ROS Proto-oncogene Research Articles

Case Series: EGFR and ROS-1 Co-Occurrence in Advanced Non–Small Cell Lung Cancer

ABSTRACT Non–small cell lung cancer (NSCLC) is a heterogeneous disease with diverse molecular alterations. Two of the most common genetic abnormalities found in advanced NSCLC are mutations in the epidermal growth factor receptor (EGFR) and rearrangements in the ROS proto-oncogene 1 (ROS-1). Although these two alterations are typically mutually exclusive, there have been reports of their co-occurrence in a small subset of NSCLC patients. The discovery of this comutation has recently become apparent due to the increased use of more sensitive whole genome sequencing. We share our experience with two cases of coexisting EGFR and ROS-1 alterations. The first case is a 60-year-old man diagnosed with advanced adenocarcinoma of the lung with metastasis to bone and left adrenal gland. The second case is a 49-year-old woman diagnosed with stage IV lung adenocarcinoma with metastasis to the contralateral lung and diffuse abdominal lymphadenopathy. The first case was treated with osimertinib, and currently has had a stable disease on this medication for more than 3 years. The second case had a short interval of stable disease on osimertinib; then she developed progressive disease with poor response to anti–ROS-1 therapy. We believe patients with advanced NSCLC may have a higher incidence of coalterations, especially in the areas of the world with higher EGFR mutations and in the era of higher usage of whole genome sequencing. The presence of comutations will allow for a good long-term response to anti-EGFR therapy. This highlights the importance of the use of next-generation sequencing whenever possible and considers variant allele frequency as a factor in directing the therapy. There are many other unanswered questions, such as the best treatment sequencing or even the combined targeted therapy approach. This case series may add some information to the current literature.

Novel therapies for pediatric low grade glioma.

Current biological findings provide new insights into the genetics driving growth of low-grade gliomas in pediatric patients. This has provided new targets for novel therapies. The purpose of this paper is to review novel therapies for pediatric low-grade gliomas that have been published in the past 24 months. Low-grade gliomas are often driven by mitogen activated protein kinase (MAPK) alterations either with BRAF V600E point mutations or BRAF fusions. Current advances have also highlighted novel fusions of fibroblast growth factor receptor (FGFR), myeloblastosis family of transcription factors (MYB), meningioma 1 tumor suppressor (MN1), neurotrophic receptor kinase family of receptors (NTRK), Kristen RAS (Rat Sarcoma Virus) oncogene homolog in mammals (KRAS), Receptor tyrosine kinase ROS proto oncogene 1 (ROS1), protein kinase C alpha (PRKCA), and platelet derive growth factor receptor (PDGFR) amplification. Novel therapies have been employed and are showing encouraging results in pediatric low-grade gliomas. Current trials are underway with newer generation pan RAF inhibitors and mitogen activated protein kinase - kinase (MEK) inhibitors. Other early phase clinical trials have provided safety data in pediatric patients targeting FGFR fusion, NTRK fusion, PDGFR amplification and ROS1 mutations. Historical treatment options in pediatric low-grade gliomas have utilized surgery, radiation therapy and conventional chemotherapy. Recently greater insight into their biology has found that alterations in MAPK driven pathways are often the hallmark of tumorigenesis. Targeting these novel pathways has led to tumor control and shrinkage without the use of conventional chemotherapy. Caution should be taken however, since these treatment options are still novel, and we do not fully appreciate the long-term effects. Nonetheless a new era of targeted medicine is here.

Clinical Utility of Circulating Tumor DNA for Detecting Lung Cancer Mutations by Targeted Next-Generation Sequencing With Insufficient Tumor Samples.

Circulating tumor deoxyribonucleic acid (ctDNA) is increasingly applied in clinical practice. This study aimed to explore clinical utility of a minimal invasive and sensitive way of ctDNA for next-generation sequencing in non-small cell lung cancer (NSCLC) with inadequate tumor samples. Targeted DNA sequencing was performed on tissue biopsies and matched plasma samples from 60 patients with NSCLC. A total of 13 driving genes were detected in 60 matched tissue DNA (tDNA) and ctDNA samples. Overall concordance rate was 75.47%, with 77.55% sensitivity and 50% specificity. Epidermal growth factor receptor (EGFR) mutations were the most common in both tDNA and ctDNA samples. Among other mutated genes were tumor protein p53 (TP53), erb-b2 receptor tyrosine kinase 2 (ERBB2), anaplastic lymphoma kinase (ALK), cyclin-dependent kinase inhibitor 2A (CDKN2A), ros proto-oncogene 1, and receptor tyrosine kinase (ROS1). Mutations in b-raf proto-oncogene, serine/threonine kinase (BRAF), cluster of differentiation 274 (CD274), neurotrophin receptor tyrosine kinase 1 (NTRK1), and rearranged during transfection (RET) occurred only in plasma. The majority of mutations in both samples were single-nucleotide variants. Deletions were found in EGFR, BRAF, and TP53 in ctDNA, whereas in tDNA, deletions were only found in EGFR. In ALK, single nucleic acid-site amplification occurred simultaneously in tissue and plasma, but insertions and copy number variations were detected only in plasma. Identifying ctDNA mutations by targeted sequencing in plasma is feasible, showing the clinical value of ctDNA-targeted sequencing in NSCLC patients when tumor tissue sampling is insufficient or even impossible.

