Prognostic Relevance of Expression of EMP1, CASP1, and NLRP3 Genes in Pediatric B-Lineage Acute Lymphoblastic Leukemia

Glucocorticoid (GC), such as prednisolone, is an essential component of multidrug chemotherapy regimen for pediatric acute lymphoblastic leukemia (ALL). Resistance to GC in leukemia cells is associated with disease progression and poor prognosis. Despite the extensive use of GC for many years, molecular mechanisms underlying its resistance in ALL have not been fully uncovered. Recent studies have shown a potential role of EMP1, CASP1, and NLRP3 genes in prednisolone response. In this study on 148 pediatric B-ALL patients, we studied these three genes to assess their association with prednisolone response measured by day 8 blast count after 7 days of induction therapy with prednisolone. Intriguingly, ALL samples exhibited higher expression of EMP1 along with a low expression of CASP1 and NLRP3 compared to disease free normal bone marrow collected from patients with solid tumors. Among the three analyzed genes, only EMP1 was found to be overexpressed in prednisolone poor responders (p=0.015). Further, a comparison of gene expression between cytogenetic subtypes revealed higher expression of EMP1 in BCR-ABL subtype. Expression of EMP1 in multiple gene expression datasets was used for gene set enrichment analysis, which revealed TNF-α, IL-2-STAT5 signaling, inflammatory responses and hypoxia as the major positively associated pathways and E2F targets as negatively associated pathways. Interestingly, the clinical remission rate was higher in CASP1 high patients (p=0.048). In univariate survival analysis, higher EMP1 expression was associated with poor prognostic measures while higher expression of NLRP3 and CASP1 was associated with better prognostic measures in our data. Further, multivariate analysis revealed an independent association of high CASP1 and NLRP3 with a better prognosis. This study strengthens the available evidence that mRNA expression of EMP1, CASP1, and NLRP3 may serve as potential biomarkers for risk stratification of pediatric B-ALL patients.

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Gene Expression Profiling in Down Syndrome Acute Lymphoblastic Leukemia Identifies Distinct Profiles Associated with CRLF2 Expression Status.

Blood ◽

10.1182/blood.v114.22.2389.2389 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2389-2389

Author(s):

Karen R. Rabin ◽

Jinhua Wang ◽

Julia Meyer ◽

Michael G. Loudin ◽

Deepa Bhojwani ◽

...

Keyword(s):

Gene Expression ◽

Down Syndrome ◽

Acute Lymphoblastic Leukemia ◽

Differentially Expressed Genes ◽

Lymphoblastic Leukemia ◽

Gene Expression Signature ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Differentially Expressed ◽

Gene Set Enrichment

Abstract Abstract 2389 Poster Board II-366 Children with Down syndrome (DS) have a 10 to 20-fold increased risk of developing acute lymphoblastic leukemia (ALL), and they have experienced poorer outcomes on recent major protocols worldwide. The cytogenetic abnormalities which are generally common in childhood ALL and contribute to risk-based treatment assignment are markedly less frequent in children with DS-ALL. Recently, activating mutations in Janus kinase 2 (JAK2) have been identified in approximately 20% of DS-ALL, and interstitial deletions involving cytokine receptor-like factor 2 (CRLF2) in approximately 50% of DS-ALL. Global gene expression profiling may provide insights into the biologic consequences of these molecular lesions. We performed microarray analysis of RNA from diagnostic bone marrow samples in 23 DS-ALL and 26 non-DS ALL cases using the Affymetrix Human Genome U133 Plus 2.0 array. CRLF2 expression was high in 10 of the 23 DS-ALL cases, 3 of which also bore JAK2 mutations, and in a single non-DS ALL case. Unsupervised hierarchical clustering analysis demonstrated clustering of non-DS ALL cases belonging to known cytogenetic subgroups such as E2A-PBX1, MLL rearrangement, and high hyperdiploidy. In contrast, neither DS-ALL cases overall nor the JAK2-mutated or high CRLF2 expressing cases formed a cohesive cluster. Supervised analysis identified 43 genes that were differentially expressed between CRLF2 high versus low cases with a false discovery rate <10%. Several of the most highly differentially expressed genes were validated by quantitative real-time PCR. These included three genes with high expression in CRLF2-high cases: chemokine (C-C motif) ligand 17 (CCL17) (p=0.01), V-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1) (p=0.007), and Iroquois homeobox 2 (IRX2) (p=0.008); and one gene with expression inversely correlated with CRLF2 expression: dual specificity phosphatase 6 (DUSP6) (p=0.0015). Our findings suggest that DS-ALL does not form a single distinct biologic subgroup, but nearly half of DS-ALL cases are defined by high CRLF2 expression, a substantial enrichment for this lesion compared to its prevalence in non-DS ALL. Identification of downstream pathways may identify opportunities for targeted intervention, including interactions with other cytokines and activation of the JAK-STAT pathway. Figure 1. Gene expression signature of top differentially expressed genes in Down syndrome (DS) acute lymphoblastic leukemia (ALL) cases with high versus low CRLF2 expression. Each column indicates a case, with CRLF2-high cases depicted in gray and CRLF2-low cases in gold. Each row indicates one of the top 100 differentially expressed genes as determined by Gene Set Enrichment Analysis. Figure 1. Gene expression signature of top differentially expressed genes in Down syndrome (DS) acute lymphoblastic leukemia (ALL) cases with high versus low CRLF2 expression. Each column indicates a case, with CRLF2-high cases depicted in gray and CRLF2-low cases in gold. Each row indicates one of the top 100 differentially expressed genes as determined by Gene Set Enrichment Analysis. Disclosures: No relevant conflicts of interest to declare.

