in limited institutions. Since Ph-like ALL patients generally exhibit poor response to standard chemotherapy, and tyrosine kinase inhibitors (TKIs) may benefit them when used in a timely manner, a fast, accurate and generalizable diagnostic method is critically needed. In the present study, we have developed a nCounter-based diagnostic method for Ph-like ALL and validated it using a cohort of Japanese adult B-ALL cases. To identify genes that are uniquely expressed (or not expressed) in Ph+ B-ALL, we first obtained publicly-available gene expression datasets comprising 1146 B-ALL cases and identified 82 differentially-expressed genes in Ph+ ALL cases. We then assessed expression levels of those genes in an independent cohort using the nCounter, which enables fast, sensitive and accurate RNA detection. We also tested whether nCounter-based methods can detect fusion transcripts relevant for Ph-like ALL pathogenesis using probes targeting ABL1, ABL2, CSF1R, PDGFRB, and JAK2. We analyzed 123 samples (Ph+ = 42, Ph- = 81, age 16 to 67) obtained from newly-diagnosed adult B-ALL patients enrolled in two clinical trials conducted by the Fukuoka Blood and Marrow Transplantation Group (FBMTG) (Nagafuji et al. Eur J Haematol 2019). Unsupervised hierarchical clustering successfully stratified 123 cases into two disease clusters: Ph+ and Ph- subgroups. As expected, Ph+ subgroup included almost all Ph+ ALL cases (40 out of 42 cases), while 18 out of 81 Ph- ALL cases (22%) were categorized into the Ph+ subgroup. We defined these cases as Ph-like ALL. To validate the nCounter-based Ph-like ALL classification, we performed RNA-seq and target-capture DNA sequencing of all Ph- ALL cases. As expected, we detected kinase-activating fusions/rearrangements, including CRLF2 rearrangements (7 cases), PDGFRB fusions (3 cases), JAK2 fusions (2 cases), EPOR rearrangements (2 cases), ABL1 fusion (1 case), and FLT3 internal tandem duplication (1 case) in 16 Ph-like ALL cases, while no genetic alterations were detected in 2 cases. Fusion genes involving PDGFRB were consistently detected by nCounter (3/3); however, detection of those involving JAK2 (1/2) and ABL1 (0/1) were inconsistent. JAK2 and/or RAS mutations were detected in 5 of 7 Ph-like ALL cases harboring CRLF2 rearrangements. Of note, CRLF2 protein expression was detected by FACS in all CRLF2-rearranged cases. We next assessed significance of the Ph-like signature on clinical outcomes using a cohort of 40 Ph- ALL cases, in which minimal/measurable residual disease (MRD) status, assessed by IgH and/or TCR rearrangements, as well as clinical data were available (Nagafuji et al. Eur J Haematol 2019). Ph-like ALL cases exclusively exhibited MRD positivity after induction therapy as compared to non-Ph-like cases (p=0.04), indicative of the chemo-resistant nature of Ph-like ALL as previously reported (Roberts et al. N Engl J Med, 2014 and Roberts et al. J Clin Oncol, 2017). As expected, Ph-like ALL cases exhibited significantly poor disease-free survival compared with non-Ph-like ALL cases (p=0.04); however, no significant difference was evident in overall survival (p=0.62) presumably due to the fact that all MRD-positive cases were subjected to allo-HSCT after induction therapy. These data indicate that MRD-based therapy stratification could overcome chemo-resistant nature of Ph-like ALL. Our data suggest that nCounter-based diagnostic method is fast and accurate to identify Ph-like ALL. Since Ph-like signature generally dictates poor clinical outcomes, and upfront TKI therapy may improve them, our method could facilitate precision medicine in the treatment of Ph- B-ALL. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Type of TP53 Mutations Affects Subclonal Configuration and Selection Pressure for Acquisition of Additional Hits in Contralateral Alleles

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-25
Author(s):  
Carmelo Gurnari ◽  
Vera Adema ◽  
Hassan Awada ◽  
Simona Pagliuca ◽  
Cassandra M Kerr ◽  
...  
Keyword(s):  
Selection Pressure ◽  
Double Mutant ◽  
Hematopoietic Cell Transplantation ◽  
De Novo ◽  
Genetic Alterations ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Tp53 Mutations ◽  
Myeloid Neoplasms ◽  
The Impact

