Immune microenvironment and lymph node yield in colorectal cancer.

BACKGROUND: Lymph node (LN) harvesting is associated with outcomes in colonic cancer. We sought to interrogate whether a distinctive immune milieu of the primary tumour is associated with LN yield. METHODS: A total of 926 treatment-naive patients with colorectal adenocarcinoma with more than 12 LNs (LN-high) were compared with patients with 12 or fewer LNs (LN-low). We performed immunohistochemistry and quantification on tissue microarrays for HLA class I/II proteins, beta-2-microglobulin (B2MG), CD8, CD163, LAG3, PD-L1, FoxP3, and BRAF V600E. RESULTS: The LN-high group was comprised of younger patients, longer resections, larger tumours, right-sided location, and tumours with deficient mismatch repair (dMMR). The tumour microenvironment showed higher CD8+ cells infiltration and B2MG expression on tumour cells in the LN-high group compared to the LN-low group. The estimated mean disease-specific survival was higher in the LN-high group than LN-low group. On multivariate analysis for prognosis, LN yield, CD8+ cells, extramural venous invasion, perineural invasion, and AJCC stage were independent prognostic factors. CONCLUSION: Our findings corroborate that higher LN yield is associated with a survival benefit. LN yield is associated with an immune high microenvironment, suggesting that tumour immune milieu influences the LN yield.

Predicting recurrence in pancreatic neuroendocrine tumours: role of ARX and alternative lengthening of telomeres (ALT).

BACKGROUND: While many pancreatic neuroendocrine tumours (PanNET) show indolent behaviour, predicting the biological behaviour of small nonfunctional PanNETs remains a challenge. Nonfunctional PanNETs with an epigenome and transcriptome that resemble islet alpha cells (ARX-positive) are more aggressive than neoplasms that resemble islet beta cells (PDX1-positive). In this study, we explore the ability of immunohistochemistry for ARX and PDX1 and telomere-specific fluorescence in situ hybridisation (FISH) for alternative lengthening of telomeres (ALT) to predict recurrence. METHODS: Two hundred fifty-six patients with PanNETs were identified, and immunohistochemistry for ARX and PDX1 was performed. Positive staining was defined as strong nuclear staining in >5% of tumour cells. FISH for ALT was performed in a subset of cases. RESULTS: ARX reactivity correlated with worse disease-free survival (DFS) (P = 0.011), while there was no correlation between PDX1 reactivity and DFS (P = 0.52). ALT-positive tumours (n = 63, 31.8%) showed a significantly lower DFS (P < 0.0001) than ALT-negative tumours (n = 135, 68.2%). ARX reactivity correlated with ALT positivity (P < 0.0001). Among nonfunctional tumours, recurrence was noted in 18.5% (30/162) of ARX-positive tumours and 7.5% (5/67) of ARX-negative tumours. Among WHO grade 1 and 2 PanNETs with ≤2 cm tumour size, 14% (6/43) of ARX-positive tumours recurred compared to 0 of 33 ARX-negative tumours and 33.3% (3/9) ALT-positive tumours showed recurrence versus 4.4% (2/45) ALT-negative tumours. CONCLUSION: Immunohistochemistry for ARX and ALT FISH status may aid in distinguishing biologically indolent cases from aggressive small low-grade PanNETs, and help to identify patients who may preferentially benefit from surgical intervention.

Correlation of clinical, pathologic, and genetic parameters with intratumoral immune milieu in mucinous adenocarcinoma of the colon.

