Probes for INS

Abstract

BACKGROUND:

Craniopharyngiomas are neoplasms of the sellar/parasellar region that are classified into adamantinomatous (ACP) and papillary (PCP) subtypes. Surgical resection of craniopharyngiomas is challenging, and recurrence is common, frequently leading to profound morbidity. BRAF V600E mutations render PCP susceptible to BRAF/MEK inhibitors, but effective targeted therapies are needed for ACP. We explored the feasibility of targeting the PD-1/PD-L1 immune checkpoint pathway in ACP and PCP.

METHODS:

We mapped and quantified PD-L1 and PD-1 expression in ACP and PCP resections using immunohistochemistry, immunofluorescence, and RNA in situ hybridization. We used tissue-based cyclic immunofluorescence (t-CyCIF) to map the spatial distribution of immune cells and characterize cell cycle and signaling pathways in ACP tumor cells which intrinsically express PD-1.

RESULTS:

All ACP (15±14% of cells, n=23, average±S.D.) and PCP (35±22% of cells, n=18) resections expressed PD-L1. In ACP, PD-L1 was predominantly expressed by tumor cells comprising the cyst-lining. In PCP, PD-L1 was highly-expressed by tumor cells surrounding the stromal fibrovascular cores. ACP also exhibited tumor cell-intrinsic PD-1 expression in whorled epithelial cells with nuclear-localized beta-catenin. These cells exhibited evidence of elevated mTOR and MAPK signaling. Profiling of immune populations in ACP and PCP showed a modest density of CD8+ T-cells.

CONCLUSIONS:

ACP exhibit PD-L1 expression in the tumor cyst-lining and intrinsic PD-1 expression in cells proposed to comprise an oncogenic stem-like population. In PCP, proliferative tumor cells express PD-L1 in a continuous band at the stromal-epithelial interface. Targeting PD-L1 and/or PD-1 in both subtypes of craniopharyngioma might therefore be an effective therapeutic strategy.

Abstract

BACKGROUND & AIMS:

The present study aimed to elucidate the clinicopathological significance of IL-6 and IL-33 expression in intrahepatic cholangiocarcinomas (iCCAs) and perihilar cholangiocarcinomas (pCCAs).

METHODS:

IL-6 and IL-33 mRNA expression was examined in iCCAs (n=55) and pCCAs (n=32) using quantitative real-time PCR and a highly sensitive in situ hybridization protocol (RNAscope™ ), and expression values were correlated with clinicopathological features. According to a recently proposed classification scheme, iCCAs were separated into small- (n=33) and large-duct types (n=22).

RESULTS:

IL-6 and IL-33 expression levels were higher in large-duct iCCAs and pCCAs than in small-duct iCCAs, with a positive correlation between the values of these cytokines. In double in situ hybridization/immunostaining, IL-6 mRNA was expressed in actin-positive (myo)fibroblasts, while IL-33 was mainly produced by CD31-positive endothelial cells. Based on the average expression value as a cut-off point, cases were classified as IL-6high and IL-6low or IL-33high and IL-33low . In the combined cohort of large-duct iCCAs and pCCAs, IL-6high and IL-6low cholangiocarcinomas shared many features, while IL-33high cases had less aggressive characteristics than IL-33low cases as evidenced by lower tumour marker concentrations, smaller tumour sizes, less common vascular invasion, lower pT stages, and higher lymphocyte-to-monocyte ratios in blood. KRAS mutations were slightly less common in IL-33high cases than in IL-33low cancers (9% vs 29%; p=0.061). The strong expression of IL-33 in tissue appeared to be an independent favourable prognostic factor.

CONCLUSIONS:

IL-33high cholangiocarcinomas may represent a unique, less aggressive carcinogenetic process of the large bile ducts.

In this study, we present the clinicopathological features associated with PD-L1 protein and mRNA expression in a large Asian cohort of patients with non-small cell lung cancer (NSCLC) and assessed the prognostic implications of PD-L1 expression, particularly in early stage NSCLC. We retrospectively analyzed 687 NSCLC specimens (476 adenocarcinoma and 211 squamous cell carcinoma) using tissue microarray. PD-L1 immunohistochemistry (IHC) was performed using Dako 22C3 pharmDx assay and PDL1 mRNA was measured using RNA in situ hybridization (RISH). The overall prevalence of PD-L1 protein expression was 25.2% in tumor cells and PDL1 mRNA expression was 11.9%. There was a strong positive correlation between PD-L1 IHC and RISH results (Spearman's rho = 0.6, p<0.001). In adenocarcinoma, PD-L1 protein and mRNA expressions significantly correlated with poorly differentiated histologic subtype (p<0.001 and p = 0.002, respectively). PD-L1 expression was also associated with genetic alteration in adenocarcinoma. High PD-L1 expression level was associated with EGFR-naïve and KRAS-mutant subgroup (p = 0.001 and p = 0.017, respectively). With a 1% cut-off value, PD-L1 protein expression showed a short overall survival duration in early stage adenocarcinoma with marginal significance (p = 0.05, Hazard ratio = 1.947). Our study revealed that PD-L1 expression varied with histologic subtype and genomic alteration status in lung adenocarcinoma, and activation of the PD-L1 pathway may be a poor prognostic factor especially in early stage lung adenocarcinoma. In addition, PDL1 RISH showed promising results in predicting PD-L1 protein expression in NSCLC.

