Immunotherapy resistance in non–small cell lung cancer (NSCLC) may be mediated by an immunosuppressive microenvironment, which can be shaped by the mutational landscape of the tumor. Here, we observed genetic alterations in the PTEN/PI3K/AKT/mTOR pathway and/or loss of PTEN expression in >25% of patients with NSCLC, with higher frequency in lung squamous carcinomas (LUSC). Patients with PTEN-low tumors had higher levels of PD-L1 and PD-L2 and showed worse progression-free survival when treated with immunotherapy. Development of a Pten-null LUSC mouse model revealed that tumors with PTEN loss were refractory to antiprogrammed cell death protein 1 (anti–PD-1), highly metastatic and fibrotic, and secreted TGFβ/CXCL10 to promote conversion of CD4+ lymphocytes into regulatory T cells (Treg). Human and mouse PTEN-low tumors were enriched in Tregs and expressed higher levels of immunosuppressive genes. Importantly, treatment of mice bearing Pten-null tumors with TLR agonists and anti-TGFβ antibody aimed to alter this immunosuppressive microenvironment and led to tumor rejection and immunologic memory in 100% of mice. These results demonstrate that lack of PTEN causes immunotherapy resistance in LUSCs by establishing an immunosuppressive tumor microenvironment that can be reversed therapeutically. Significance: PTEN loss leads to the development of an immunosuppressive microenvironment in lung cancer that confers resistance to anti–PD-1 therapy, which can be overcome by targeting PTEN loss–mediated immunosuppression.

Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312.

Early alterations in cancer include the deregulation of epigenetic events such as changes in DNA methylation and abnormal levels of non-coding (nc)RNAs. Although these changes can be identified in tumors, alternative sources of samples may offer advantages over tissue biopsies. Because tumors shed DNA, RNA, and proteins, biological fluids containing these molecules can accurately reflect alterations found in cancer cells, not only coming from the primary tumor, but also from metastasis and from the tumor microenvironment (TME). Depending on the type of cancer, biological fluids encompass blood, urine, cerebrospinal fluid, and saliva, among others. Such samples are named with the general term “liquid biopsy” (LB). With the advent of ultrasensitive technologies during the last decade, the identification of actionable genetic alterations (i.e., mutations) in LB is a common practice to decide whether or not targeted therapy should be applied. Likewise, the analysis of global or specific epigenetic alterations may also be important as biomarkers for diagnosis, prognosis, and even for cancer drug response. Several commercial kits that assess the DNA promoter methylation of single genes or gene sets are available, with some of them being tested as biomarkers for diagnosis in clinical trials. From the tumors with highest incidence, we can stress the relevance of DNA methylation changes in the following genes found in LB: SHOX2 (for lung cancer); RASSF1A, RARB2, and GSTP1 (for lung, breast, genitourinary and colon cancers); and SEPT9 (for colon cancer). Moreover, multi-cancer high-throughput methylation-based tests are now commercially available. Increased levels of the microRNA miR21 and several miRNA- and long ncRNA-signatures can also be indicative biomarkers in LB. Therefore, epigenetic biomarkers are attractive and may have a clinical value in cancer. Nonetheless, validation, standardization, and demonstration of an added value over the common clinical practice are issues needed to be addressed in the transfer of this knowledge from “bench to bedside”.

The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm.