Journal: Journal of Translational Medicine

Article Title: Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

doi: 10.1186/s12967-024-04869-6

Figure Lengend Snippet: Mismatch repair deficiency due to concurring MLH1/PMS2 loss is a prevalent feature of naturally occurring CRC in rhesus macaques and results in functional microsatellite instability. Exemplary IHC staining of CRC_1 for MMR proteins A MLH1, B MSH2, C MSH6, and D PMS2. The consistent nuclear expression of these proteins in healthy colon and infiltrating lymphocytes served as an internal positive control. As clearly depicted, MLH1 and PMS2 staining was lost in the neoplastic cells but MSH2 and MSH6 were highly expressed. E This pattern of MLH1/PMS2 loss was observed cohort-wide in all 24 rhesus CRCs. MSH2 expression was maintained in 23/24 CRCs and MSH6 expression was present in all 24/24 CRCs. The emphasized mismatch repair deficiency translates to an instable genetic phenotype as corroborated by the widespread microsatellite instability. In summary, 13/16 CRCs were classified as MSI-high (≥ 2 unstable loci), 3/16 as MSI-low (1 unstable loci), and none as MSS

Article Snippet: We adapted staging and grading guidelines for NHP CRCs from established human CRC guidelines as published by the College of American Pathologists (CAP) [ ] and the American Joint Cancer Commission AJCC (AJCC 8th Edition).

Techniques: Functional Assay, Immunohistochemistry, Expressing, Positive Control, Staining

Journal: Journal of Translational Medicine

Article Title: Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

doi: 10.1186/s12967-024-04869-6

Figure Lengend Snippet: The mutational signature of rhesus CRCs is congruent to the human SBS6 signature with dMMR as proposed etiology. Most base substitutions observed in rhesus CRC are C > T transitions with a clear enrichment for ACG, CCG, GCG, and TCG. This pattern is highly similar to the human SBS6 signature , which is strongly associated with mismatch repair deficiency

Article Snippet: We adapted staging and grading guidelines for NHP CRCs from established human CRC guidelines as published by the College of American Pathologists (CAP) [ ] and the American Joint Cancer Commission AJCC (AJCC 8th Edition).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

doi: 10.1186/s12967-024-04869-6

Figure Lengend Snippet: Transcriptomics reveals similar DEG signatures in rhesus and human CRCs and indicates transcriptional downregulation of MLH1. A RNAseq revealed 1965 significantly differentially expressed genes (985 up, 980 down) in rhesus CRC compared to adjacent healthy colon. B Genes frequently dysregulated in human cancers are similarly altered in rhesus CRC including upregulation of MMPs, FAP, telomerase, tumor-promoting or associated pathways and reduction of GZMA, FRZB, CD69, and PPARG. A statistically significant downregulation of MLH1 was determined in rhesus CRC by ( C ) RNAseq and corroborated by ( D ) RT-qPCR. E In contrast, no difference was observed for the other MMR proteins MSH2, MSH6, and even PMS2. Statistical analysis was performed by Mann–Whitney U test and a p < 0.05 was considered statistically significant

Article Snippet: We adapted staging and grading guidelines for NHP CRCs from established human CRC guidelines as published by the College of American Pathologists (CAP) [ ] and the American Joint Cancer Commission AJCC (AJCC 8th Edition).