Somatic TP53 mutations are found in 10% of adult patients with MDS and de novo AML and in up to 20% of patients with therapy-related myeloid neoplasms. TP53 status is associated with complex karyotype (CK), aberrations of chromosome 5 and poor survival. Moreover, mutated TP53 (TP53MT) may be an indication for hematopoietic cell transplantation, but also predictive of relapse following the procedure, making this particular category of myeloid neoplasms (MN) a conundrum of clinical management. Unlike other tumor suppressor genes, missense (ms) mutations within the DNA-binding domain (DBD) are the most common genetic alterations in TP53 gene representing up to 80% of somatic hits, with involvement of canonical hotspots (R175H, Y220C, M237I, R248Q, R273H, R282W) in around 30% of cases. A loss-of-function (LOF) dominant-negative effect (DN) may explain the ability of TP53MT to interfere with wild type (WT) functions. Moreover, TP53 germ line (GL) mutations are responsible for Li-Fraumeni syndrome, and GL contamination may also exist in adult MN. Here we comprehensively characterize TP53MT MNs to better dissect the role of specific mutational configurations and identify the selective forces affecting outcomes in this poor prognostic MN category. A total of 764 TP53 mutations were found in 632 MN patients. Ms mutations were the most common (75%) followed by frameshift (11%), splice site (7%), nonsense (5%) and insertion/deletions (2%), with 20% of patients harboring more than 1 lesion. Topographical annotation revealed that ms mutations typically (98%) occurred within the DBD (residues 102-292) and only 2% occurred outside this region (vs. 28% in case of truncating mutations, p<.0001). Overall, 22% of patients displayed a mutation in the canonical hotspot regions. Among TP53MT, 36 cases (6%) were of possible GL origin. Focusing on the somatic lesions, a male preponderance (1.42 vs. 1.1 M:F, p=.0069) and a younger age at presentation (median 68.9 vs 71, p<.00001) were found in WT vs. mutant cases, which were also less enriched in coincident de novo leukemia-driver genes mutations (e.g. NPM1, FLT3). When compared to WT MN, TP53MT cases were more likely to have CK (8% vs. 70%, p<.00001), del(5q) (4% vs. 40%, p<.00001), del(7q)/-7 (6% vs. 18%, p<.00001) and trisomy 8 (8% vs. 49%, p<.00001). Of note, deletion of the TP53 locus was found in 27% of mutated cases vs only 1% of WT counterparts (p<.00001). Conversely, 77% of all MN cases with CK had either TP53 mutations (61%), del(17p) (3%) or both (36%). When classifying patients according to TP53 genomic context (30% single vs. 70% double hit, defined as a presence of biallelic, hemizigous or UPD configuration) progressive inactivation had an adverse impact on survival (p<.0001). We then hypothesized that truncating (frameshift/nonsense/deletion) alterations require additional hits as the presence of one allele may be partially protective. Consequently, the VAF for these lesions may be a result of a UPD in a smaller fraction of cells; biallelic truncation hits thus may be truly biallelic rather than clonally mosaic, which can be demonstrated only by single cell DNA sequencing. In contrast, DN mutations in canonical hotspots decrease the function beyond 50% of the activity, with further inactivation would having less of an effect, thus exerting less selection pressure for acquisition of additional lesions. Indeed, second truncating hits (including UPD and del(17p)) were common (30%), while none of the dominant ms hits had a double-mutant hotspot configuration (vs. 14% of non-canonical ms double mutant), and these canonical dominant hits were less likely to be paired with del(17p) or truncating mutations (8%). Only 25% of CK had a WT configuration of TP53, consistent with our theory that dominant ms hits were more likely to be present without del(17p). It is possible that the inability to assert clear survival differences according to the number or types of TP53 lesions may be due to an inability to resolve the intraclonal configuration of mutations using VAF calculations. We also conclude that non-canonical ms mutations (many of them classified as VUS) may have a variable impact, with functional consequences ranging from those that are less severe than truncations to various degrees of negative dominance. Analyses of the impact of ms mutations on TP53 tetramers (which may contain various doses of mutant vs WT monomer), will shed further light on the biology of TP53MT MN. Disclosures Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sekeres:Takeda/Millenium: Consultancy; BMS: Consultancy; Pfizer: Consultancy. Carraway:Abbvie: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; ASTEX: Other: Independent Advisory Committe (IRC); BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC). Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Comprehensive genomic profiling (CGP) in patients with relapsed/refractory germ cell tumors (GCT).