Association of PD-L1 expression and clinical outcomes in ROS1 - rearranged advanced non-small cell lung cancer treated with crizotinib.

Programmed cell death ligand 1 (PD-L1) is more readily expressed in ROS proto-oncogene 1 (ROS1) rearranged non-small cell lung cancer (NSCLC) compared to NSCLC cases lacking driver gene mutations. Prior research has established a link between PD-L1 expression and reduced effectiveness of EGFR or ALK inhibitors in EGFR or ALK-positive NSCLC. Nonetheless, the relationship between initial PD-L1 levels and the clinical impact of first-line crizotinib therapy in ROS1-rearranged NSCLC is still uncertain. From January 2016 to December 2021, a total of 246 patients with ROS1 positive tumors were collected. Out of these, 82 patients with advanced ROS1-rearranged NSCLC, who were treated with crizotinib as their initial therapy, were selected for the study. The study aimed primarily to evaluate the objective response rate (ORR) and progression-free survival (PFS), and secondarily to assess disease control rate (DCR) and overall survival (OS). Of the 82 advanced ROS1-rearranged NSCLC patients, 38 exhibited PD-L1 positivity, subdivided into 11 with high and 27 with low expression levels, while the remaining 44 showed no PD-L1 expression. The ORR for all included patients was 80.5%. No statistically significant variance in ORR was observed among ROS1-rearranged NSCLC patients across differing PD-L1 expression statuses. However, there was a statistically significant difference in DCR between PD-L1 negative group (100%) and high expression group (90.9%) (p=0.04). The median PFS spanned 26.4 months for the PD-L1 negative group, 16.6 for the low expression group, and 13.7 for the high expression group (p=0.001). Additionally, a notable statistical disparity was also observed in median PFS between the PD-L1 negative and positive groups (p=0.02). For the entire study population, the median OS was 53.0 months (95% CI 43.8 - 62.2). In the PD-L1-negative group, the median OS reached 57.2 months, compared to 53.0 months in the PD-L1-positive group, a difference lacking statistical significance (p=0.43). Our results suggest that for ROS1-positive NSCLC patients receiving crizotinib as first-line therapy, PD-L1 expression may serve as a negative prognostic marker for PFS rather than OS.

Efficacy and safety of crizotinib in the treatment of advanced non-small cell lung cancer with ROS1 gene fusion: a systematic literature review and meta-analysis of real-world evidence

BackgroundCrizotinib was approved to treat patients with advanced non-small cell lung cancer (aNSCLC) with ROS proto-oncogene 1 (ROS1) gene fusion in 2016. We conducted a systematic literature review to identify real-world evidence (RWE) studies and estimated the efficacy and safety of crizotinib using meta–analyses (MA) for objective response rate (ORR), real-world progression-free survival (PFS), and overall survival (OS). MethodsWe searched MEDLINE®, Embase, and Cochrane CENTRAL from January 2016 to March 2023 using Ovid® for published single-arm or comparative RWE studies evaluating patients (N ≥ 20) receiving crizotinib monotherapy for aNSCLC with ROS1 gene fusion. Pooled estimates for ORR and grade 3/4 adverse events (AEs) were derived using the metafor package in R while pooled estimates for median real-world PFS (rwPFS) and OS were derived using reconstructed individual patient data from published Kaplan-Meier curves. The primary analysis included all studies regardless of crizotinib line of therapy; a subgroup analysis (SA) was conducted using studies evaluating patients receiving first-line crizotinib. ResultsFourteen studies met the eligibility criteria and were considered feasible for MA. For the primary analysis, the pooled ORR (N = 9 studies) was 70.6 % (95 % confidence interval [CI]: 57.0, 81.3), median rwPFS was 14.5 months (N = 11 studies), and OS was 40.2 months (N = 9 studies). In the SA, the pooled ORR (N = 4 studies) was 81.1 % (95 % CI: 76.1, 85.2) and the median rwPFS (N = 4 studies) and OS (N = 2 studies) were 18.1 and 60 months, respectively. All MAs were associated with significant heterogeneity (I2 > 25 %). Grade 3/4 AEs occurred in 18.7 % of patients (pooled estimate). ConclusionThe results from this study are consistent with clinical trial data and, taken collectively, supports crizotinib as a safe and effective treatment across different lines of therapy in patients with ROS1 aNSCLC in the real-world setting.

