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.

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 Acute Lymphoblastic Leukemia with Zinc-Finger Protein 384 (ZNF384)-Related Rearrangements: A Retrospective Analysis from the Ponte Di Legno Childhood ALL Working Group

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 652-652 ◽  
Author(s):  
Shinsuke Hirabayashi ◽  
Ellie Butler ◽  
Kentaro Ohki ◽  
Nobutaka Kiyokawa ◽  
Anke K. Bergmann ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Clinical Features ◽  
Relapse Rate ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Cells ◽  
Response To Therapy ◽  
Biological Characteristics ◽  
Fusion Partner ◽  
Cell Transplant ◽  
Genetic Abnormalities

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease that can be subdivided according to primary recurrent genetic abnormalities that are strongly associated with characteristic biological and clinical features. The first few cases with ZNF384-related rearrangements were described as early as 2002. The leukemic phenotype of these cases was not only BCP-ALL but also mixed phenotype acute leukemia (MPAL) and acute myeloid leukemia (AML) switched from ALL. The number of patients was small because this type of leukemia is rare and many of the fusions are cytogenetically cryptic. RNA sequencing revealed that 1% to 6% of childhood BCP-ALL cases, 5% to 15% of adult BCP-ALL cases and 48% of B/Myeloid MPAL cases harbored ZNF384 rearrangements. Of note, ZNF384 has a variety of partner genes such as TCF3, EP300 and TAF15. Their biological characteristics showed distinct expression profiles, and the cell origin might arise from primitive hematopoietic cells. The clinical features associated with ZNF384-related rearrangements have not been analyzed in a large cohort of patients. To identify the clinical characteristics of ZNF384-related rearrangements in childhood BCP-ALL (MPAL was excluded), we studied a total with 226 cases of ZNF384-related rearrangements from 15 international consortia who participate in the Ponte di Legno study group registered between 1987 and 2018. We analyzed the impact of outcome in association with clinical and biological characteristics. ZNF384-related rearrangements were detected by fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) and/or next generation sequencing (NGS), according to local selection policies, or because of poor response to therapy. Additional genetic abnormalities were detected by multiplex ligation-dependent probe amplification (MLPA), single nucleotide polymorphism array (SNP array) and/or NGS. The median age of presentation was 9 years old (range, 1 - 25 years old). The female and male ratio was 1:1. Immunophenotypic characteristics were classified as BCP-ALL exclusively In addition, 33% were CD10 negative cases (cutoff 20%); 71% were CD13 positive; and 86% were CD33 positive. Complete hematological remission was achieved in 99% of cases. One third (31%) of patients were treated as high risk and one quarter (23%) of patients received a stem cell transplant in first remission. After a median follow-up of 5.3 years, the 5-year event free survival (EFS) rate was 84% (95%CI, 77 to 89%), and the 5-year overall survival (OS) rate was 91% (95% CI, 85 to 94%). There was no difference in survival rate by treatment era or by country or region of origin. The proportion of partner genes with ZNF384 was as follows: EP300 (37%, n=84), TCF3 (27%, n=60), TAF15 (8%, n=17), CREBBP (7%, n=16), others (8%, n=18) of identifiable partners, and unknown (14%, n=31), although a prospective unselected analysis is needed for an appropriate estimate of the partners distribution. Patients with an EP300-ZNF384 fusion had a significantly lower relapse rate at 5 years compared with the remaining patients: 5% (95% CI 2-14) versus 20% (12-32), hazard ratio 4.58 (1.56-13.45), p=0.006), respectively. The corresponding EFS and OS rates were 91% (81-96) vs. 76% (64-85), p=0.024 and 92% (81-96) vs. 90% (80-95), p=0.3, suggesting that the non-EP300 relapses were salvageable (Figure). Multivariate analysis adjusting for sex, age, WBC and treatment era did not alter these results. Of note, in cases of TCF3 and TAF15, relapse occurred very late even after 5 years from diagnosis. Additional genetic abnormalities such as IKZF1, PAX5, CDKN2A/2B deletions were also analyzed. The distribution of deletions by partner genes was different between fusion partners but were not significant as prognostic factors. We confirm that ZNF384 rearrangement is a biologically and clinically distinct subtype of BCP-ALL. Immunophenotype abnormalities imply that ZNF384 rearrangements arise from primitive hematopoietic cells. Even considering a potential selection bias for the retrospective nature of the study, the OS was excellent in this subtype, although, relapse events did not reach a plateau among patients with TCF3-ZNF384 and TAF15-ZNF384. On the other hand, EP300-ZNF384 showed good prognosis with a low relapse rate. The biological background in each fusion partner warrants further investigation. Disclosures Loh: Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Different Activation of WNT Signaling Pathway in B-Cell and T-Cell Acute Leukemias.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4317-4317
Author(s):  
Muge Sayitoglu ◽  
Ozden Hatirnaz ◽  
Yucel Erbilgin ◽  
Fatmahan Atalar ◽  
Ugur Ozbek
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Wnt Signaling ◽  
B Cell ◽  
Signaling Pathway ◽  
Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
Wnt Signaling Pathway ◽  
Hematopoietic Cells ◽  
Expression Levels