Techniques: Quantitative RT-PCR, MANN-WHITNEY

Journal: Journal of Translational Medicine

Article Title: Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

doi: 10.1186/s12967-024-04869-6

Figure Lengend Snippet: MLH1 hypermethylation occurs in all examined rhesus CRCs and is independent of the more global CIMP status. A marker was considered hypermethylated in CRC upon an increase of 20% in DNA methylation (red dotted line) in comparison to paired healthy colon as reference. A As a result, 5/16 CRCs were considered CIMP-high (orange, ≥ 4/5 methylated marker), 4/16 CRCs CIMP-low (yellow, 3/5 M), and 7/16 CRCs CIMP-negative (gray, ≤ 2/5 M). Interestingly, the MLH1 promoter region was hypermethylated in the entire CRC cohort ( B , MLH1 probe 1) which was corroborated by an independently designed second probe ( C , MLH1 probe 2). Importantly, this striking hypermethylation of MLH1 was observed in all CRCs and independent of the more global CIMP status. In contrast to MLH1, the other CIMP markers D CACNA1G, E CDKN2A, F CRABP1, and G NEUROG1 followed a clear trend and delineated CIMP high CRCs with higher methylation levels from CIMP negative tumors. Nonparametric Mann–Whitney U test was used for statistical comparisons and a p < 0.05 considered statistically significant

Article Snippet: We adapted staging and grading guidelines for NHP CRCs from established human CRC guidelines as published by the College of American Pathologists (CAP) [ ] and the American Joint Cancer Commission AJCC (AJCC 8th Edition).

Techniques: Marker, DNA Methylation Assay, Comparison, Methylation, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Vascular endothelial growth factor A amplification in colorectal cancer is associated with reduced M1 and M2 macrophages and diminished PD-1-expressing lymphocytes

doi: 10.1371/journal.pone.0175563

Figure Lengend Snippet: Boxplots depict the number of (A, C) CD68+ and (B, D) CD163+ cells (A, B) in CRCs with and without VEGFA amplification/polysomy and (C, D) in CRCs of intermediate and high grade.

Article Snippet: However, we observed that CRCs with VEGFA amplification or Chr6 polysomy had lower PD-L1 expression in the stroma than in VEGFA copy number neutral CRCs (p = 0.0079, Chi-square test; ).

Techniques: Amplification

Journal: PLoS ONE

Article Title: Vascular endothelial growth factor A amplification in colorectal cancer is associated with reduced M1 and M2 macrophages and diminished PD-1-expressing lymphocytes

doi: 10.1371/journal.pone.0175563

Figure Lengend Snippet: Barplots depict the number of samples with high, low and negative expression of (A, B) PD-1 and (C, D) PD-L1 in (A, C) tumoral and (B, D) stromal areas of CRCs.

Article Snippet: However, we observed that CRCs with VEGFA amplification or Chr6 polysomy had lower PD-L1 expression in the stroma than in VEGFA copy number neutral CRCs (p = 0.0079, Chi-square test; ).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: NGS Evaluation of Colorectal Cancer Reveals Interferon Gamma Dependent Expression of Immune Checkpoint Genes and Identification of Novel IFNγ Induced Genes

doi: 10.3389/fimmu.2020.00224

Figure Lengend Snippet: Expression of IFNγ in 79 CRC pairs. (A) Subtyping of 79 CRCs into CRC with high IFNγ (FPKM > 1) and CRC with low IFNγ (FPKM < 1). (B) Box and Whisker plot of six ICPs in INFγ positive CRC. More upregulation of all six ICPs and IFNγ (IDO1, 7.8-fold; Tim3, 2.3-fold, LAG3, 1.6-fold; CTLA4, 2.9-fold; PDL1, 3.0-fold; PD1, 2.1-fold; and IFNγ, 24.1-fold) in tumor vs. normal ( P < 0.01). (C) Box and Whisker plot of six ICPs in INFγ negative CRC. Less upregulation ( P < 0.05) of four ICPs and IFNγ (IDO1, 1.4-fold; Tim3, 1.4-fold; CTLA4, 1.7-fold; PDL1, 1.3-fold; and IFNγ, 4.4-fold), downregulation of LAG3 (0.54-fold) ( P = 1.7E-05), and no dysregulation of PD1 (1.0-fold) ( P = 0.57) in tumor vs. normal.