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Folate Pathway Gene Expression Differs in Genetic Subtypes of Acute Lymphoblastic Leukemia and Influences Methotrexate Pharmacodynamics.

Blood ◽

10.1182/blood.v104.11.452.452 ◽

2004 ◽

Vol 104 (11) ◽

pp. 452-452

Author(s):

Leo Kager ◽

Meyling H. Cheok ◽

Wenjian Yang ◽

Gianluigi Zaza ◽

Ching-Hon Pui ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Leukemia Cells ◽

Cancer Resistance ◽

Childhood All ◽

Pathway Gene ◽

Folate Pathway ◽

B Lineage ◽

Lower Expression

Abstract Methotrexate (MTX) is an essential treatment component for acute lymphoblastic leukemia (ALL). The ability of leukemia cells to accumulate MTX in its polyglutamylated form (MTXPG) is recognized as an important determinant of its antileukemic effect. We measured in vivo MTXPG accumulation in leukemia cells from 101 children with ALL, and established that blasts of B-lineage ALL with either the TEL-AML1 (n=24 patients, median 911, range 338 to 5906 pmol/109 blasts) or E2A-PBX1 gene fusion (n=5, median 553, range 364 to 800 pmol/109 blasts) or T-lineage ALL (n=14, median 572, range 284 to 1468 pmol/109 blasts) accumulate significantly lower MTXPG, compared to those of other B-lineage ALL (BNHD, n=39, median 2210, range 186 to 9722 pmol/109 blasts) or hyperdiploid ALL (BHD, n=19, median 4375, range 377 to 9206 pmol/109 blasts) (E2A-PBX1 versus BHD, p=0.008; E2A-PBX1 vs. BNHD, p=0.010; TEL-AML1 vs. BHD, p<0.001; TEL-AML1 vs. BNHD, p=0.004; T-ALL vs. BHD and BNHD, p<0.001; p-values are from pair-wise comparisons using Wilcoxon rank sum test, adjusted for multiple testing using Holm’s method). To elucidate mechanisms underlying these differences in MTXPG accumulation, we used oligonucleotide microarrays (Affymetrix® HG-U133A) to analyze expression of 32 folate pathway genes (53 probe sets) in diagnostic bone marrow blasts from 197 children with ALL. This revealed ALL subtype-specific patterns of folate metabolism gene expression and identified differences in gene expression that discriminated the MTXPG accumulation phenotype in ALL cells. We found significantly lower expression of the reduced folate carrier (SLC19A1, MTX uptake transporter) in E2A-PBX1 ALL; significantly higher expression of breast cancer resistance protein (ABCG2, MTX efflux transporter) in TEL-AML1 ALL; and lower expression of FPGS (catalyzes formation of MTXPG) in T-ALL; consistent with lower MTXPG accumulation in these ALL subtypes. These findings reveal distinct mechanisms of subtype-specific differences in MTXPG accumulation and point to new strategies to overcome this potential cause of treatment failure in childhood ALL.