Abstract WNT signaling pathway proteins function as hematopoietic growth factors and regulate proliferation in normal T-cell and B-cell development. Recent experimental evidence demonstrated that oncogenic transformation in leukemias of both lymphoid and myeloid lineages is dependent on WNT signaling. Not much is known about activation of WNT signaling pathway, its ligands and receptors in hematopoiesis and leukemia pathogenesis. To define its role in leukemia, we aimed to determine mRNA levels of the critical members of WNT pathway (WNT5A, WNT10B, FZ5, β catenin, APC, TCF-1 and LEF-1) by using quantitative real time PCR in acute lymphoblastic leukemia (ALL) patients (T-cell n=42, B-cell n=46 and pre B-cell n=30) and normal hematopoietic cells (bone marrow n=6, peripheral blood n=10, and CD19+ cells from peripheral blood). These genes expressed varying levels in B-cells, preB-cells and T-cells. In the B-cell leukemia patients, WNT5A was expressed notably (OR=58.05 CI 95% 1.63–1219.55, p>0,001). WNT5A directs Ca++ dependent signaling by PKC and a G protein dependent manner which is an alternative pathway for beta-catenin mediated signaling. Also LEF-1 levels were higher in B-ALL patients and APC expression was down regulated when compared to normal tissue (OR=18.81 CI 95% 0.34–5703, p>0.001 and OR=0.212 CI 95% 0.006–8.816, p=0.001, respectively). It is known that LEF-1 blocks APC mediated β catenin nuclear export and activates transcription of various transforming genes, including cyclin, D1, c-myc, MMP7, and LEF-1 itself. WNT5A or WNT10B proteins were not found to be up regulated in preB-ALL whereas APC and LEF-1 gene expressions were increased compared to normal hematopoietic cells (OR=32.97 CI 95% 0.27–1281, 38 p>0.001 and OR=5.57 CI 95% 0.28–89.51, p=0.01, respectively). We found increased TCF-1 expression (7.4 fold) without any β catenin accumulation in T-ALL patients. It is known that TCF-1 in absence of β catenin functions as a tumor suppressor gene. WNT5A, APC and LEF-1 gene expression levels were also different between T-cell, B-cell and preB cell ALL cases. WNT5A expression had the highest levels in B-ALL compared to T-ALL cases, whereas the highest APC expression levels were observed in preB and T-ALL patients. Also LEF-1 expression levels were significantly different between preB and T-cell ALL patients. Taken together these results indicate that WNT signaling genes have abnormal expression and are active in acute lymphoblastic leukemia. This data suggests different WNT activation mechanisms exist in the leukemic transformation in different hematopoietic cells.

Molecular and Clinical Characterization of Patients with Myeloid Neoplasms Carrying the 12p Deletion

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2007-2007
Author(s):  
Vera Adema ◽  
Cassandra M. Hirsch ◽  
Bartlomiej P Przychodzen ◽  
Andrea Pellagatti ◽  
Jacqueline Boultwood ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Board Of Directors ◽  
Transcriptional Factors ◽  
Chromosome 12 ◽  
Complex Karyotype ◽  
Advisory Committees ◽  
Expression Levels ◽  
Myeloid Neoplasms ◽  
Mrna Expression Levels