Mutational Profiling by Next-Generation Sequencing in Patients with Metastatic Non-Small Cell Lung Carcinoma: Our Experience

Introduction According to GLOBOCAN 2020, overall lung cancer is the second most common cancer in both sexes (11.4%) accounting for highest cancer-related mortality (18%).1 Adenocarcinoma subtype of non-small cell lung cancer (NSCLC) is the most common subtype and is divided into further molecular subtypes based on oncogenic driver mutations. Overall survival in these patients is poor with the use of conventional platinum-based double chemotherapy and various recent studies on targeted therapy studies have showed improved survival. Therefore, broad panel-based testing like next-generation sequencing (NGS) is strongly recommended to identify these targetable driver mutations. Aims and Objectives The aim of this study was to evaluate the mutational profile in patients with metastatic NSCLC (mNSCLC) by NGS method. Materials and Methods A hospital-based prospective observational study done on 88 patients under the Department of Medical Oncology, State Cancer Institute during a period of 1 year. All patients above 18 years of age diagnosed as mNSCLC having Eastern Cooperative Oncology Group performance status 0 to 2 and evaluated for mutational profiling by NGS method were included. Five gene panel tests including endothelial growth factor receptor (EGFR), echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK), BRAF, mesenchymal epithelial transition (MET), and ROS proto-oncogene 1 (ROS1) were used. Results and Observations Majority of mNSCLC cases were in the age group of 41 to 50 years (n = 30, 34.1%) with average age at presentation being 53.74 years. Male: female ratio was 1.14:1 and most patients were nonsmokers. Adenocarcinoma subtype of mNSCLC cases had the highest mutational burden (n = 55, 62.5%). EGFR (n = 32, 56.14%) was the most common mutation followed by EML4-ALK (n = 19, 33.33%). Most common EGFR mutation was in Exon 19. Other rare mutations were ROS1 (n = 4), BRAF V600E (n = 1), and MET (n = 1). Skeleton was the most common site of metastasis across all driver mutations. Conclusion EGFR and EML4-ALK were the commonest targetable mutations detected in the study. As there is very limited data from North Eastern region of India regarding mutational status in mNSCLC, this study opens up possibilities for further studies targeting multiple mutations to give us more comprehensive understanding of the mutational landscape of mNSCLC in this era of precision medicine.

Abstract 5839: Identification of low-dose ligand dependent drug-resistance in ALK/ROS1 positive NSCLC via CRISPR library screening

Abstract Tyrosine kinase inhibitor (TKI) therapy has displayed significant effectiveness in treating patients with anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1)-positive non-small cell lung cancer (NSCLC). Nonetheless, the emergence of acquired resistance in most patients presents a major challenge. Drug-tolerant persister (DTP) cells have recently gained attention as potential sources of resistance, highlighting the need for a deeper understanding of drug resistance mechanisms. To address this issue, a comprehensive genome-wide CRISPR-Cas9 knockout screening was conducted using an ALK-positive NSCLC cell line derived from pleural effusion in a patient who had not been exposed to ALK-TKI treatment. Following a 9-day ALK-TKI treatment, sequencing analysis was performed to identify sgRNAs in DTP cells. Notably, this analysis detected the involvement of tumor suppressor genes, including NF2, BAX, and ERRFI1.NF2 depletion was found to induce a substantial resistance to ALK-TKIs, and a combination therapy involving mTOR inhibitors showed promise in partially overcoming this resistance. Moreover, knockout of ERRFI1 or BAX led to an increase in DTP cells, drawing particular attention to ERRFI1, also known as MIG6, which is known for negatively regulating EGFR signaling. One intriguing discovery was that the loss of MIG6 induced resistance to ALK-TKIs when exposed to a relatively low dose of EGF, comparable to concentrations in plasma of healthy adults. This resistance was attributed to the upregulation of the MAPK and PI3K/Akt/mTOR pathways. Similar to ALK-rearranged NSCLC, ROS1 fusion-positive NSCLC cell lines also showed resistance by MIG6 knockout. Interestingly, some cell lines with reduced MIG6 expression tended to develop resistance with low-dose EGF, even without prior treatment, and overexpression of MIG6 restored sensitivity to TKIs. We further explored the potential of combination therapy, combining ALK-TKIs with anti-EGFR antibodies, and this approach effectively addressed acquired resistance in both in vivo and in vitro models. Furthermore, an analysis of clinical samples revealed that individuals resistant to ALK-TKIs displayed decreased mRNA MIG6 levels in comparison to their sensitive counterparts. Investigating the mechanisms behind MIG6 expression, we confirmed that ALK-TKI therapy suppressed MIG6 expression levels, while factors such as corticosteroid usage or exposure to hypoxia induced the upregulation of MIG6, potentially influencing the formation of DTP cells. In conclusion, this research has unveiled a novel factor contributing to resistance in ALK and ROS1-TKI therapies by activating the EGFR pathway through exposure to low-dose ligands, shedding light on potential therapeutic strategies to combat acquired resistance. Citation Format: Nobuyuki Kondo, Takahiro Utsumi, Ken Uchibori, Yasunari Miyazaki, Ryohei Katayama. Identification of low-dose ligand dependent drug-resistance in ALK/ROS1 positive NSCLC via CRISPR library screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5839.

Genomic testing and targeted therapy of non-small cell lung cancer in China: a nationwide survey of physicians and clinical pathologists.

Genomic diagnostic testing is necessary to guide optimal treatment for non-small cell lung cancer (NSCLC) patients. The proportion of NSCLC patients whose treatment was selected based on genomic testing is still unknown in many countries or needs further improvement. This survey aimed to assess perception of genomic testing and targeted therapy for NSCLC in clinical pathologists and physicians across China. The web-based survey was conducted with 150 clinical pathologists and 450 physicians from oncology, respiratory and thoracic surgery departments from May to September 2020, across 135 cities in China. The participants had >5 years of clinical experience in genomic testing, diagnosis or treatment of NSCLC. Clinical pathologists reported capability of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and ROS proto-oncogene 1 (ROS-1) testing as 95.3%, 94.7%, and 84.7%, respectively, but only 81.9%, 75.5%, and 65.6% of physicians believed that the pathology department of the hospital is capable of performing the testing. The proportions of sending out specimens for testing were 21.0% and 49.7% as reported from clinical pathologists and physicians, respectively. Testing for EGFR mutation was recommended by physicians most often, followed by ALK and ROS-1 rearrangement. As first-line treatment, among the newly diagnosed patients with EGFR mutation, 77% received tyrosine kinase inhibitors (TKIs) therapy (49% treated with gefitinib); among patients with ALK rearrangement, 71% received TKI (64% treated with crizotinib); among patients with ROS-1 fusion, 65% received TKI (88% treated with crizotinib). The improvement of the non-tertiary hospital pathology departments' detection capabilities and the physicians' awareness are needed for enhancing the rate of genomic testing and targeted therapy in NSCLC patients in China.

PD-L1 expression and its correlation with clinicopathological and molecular characteristics in Chinese patients with non-small cell lung cancer.

Little is known about the relationship between programmed cell death-ligand 1 (PD-L1) expression and histologic and genetic features in real-world Chinese non-small cell lung cancer patients. From November 2017 to June 2019, tumor tissues were collected from 2674 non-small cell lung cancer patients. PD-L1 expression was detected with immunohistochemistry using the 22C3 and SP263 antibodies, and patients were stratified into subgroups based on a tumor proportion score of 1%, 1% to 49%, and ≥ 50%. Genetic alterations were profiled using targeted next-generation sequencing. In the total population, 50.5% had negative PD-L1 expression (tumor proportion score < 1%), 32.0% had low-positive expression (1%-49%), and 17.5% had high-positive expression (≥50%). The PD-L1 positive rate was 39.0% in squamous cell carcinomas and 53.6% in adenocarcinomas. PD-L1 expression was higher in squamous cell carcinomas (P < .001) and lower in adenocarcinomas (P < .001). Of the overall patient population, 11.2% had Kirsten rat sarcoma viral oncogene (KRAS) mutations, 44.9% had epidermal growth factor receptor (EGFR) mutations, 2.1% had BRAF V600E mutations, 0.3% had MET exon 14 skipping mutations, 5.4% had anaplastic lymphoma kinase translocations, and 0.9% had ROS proto-oncogene 1 translocations. Patients carrying ROS proto-oncogene 1 translocations (P = .006), KRAS (P < .001), and MET (P = .023) mutations had significantly elevated expression of PD-L1, while those harboring EGFR (P < .001) mutations had lower PD-L1 expression. In our study, PD-L1 expression was significantly higher in squamous cell carcinomas and lower in adenocarcinomas, and was positively associated with MET and KRAS mutations, as well as the wild-type EGFR gene state. Nonetheless, additional studies are needed to further validate those associations and determine the clinical significance for immune checkpoint inhibitors of these factors.

The Epidermal Growth Factor, Anaplastic Lymphoma Kinase, and ROS Proto-oncogene 1 Mutation Profile of Non-Small Cell Lung Carcinomas in the Turkish Population: A Single-Center Analysis.

The management of non-small cell lung carcinomas (NSCLC) has changed with the identification of molecular pathways. We aimed to reveal the 3-year epidermal growth factor (EGFR), anaplastic lymphoma kinase (ALK), and ROS proto-oncogene 1 (ROS1) mutation profile in the Turkish population. The histopathological and molecular data of all NSCLC cases from our department between May 2019 and April 2022 were evaluated. Molecular testing was performed in 197 NSCLC cases, and results were obtained in 182 (92.4%) (M/F: 144/38, aged 39-86). Of these, 121 were diagnosed with adenocarcinoma, 36 with squamous cell carcinoma, and 25 with NSCLC-not otherwise specified. The EGFR mutation was seen in 21 (11.5%) cases (6 exon 19 deletions, 3 exon 18 [all codon 719], 2 exon 20, 8 exon 21 point mutations, 1 concurrent exon 19 deletion and exon 20 codon 790 M point mutation, and 1 concurrent exon 19 deletion and exon 21 point mutation). The double mutation rate of EGFR was 1.1%. The mean age of these patients was 63.4 (40-79), with 24% of all females (n = 9) and 8.3% of all males (n = 12). The ALK mutation was detected in 6 (3.3%) patients (M/F: 4/2, aged 45-82), whereas the ROS1 mutation was detected in 3 (1.7%) (M/F: 2/1, aged 40-64). It is well established in the literature that EGFR-activating mutation rates vary depending on regions and ethnic groups. We concluded that the EGFR-activating mutation rates of the Turkish population are similar to the European molecular data instead of the Asian. The ALK and ROS1 mutation rates also seem concordant with the literature.