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Bortezomib Combined with VXLD Chemotherapy Is Highly Effective In Advanced B-Lineage Acute Lymphoblastic Leukemia Allowing Early Study Termination Due to Efficacy. A Therapeutic Advances in Childhood Leukemia (TACL) Consortium Phase II Study

Blood ◽

10.1182/blood.v118.21.251.251 ◽

2011 ◽

Vol 118 (21) ◽

pp. 251-251

Author(s):

Yoav H Messinger ◽

Paul Gaynon ◽

Richard Sposto ◽

Jeannette van der Giessen ◽

Elena Eckroth ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Complete Remission ◽

Phase Ii ◽

Response Rate ◽

Remission Rate ◽

Lymphoblastic Leukemia ◽

Platelet Recovery ◽

Off Label ◽

B Lineage

Abstract Abstract 251 Literature review and TACL experience support an expected less than 40% complete remission rate with variety of regimens in patients with ALL in second and subsequent relapse (Ko, J Clin Oncol 2010; 28: 648–654). We had shown that bortezomib might be safely combined with vincristine, dexamethasone, pegylated asparaginase, and doxorubicin (VXLD) in the phase I portion of our study (Messinger, Pediatr Blood Cancer 2010;55:254–9). We now report the phase II expansion of that study. ALL patients who relapsed or were refractory after 2 or 3 regimens were treated with bortezomib 1.3 mg/m2/dose on days 1, 4, 8 and 11, combined with VXLD. Patients between ages 1 to 21 years old, with more than 25% bone marrow blasts, were eligible. One patient from the phase I cohort with these criteria was included in the phase II extension. In the phase II extension 22 patients were treated with this combination and all are included in analyses. All patients had relapsed or failed at least 2 prior regimens. Overall 14 achieved complete remission (CR; M1 marrow with ANC and platelet recovery and no extramedullary disease or circulating blasts) and 2 achieved CRp (CR with no platelet recovery) for total 73% remission rate (Table). This level of response exceeded the predefined criteria, allowing for early termination of the study. Three patients (14%) died from bacterial infections and two patients (9%) had no response (Table). One patient (4.5%) was not evaluable for response due to protocol violation, when additional therapy was administered before CR was confirmed with peripheral blood count recovery. B-Lineage ALL patients fared best, with 16/20 achieving CR + CRp (overall response rate 80%), whereas the two patients with T-cell ALL did not respond. Similarly, B-Lineage ALL had superior bone marrow response (M1 marrow): B-Lineage = 17/20 (85%) vs. T-cell = 0/2 (0%). Severe grade 3 or more peripheral neuropathy (PN) was seen in 2 (9%) patients, (one had prior vincristine PN). One patient has developed mucor invasive sinus and orbital infection, requiring halting therapy after day 14 but achieved CRp. After the 3 (14%) septic deaths, the use of vancomycin, levofloxacin and voriconazole or posaconazole prophylaxis in the last 6 patients has prevented further infectious mortality.ResponseAllBTn22202CR14 (64%)14 (70%)0CRp2 (9%)2 (10%)0Total Response16 (73%)16 (80%)0Deaths3 (14%)3 (15%)0SD/PD2 (9%)02 (100%)N/E1 (4.5%)1 (5%)0 In conclusion, the regimen of bortezomib + VXLD is exceptionally effective in multiple relapsed B-Lineage ALL with the highest response rate for any multiply relapsed ALL trial reported thus far. The use of prophylactic antibiotics may be effective in reducing mortality. Bortezomib with VXLD should be further evaluated in randomized fashion on frontline relapse and high-risk pediatric B-Lineage ALL clinical studies. Disclosures: Messinger: Genzyme: Consultancy. Off Label Use: Bortezomib (Velcade®) is approved for multiple myeloma and mantle cell lymphoma both B cell malignancies. We are describing use in relapsed B cell Acute Lymphoblastic Leukemia which is off label.”

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Establishment of an Autophagy-Related Clinical Prognosis Model for Predicting the Overall Survival of Osteosarcoma

BioMed Research International ◽

10.1155/2021/5428425 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Jianyi Li ◽

Xiaojie Tang ◽

Yukun Du ◽

Jun Dong ◽

Zheng Zhao ◽

...

