Novel ALK-specific mRNA in situ hybridization assay for non-small-cell lung carcinoma.

BACKGROUND: A recent technical advance in mRNA in situ hybridization (mRNA-ISH) assays provides simultaneous signal amplification and background suppression with a unique probe design that enables single-molecule visualization. We assessed the utility of the mRNA-ISH assay as a diagnostic tool for detecting anaplastic lymphoma receptor tyrosine kinase (ALK) mRNA in non-small-cell lung carcinoma (NSCLC). We compared the mRNA-ISH assay with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). METHODS: The study included 279 surgically resected lung adenocarcinomas and 44 transbronchial-biopsied (TBB) adenocarcinomas. mRNA-ISH was conducted using the RNAscope 2.0 system, which includes pre-designed probes for detecting the tyrosine kinase domain encoded in ALK mRNA. IHC was conducted on all 323 samples using ALK-specific antibodies. mRNA-ISH was performed on 279 surgical samples and 6 TBB samples. Break-apart FISH was used to examine samples that were mRNA-ISH-positive or IHC-positive. RESULTS: ALK protein expression was detected in 11 of 279 specimens (3.9%). ALK mRNA was also detected with mRNA-ISH in ALK-positive samples, and 9 of the 11 specimens (81%) were also positive for ALK using break-apart FISH. Using the IHC results as a reference, the sensitivity and specificity of mRNA-ISH was 100%. In the TBB cohort, ALK protein expression was observed in 3 of 44 specimens (6.8%), in which ALK mRNA expression was also detected. CONCLUSIONS: The ALK mRNA-ISH data were highly correlated with the IHC data, and ALK mRNA-ISH detected ALK mRNA expression in every FISH-positive sample. We conclude that mRNA-ISH could serve as an alternative or complementary method for diagnosing ALK rearrangements in NSCLC.

Activation of Src signaling mediates acquired resistance to ALK inhibition in lung cancer.

Anaplastic lymphoma kinase (ALK) fusion oncogenes occur in approximately 3-5% of non-small cell lung cancer (NSCLC) cases. Various ALK inhibitors are in clinical use for the treatment of ALK-NSCLC, including the first generation ALK inhibitor, crizotinib, and recently the more highly potent alectinib and ceritinib. However, most tumors eventually become resistant to ALK specific inhibitors. To address the mechanisms underlying the development of ALK inhibitor resistance, we used iTRAQ quantitative mass spectrometry and phosphor-receptor tyrosine kinase arrays to investigate intracellular signaling alterations in ALK inhibitor resistant NSCLC cell lines. Src signaling was identified as an alectinib resistance mechanism, and combination treatment with ALK and Src inhibitors was highly effective for inhibiting the growth of ALK inhibitor resistant cells in vitro and in mouse xenograft models. Furthermore, phospho-receptor tyrosine kinase activation and downstream PI3K/AKT signaling was effectively blocked by inhibiting Src in alectinib resistant cells. Finally, we showed that the combined use of ALK and Src inhibitors inhibited the growth of other ALK-NSCLC cell lines, including those that were ceritinib or lorlatinib resistant. Our data suggest that targeting Src signaling may be an effective approach to the treatment of ALK-NSCLC with acquired resistance to ALK inhibitors.

Programmed death-ligand 1 and its soluble form are highly expressed in nasal natural killer/T-cell lymphoma: a potential rationale for immunotherapy.

Nasal natural killer/T-cell lymphoma (NNKTL) is an aggressive neoplasm with poor therapeutic responses and prognosis. The programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway plays an important role in immune evasion of tumor cells through T-cell exhaustion. The aim of the present study was to examine the expression of PD-L1 and PD-1 molecules in NNKTL. We detected the expression of PD-L1 in biopsy samples from all of the NNKTL patients studied. PD-L1 was found on both malignant cells and tumor-infiltrating macrophages, while PD-1-positive mononuclear cells infiltrated the tumor tissues in 36% of patients. Most significantly, soluble PD-L1 (sPD-L1) was present in sera of NNKTL patients at higher levels as compared to healthy individuals and the levels of serum sPD-L1 in patients positively correlated with the expression of PD-L1 in lymphoma cells of tumor tissues. In addition, the high-sPD-L1 group of patients showed significantly worse prognosis than the low-sPD-L1 group. Furthermore, we confirmed that membrane and soluble PD-L1 was expressed on the surface and in the culture supernatant, respectively, of NNKTL cell lines. The expression of PD-L1 was observed in tumor tissues and sera from a murine xenograft model inoculated with an NNKTL cell line. Our results suggest that sPD-L1 could be a prognostic predictor for NNKTL and open up the possibility of immunotherapy of this lymphoma using PD-1/PD-L1 axis inhibitors.

Epigenetic modification augments the immunogenicity of human leukocyte antigen G serving as a tumor antigen for T cell-based immunotherapy.

Tumor immune escape has been a major problem for developing effective immunotherapy. The human leukocyte antigen G (HLA-G) is a non-classical MHC class I molecule whose primary function is to protect the fetus from the mother's immune system. While HLA-G is hardly found in normal adult tissues, various tumor cells are known to express it, aiding their escape from the immune system. Thus, HLA-G is an attractive immunotherapy target. CD4(+) helper T lymphocytes (HTLs) play an important role in the immune reaction against tumors by assisting in the generation and persistence of CD8(+) cytotoxic T lymphocytes (CTLs) or by displaying direct antitumor effects. We report here that HLA-G expression in breast cancer significantly correlates with a poor prognosis. Also, we describe that the MHC class II-binding peptide HLA-G26-40 was effective in eliciting tumor-reactive CD4(+) T cell responses. Furthermore, treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased HLA-G expression in tumors and subsequently enhanced recognition by HLA-G26-40-specific HTLs. These findings predict that a combination immunotherapy targeting HLA-G together with a DNA methyltransferase inhibitor could be useful against some cancers.