Probes for INS

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.

Gastric cancer is the fifth most common cancer and third leading cause of cancer-related death worldwide. Several studies on angiogenic blocking agents in gastric cancer revealing promising results by the use of monoclonal antibodies against VEGFA or its receptor VEGFR2 or against VEGFA activating pathway. The validation of biomarkers useful to better organize the clinical trials involving anti-angiogenic therapies is crucial. Molecular markers such as RNA are increasingly used for cancer diagnosis, prognosis, and therapy guidance as in the case of the targeted therapies concerning the inhibition of angiogenesis. The aim of this study is to set the conditions for evaluating the expression of VEGFA and VEGFR2 in gastric cancer specimens and in healthy gastric mucosa by the use of RNAscope, a novel RNA in situ hybridization (ISH) method that allows the visualization of a specific gene expression in individual cells. We found the increased expression of VEGFA in the tubular glands and VEGFR2 in the endothelium of gastric cancer samples mainly in the T2, T3 and T4 stages of tumor progression as compared to the healthy controls. These results obtained by the application of this highly sensitive method for oligonucleotide detection the role of angiogenesis in gastric cancer progression already highlighted by conventional immunohistochemical methods, and offer significant promise as a new platform for developing and implementing RNA-based molecular diagnostics also in the conditions in which immunohistochemistry is not applicable.

Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3-4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.

Angiostasis mediated by IFNγ is a key mechanism of anti-tumor immunity; however, the effect of IFNγ on host VEGFA-expressing cells during tumor progression is still elusive. Here, we developed transgenic mice with IFNγ receptor (IFNγR) expression under control of the Vegfa promoter (V-γR). In these mice, the IFNγ responsiveness of VEGFA -expressing cells led to a dramatic growth suppression of transplanted lung carcinoma cells. Surprisingly, increased mortality and tumor metastasis were observed in the tumor-bearing V-γR mice, in comparison to the control wild type and IFNγR-deficient mice. Further study showed that perivascular cells were VEGFA-expressing cells and potential IFNγ targets. In vivo, tumor vascular perfusion and pericyte association with blood vessels were massively disrupted in V-γR mice. In vitro, IFNγ inhibited TGF-β-signaling through upregulating SMAD7 and therefore, down-regulated N-cadherin expression in pericytes. Importantly, IFNγ neutralization in vivo using a monoclonal antibody reduced tumor metastasis. Together, the results suggest that IFNγR-mediated dissociation of perivascular cells from blood vessels contributes to the acceleration of tumor metastasis. Thus the inhibition of tumor growth via IFNγ-induced angiostasis might also accelerate tumor metastasis.

Primary mediastinal large B-cell lymphoma (PMLBCL) is recognized as a distinct clinicopathological entity in the current World Health Organization classification of lymphoid neoplasms (Swerdlow et al, 2016). Gene expression profiling studies have confirmed a distinct signature in PMLBCL that differs from diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) and partially overlaps with that found in classical Hodgkin lymphoma (Savage et al, 2003; Bea et al, 2005). In routine clinical practice, however, distinguishing between PMLBCL and DLBCL, NOS is frequently difficult, due partly to a paucity of sensitive and specific biomarkers (Martelli et al, 2008; Dorfman et al, 2012). Recent studies have shown that PMLBCL shows frequent copy number alterations or translocations involving the CD274 (PD-L1) or PDCD1LG2 (PD-L2) genes at chromosome 9p24.1, leading to overexpression of CD274 (PD-L1) and, especially, PDCD1LG (PD-L2) proteins (Shi et al, 2014; Twa & Steidl, 2015). Anti-PDCD1LG2 antibodies suitable for immunohistochemical analysis in formalin-fixed paraffin-embedded (FFPE) tissue are not currently commercially available, limiting the utility of this potential marker for routine diagnostic practice. In this study, we have performed RNA in situ hybridization (RISH) for CD274 and PDCD1LG2 RNA expression, using a standard automated immunohistochemistry (IHC) platform, and have compared the results to IHC using a commercially available anti-CD274 antibody.