Simultaneous inhibition of FAK and ROS1 synergistically repressed triple-negative breast cancer by upregulating p53 signalling

BackgroundTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking effective targeted therapies, necessitating innovative treatment approaches. While targeting ROS proto-oncogene 1 (ROS1) with crizotinib has shown promise, resistance remains a limitation. Recent evidence links focal adhesion kinase (FAK) to drug resistance, prompting our study to assess the combined impact of FAK inhibitor IN10018 and crizotinib in TNBC and elucidate the underlying mechanisms.MethodsWe employed the Timer database to analyze FAK and ROS1 mRNA levels in TNBC and adjacent normal tissues. Furthermore, we investigated the correlation between FAK, ROS1, and TNBC clinical prognosis using the GSE database. We conducted various in vitro assays, including cell viability, colony formation, flow cytometry, EdU assays, and western blotting. Additionally, TNBC xenograft and human TNBC organoid models were established to assess the combined therapy’s efficacy. To comprehensively understand the synergistic anti-tumor mechanisms, we utilized multiple techniques, such as RNA sequencing, immunofluorescence, cell flow cytometry, C11-BODIPY staining, MDA assay, and GSH assay.ResultsThe Timer database revealed higher levels of FAK and ROS1 in TNBC tissues compared to normal tissues. Analysis of GEO databases indicated that patients with high FAK and ROS1 expression had the poorest prognosis. Western blotting confirmed increased p-FAK expression in crizotinib-resistant TNBC cells. In vitro experiments showed that the combination therapy down-regulated cyclin B1, p-Cdc2, and Bcl2 while up-regulating BAX, cleaved-Caspase-3, cleaved-Caspase-9, and cleaved PARP. In TNBC xenograft models, the tumor volume in the combination therapy group was 73% smaller compared to the control group (p < 0.0001). Additionally, the combination therapy resulted in a 70% reduction in cell viability in human TNBC organoid models (p < 0.0001). RNA sequencing analysis of TNBC cells and xenograft tumor tissues highlighted enrichment in oxidative stress, glutathione metabolism, and p53 pathways. The combined group displayed a fivefold rise in the reactive oxygen species level, a 69% decrease in the GSH/GSSG ratio, and a sixfold increase in the lipid peroxidation in comparison to the control group. Western blotting demonstrated p53 upregulation and SCL7A11 and GPX4 downregulation in the combination group. The addition of a p53 inhibitor reversed these effects.ConclusionOur study demonstrates that the combination of IN10018 and crizotinib shows synergistic antitumor effects in TNBC. Mechanistically, this combination inhibits cell proliferation, enhances apoptosis, and induces ferroptosis, which is associated with increased p53 levels.

ROS1 kinase inhibition reimagined: identifying repurposed drug via virtual screening and molecular dynamics simulations for cancer therapeutics.

Precision medicine has revolutionized modern cancer therapeutic management by targeting specific molecular aberrations responsible for the onset and progression of tumorigenesis. ROS proto-oncogene 1 (ROS1) is a receptor tyrosine kinase (RTK) that can induce tumorigenesis through various signaling pathways, such as cell proliferation, survival, migration, and metastasis. It has emerged as a promising therapeutic target in various cancer types. However, there is very limited availability of specific ROS1 inhibitors for therapeutic purposes. Exploring repurposed drugs for rapid and effective treatment is a useful approach. In this study, we utilized an integrated approach of virtual screening and molecular dynamics (MD) simulations of repurposing existing drugs for ROS1 kinase inhibition. Using a curated library of 3648 FDA-approved drugs, virtual screening identified drugs capable of binding to ROS1 kinase domain. The results unveil two hits, Midostaurin and Alectinib with favorable binding profiles and stable interactions with the active site residues of ROS1. These hits were subjected to stability assessment through all-atom MD simulations for 200 ns. MD results showed that Midostaurin and Alectinib were stable with ROS1. Taken together, the study showed a rational framework for the selection of repurposed Midostaurin and Alectinib with ROS1 inhibitory potential for therapeutic management after further validation.

Involvement of ICAM5 in Carcinostasis Effects on LUAD Based on the ROS1-Related Prognostic Model.