Mucinous adenocarcinoma (MAD), the most common subtype of colonic adenocarcinoma (CA), requires >50% intratumoral mucin. There is limited data regarding the impact of MAD on key lymphocyte subsets and therapeutically critical immune elements. In this study we address: (1) the definition of MAD, (2) grading of MAD, and (3) the impact of MAD and extracellular mucin on intratumoral immune milieu. Estimation of the percentage of intratumoral mucin was performed by two pathologists. Tissue microarrays were stained for immune markers including CD8, CD163, PD-L1, FoxP3, ß2 microglobulin, HLA class I, and HLA class II. Immunohistochemistry for BRAF V600E was performed. MMR status was determined on immunohistochemistry for MSH2, MSH6, MLH1, PMS2. Manual and automated HALO platforms were used for quantification. The 903 CAs included 62 (6.9%) MAD and 841 CA with ≤ 50% mucin. We identified 225 CAs with mucinous differentiation, defined by ≥10% mucin. On univariate analysis neither cut point, 50% (p = 0.08) and 10% (p = 0.08) mucin, correlated with disease-specific survival (DSS). There were no differences in key clinical, histological and molecular features between MAD and CA with mucinous differentiation. On univariate analysis of patients with MAD, tumor grade correlated with DSS (p = 0.0001) while MMR status did not (p = 0.86). There was no statistically significant difference in CD8 (P = 0.17) and CD163 (P = 0.05) positive immune cells between MAD and conventional CA. However, deficient (d) MMR MADs showed fewer CD8 (P = 0.0001), CD163 (P = 0.0001) and PD-L1 (P = 0.003) positive immune cells compared to proficient (p)MMR MADs, a finding also seen with at 10% mucin cut point. Although MAD does not impact DSS, this study raises the possibility that the immune milieu of dMMR MADs and tumors with > =10% mucin may differ from pMMR MADs and tumors with <10% mucin, a finding that may impact immune-oncology based therapeutics.

Programmed death-ligand 1 expression in the immune compartment of colon carcinoma.

Programmed cell death ligand 1 (PD-L1) on tumor cells is a significant prognostic biomarker for a number of malignancies, although less is known about the significance of PD-L1 positive immune cells in colon carcinoma. The purpose of this study is to evaluate the role of PD-L1 in a large cohort of colon carcinomas to identify patterns of PD-L1 expression in the tumor microenvironment and its correlation with other key immune subsets to better understand the impact of these immune cells. We assessed 1218 colon carcinomas on representative tissue microarray sections, gathered relevant clinicopathologic information, and performed immunohistochemical staining for mismatch repair proteins, CD8, CD163, LAG3, PD-L1, FoxP3, and BRAF V600E. We then performed automated quantification; manual quantification was used for PD-L1 tumor cells and immune cells. Dual PD-L1/PU.1 immunostain was also performed. The majority of PD-L1 positive cells expressed PU.1 thus representing tumor-associated macrophages. Based on the median number of PD-L1 positive immune cells (7.6/mm2), we classified tumors into two classes: (1) PD-L1 immune cell low and (2) PD-L1 immune cell high. PD-L1 immune cell high colon carcinomas showed favorable prognostic pathologic features including less frequent extramural venous invasion (p = 0.0001) and lower AJCC stage (p = 0.0001); they were also more commonly associated with deficient mismatch repair (dMMR) (p = 0.0001) and BRAF V600E reactivity. PD-LI immune cell high tumors were associated with high CD8, CD163, and FoxP3 positive cells (p = 0.0001, respectively). PD-L1 immune cell high and LAG3 high colon carcinomas were associated with improved disease-specific survival (p = 0.0001 and 0.001, respectively). PD-L1 expression on tumor cells was not associated with disease-specific survival. On multivariate analysis of chemotherapy naïve stage 2 colon carcinomas, only extramural venous invasion (p = 0.002), perineural invasion (p = 0.001) and PD-L1 immune cell expression (p = 0.032) correlated with disease-specific survival. Resected colonic carcinomas with high expression of PD-L1 and LAG3 proteins on immune cells were associated with improved prognosis in colon carcinoma. The mechanism underlying the improved prognosis of colon carcinomas bearing high numbers of immunoregulatory cells needs further investigation.

Molecular Basis of Extramural Vascular Invasion (EMVI) in Colorectal Carcinoma.