Keyword(s):

Gene Expression ◽

Overall Survival ◽

Molecular Mechanisms ◽

Cox Regression ◽

Enrichment Analysis ◽

Risk Groups ◽

Gene Set Enrichment Analysis ◽

Data Set ◽

Primary Tumor Site ◽

Clinical Prognosis

Purpose. Osteosarcoma is the most common primary and highly invasive bone tumor in children and adolescents. The purpose of this study is to construct a multi-gene expression feature related to autophagy, which can be used to predict the prognosis of patients with osteosarcoma. Materials and methods. The clinical and gene expression data of patients with osteosarcoma were obtained from the target database. Enrichment analysis of autophagy-related genes related to overall survival (OS-related ARGs) screened by univariate Cox regression was used to determine OS-related ARGs function and signal pathway. In addition, the selected OS-related ARGs were incorporated into multivariate Cox regression to construct prognostic signature for the overall survival (OS) of osteosarcoma. Use the dataset obtained from the GEO database to verify the signature. Besides, gene set enrichment analysis (GSEA) were applied to further elucidate the molecular mechanisms. Finally, the nomogram is established by combining the risk signature with the clinical characteristics. Results. Our study eventually included 85 patients. Survival analysis showed that patients with low riskScore had better OS. In addition, 16 genes were included in OS-related ARGs. We also generate a prognosis signature based on two OS-related ARGs. The signature can significantly divide patients into low-risk groups and high-risk groups, and has been verified in the data set of GEO. Subsequently, the riskScore, primary tumor site and metastasis status were identified as independent prognostic factors for OS and a nomogram were generated. The C-index of nomogram is 0.789 (95% CI: 0.703~0.875), ROC curve and calibration chart shows that nomogram has a good consistency between prediction and observation of patients. Conclusions. ARGs was related to the prognosis of osteosarcoma and can be used as a biomarker of prognosis in patients with osteosarcoma. Nomogram can be used to predict OS of patients and improve treatment strategies.

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Altered Genes and Biological Functions in Response to Severe Burns

BioMed Research International ◽

10.1155/2021/8836243 ◽

2021 ◽

Vol 2021 ◽

pp. 1-19

Author(s):

Xinheng Liu ◽

Yongxian Rong ◽

Donglin Huang ◽

Zhijie Liang ◽

Xiaolin Yi ◽

...

Keyword(s):

Gene Expression ◽

Roc Curve ◽

Molecular Mechanisms ◽

Metabolic Response ◽

Heat Exposure ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Ppi Network ◽

Gene Set ◽

Severe Burns

Severe burns are acute wounds caused by local heat exposure, resulting in life-threatening systemic effects and poor survival. However, the specific molecular mechanisms remain unclear. First, we downloaded gene expression data related to severe burns from the GEO database (GSE19743, GSE37069, and GSE77791). Then, a gene expression analysis was performed to identify differentially expressed genes (DEGs) and construct protein-protein interaction (PPI) network. The molecular mechanism was identified by enrichment analysis and Gene Set Enrichment Analysis. In addition, STEM software was used to screen for genes persistently expressed during response to severe burns, and receiver operating characteristic (ROC) curve was used to identify key DEGs. A total of 2631 upregulated and 3451 downregulated DEGs were identified. PPI network analysis clustered these DEGs into 13 modules. Importantly, module genes mostly related with immune responses and metabolism. In addition, we identified genes persistently altered during the response to severe burns corresponding to survival and death status. Among the genes with high area under the ROC curve in the PPI network gene, CCL5 and LCK were identified as key DEGs, which may affect the prognosis of burn patients. Gene set variation analysis showed that the immune response was inhibited and several types of immune cells were decreased, while the metabolic response was enhanced. The results showed that persistent gene expression changes occur in response to severe burns, which may underlie chronic alterations in physiological pathways. Identifying the key altered genes may reveal potential therapeutic targets for mitigating the effects of severe burns.

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Bioinformatics Analysis Reveals the Potential Diagnostic Biomarkers for Abdominal Aortic Aneurysm

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.656263 ◽

2021 ◽

Vol 8 ◽

Author(s):

Xinsheng Xie ◽

En ci Wang ◽

Dandan Xu ◽

Xiaolong Shu ◽

Yu fei Zhao ◽

...