Four immunohistochemistry (IHC) diagnostic assays have been approved for tumour PD-L1 protein assessment in the clinic. However, mRNA detection by in situ hybridisation (ISH) could be utilised as an alternative to protein detection. Detecting spatial changes in gene expression provides vital prognostic and diagnostic information, particularly in immune oncology where the phenotype, cellular infiltration and immune activity status may be associated with patient survival. Translation of mRNA expression to a clinically relevant cut off or threshold is challenging due to variability between assays and the detection of different analytes. These studies aim to confirm the suitability of formalin fixed paraffin embedded (FFPE) tissue sections for use with RNA ISH. A comparison of mRNA expression and protein expression may inform the suitability of mRNA as a patient selection biomarker in a similar manner to IHC and provide evidence of a suitable scoring algorithm. Ninety patient samples, thirty for each indication of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC), previously assessed using the VENTANA PD-L1 (SP263) Assay were chosen to represent a wide dynamic range of percentage tumour cell staining (TCIHC). Expression of mRNA was assessed by ISH using the RNAScope 2.5 assay and probe CD274/PD-L1 (Advanced Cell Diagnostics) including kit provided positive and negative control probes. Brightfield whole slide images of tissues were captured. The percentage of tumour cells with PD-L1 mRNA expression (%TCmRNA) and mean punctate dots/tumour cell were determined using image analysis. Differences in RNA expression between the IHC derived TCIHC≥25% and <25% groups were assessed using t-tests. For each indication, a receiver-operating characteristic (ROC) analysis identified thresholds for patient classification using %TCmRNA and dots/tumour cell, with reference to TCIHC≥25%. Eighty-six samples were successfully tested; 3 failed due to insufficient control probe staining, 1 due to lack of tumour. Percent TCmRNA staining using RNAScope demonstrated statistical significance (at α = 0.05) in the PD-L1 high (TCIHC ≥25%) vs the PD-L1 low (TCIHC <25%) groups for NSCLC, HNSCC, and UC. The number of punctate dots/tumour cell was significantly higher in the PD-L1 high vs the PD-L1 low groups for NSCLC and HNSCC but not UC. For %TCmRNA; ROC analysis identified thresholds of: NSCLC 18.0%, HNSCC 31.8%, UC 25.8%. For dots/tumour cell, thresholds were: NSCLC 0.26, HNSCC 0.53, UC 0.45. Routine tissue fixation and processing is suitable for RNA detection using RNAScope. PD-L1 mRNA extent and level is associated with PD-L1 status determined by IHC. Threshold optimisation for %TCmRNA and mean dots/tumour cell results in high specificity to IHC PD-L1 classification, but only moderate sensitivity.

Medulloblastomas comprise a heterogeneous group of tumours and can be subdivided into four molecular subgroups (WNT, SHH, Group 3 and Group 4) with distinct prognosis, biological behaviour and implications for targeted therapies. Few experimental models exist of the aggressive and poorly characterized Group 3 tumours. In order to establish a reproducible transplantable Group 3 medulloblastoma model for preclinical therapeutic studies, we acquired a patient-derived tumour sphere culture and inoculated low-passage spheres into the cerebellums of NOD-scid mice. Mice developed symptoms of brain tumours with a latency of 17-18 weeks. Neurosphere cultures were re-established and serially transplanted for 3 generations, with a negative correlation between tumour latency and numbers of injected cells. Xenografts replicated the phenotype of the primary tumour, including high degree of clustering in DNA methylation analysis, high proliferation, expression of tumour markers, MYC amplification and elevated MYC expression, and sensitivity to the MYC inhibitor JQ1. Xenografts maintained maintained expression of tumour-derived VEGFA and stromal-derived COX-2. VEGFA, COX-2 and c-Myc are highly expressed in Group 3 compared to other medulloblastoma subgroups, suggesting that these molecules are relevant therapeutic targets in Group 3medulloblastoma.