BACKGROUND: Extramural vascular invasion (EMVI) is a known poor prognostic factor in colorectal carcinoma; however, its molecular basis has not been defined. This study aimed to assess the expression of molecular markers in EMVI positive colorectal carcinoma to understand their tumor microenvironment. METHODS: Immunohistochemistry was performed on tissue microarrays of surgically resected colorectal cancer specimens for immunological markers, and BRAFV600E mutation (and on the tissue blocks for mismatch repair proteins). Automated quantification was used for CD8, LAG3, FOXP3, PU1, and CD163, and manual quantification was used for PDL1, HLA I markers (beta-2 microglobulin, HC10), and HLA II. The Wilcoxon rank-sum test was used to compare EMVI positive and negative tumors. A logistic regression model was fitted to assess the predictive effect of biomarkers on EMVI. RESULTS: There were 340 EMVI positive and 678 EMVI negative chemo naïve tumors. PDL1 was barely expressed on tumor cells (median 0) in the entire cohort. We found a significantly lower expression of CD8, LAG3, FOXP3, PU1 cells, PDL1 positive macrophages, and beta-2 microglobulin on tumor cells in the EMVI positive subset (p ≤ 0.001). There was no association of BRAFV600E or deficient mismatch repair proteins (dMMR) with EMVI. PU1 (OR 0.8, 0.7-0.9) and low PDL1 (OR 1.6, 1.1-2.3) independently predicted EMVI on multivariate logistic regression among all biomarkers examined. CONCLUSION: There is a generalized blunting of immune response in EMVI positive colorectal carcinoma, which may contribute to a worse prognosis. Tumor-associated macrophages seem to play the most significant role in determining EMVI.

IgG4-related disease is characterised by the overexpression of immunomodulatory proteins.

AIMS: Immunoglobulin 4-related disease (IgG4-RD) is a multisystem disease, characterised by tumefactive lesions and a swift response to immunosuppressive therapy. Although elevated serum and tissue IgG4 are characteristic, T cells appear to be the primary driver of this immunologically mediated disease. The overarching goal was to examine the role of immunomodulatory cells in IgG4-RD. METHODS AND RESULTS: Biopsies from patients with IgG4-RD (n = 39) and mimics of this disease (n = 78) were evaluated for IgG4, IgG, CD8, programmed cell death ligand 1 (PD-L1) and a subset (n = 18) evaluated for CD4, purine rich box 1 (PU.1), forkhead box protein 3 (FoxP3), PD-L1, programmed cell death 1 (PD-1), indoleamine 2,3-dioxygenase 1 (IDO1) and lymphocyte-activation gene 3 (LAG3). Data pertaining to demographics and laboratory findings at baseline evaluation was extracted from electronic medical records. When compared to mimics, IgG4-RD showed increased numbers of PD-L1- (P = 0.0001), PD-1- (P = 0.001), IDO1- (P = 0.03), LAG3- (P = 0.04) and FoxP3- (P = 0.04)-positive immune cells. The PD-L1-positive cells were enriched within aggregates of CD4 and CD8-positive T cells. Thirty-one of 39 (80%) IgG4-RD cases showed greater than five PD-L1-positive cells per high-power field (HPF), while four of 78 (5%) mimics of this disease exceeded this cut-point. In IgG4-RD, PD-L1-positive macrophages correlated with PD-1- (P = 0.002), LAG3- (P = 0.001) and IDO1-positive cells (P = 0.001); a-positive correlation was also noted between IgG4/IgG ratio and PD-L1-, PD-1- and IDO1-positive cells. CONCLUSIONS: IgG4-RD shows expansion of mechanisms that maintain peripheral tolerance. The spatial and temporal relationship between T cells and the PD-L1-PD-1 axis and the up-regulation of multiple immunomodulatory proteins suggests that these immunoregulatory mechanisms play a significant role in IgG4-RD.

Clinical, pathological genetics and intratumoral immune milieu of serrated adenocarcinoma of the colon.