Article Snippet: To further define the dosage impact of IFNγ on the expression of six ICPs and three ICPRGs, we generated six IFNγ expression level gradients IFNγ: FPKM > 5 (4 CRCs); ( ) IFNγ: FPKM = 4.9–2 (20 CRCs); ( ) IFNγ: FPKM = 1.99–1(44 CRCs); ( ) IFNγ: FPKM = 0.99–0.5 (73 CRCs); ( ) IFNγ: FPKM = 0.49–0.01 (467 CRC); and ( ) IFNγ: FPKM < 0.009 (107 CRCs) in 716 CRCs (Indivumed [79 CRCs] and TCGA [637 CRCs]) ( ) and examined the impact of the levels on expression of the ICPs and ICPRGs examined in our more limited cohort.

Techniques: Expressing, Whisker Assay

Journal: Frontiers in Immunology

Article Title: NGS Evaluation of Colorectal Cancer Reveals Interferon Gamma Dependent Expression of Immune Checkpoint Genes and Identification of Novel IFNγ Induced Genes

doi: 10.3389/fimmu.2020.00224

Figure Lengend Snippet: Co-expression (cc > 0.8) of IFNγ with six ICPs in CRC. (A) IFNγ and all six ICPs co-expressed with CD8A/CD8B/CD4 within one 190 gene network in 32 IFNγ positive CRCs. (B) Tim3 and PDL1 co-expressed with CD4 within one 83 gene network in 47 IFNγ negative CRCs. (C) CTLA4 co-expressed with PD1 within one 88 gene expression network, but not Tim3, in normal colon without CD8/CD4.

Article Snippet: To further define the dosage impact of IFNγ on the expression of six ICPs and three ICPRGs, we generated six IFNγ expression level gradients IFNγ: FPKM > 5 (4 CRCs); ( ) IFNγ: FPKM = 4.9–2 (20 CRCs); ( ) IFNγ: FPKM = 1.99–1(44 CRCs); ( ) IFNγ: FPKM = 0.99–0.5 (73 CRCs); ( ) IFNγ: FPKM = 0.49–0.01 (467 CRC); and ( ) IFNγ: FPKM < 0.009 (107 CRCs) in 716 CRCs (Indivumed [79 CRCs] and TCGA [637 CRCs]) ( ) and examined the impact of the levels on expression of the ICPs and ICPRGs examined in our more limited cohort.

Techniques: Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: NGS Evaluation of Colorectal Cancer Reveals Interferon Gamma Dependent Expression of Immune Checkpoint Genes and Identification of Novel IFNγ Induced Genes

doi: 10.3389/fimmu.2020.00224

Figure Lengend Snippet: Characterization of three ICPRGs in CRC. (A) Box and Whisker plot of three ICPRGs in IFNγ positive CRC. More upregulation ( P < 6.0E-09) of all three ICPRGs (IFI30: 2.3-fold; GBP1: 3.1-fold, GBP4; 12.9-fold in tumor vs. normal). (B) Box and Whisker plot of three ICPRGs in IFNγ negative CRC. Less upregulation ( P < 0.05) of two ICPRGs (IFI30: 1.4-fold and GBP1: 1.2-fold) and GBP4 (1.1-fold) ( P = 0.23) in tumor vs. normal. (C) Co-expression of IFI30, GBP1, and GBP4 with IFNγ, IDO1, Tim3, LAG3 and CD8A within a 119 gene network in IFNγ positive CRCs. (D) Co-expression of IFI30 with PDL1 and Tim3 within a 101 network without CD8/CD4 in IFNγ negative CRCs. (E) GBP1 co-expressed with GBP4 within an eight-gene network in normal colon. (F) Identification of unique genes co-expressed with 10 genes (INFγ, six ICPs, and three ICPRGs) between INFγ positive and negative tumors. There are 151 unique genes in IFNγ positive tumors, 73 unique genes in IFNγ negative tumors, and 67 unique genes in normal tissue.