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Bioinformatics Analysis ◽

Inflammatory Responses ◽

Expression Profiles ◽

Enrichment Analysis ◽

Aortic Aneurysms ◽

Differentially Expressed ◽

Pathway Enrichment Analysis ◽

Abdominal Aortic

Objectives: Abdominal aortic aneurysms (AAAs) are associated with high mortality rates. The genes and pathways linked with AAA remain poorly understood. This study aimed to identify key differentially expressed genes (DEGs) linked to the progression of AAA using bioinformatics analysis.Methods: Gene expression profiles of the GSE47472 and GSE57691 datasets were acquired from the Gene Expression Omnibus (GEO) database. These datasets were merged and normalized using the “sva” R package, and DEGs were identified using the limma package in R. The functions of these DEGs were assessed using Cytoscape software. We analyzed the DEGs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Protein–protein interaction networks were assembled using Cytoscape, and crucial genes were identified using the Cytoscape plugin, molecular complex detection. Data from GSE15729 and GSE24342 were also extracted to verify our findings.Results: We found that 120 genes were differentially expressed in AAA. Genes associated with inflammatory responses and nuclear-transcribed mRNA catabolic process were clustered in two gene modules in AAA. The hub genes of the two modules were IL6, RPL21, and RPL7A. The expression levels of IL6 correlated positively with RPL7A and negatively with RPL21. The expression of RPL21 and RPL7A was downregulated, whereas that of IL6 was upregulated in AAA.Conclusions: The expression of RPL21 or RPL7A combined with IL6 has a diagnostic value for AAA. The novel DEGs and pathways identified herein might provide new insights into the underlying molecular mechanisms of AAA.

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Alterations in the host transcriptome in vitro and in vivo following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection

10.21203/rs.3.rs-37567/v1 ◽

2020 ◽

Author(s):

Xiaomei Lei ◽

Zhijun Feng ◽

Xiaojun Wang ◽

Xiaodong He

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Microarray Data ◽

Molecular Mechanisms ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Gene Set ◽

Gene Sets ◽

Core Genes

Abstract Background. Exploring alterations in the host transcriptome following SARS-CoV-2 infection is not only highly warranted to help us understand molecular mechanisms of the disease, but also provide new prospective for screening effective antiviral drugs, finding new therapeutic targets, and evaluating the risk of systemic inflammatory response syndrome (SIRS) early.Methods. We downloaded three gene expression matrix files from the Gene Expression Omnibus (GEO) database, and extracted the gene expression data of the SARS-CoV-2 infection and non-infection in human samples and different cell line samples, and then performed gene set enrichment analysis (GSEA), respectively. Thereafter, we integrated the results of GSEA and obtained co-enriched gene sets and co-core genes in three various microarray data. Finally, we also constructed a protein-protein interaction (PPI) network and molecular modules for co-core genes and performed Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis for the genes from modules to clarify their possible biological processes and underlying signaling pathway. Results. A total of 11 co-enriched gene sets were identiﬁed from the three various microarray data. Among them, 10 gene sets were activated, and involved in immune response and inflammatory reaction. 1 gene set was suppressed, and participated in cell cycle. The analysis of molecular modules showed that 2 modules might play a vital role in the pathogenic process of SARS-CoV-2 infection. The KEGG enrichment analysis showed that genes from module one enriched in signaling pathways related to inflammation, but genes from module two enriched in signaling of cell cycle and DNA replication. Particularly, necroptosis signaling, a newly identified type of programmed cell death that differed from apoptosis, was also determined in our findings. Additionally, for patients with SARS-CoV-2 infection, genes from module one showed a relatively high-level expression while genes from module two showed low-level. Conclusions. We identified two molecular modules were used to assess severity and predict the prognosis of the patients with SARS-CoV-2 infection. In addition, these results provide a unique opportunity to explore more molecular pathways as new potential targets on therapy in COVID 19.