BACKGROUND: Serrated adenocarcinoma (SAC), a recognised WHO variant of colonic adenocarcinoma, is the purported end-product of serrated neoplasia. However, the diagnosis of SAC is infrequently rendered, and little is known about its prognosis, immune microenvironment and molecular alterations. MATERIALS AND METHODS: We assessed 903 consecutive colon carcinomas and recognised tumours with ≥ 5% (n = 77) serrated and ≥ 50% serrated patterns (n = 13). We assessed precursor polyps and synchronous polyps. We recorded demographic/clinical parameters, histological features and mismatch repair (MMR) status. We performed immunohistochemistry and quantification on tissue microarray for HLA class I/II proteins, B2MG, CD8, CD163, LAG3, FoxP3, PD-L1 and BRAF V600E. RESULTS: We identified ≥ 5% epithelial serration prevalence in 8.5% of cases and ≥ 50% epithelial serration prevalence in 1.4% of cases. Precursor lesions were present in 21.4% of cases; these were mostly tubular adenomas with two traditional serrated adenomas identified. SAC with ≥ 5% serrations exhibited lower numbers of CD8-positive lymphocytes (P = 0.002) and lower B2MG expression (P = 0.048), although neither value was significant at ≥ 50% serration threshold. There was no difference in HLA class I/II, or PD-L1 expression on tumour cells and no difference in PD-L1, LAG3, FoxP3 and CD163 expression on immune cells. There was no association with MMR status, or BRAFV600E relative to conventional adenocarcinoma. There was improved disease-specific survival on univariate (but not multivariate) analysis between carcinomas with serrated pattern and non-mucinous conventional colonic carcinomas at ≥ 5% epithelial serrations (P = 0.04). CONCLUSION: SAC category shows a limited impact on survival, and this phenotype may harbour a unique immunological milieu.

Microgranulomas at Diagnosis Are Associated With Need for Antitumor Necrosis Factor Escalation in Pediatric Crohn Disease.

INTRODUCTION: Granulomas are a pathologic hallmark of Crohn disease (CD) although they are found in only a subset of patients. Well-formed granulomas are associated with an aggressive phenotype although it is unknown if microgranulomas confer a similar phenotype. This study sought to define the incidence of microgranulomas in pediatric CD and compare the clinical course with cases with granulomas and those without granulomatous inflammation. METHODS: We performed a single-center, retrospective study of pediatric CD patients who had at least 3 years of follow-up. initial diagnostic biopsies were systematically re-examined by a gastrointestinal pathologist. A priori definitions of granuloma (10+ histiocytes) and microgranuloma (4-9 histiocytes) were used. Disease outcomes of hospitalization, development of complicated disease behavior, perianal disease, and the use of anti-tumor necrosis factor (anti-TNF) therapy were assessed by Kaplan-Meier survival plots. RESULTS: This study included 138 subjects with an average follow-up of 4.6 years. Granulomas were seen in 38 of 138 subjects (27.5%) and an additional 38 subjects (27.5%) had at least 1 microgranuloma (in the absence of granulomas). Escalation to anti-TNF therapy was higher in CD with granulomas (P  = 0.001) and microgranulomas (P  = 0.0001) compared with those without granulomatous inflammation. CONCLUSIONS: A significant subset of pediatric CD patients have microgranulomas (in the absence of well-defined granulomas). Children with CD who have microgranulomas are escalated to anti-TNF therapy more frequently than those without granulomatous inflammation (and at a similar rate to those with granulomas). Pathologists should have a low threshold to report microgranulomas as they may help to predict disease behavior.

Paediatric Crohn's disease: histologic findings at initial presentation.