Abstract Background: Cytogenetic abnormalities have been described in almost 50% of patients with MDS and area strong and independent risk factor for prognosis. The interstitial deletion in the short arm of the chromosome 12 [del(12p)], is a characteristic but rare abnormality in MDS patients. Del(12p) abnormality has been described in approximately 1-5% of patients as a sole anomaly and is also found in up to 4% of patients along with an additional cytogenetic alteration. Isolated del(12p) is classified as a good risk abnormality according to the Revised International Prognostic Scoring Systems (IPSS-R). The commonly deleted region between 12p12.2 and 12p13.1 encompasses the ETV6 gene. To date, besides mutations in the transcriptional factor ETV6 and in the cell signaling KRAS gene, no other molecular mutations have been associated with del(12p). Murine studies have highlighted a role of the transcriptional factors ETV6 and RUNX1 in the impairment of both erythroid and platelets maturation. Here we investigated the presence of alternative molecular factors associated with del(12p) possibly influencing clinical outcomes and disease phenotypes. Methods: We studied the molecular and clinical data of a total of 2834 patients with myeloid neoplasms and found that 3% (93/2834) had alterations in chromosome 12. The median age was 67 years (24-84), with a male: female ratio of 56:37. Del(12p) occurred in 71% of cases (66/93); among them 14% (9/66) had isolated del(12p), 9% (6/66) had del(12p) + 1 additional alteration and 77% (51/66) carried a complex karyotype. The additional alteration to del(12p) included -7/del 7q (N=3), del(5q) (N=1) and t(X;20) (N=1). Cases with del(12p) were also classified according to disease type (MDS=40, AML=16; MDS/MPN=10; P=.057) and according to MDS risk group [lower-risk (33%, 22/66) and higher-risk (45%, 30/66)]. We applied whole exome sequencing and a targeted deep sequencing panel of 64 most frequently mutated genes in myeloid neoplasms. The ETV6 (12p13.2) gene was deleted in 55% (36/66) of cases while the KRAS (12p12) gene was deleted in 32% (21/66) of cases. One-third (32%, 21/66) of patients had deleted both genes. Two patients were hemizygous for KRAS. Results: Comparing patients with del(12p) (isolated, +1 alteration) to patients without alterations in chromosome 12 (n=2741), those with del(12p) had lower hemoglobin levels compared to patients without 12p aberrations (9.2 g/dL (6-16) vs. 9.7 g/dL (3-17); P=.009) and lower platelets counts (47 x109/L (8-577) vs. 73 x109/L (2-2336); P=.04). We noted that patients with isolated del(12p) had a longer median OS compared to patients with del(12p) associated with a complex karyotype [14 months (1-27) vs. 7 months (5-8)] although this difference was not significant. We then analyzed the mutational profile of the del(12p) cohort (isolated, +1 alteration) and compared their mutational spectrum with that of cases diploid for 12p. The most recurrently mutated genes in cases with del(12p) compared to cases diploid for 12p included RUNX1 (27% vs. 7%; P=.01) and DNMT3A (27% vs. 9%; P=.04). When we analyzed all the cases with del(12p) abnormalities (isolated, +1 alteration and complex) the significantly mutated genes were the transcriptional factors TP53 (38% vs. 4%; P=.0001) and RUNX 1 (14% vs. 7%; P=.04) and the histone modifier ASXL1 (21% vs. 10%; P=.01) We then analyzed the gene expression profile of patients carrying the del(12p) abnormality and found that KRAS mRNA expression levels of patients with MDS with del(12p) had a 2-fold reduction compared to the levels of healthy subjects (P=.017). Similarly, we observed also a decrease in ETV6 mRNA expression levels in patients with del(12p) (P=.07). Conclusions: Patients with del(12p) had lower levels of hemoglobin and platelets counts compared to patients without this cytogenetic abnormality. Mutations in transcriptional factors such as RUNX1 were commonly detected in this cohort, suggesting a possible mechanism contributing to the role of ETV6 in the impairment of erythroid and megakaryocytic cell maturation. Disclosures Sole: Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.

MicroRNAs Characterize Genetic Diversity and Drug Sensitivity in Pediatric Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 89-89 ◽  
Author(s):  
Diana Schotte ◽  
Renee X de Menezes ◽  
Farhad Akbari Moqadam ◽  
Ellen Lange-Turenhout ◽  
Caifu Chen ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Mirna Expression ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Cells ◽  
Mirna Expression Profile ◽  
Leukemic Cells ◽  
Expression Levels ◽  
Genetic Subtypes