2021 ◽  
Vol 39 (6_suppl) ◽  
pp. 388-388
Author(s):  
Reem Akel ◽  
Bilal Anouti ◽  
Sean Kern ◽  
Clint Cary ◽  
Timothy A. Masterson ◽  
...  
Keyword(s):  
Primary Tumor ◽  
Germ Cell Tumors ◽  
Genetic Alterations ◽  
Immune Checkpoint Blockade ◽  
High Status ◽  
First Line ◽  
Genomic Alterations ◽  
Primary Tumors ◽  
Entire Cohort ◽  
Pure Seminoma

388 Background: Understanding the genetic alterations in patients with relapsed/refractory GCT (rrGCT) could delineate the pathogenesis of cisplatin resistance. Our study uses CGP to characterize genomic alterations (GA) in refractory GCT and correlate with clinical outcomes. Methods: 432 patients with rrGCT were seen at Indiana University between Jan 2016 to Sep 2019 of whom 52 patients underwent CGP using a hybrid-capture based commercial assay to evaluate all classes of GA. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and reported as mutations/Mb and microsatellite instability (MSI) was determined on 114 loci. PDL1 expression was determined by IHC (DAKO 22C3 antibody). Results: All patients relapsed after first-line cisplatin-based combination chemotherapy. Median age at diagnosis was 33 (range 15-68). Primary site of GCT was testicular in 85% and mediastinum in 8%. 6 patients had pure seminoma and 46 had non-seminoma. Platinum refractory disease, defined as serologic or radiographic progression within 4 weeks of first-line chemotherapy comprised 23% of patients. The primary tumor was used for sequencing in 6 cases (12%) and non-primary tumor metastatic site (lymph node, lung, liver, brain, omentum) in 46 cases (88%). The most common GA in the entire cohort were FGF6 (27%), FGF23 (27%), KDM5A (27%), CCND2 (27%), KRAS (18%), TP53 (14%), KIT (8%), APC (8%), ZNF217 (6%), MUTYH (6%), AURKA (6%), NRAS (6%), EGFR (6%), CTNNB1 (6%), GNAS (6%). Most common alterations for testicular primary tumors were FGF6, FGF23, KDM5A, CCND2, KRAS, TP53, KIT. For non-testicular primary GCT, most common GA were APC, TP53, EGFR. Most common GA for non-seminoma were FGF6, FGF23, KDM5A, CCND2, KRAS, TP53, APC. Most common GA for pure seminoma was KIT. Potentially targetable genomic alterations were found in 17 patients (33%). 10 of 17 patients (59%) tested had PDL1 score ≥1% and 3 patients had PDL1 ≥50%. Median TMB was 3.5 mutations/MB. There were 4 patients (8%) with TMB ≥ 10 mutations/Mb and 2 patients (4%) with TMB ≥ 20 mutations/Mb. 1 of 48 patients (2%) evaluated for MSI had MSI-High status. Isochromosome 12p was detected in the majority of samples where it was tested. Outcomes with GA-directed therapy will be presented at the conference. Conclusions: CGP can reveal potential therapeutic targets in patients with rrGCT including EGFR, ERBB3, KIT, and MET. Consistent with reported clinical trials in rrGCT, biomarkers predicting response to immune checkpoint blockade are uncommon with most patients having low TMB, absence of MSI-H status, and low expression of PDL1.

scholarly journals Whole-transcriptome analysis in acute lymphoblastic leukemia: a report from the DFCI ALL Consortium Protocol 16-001

2021 ◽  
Author(s):  
Thai Hoa Tran ◽  
Sylvie Langlois ◽  
Caroline Meloche ◽  
Maxime Caron ◽  
Pascal St-Onge ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mapk Pathway ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Molecular Classification ◽  
Genetic Alterations ◽  
Rna Seq ◽  
Childhood All ◽  
Next Generation Sequencing Technology

The molecular hallmark of childhood ALL is characterized by recurrent, prognostic genetic alterations, many of which are cryptic by conventional cytogenetics. RNA-seq is a powerful next-generation sequencing technology with the ability to simultaneously identify cryptic gene rearrangements, sequence mutations and gene expression profiles in a single assay. We examined the feasibility and utility of incorporating RNA-seq into a prospective multi-center phase 3 clinical trial for children with newly-diagnosed ALL. Patients enrolled on the DFCI ALL Consortium Protocol 16-001 who consented to optional studies and had available material underwent RNA-seq. RNA-seq was performed in 173 ALL patients. RNA-seq identified at least one alteration in 157 (91%) patients. Fusion detection was 100% concordant with results obtained from conventional cytogenetic analyses. An additional 56 gene fusions were identified by RNA-seq, many of which confer prognostic or therapeutic significance. Gene expression profiling enabled further molecular classification into the following B-ALL subgroups: High hyperdiploid (n=36), ETV6-RUNX1/-like (n=31), TCF3-PBX1 (n=7), KMT2A-rearranged (n=5), iAMP21 (n=1), hypodiploid (n=1), BCR-ABL1/-like (n=16), DUX4-rearranged (n=11), PAX5 alterations (n=11), PAX5 P80R (n=1), ZNF384-rearranged (n=4), NUTM1-rearranged (n=1), MEF2D-rearranged (n=1) and Others (n=10). RNA-seq identified 141 nonsynonymous mutations in 93 (54%) patients; the most frequent were RAS-MAPK pathway mutations. Among 79 patients with both low-density array and RNA-seq data for the Ph-like gene signature prediction, results were concordant in 74 (94%) patients. In conclusion, RNA-seq identified several clinically-relevant genetic alterations not detected by conventional methods, supporting the integration of this technology in frontline pediatric ALL trials.