Abstract

BACKGROUND:
In celiac disease (CD), intestinal epithelium damage occurs secondary to an immune insult and is characterized by blunting of the villi and crypt hyperplasia. Similarities between Hedgehog (Hh)/BMP4 downregulation, as reported in a mouse model, and CD histopathology, suggest mechanistic involvement of Hh/BMP4/WNT pathways in proliferation and differentiation of immature epithelial cells in the context of human intestinal homeostasis and regeneration after damage. Herein we examined the nature of intestinal crypt hyperplasia and involvement of Hh/BMP4 in CD histopathology.

METHODS AND FINDINGS:
Immunohistochemistry, qPCR and in situ hybridization were used to study a cohort of 24 healthy controls (HC) and 24 patients with diagnosed acute celiac disease (A-CD) intestinal biopsies. In A-CD we observed an increase in cells positive for Leucin-rich repeat-containing G protein-coupled receptor 5 (LGR5), an epithelial stem cell specific marker and expansion of WNT responding compartment. Further, we observed alteration in number and distribution of mesenchymal cells, predicted to be part of the intestinal stem cells niche. At the molecular level we found downregulation of indian hedgehog (IHH) and other components of the Hh pathway, but we did not observe a concurrent downregulation of BMP4. However, we observed upregulation of BMPs antagonists, gremlin 1 and gremlin 2.

CONCLUSIONS:
Our data suggest that acute CD histopathology partially recapitulates the phenotype reported in Hh knockdown models. Specifically, Hh/BMP4 paradigm appears to be decoupled in CD, as the expansion of the immature cell population does not occur consequent to downregulation of BMP4. Instead, we provide evidence that upregulation of BMP antagonists play a key role in intestinal crypt hyperplasia. This study sheds light on the molecular mechanisms underlying CD histopathology and the limitations in the use of mouse models for celiac disease.

Amplifications at 9p24 have been identified in breast cancer and other malignancies, but the genes within this locus causally associated with oncogenicity or tumor progression remain unclear. Targeted next-generation sequencing of postchemotherapy triple-negative breast cancers (TNBCs) identified a group of 9p24-amplified tumors, which contained focal amplification of the Janus kinase 2 (JAK2) gene. These patients had markedly inferior recurrence-free and overall survival compared to patients with TNBC withoutJAK2amplification. Detection ofJAK2/9p24 amplifications was more common in chemotherapy-treated TNBCs than in untreated TNBCs or basal-like cancers, or in other breast cancer subtypes. Similar rates ofJAK2amplification were confirmed in patient-derived TNBC xenografts. In patients for whom longitudinal specimens were available,JAK2amplification was selected for during neoadjuvant chemotherapy and eventual metastatic spread, suggesting a role in tumorigenicity and chemoresistance, phenotypes often attributed to a cancer stem cell-like cell population. In TNBC cell lines withJAK2copy gains or amplification, specific inhibition of JAK2 signaling reduced mammosphere formation and cooperated with chemotherapy in reducing tumor growth in vivo. In these cells, inhibition of JAK1-signal transducer and activator of transcription 3 (STAT3) signaling had little effect or, in some cases, counteracted JAK2-specific inhibition. Collectively, these results suggest that JAK2-specific inhibitors are more efficacious than dual JAK1/2 inhibitors against JAK2-amplified TNBCs. Furthermore,JAK2amplification is a potential biomarker for JAK2 dependence, which, in turn, can be used to select patients for clinical trials with JAK2 inhibitors.

Overexpression of mutant p53 is a common finding in most cancers but testicular tumours accumulate wild-type p53 (wtp53). In contrast to the accepted concept that p53 homozygous mutant mice do not accumulate mutant p53 in normal cells, our study on a mutant p53 mouse model of Li-Fraumeni syndrome harbouring the hot-spot p53R172H mutation described an elevated level of mutant p53 in non-cancerous mouse tissues. Here we use detailed immunohistochemical analysis to document the expression of p53R172H in mouse testis. In developing and adult testes, p53R172H was expressed in gonocytes, type A, Int, B spermatogonia as well as in pre-Sertoli cells and Leydig cells but was undetectable in spermatocytes and spermatids. A similar staining pattern was demonstrated for wtp53. However, the intensity of wtp53 staining was generally weaker than that of p53R172H, which indicates that the expression of p53R172H can be a surrogate marker of p53 gene transcription. Comparing the responses of wtp53 and p53R172H to irradiation, we found persistent DNA double-strand breaks in p53R172H testes and the formation of giant spermatogonia (GSG) following persistent DNA damage in p53R172H and p53-null mice. Strikingly, we found that p53R172H promotes spontaneous formation of GSG in non-stressed p53R172H ageing mice. Two types of GSG: Viable and Degenerative GSG were defined. We elucidate the factors involved in the formation of GSG: the loss of p53 function is a requirement for the formation of GSG whereas DNA damage acts as a promoting trigger. The formation of GSG does not translate to higher efficacy of testicular tumorigenesis arising from mutant p53 cells, which might be due to the presence of delayed-onset of p53-independent apoptosis.

Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11-13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.