Abstract Abstract 89 MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate the activity of protein-coding genes, including those involved in cancer. The function of a miRNA depends on the cellular context and hence prominent miRNAs in lymphoma may not be important in acute lymphoblastic leukemia (ALL). To understand which miRNAs may be relevant in pediatric ALL, the expression levels of 397 miRNAs including seven newly cloned miRNAs were measured in seven genetic subtypes of ALL and normal hematopoietic cells. Except for BCR-ABL-positive and B-other ALL all major subtypes, i.e. T-ALL, MLL-rearranged, TEL-AML1-positive, E2A-PBX1-positive and hyperdiploid ALL, have unique miRNA signatures that differ from each other and from those in healthy hematopoietic cells. The expression of miR-383, miR-125b, miR-99a, miR-100 and let-7c was increased by a five to 1700-fold (P < 0.001) in TEL-AML1-positive cases. Hyperdiploid patients demonstrated a three to 24-fold upregulation (P < 0.001) of miR-222/222*, miR-223, miR-511 and miR-660, encoded on either chromosome X or 10 which is often duplicated in hyperdiploid cases. Some ALL subtypes shared similarities in their miRNA expression signature suggesting a common underlying biology e.g. both E2A-PBX1 and T-ALL cases demonstrated a downregulation of eight miRNAs (P ≤ 0.02) and within the TEL-AML1-positive subtype, two distinct groups were identified of which one showed an overlapping miRNA expression profile with hyperdiploid cases. Aberrant miRNA expression may result in dysregulated expression of their targeted proteins. Here we observed that the 70-fold downregulation of let-7b in MLL-rearranged ALL was associated with a 2-fold upregulation of oncoprotein c-Myc (P< 0.0001). Furthermore, a classifier built with a selection of 28 miRNAs predicted the MLL-rearranged, TEL-AML1-positive, E2A-PBX-positive and T-ALL subtypes with 100% sensitivity and specificity. Besides the genetic subtype, cellular drug resistance determines outcome of ALL. In vitro resistance of patients to vincristine, daunorubicin and L-asparginase was characterized by abnormal expression of 27 miRNAs (P < 0.05) whereas no discriminative miRNAs were found for resistance to prednisolone. Most striking was the 14- to 25-fold upregulation (P ≤ 0.002) of miR-125b, miR-99a and miR-100 in cases resistant to vincristine or daunorubicin. In conclusion, genetic subtypes and drug resistant leukemic cells display characteristic miRNA expression levels. Functional studies are indicated for discriminative miRNAs and may provide new insights into leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of growth factors and tyrosine kinase receptors in the primary tumor and tumor thrombus cells in patients with renal cell carcinoma

2020 ◽  
Vol 16 (1) ◽  
pp. 17-26
Author(s):  
M. I. Volkova ◽  
A. S. Olshanskaya ◽  
I. V. Tsimafeyeu ◽  
N. L. Vashakmadze ◽  
Yu. A. Khochenkova ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Growth Factors ◽  
Tumor Cells ◽  
Primary Tumor ◽  
Tumor Thrombus ◽  
Primary Tumors ◽  
Expression Levels ◽  
Venous Wall ◽  
Lower Expression ◽  
Tumor Thrombi

Objective: to assess the expression and prognostic value of vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2) and their receptors VEGFR-1, -2; FGFR-1, -2, as well as platelet-derived growth factor receptors (PDGFR-α, PDGFR-β) in paired samples of primary tumors and tumor thrombi in renal cell carcinoma (RCC).Materials and methods. Expression of VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β was studied in paired surgical samples of primary tumors and tumor thrombi in 25 patients with clear cell RCC pT3a–T4N0–1M0–1 and tumor venous thrombosis by immunohistochemical assay using the appropriate Abcam/Santa Cruz Biotech antibodies from the immunohistochemical staining kit Invitrogen. Expression levels were evaluated by a semi-quantitative method (H-score). The analysis of the correlation between expression levels of VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β and RCC characteristics, as well as evaluation of their influence on the outcome of RCC were performed.Results. VEGF-A, FGF-2, as well as VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β were expressed in the cytoplasm and on the membrane of the primary tumor and tumor thrombus cells in RCC patients. Tumor thrombus cells were characterized by lower expression of VEGFR-1, VEGFR-2, PDGFR-α (p <0.05 for all) and tendency to lower expression of VEGF-A (p = 0.060), FGF-2 (p = 0.046), FGFR-1 (p = 0.077) and FGFR-2 (p = 0.090) compared with primary tumor cells. RCC Furman grade correlated with the expression levels of VEGFR-1 (p = 0.035) and FGFR-1 (p = 0.022) in the primary tumor cells, tumor invasion into venous wall correlated with the expression levels of VEGFR-1 (p = 0.023) and FGFR-2 (p = 0.005) on the thrombus cells. VEGFR-2 overexpression in the primary tumor cells was associated with significant decrease of overall survival (OS) rate (p = 0.011). There was a tendency to OS deterioration in cases with overexpression of VEGFR-2 (p = 0.093) and VEGF-A (p = 0.095) in the tumor thrombus cells. One-year OS in patients with ³2 identified risk factors was 27.3 %, <2 risk factors – 87.5 % (p = 0.004).Conclusion. Tumor thrombus cells in RCC patients expressed VEGF-A, FGF-2, VEGFR-1, -2; FGFR-1, -2; PDGFR-α, -β less active than the cells of the primary tumor. Overexpression of growth factors and tyrosine kinases correlated with RCC Furman grade and tumor venous wall invasion. Overexpression of VEGFR-2 in both primary tumor and thrombus cells in combination with hypoexpression of VEGF-A in the thrombus negatively influenced on OS.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

scholarly journals Intravitreal brimonidine inhibits form-deprivation myopia in guinea pigs