BACKGROUND: Diagnosing paediatric Crohn's disease (CD) based on histology can present challenges. We evaluate the histological spectrum of treatment-naïve biopsies from children with CD and assess these findings' diagnostic and predictive value. METHODS: Three cohorts were identified: (1) 137 patients with CD, (2) 116 patients with ulcerative colitis (UC) and (3) 50 patients without inflammatory bowel disease. Biopsies from the gastrointestinal (GI) tract were re-examined for signs of active and chronic inflammation, including lymphocyte-pattern oesophagitis, focal enhancing gastritis and indicators of chronicity. Additionally, granulomas and microgranulomas (defined as clusters of 4-9 epithelioid histiocytes) were evaluated. RESULTS: Lymphocyte-pattern oesophagitis was observed in 15% of patients (n=20). Moderate-to-severe diffuse gastritis was noted in 50.4% of patients (n=68), while focal enhancing gastritis was identified in 11.1% (n=15). In terminal ileal biopsies, 46.1% exhibited activity and 5.3% showed features of chronicity. Active colitis was present in 73% of patients (n=100), with chronic colitis seen in 11.7% (n=16). Granulomas and microgranulomas were observed in 31.4% (43/137) and 48.9% (67/137) of patients, respectively. Notably, 30.7% (42/137) of patients with microgranulomas were without granulomas. Previously undetected microgranulomas were found in 20 of 27 cases. 2.5% of patients with UC and none of the control cohort showed microgranulomas. Lymphocyte-pattern oesophagitis was associated with an increased need for anti-tumor necrosis factor (TNF) therapy (p=0.007). CONCLUSIONS: GI microgranulomas, often overlooked, are specific to CD in the proper clinical context. Oesophageal lymphocytosis may predict a need for more aggressive treatment. The study brings to light under-recognised aspects of CD's histological diagnosis, including the oversight of microgranulomas, the high prevalence of diffuse gastritis and low prevalence of focal enhancing gastritis, the frequent absence of terminal ileitis and the infrequent occurrence of chronic colitis.

ß2-microglobulin expression is associated with aggressive histology, activated tumor immune milieu, and outcome in colon carcinoma.

OBJECTIVES: We sought to assess the expression of human leukocyte antigen (HLA) proteins and ß2-microglobulin (B2M) in tumor cells and the relationship with immune microenvironment and outcome in colorectal cancer (CRC). METHODS: A total of 953 CRC cases were evaluated by immunohistochemistry for HLA class I, HLA class II, and B2M. The expression level of these biomarkers was correlated with clinicopathologic information, BRAF V600E and mismatch repair (MMR) proteins, and the quantitated expression levels of immune cells (CD8 and CD163) and immune regulatory proteins (FoxP3, programmed cell death 1 ligand 1 [PD-L1], and LAG3). RESULTS: We found that B2M-low tumors were statistically correlated with aggressive histologic features, including higher stage, higher grade, extramural venous invasion, perineural invasion, and distant metastasis. Expression of B2M was positively correlated (R2 = 0.3) and significantly associated with MMR-deficient tumors (P < .001); B2M-low tumors were also associated with an "immune cold"' microenvironment, including a reduced number of immune cells (CD8 and CD163), reduced expression of immune regulatory proteins by immune cells (PD-L1, FoxP3, and LAG3), and reduced tumor cell expression of PD-L1. These B2M-low tumors correlated with lower disease-specific survival (P = .018), a finding that maintained significance only for the proficient MMR cohort (P = .037). CONCLUSIONS: Our findings suggest that B2M expression may support predictive models for both outcome and checkpoint inhibitor therapy treatment response for colorectal adenocarcinoma.

HCC spatial transcriptomic profiling reveals significant and potentially targetable cancer-endothelial interactions.

BACKGROUND: HCC is a highly vascular tumor, and many effective drug regimens target the tumor blood vessels. Prior bulk HCC subtyping data used bulk transcriptomes, which contained a mixture of parenchymal and stromal contributions. METHODS: We utilized computational deconvolution and cell-cell interaction analyses to cell type-specific (tumor-enriched and vessel-enriched) spatial transcriptomic data collected from 41 resected HCC tissue specimens. RESULTS: We report that the prior Hoshida bulk transcriptional subtyping schema is driven largely by an endothelial fraction, show an alternative tumor-specific schema has potential prognostic value, and use spatially paired ligand-receptor analyses to identify known and novel (LGALS9 tumor-HAVCR2 vessel) signaling relationships that drive HCC biology in a subtype-specific and potentially targetable manner. CONCLUSIONS: Our study leverages spatial gene expression profiling technologies to dissect HCC heterogeneity and identify heterogeneous signaling relationships between cancer cells and their endothelial cells. Future validation and expansion of these findings may validate novel cancer-endothelial cell interactions and related drug targets.

Immunological and Clinico-Molecular Features of Tumor Border Configuration in Colorectal Cancer.