Lung cancer is the most common type of cancer in the world. In lung adenocarcinoma (LUAD), studies on receptor tyrosine kinase ROS proto-oncogene 1 (ROS1) have mainly focused on the oncogenic effects of its fusion mutations, whereas ROS1 has been reported to be aberrantly expressed in a variety of cancers and can extensively regulate the growth, survival, and proliferation of tumor cells through multiple signaling pathways. The comprehensive analysis of ROS1 expression has not been fully investigated regarding its predictive value for LUAD patients. Gene expression profiles collected from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases were used to build and validate prognostic risk models. The association of ROS1 with overall survival and the immune landscape was obtained from the Tumor Immune Estimation Resource (TIMER) database. The following analyses were performed using the R package to determine the model's validity: pathway dysregulation analysis, gene set enrichment analysis, Gene Oncology analysis, immune invasion analysis, chemotherapy, radiotherapy, and immunotherapy sensitivity analysis. Finally, we conducted a pan-cancer analysis and performed in vitro experiments to explore the regulatory role of intercellular adhesion molecule 5 (ICAM5) in the progression of LUAD. We constructed a 17-gene model that categorized patients into two risk groups. The model had predictive accuracy for tumor prognosis and was specific for patients with high ROS1 expression. Comprehensive analysis showed that patients in the high-risk group were characterized by marked dysregulation of multiple pathways (eg, unfolded protein response), immune suppression of the tumor microenvironment, and poor benefit from immunotherapy and radiotherapy compared with patients in the low-risk group. PLX4720 may be a suitable treatment for the high-risk patient population. The ICAM5 gene has been demonstrated to inhibit the proliferation, cell cycle, invasion, and migration of LUAD cells. We constructed a 17-gene prognostic risk model and found differences in immune-related cells, biological processes, and prognosis among patients in different risk groups based on the correlation between ROS1 and immunity. Personalized therapy may play an essential role in treatment. We further investigated the role of ICAM5 in inhibiting the malignant bioactivity of LUAD cells.

Understanding the treatment response and resistance to targeted therapies in non-small cell lung cancer: clinical insights and perspectives.

Lung cancer remains the leading cause of mortality worldwide. Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer with a generally poor prognosis. In recent years, advances in targeted therapy and sequencing technology have brought significant improvement in the therapeutic outcomes of patients with advanced NSCLC. Targeted inhibitors directed against specific mutated or rearranged oncogenes, such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and receptor tyrosine kinase ROS proto-oncogene 1(ROS1) among others, exhibit promising anti-tumor activity. Unfortunately, some patients develop acquired resistance and disease progression soon after initial remission. Despite the continuous development of new drugs and strategies to overcome drug resistance, it is still a major challenge in the treatment of NSCLC. The landscape of targeted therapy for NSCLC is evolving rapidly in response to the pace of scientific research. This study aimed to provide a comprehensive review of tumor target antigens and agents related to targeted therapy in NSCLC.

Different effects of crizotinib treatment in two non-small cell lung cancer patients with SDC4::ROS1 fusion variants.

The possibility of stratifying patients according to differences in ROS proto-oncogene 1 (ROS1) fusion partners has been discussed. This study aimed to clarify the clinicopathological differences between two SDC4::ROS1 positive NSCLC cases who had different responses to crizotinib. Cytology and pathology samples from two NSCLC cases with SDC4::ROS1 who were diagnosed and treated with crizotinib at Nihon University Itabashi Hospital were obtained. Case 1 has been well-controlled with crizotinib for over 5 years, but case 2 was worse and overall survival was 19 months. Sequencing analysis of ROS1 fusion genes was performed by reverse-transcription-PCR and Sanger's sequencing methods. In addition, thyroid transcription factor (TTF)-1, ROS-1, Ki67, and phosphorylated extracellular signal-regulated kinase (pERK)1/2 expression were investigated using immunohistochemistry. Sequencing analysis showed SDC4 exon2::ROS1 exon 32 (exon33 deleted) in case 1, and coexistence of SDC4 exon2::ROS1 exon 34 and SDC4 exon2::ROS1 exon35 in case 2. The Ki67 index was not different, but ROS1 and pERK1/2 expression levels tended to be higher in the tumor cells of case 2 than in case 1. Therapeutic response to crizotinib and patients' prognosis in ROS1 rearranged NSCLC may be related to the activation of ROS1 signaling, depending on ROS1 and pERK1/2 overexpression status, even if the ROS1 fusion partner is the same.

Concordance of Immunohistochemistry and Fluorescence In Situ Hybridization in the Detection of Anaplastic Lymphoma Kinase (ALK) and Ros Proto-oncogene 1 (ROS1) Gene Rearrangements in Non-Small Cell Lung Carcinoma: A 4.5-Year Experience Highlighting Challenges and Pitfalls.