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Yifang Yang ◽  
Junshu Wu ◽  
Defu Wu ◽  
Qi Wei ◽  
Tan Zhong ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Intravitreal Injection ◽  
Guinea Pigs ◽  
Intravitreal Injections ◽  
Eye Drops ◽  
Rna Seq ◽  
Myopia Progression ◽  
Expression Levels ◽  
Guinea Pig Model ◽  
Administration Methods

Abstract Background The use of ocular hypotensive drugs has been reported to attenuate myopia progression. This study explores whether brimonidine can slow myopia progression in the guinea pig form-deprivation (FD) model. Methods Three-week-old pigmented male guinea pigs (Cavia porcellus) underwent monocular FD and were treated with 3 different methods of brimonidine administration (eye drops, subconjunctival or intravitreal injections). Four different concentrations of brimonidine were tested for intravitreal injection (2 μg/μL, 4 μg/μL, 20 μg/μL, 40 μg/μL). All treatments continued for a period of 21 days. Tonometry, retinoscopy, and A-scan ultrasonography were used to monitor intraocular pressure (IOP), refractive error and axial length (AL), respectively. On day 21, guinea pigs were sacrificed for RNA sequencing (RNA-seq) to screen for associated transcriptomic changes. Results The myopia model was successfully established in FD animals (control eye vs. FD eye, respectively: refraction at day 20, 0.97 ± 0.18 D vs. − 0.13 ± 0.38 D, F = 6.921, P = 0.02; AL difference between day 0 and day 21, 0.29 ± 0.04 mm vs. 0.45 ± 0.03 mm, F = 11.655, P = 0.004). Among the 3 different brimonidine administration methods, intravitreal injection was the most effective in slowing myopia progression, and 4 μg/μL was the most effective among the four different concentrations of brimonidine intravitreal injection tested. The AL and the refraction of the brimonidine intravitreal injection group was significantly shorter or more hyperopic than those of other 2 groups. Four μg/μL produced the smallest difference in AL and spherical equivalent difference values. FD treatment significantly increased the IOP. IOP was significantly lower at 1 day after intravitreal injections which was the lowest in FD eye of intravitreal injection of brimonidine. At day 21, gene expression analyses using RNA-seq showed upregulation of Col1a1 and Mmp2 expression levels by intravitreal brimonidine. Conclusions Among the 3 different administration methods, intravitreal injection of brimonidine was the most effective in slowing myopia progression in the FD guinea pig model. Intravitreal brimonidine at 4 μg/μL significantly reduced the development of FD myopia in guinea pigs. Expression levels of the Col1a1 and Mmp2 genes were significantly increased in the retinal tissues of the FD-Inj-Br group.

scholarly journals High heterogeneity undermines generalization of differential expression results in RNA-Seq analysis

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Weitong Cui ◽  
Huaru Xue ◽  
Lei Wei ◽  
Jinghua Jin ◽  
Xuewen Tian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Small Sample ◽  
Differentially Expressed ◽  
Cancer Type ◽  
Rna Seq ◽  
Sample Sizes ◽  
Large Sample ◽  
Expression Levels ◽  
Gene Expression Levels

Abstract Background RNA sequencing (RNA-Seq) has been widely applied in oncology for monitoring transcriptome changes. However, the emerging problem that high variation of gene expression levels caused by tumor heterogeneity may affect the reproducibility of differential expression (DE) results has rarely been studied. Here, we investigated the reproducibility of DE results for any given number of biological replicates between 3 and 24 and explored why a great many differentially expressed genes (DEGs) were not reproducible. Results Our findings demonstrate that poor reproducibility of DE results exists not only for small sample sizes, but also for relatively large sample sizes. Quite a few of the DEGs detected are specific to the samples in use, rather than genuinely differentially expressed under different conditions. Poor reproducibility of DE results is mainly caused by high variation of gene expression levels for the same gene in different samples. Even though biological variation may account for much of the high variation of gene expression levels, the effect of outlier count data also needs to be treated seriously, as outlier data severely interfere with DE analysis. Conclusions High heterogeneity exists not only in tumor tissue samples of each cancer type studied, but also in normal samples. High heterogeneity leads to poor reproducibility of DEGs, undermining generalization of differential expression results. Therefore, it is necessary to use large sample sizes (at least 10 if possible) in RNA-Seq experimental designs to reduce the impact of biological variability and DE results should be interpreted cautiously unless soundly validated.

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