INTRODUCTION: Infiltrating tumor border configuration (ITBC) portends a poor prognosis compared with pushing tumor border configuration (PTBC) in colorectal cancer. The tumor and its surrounding immune microenvironment of tumor border configuration is not well-characterized. We aim to elucidate the differences in expression of molecular markers between the 2 groups using tissue microarray (TMA). STUDY DESIGN: Immunohistochemistry was performed on TMAs of surgical pathology specimens obtained from colorectal cancer patients consecutively operated at our institution from 2004 to 2015. TMAs were stained for immune cells (CD8, FOXP3, LAG3, PU1, CD163, and PDL1); HLA II, beta 2 microglobulin, and HC10 on tumor cells; BRAFV600E mutation; and DNA mismatch repair proteins (MMR) status. Patients who received neoadjuvant therapy were excluded. RESULTS: There were 646 tumors with ITBC and 310 tumors with PTBC. There was a significantly lower expression (p < 0.05) of immune components, namely CD8, FOXP3, LAG3, PU1, PDL1 immune cells, and Beta-2 Microglobulin on tumor cells in the tumors with ITBC compared with PTBC, except CD163 immune cells, and HC10 and HLAII on tumor cells. Tumors with ITBC were less likely to be associated with BRAFV600E mutations and deficient MMR proteins (p < 0.001). On analyzing MMR-proficient tumors separately, we could not find any difference in the expression of any molecular marker (including BRAF), except a lower expression of PDL1 immune cells in tumors with ITBC (p < 0.001). CONCLUSIONS: Colorectal tumors with ITBC are associated with a generalized low immune microenvironment and low rates of BRAFV600E mutation compared with tumors with PTBC. However, the molecular expression of tumor border configuration seems confounded by the MMR molecular signature. MMR-proficient colorectal tumors with ITBC are associated with a lower expression of only PDL1 immune cells among all immune markers examined.

Clinical, pathological, genetics and intratumoural immune milieu of micropapillary carcinoma of the colon.

AIM: Micropapillary carcinoma (MPC) is a recognised WHO variant of colonic carcinoma (CC), although little is known about its prognosis, immune microenvironment and molecular alterations. We investigated its clinical, pathological and immunological characteristics. METHODS: We assessed 903 consecutive CCs and used the WHO definition to identify MPC. We recorded serrated and mucinous differentiation and mismatch repair (MMR) status. We performed immunohistochemistry and quantification on tissue microarrays for HLA class I/II proteins, beta-2-microglobulin (B2MG), CD8, CD163, LAG3, PD-L1, FoxP3, PD-L1and BRAF V600E. RESULTS: We classified 8.6% (N=78) of CC as MPC. Relative to non-MPC, MPC was more often high grade (p=0.03) and showed serrated morphology (p<0.01); however, we found no association with extramural venous invasion (p=0.41) and American Joint Committee on Cancer stage (p=0.95). MPCs showed lower numbers of CD8 positive lymphocytes (p<0.01), lower tumour cell B2MG expression (p=0.04) and lower tumour cell PD-L1 expression (p<0.01). There was no difference in HLA class I/II, LAG3, FOXP3, CD163 and PD-L1 positive histiocytes. There was no association with MMR status or BRAF V600E relative to non-MPC. MPC was not associated with decreased disease-specific survival (p=0.36). CONCLUSION: MPCs are associated with high-grade differentiation and a less active immune microenvironment than non-MPC. MPC is not associated with inferior disease-specific survival.

Dysregulated Repeat Element Viral-like Immune Response in Hepatocellular Carcinoma.