Article Snippet: To further define the dosage impact of IFNγ on the expression of six ICPs and three ICPRGs, we generated six IFNγ expression level gradients IFNγ: FPKM > 5 (4 CRCs); ( ) IFNγ: FPKM = 4.9–2 (20 CRCs); ( ) IFNγ: FPKM = 1.99–1(44 CRCs); ( ) IFNγ: FPKM = 0.99–0.5 (73 CRCs); ( ) IFNγ: FPKM = 0.49–0.01 (467 CRC); and ( ) IFNγ: FPKM < 0.009 (107 CRCs) in 716 CRCs (Indivumed [79 CRCs] and TCGA [637 CRCs]) ( ) and examined the impact of the levels on expression of the ICPs and ICPRGs examined in our more limited cohort.

Techniques: Whisker Assay, Expressing

Journal: Frontiers in Immunology

Article Title: NGS Evaluation of Colorectal Cancer Reveals Interferon Gamma Dependent Expression of Immune Checkpoint Genes and Identification of Novel IFNγ Induced Genes

doi: 10.3389/fimmu.2020.00224

Figure Lengend Snippet: Close association of IFNγ with T cells in tumor but not in normal tissues. (A) Box and Whisker plot of immune cell genes in IFNγ positive CRCs. Upregulation ( P < 0.05) of T cells (CD8A, 3.5-fold; CD3E, 2.0-fold; CD4, 1.3-fold; FOXP3, 1.5-fold) but not B cell (CD19) (1.2-fold, P = 0.38), neutrophil (CD11b) (1.4-fold, P = 0.079), M1 (ARG1) (0.68-fold, P = 0.62), (ARG2) (1.0-fold, P = 0.87), or M2 (ARG2, CCR7) (1.2-fold, P = 0.30) leukocyte-related genes in IFNγ positive CRC vs. negative CRC. (B) Box and Whisker plot of immune cell genes in normal colon tissue. No upregulation ( P > 0.05) of T cells (CD8A, 0.93-fold; CD3E, 1.0-fold; CD4, 1.0-fold; FOXP3, 1.1-fold), B cell (CD19) (1.4-fold), neutrophil (CD11b) (1.1-fold), MDSC1 (ARG1) (1.0-fold), (ARG2) (0.97-fold), or MDSC2 (ARG2, CCR7) (1.2-fold) leukocytes related genes in tissues adjacent to IFNγ positive and negative CRCs.

Article Snippet: To further define the dosage impact of IFNγ on the expression of six ICPs and three ICPRGs, we generated six IFNγ expression level gradients IFNγ: FPKM > 5 (4 CRCs); ( ) IFNγ: FPKM = 4.9–2 (20 CRCs); ( ) IFNγ: FPKM = 1.99–1(44 CRCs); ( ) IFNγ: FPKM = 0.99–0.5 (73 CRCs); ( ) IFNγ: FPKM = 0.49–0.01 (467 CRC); and ( ) IFNγ: FPKM < 0.009 (107 CRCs) in 716 CRCs (Indivumed [79 CRCs] and TCGA [637 CRCs]) ( ) and examined the impact of the levels on expression of the ICPs and ICPRGs examined in our more limited cohort.

Techniques: Whisker Assay

Journal: Frontiers in Immunology

Article Title: NGS Evaluation of Colorectal Cancer Reveals Interferon Gamma Dependent Expression of Immune Checkpoint Genes and Identification of Novel IFNγ Induced Genes

doi: 10.3389/fimmu.2020.00224

Figure Lengend Snippet: IFNy dependent expression of six ICPs (PDl, PDLl, CTLA4, IDOl, LAG3, Tim3) and three ICPRGs (IFI30, GBPl, GBP4) in 716 CRCs. (A) Classification of INFγ into six expression gradients in 716 CRCs. (B) IFNγ dosage dependent expression positive correlation (cc > 0.94) with six ICPs and three ICPRGs across six IFNγ expression level gradients (six CRC subsets).