Genetic Architecture of Relapsed Pediatric B Cell Precursor Acute Lymphoblastic Leukemia Are Related to the Intensity of Upfront Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 247-247
Author(s):  
Jiangyan Yu ◽  
Esmé Waanders ◽  
Simon V. van Reijmersdal ◽  
Edwin Sonneveld ◽  
Frank N. van Leeuwen ◽  
...  
Keyword(s):  
B Cell ◽  
Genetic Architecture ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Treatment Groups ◽  
Significant Difference ◽  
Additional Chemotherapy ◽  
Group 3 ◽  
Group 2 ◽  
Group 1

Abstract B cell precursor acute lymphoblastic leukemia (BCP-ALL) is one of the most common malignancies in children. In the period 1991-2013, the Dutch Childhood Oncology Group (DCOG) has completed three treatment trials for childhood ALL: ALL8, 9 and 10, each protocol with stratifications into risk-groups (details: www.skion.nl). Although the cure rates increased in these subsequent trials, relapses still occurred in a significant number of children. Since consecutive upfront treatment protocols usually change at multiple levels, genomic alterations that are associated with relapse may also be variable, which could provide insight into the biology underlying therapy failure and relapse. In this study, we characterized the genetic architecture of relapsed BCP-ALL patients within the context of these three Dutch upfront protocols. We identified 3 patient groups based on upfront treatment as follows: Group-1: patients treated upfront with high-amounts of corticosteroids (CS) and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). The number of high-risk patients that relapsed after ALL8 HR and ALL10 HR chemotherapy courses was too low to be included for analysis. We determined, at relapse, the presence of copy number alterations and sequence mutations in 21 recurrently affected genes involved in B-cell development, cell cycle regulation and RAS signaling, in 123 patients that relapsed after treatment in group-1 (n=56), group-2 (n=20) and group-3 (n=47). The number of CREBBP mutations in patients that relapsed after treatment according to group-1 (ALL9) was significantly lower compared to the other two groups, whereas B-cell development alterations were most common in patients that relapsed after treatment according to group-1, mainly due to a higher number of IKZF1 alterations (Figure 1). The high number of relapsed patients with leukemic clones carrying IKZF1 alterations in patients treated with high-amounts of CS and relatively mild additional chemotherapy is in line with our recent finding that IKZF1 is a key determinant of GC-induced apoptosis in normal and leukemic B-cells, and that loss of IKZF1 function confers resistance to dexamethasone, the major treatment component in group-1 (Marke et al., submitted). Additionally, in the group-2 patients treated with high-amounts of CS and highly intensive additional chemotherapy, a lower percentage IKZF1-deleted clones was detected at relapse, indicating that more GC-resistant, IKZF1-deleted clones are killed by the intense chemotherapy given in addition to CS in group-2 patients. Similarly, in the group-3 patients relapsing after treatment with lower amounts of CS and moderately-intensive additional chemotherapy, the percentage of surviving IKZF1-deleted clones was lower than in patients treated with high-amounts of CS. Taken together, our data indicate that the genetic architecture of relapsed BCP-ALL patients depends on the upfront treatment and, in addition, that the poor-prognostic feature of IKZF1-deletions may be more prominent in upfront treatment with high-amounts of CS and relatively mild additional chemotherapy. Figure 1. The frequency of genetic alterations in studied genes in patients that relapsed after treatment according to group-1, 2 and 3. Genes were grouped by their corresponding pathways. Group-1: patients treated upfront with high-amounts of CS and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). Asterisk showed significant difference between upfront treatment groups, **p<0.001. Figure 1. The frequency of genetic alterations in studied genes in patients that relapsed after treatment according to group-1, 2 and 3. Genes were grouped by their corresponding pathways. Group-1: patients treated upfront with high-amounts of CS and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). Asterisk showed significant difference between upfront treatment groups, **p<0.001. Disclosures No relevant conflicts of interest to declare.

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