Purpose: Dysregulation of viral-like repeat RNAs are a common feature across many malignancies that are linked with immunological response, but the characterization of these in hepatocellular carcinoma (HCC) is understudied. In this study, we performed RNA in situ hybridization (RNA-ISH) of different repeat RNAs, immunohistochemistry (IHC) for immune cell subpopulations, and spatial transcriptomics to understand the relationship of HCC repeat expression, immune response, and clinical outcomes. Experimental Design: RNA-ISH for LINE1, HERV-K, HERV-H, and HSATII repeats and IHC for T-cell, Treg, B-cell, macrophage, and immune checkpoint markers were performed on 43 resected HCC specimens. Spatial transcriptomics on tumor and vessel regions of interest was performed on 28 specimens from the same cohort. Results: High HERV-K and high LINE1 expression were both associated with worse overall survival. There was a positive correlation between LINE1 expression and FOXP3 T-regulatory cells (r = 0.51 p < 0.001) as well as expression of the TIM3 immune checkpoint (r = 0.34, p = 0.03). Spatial transcriptomic profiling of HERV-K high and LINE-1 high tumors identified elevated expression of multiple genes previously associated with epithelial mesenchymal transition, cellular proliferation, and worse overall prognosis in HCC including SSX1, MAGEC2, and SPINK1. Conclusion: Repeat RNAs may serve as useful prognostic biomarkers in HCC and may also serve as novel therapeutic targets. Additional study is needed to understand the mechanisms by which repeat RNAs impact HCC tumorigenesis.

Mutant IDH Inhibits IFNγ-TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma.

Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate-dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate-mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell-autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell-specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ-TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. SIGNIFICANCE: Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587.

Satellite repeat RNA expression in epithelial ovarian cancer associates with a tumor-immunosuppressive phenotype.

Aberrant expression of viral-like repeat elements is a common feature of epithelial cancers, and the substantial diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering independent of tissue origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that human satellite II (HSATII) satellite repeat expression was highly associated with epithelial-mesenchymal transition (EMT) and anticorrelated with IFN-response genes indicative of a more aggressive phenotype. SATII expression - and its correlation with EMT and anticorrelation with IFN-response genes - was also found in ovarian cancer RNA-Seq data and was associated with significantly shorter survival in a second independent cohort of patients with ovarian cancer. Repeat RNAs were enriched in tumor-derived extracellular vesicles capable of stimulating monocyte-derived macrophages, demonstrating a mechanism that alters the tumor microenvironment with these viral-like sequences. Targeting of HSATII with antisense locked nucleic acids stimulated IFN response and induced MHC I expression in ovarian cancer cell lines, highlighting a potential strategy of modulating the repeatome to reestablish antitumor cell immune surveillance.

Fibrohistiocytic Variant of Hepatic Pseudotumor: An Antibiotic Responsive Tumefactive Lesion.

Inflammatory pseudotumor is a term used to designate inflammation-rich tumefactive lesions. Following the exclusion of specific entities such as IgG4-related disease and other neoplastic entities previously included in this entity, the majority of hepatic pseudotumors show a prominent fibrohistiocytic inflammatory reaction and have been previously categorized as fibrohistiocytic variant of hepatic pseudotumor (FHVHPT). The goal of this study was to examine the clinical, radiologic, histologic, and etiologic aspects of this entity. After excluding neoplastic diseases, we identified 30 patients with FHVHPT from 3 institutions between 2009 and 2019. We extracted demographic and clinical data, liver function tests as well as culture results and radiologic information. Hematoxylin and eosin-stained slides were reviewed for pattern of inflammation as well as its cellular composition. Immunohistochemistry for IgG4 and IgG was performed in all cases. The mean age of the 30 lesions characterized as FHVHPT was 56 years (range: 23 to 79 y). Nineteen patients showed solitary lesions; 11 were multiple. The mean size of the lesion was 3.8 cm (range: 1 to 7.5 cm). On imaging, a neoplastic process or metastasis was the leading diagnostic consideration (n=15, 50%). The most common symptom was abdominal pain (n=14/30); 8 patients presented with symptoms compatible with an infectious process, including fever. The inflammatory infiltrate was dominated by lymphocytes and plasma cells, and in most cases, a prominent histiocytic infiltrate (22/30). Neutrophils were identified in 12 cases, with microabscess noted in 8. Storiform pattern of fibrosis was seen in 14/30 cases; obliterative phlebitis was not identified. Culture identified a microorganism in 4 of 9 cases evaluated. The mean IgG4 count was 9.3 per HPF (range: 0 to 51) with 9 of the 26 (35%) biopsies showing >10 IgG4 positive plasma cells per HPF. The mean IgG4 to IgG ratio was 8% (range: 8% to 46%). A hepatectomy was performed in 4 cases. On broad spectrum antibiotics (n=14) the lesions either resolved or decreased in size. Eight patients did not receive specific therapy, nevertheless, the lesion(s) resolved spontaneously in 6 cases, remained stable or decreased in size in 2 cases. Notably, none of these patients showed evidence of a hepatic recurrence. FHVHPT, a tumefactive lesion that mimics hepatic neoplasia, is histologically characterized by a fibrohistiocytic infiltrate. In the majority of patients FHVHPT represents the organizing phase of hepatic abscess and can be successfully managed with antibiotic therapy.