Article Snippet: To further define the dosage impact of IFNγ on the expression of six ICPs and three ICPRGs, we generated six IFNγ expression level gradients IFNγ: FPKM > 5 (4 CRCs); ( ) IFNγ: FPKM = 4.9–2 (20 CRCs); ( ) IFNγ: FPKM = 1.99–1(44 CRCs); ( ) IFNγ: FPKM = 0.99–0.5 (73 CRCs); ( ) IFNγ: FPKM = 0.49–0.01 (467 CRC); and ( ) IFNγ: FPKM < 0.009 (107 CRCs) in 716 CRCs (Indivumed [79 CRCs] and TCGA [637 CRCs]) ( ) and examined the impact of the levels on expression of the ICPs and ICPRGs examined in our more limited cohort.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: NGS Evaluation of Colorectal Cancer Reveals Interferon Gamma Dependent Expression of Immune Checkpoint Genes and Identification of Novel IFNγ Induced Genes

doi: 10.3389/fimmu.2020.00224

Figure Lengend Snippet: IFNγ dependent expression of three ICPRGs among five other solid cancers in Indivmed and TCGA cohort. Box and Whisker analysis of three ICPRGs in six types of cancer. Higher three ICPRGs and IFNγ expression in IFNγ (+) 69 CRCs (IFI30, 2.9-fold; GBP1, 4.6-fold; GBP4, 7.9-fold; and IFNγ, 44-fold), 13 SKCMs (IFI30, 4.6-fold; GBP1, 8.5-fold; GBP4, 8.3-fold; and IFNγ, 55-fold), 85 BCs (IFI30, 2.3-fold; GBP1, 4.9-fold; GBP4, 4.5-fold; and IFNγ, 19-fold), 13 ESCs (IFI30, 2.0-fold; GBP1, 4.5-fold; GBP4, 4.1-fold; and IFNγ 28-fold), 71 STCs (IFI30, 2.5-fold; GBP1, 4.5-fold; GBP4, 6.2-fold; and IFNγ 23-fold), and 68 LUSCs (IFI30, 2.6-fold; GBP1, 3.4-fold; GBP4, 4.1-fold; and IFNγ 17-fold), vs. IFNγ (–) 647 CRCs, 90 SKCMs, 1,020 BCs, 171 ESCs, 345 STCs, and 433 LUSCs.

Article Snippet: To further define the dosage impact of IFNγ on the expression of six ICPs and three ICPRGs, we generated six IFNγ expression level gradients IFNγ: FPKM > 5 (4 CRCs); ( ) IFNγ: FPKM = 4.9–2 (20 CRCs); ( ) IFNγ: FPKM = 1.99–1(44 CRCs); ( ) IFNγ: FPKM = 0.99–0.5 (73 CRCs); ( ) IFNγ: FPKM = 0.49–0.01 (467 CRC); and ( ) IFNγ: FPKM < 0.009 (107 CRCs) in 716 CRCs (Indivumed [79 CRCs] and TCGA [637 CRCs]) ( ) and examined the impact of the levels on expression of the ICPs and ICPRGs examined in our more limited cohort.

Techniques: Expressing, Whisker Assay

Journal: Frontiers in Immunology

Article Title: NGS Evaluation of Colorectal Cancer Reveals Interferon Gamma Dependent Expression of Immune Checkpoint Genes and Identification of Novel IFNγ Induced Genes

doi: 10.3389/fimmu.2020.00224

Figure Lengend Snippet:

Article Snippet: To further define the dosage impact of IFNγ on the expression of six ICPs and three ICPRGs, we generated six IFNγ expression level gradients IFNγ: FPKM > 5 (4 CRCs); ( ) IFNγ: FPKM = 4.9–2 (20 CRCs); ( ) IFNγ: FPKM = 1.99–1(44 CRCs); ( ) IFNγ: FPKM = 0.99–0.5 (73 CRCs); ( ) IFNγ: FPKM = 0.49–0.01 (467 CRC); and ( ) IFNγ: FPKM < 0.009 (107 CRCs) in 716 CRCs (Indivumed [79 CRCs] and TCGA [637 CRCs]) ( ) and examined the impact of the levels on expression of the ICPs and ICPRGs examined in our more limited cohort.

Techniques: Control