ALK and ROS1 rearrangements are essential biomarkers to be tested in advanced lung adenocarcinomas. While D5F3 Ventana assay is a companion diagnostic for anaplastic lymphoma kinase-targeted therapy, immunohistochemistry is only a screening tool for detecting ROS1 rearrangement. Confirmation by cytogenetic or molecular techniques is necessary. To evaluate the utility of ALK and ROS1 fluorescence in situ hybridization as a complement to immunohistochemistry in routine predictive biomarker testing algorithms. The study was ambispective, spanning 4.5 years during which lung adenocarcinoma samples were subjected to EGFR mutation testing by real-time polymerase chain reaction and ALK/ROS1 rearrangement testing by immunohistochemistry (Ventana D5F3 assay for anaplastic lymphoma kinase protein; manual assay with D4D6 clone for Ros proto-oncogene 1 protein). Fluorescence in situ hybridization was performed in all anaplastic lymphoma kinase equivocal and Ros proto-oncogene 1 immunopositive cases. Of 1874 samples included, EGFR mutations were detected in 27% (481 of 1796). Anaplastic lymphoma kinase immunohistochemistry was positive in 10% (174 of 1719) and equivocal in 3% (58 of 1719) of samples tested. ALK fluorescence in situ hybridization showed 81% (77 of 95) concordance with immunohistochemistry. Ros proto-oncogene 1 immunopositivity was noted in 13% (190 of 1425) of cases, with hybridization-confirmed rearrangements in 19.3% (26 of 135) of samples, all of which showed diffuse, strong- to moderate-intensity, cytoplasmic staining in tumor cells. Ros proto-oncogene 1 protein overexpression without rearrangement was significantly common in EGFR-mutant and ALK-rearranged adenocarcinomas. Immunostaining is a robust method for ALK-rearrangement testing, with fluorescence in situ hybridization adding value in the rare equivocal stained case. ROS1-rearrangement testing is more cost-effective if immunohistochemistry is followed by fluorescence in situ hybridization after excluding EGFR-mutant and ALK-rearranged adenocarcinomas.

Genetic Alterations and Risk Factors for Recurrence in Patients with Non-Small Cell Lung Cancer Who Underwent Complete Surgical Resection

A definitive surgical resection is the preferred treatment for early-stage non-small cell lung cancer (NSCLC). Research on genetic alterations, including epidermal growth factor receptor (EGFR) mutations, in early-stage NSCLC remains insufficient. We investigated the prevalence of genetic alterations in early-stage NSCLC and the association between EGFR mutations and recurrence after a complete resection. Between January 2019 and December 2021, 659 patients with NSCLC who underwent curative surgical resections at a single regional cancer center in Korea were recruited. We retrospectively compared the clinical and pathological data between the recurrence and non-recurrence groups. Among the 659 enrolled cases, the median age was 65.86 years old and the most common histology was adenocarcinoma (74.5%), followed by squamous cell carcinoma (21.7%). The prevalence of EGFR mutations was 43% (194/451). Among them, L858R point mutations and exon 19 deletions were 52.3% and 42%, respectively. Anaplastic lymphoma kinase (ALK) rearrangement was found in 5.7% of patients (26/453) and ROS proto-oncogene 1 (ROS1) fusion was found in 1.6% (7/441). The recurrence rate for the entire population was 19.7%. In the multivariate analysis, the presence of EGFR mutations (hazard ratio (HR): 2.698; 95% CI: 1.458–4.993; p = 0.002), stage II (HR: 2.614; 95% CI: 1.29–5.295; p = 0.008) or III disease (HR: 9.537; 95% CI: 4.825–18.852; p &lt; 0.001) (vs. stage I disease), and the presence of a pathologic solid type (HR: 2.598; 95% CI: 1.405–4.803; p = 0.002) were associated with recurrence. Among the recurrence group, 86.5% of the patients with EGFR mutations experienced distant metastases compared with only 66.7% of the wild type (p = 0.016), with no significant difference in median disease-free survival (52.21 months vs. not reached; p = 0.983). In conclusion, adjuvant or neoadjuvant targeted therapy could be considered more actively because EGFR mutations were identified as an independent risk factor for recurrence and were associated with systemic recurrence. Further studies on perioperative therapy for other genetic alterations are necessary.

Detection of Novel Tyrosine Kinase Fusion Genes as Potential Therapeutic Targets in Bone and Soft Tissue Sarcomas Using DNA/RNA-based Clinical Sequencing.