Recreational Cannabis Use and Risk of Prescription Opioid Overdose: Insights from Pediatric Inpatients.

Objectives Our first goal is to evaluate the prevalence of hospital admissions for prescription opioid overdose (POD) in pediatric inpatients, and next goal is to measure the independent association between cannabis use disorders (CUD) and POD. Methods We used the nationwide inpatient sample (NIS) and included 27,444,239 pediatric inpatients (age ≤ 18 years), and 10,562 (0.04%) were managed primarily for POD. The odds ratio (OR) of the association of variables in POD inpatients was measured using the binomial logistic regression model that was adjusted for demographic confounders and psychiatric comorbidities. Results Adolescents have higher odds (OR 10.75, 95% CI 10.16-11.36) of POD-related hospitalization compared to children ≤ 12 years. Whites formed the significant proportion (67%), and those from low-income families (<50th percentile) had higher likelihood for POD-related hospitalization. The most prevalent psychiatric comorbidities were mood disorders (44.3%) and anxiety disorders (14.6%). Prevalent comorbid substance use disorders (SUDs) included cannabis (14.2%), tobacco (13.1%), and opioid (9.4%). A higher odds of association with POD-related hospitalizations were seen in pediatric inpatients with comorbid opioid (OR 8.79, 95% CI 8.08-9.56), tobacco (OR 1.58, 95% CI 1.47-1.70), and cannabis (OR 1.68, 95% CI 1.57-1.81) use disorders. Conclusion The prescription opioid is a bridge to opioid abuse/dependence, thereby increasing the risk of other SUDs like tobacco (by 58%) and cannabis (by 68%). Regulating the easy availability of prescription opioids and also improving the existing prescription trends are an essential way to reduce this problem. Finally, awareness and counseling are recommended strategies for harm reduction/rehabilitation among the pediatric population.

Conduct Disorder-Related Hospitalization and Substance Use Disorders in American Teens.

OBJECTIVE: Our study aimed to compare the demographic characteristics of conduct disorder (CD) inpatients versus other psychiatric inpatients in children and adolescents, and assess the association between conduct disorder patients and the spectrum of substance use disorders (SUD). METHODS: We included 800,614 psychiatric adolescent (12-18 years) inpatients, and this included 8885 inpatients (1.1%) primarily for conduct disorder in the Nationwide Inpatient Sample (2010-2014). ICD-9 codes were used to detect SUD, and a logistic regression model was used to evaluate the odds ratio (OR) for SUD in conduct disorder inpatients. RESULTS: A higher proportion of conduct disorder inpatients were of 12-15 years of age (62.6%), male (64.4%), and White (45.7%). The lower median household income was correlated with a higher prevalence of conduct disorder (36.4%). Among SUD, cannabis use (23.7%) was most prevalent in conduct disorder inpatients followed by tobacco and alcohol use (10.1% each). Conduct disorder inpatients have 1.7-fold higher odds (95% confidence interval (CI) 1.52-1.82) for alcohol use and 1.4-fold higher odds (95% CI 1.31-1.49) for cannabis use compared to the non-conduct disorder inpatients. Cannabis use was seen significantly in adolescents (49.1%, 12-15 years), male (75.6%), and African Americans (45.6%). CONCLUSION: Conduct disorder inpatients have a higher risk of comorbid SUD compared to other psychiatric illnesses. The most common substance to be abused is cannabis followed by tobacco and alcohol. Varying pattern of substance use was seen by demographics and these predictors may help the clinicians for early diagnosis and treatment to improve overall health-related quality of life.