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Utilization of Ig heavy chain variable, diversity, and joining gene segments in children with B-lineage acute lymphoblastic leukemia: implications for the mechanisms of VDJ recombination and for pathogenesis

Blood ◽

10.1182/blood-2003-11-3857 ◽

2004 ◽

Vol 103 (12) ◽

pp. 4602-4609 ◽

Cited By ~ 31

Author(s):

Aihong Li ◽

Montse Rue ◽

Jianbiao Zhou ◽

Hongjun Wang ◽

Meredith A. Goldwasser ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Heavy Chain ◽

Molecular Mechanisms ◽

Lymphoblastic Leukemia ◽

Tumor Biology ◽

Vdj Recombination ◽

Childhood All ◽

Increased Risk ◽

B Lineage

Abstract Sequence analysis of the immunoglobulin heavy chain genes (IgH) has demonstrated preferential usage of specific variable (V), diversity (D), and joining (J) genes at different stages of B-cell development and in B-cell malignancies, and this has provided insight into B-cell maturation and selection. Knowledge of the association between rearrangement patterns based on updated databases and clinical characteristics of pediatric acute lymphoblastic leukemia (ALL) is limited. We analyzed 381 IgH sequences identified at presentation in 317 children with B-lineage ALL and assessed the VHDHJH gene utilization profiles. The DHJH-proximal VH segments and the DH2 gene family were significantly overrepresented. Only 21% of VH-JH joinings were potentially productive, a finding associated with a trend toward an increased risk of relapse. These results suggest that physical location at the VH locus is involved in preferential usage of DHJH-proximal VH segments whereas DH and JH segment usage is governed by position-independent molecular mechanisms. Molecular pathophysiology appears relevant to clinical outcome in patients who have only productive rearrangements, and specific rearrangement patterns are associated with differences in the tumor biology of childhood ALL. (Blood. 2004;103:4602-4609)

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Differential and longitudinal immune gene patterns associated with reprogrammed microenvironment and viral mimicry in response to neoadjuvant radiotherapy in rectal cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001717 ◽

2021 ◽

Vol 9 (3) ◽

pp. e001717

Author(s):

Anna Wilkins ◽

Elisa Fontana ◽

Gift Nyamundanda ◽

Chanthirika Ragulan ◽

Yatish Patil ◽

...

Keyword(s):

Gene Expression ◽

Rectal Cancer ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Poor Responders ◽

Type I ◽

Immune Gene ◽

Neoadjuvant Radiotherapy ◽

The Impact ◽

Crt Response

BackgroundRectal cancers show a highly varied response to neoadjuvant radiotherapy/chemoradiation (RT/CRT) and the impact of the tumor immune microenvironment on this response is poorly understood. Current clinical tumor regression grading systems attempt to measure radiotherapy response but are subject to interobserver variation. An unbiased and unique histopathological quantification method (change in tumor cell density (ΔTCD)) may improve classification of RT/CRT response. Furthermore, immune gene expression profiling (GEP) may identify differences in expression levels of genes relevant to different radiotherapy responses: (1) at baseline between poor and good responders, and (2) longitudinally from preradiotherapy to postradiotherapy samples. Overall, this may inform novel therapeutic RT/CRT combination strategies in rectal cancer.MethodsWe generated GEPs for 53 patients from biopsies taken prior to preoperative radiotherapy. TCD was used to assess rectal tumor response to neoadjuvant RT/CRT and ΔTCD was subjected to k-means clustering to classify patients into different response categories. Differential gene expression analysis was performed using statistical analysis of microarrays, pathway enrichment analysis and immune cell type analysis using single sample gene set enrichment analysis. Immunohistochemistry was performed to validate specific results. The results were validated using 220 pretreatment samples from publicly available datasets at metalevel of pathway and survival analyses.ResultsΔTCD scores ranged from 12.4% to −47.7% and stratified patients into three response categories. At baseline, 40 genes were significantly upregulated in poor (n=12) versus good responders (n=21), including myeloid and stromal cell genes. Of several pathways showing significant enrichment at baseline in poor responders, epithelial to mesenchymal transition, coagulation, complement activation and apical junction pathways were validated in external cohorts. Unlike poor responders, good responders showed longitudinal (preradiotherapy vs postradiotherapy samples) upregulation of 198 immune genes, reflecting an increased T-cell-inflamed GEP, type-I interferon and macrophage populations. Longitudinal pathway analysis suggested viral-like pathogen responses occurred in post-treatment resected samples compared with pretreatment biopsies in good responders.ConclusionThis study suggests potentially druggable immune targets in poor responders at baseline and indicates that tumors with a good RT/CRT response reprogrammed from immune “cold” towards an immunologically “hot” phenotype on treatment with radiotherapy.

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