Approximately 1% of clinically treatable tyrosine kinase fusions, including anaplastic lymphoma kinase, neurotrophic tyrosine receptor kinase, RET proto-oncogene, and ROS proto-oncogene 1, have been identified in soft tissue sarcomas via comprehensive genome profiling based on DNA sequencing. Histologic tumor-specific fusion genes have been reported in approximately 20% of soft tissue sarcomas; however, unlike tyrosine kinase fusion genes, these fusions cannot be directly targeted in therapy. Approximately 80% of tumor-specific fusion-negative sarcomas, including myxofibrosarcoma and leiomyosarcoma, that are defined in complex karyotype sarcomas remain genetically uncharacterized; this mutually exclusive pattern of mutations suggests that other mutually exclusive driver oncogenes are yet to be discovered. Tumor-specific, fusion-negative sarcomas may be associated with unique translocations, and oncogenic fusion genes, including tyrosine kinase fusions, may have been overlooked in these sarcomas. (1) Can DNA- or RNA-based analysis reveal any characteristic gene alterations in bone and soft tissue sarcomas? (2) Can useful and potential tyrosine kinase fusions in tumors from tumor-specific, fusion-negative sarcomas be detected using an RNA-based screening system? (3) Do the identified potential fusion tumors, especially in neurotrophic tyrosine receptor kinase gene fusions in bone sarcoma, transform cells and respond to targeted drug treatment in in vitro assays? (4) Can the identified tyrosine kinase fusion genes in sarcomas be useful therapeutic targets? Between 2017 and 2020, we treated 100 patients for bone and soft tissue sarcomas at five institutions. Any biopsy or surgery from which a specimen could be obtained was included as potentially eligible. Ninety percent (90 patients) of patients were eligible; a further 8% (8 patients) were excluded because they were either lost to follow-up or their diagnosis was changed, leaving 82% (82 patients) for analysis here. To answer our first and second questions regarding gene alterations and potential tyrosine kinase fusions in eight bone and 74 soft tissue sarcomas, we used the TruSight Tumor 170 assay to detect mutations, copy number variations, and gene fusions in the samples. To answer our third question, we performed functional analyses involving in vitro assays to determine whether the identified tyrosine kinase fusions were associated with oncogenic abilities and drug responses. Finally, to determine usefulness as therapeutic targets, two pediatric patients harboring an NTRK fusion and an ALK fusion were treated with tyrosine kinase inhibitors in clinical trials. DNA/RNA-based analysis demonstrated characteristic alterations in bone and soft tissue sarcomas; DNA-based analyses detected TP53 and copy number alterations of MDM2 and CDK4 . These single-nucleotide variants and copy number variations were enriched in specific fusion-negative sarcomas. RNA-based screening detected fusion genes in 24% (20 of 82) of patients. Useful potential fusions were detected in 19% (11 of 58) of tumor-specific fusion-negative sarcomas, with nine of these patients harboring tyrosine kinase fusion genes; five of these patients had in-frame tyrosine kinase fusion genes ( STRN3-NTRK3, VWC2-EGFR, ICK-KDR, FOXP2-MET , and CEP290-MET ) with unknown pathologic significance. The functional analysis revealed that STRN3-NTRK3 rearrangement that was identified in bone had a strong transforming potential in 3T3 cells, and that STRN3-NTRK3 -positive cells were sensitive to larotrectinib in vitro. To confirm the usefulness of identified tyrosine kinase fusion genes as therapeutic targets, patients with well-characterized LMNA-NTRK1 and CLTC-ALK fusions were treated with tyrosine kinase inhibitors in clinical trials, and a complete response was achieved. We identified useful potential therapeutic targets for tyrosine kinase fusions in bone and soft tissue sarcomas using RNA-based analysis. We successfully identified STRN3-NTRK3 fusion in a patient with leiomyosarcoma of bone and determined the malignant potential of this fusion gene via functional analyses and drug effects. In light of these discoveries, comprehensive genome profiling should be considered even if the sarcoma is a bone sarcoma. There seem to be some limitations regarding current DNA-based comprehensive genome profiling tests, and it is important to use RNA testing for proper diagnosis and accurate identification of fusion genes. Studies on more patients, validation of results, and further functional analysis of unknown tyrosine kinase fusion genes are required to establish future treatments. DNA- and RNA-based screening systems may be useful for detecting tyrosine kinase fusion genes in specific fusion-negative sarcomas and identifying key therapeutic targets, leading to possible breakthroughs in the treatment of bone and soft tissue sarcomas. Given that current DNA sequencing misses fusion genes, RNA-based screening systems should be widely considered as a worldwide test for sarcoma. If standard treatments such as chemotherapy are not effective, or even if the sarcoma is of bone, RNA sequencing should be considered to identify as many therapeutic targets as possible.

Analysis of Clinicopathological Features on Spread Through Air Spaces of Lung Adenocarcinoma

The biological and molecular characteristics of spread through air spaces (STAS), a newly recognized invasive mode of lung cancer, remain controversial. The aim of this study was to investigate the clinicopathological features and molecular characteristics of STAS in patients with pulmonary adenocarcinoma. A total of 694 resected invasive non-mucinous lung adenocarcinomas diagnosed by clinicopathology from July 2019 to March 2021 in the First Affiliated Hospital of Guangzhou Medical University were collected, and the relationship between STAS and clinicopathological factors was analyzed. The state of protein expression of anaplastic lymphoma kinase (ALK) was detected by immunohistochemical method. Epidermal growth factor receptor (EGFR) was detected by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). ROS proto-oncogene 1-receptor (ROS1) was detected by reverse transcription-PCR (RT-PCR). A total of 344 STAS positive cases and 350 STAS negative cases were collected. By univariate analysis, STAS positivity was statistically associated with tumor maximum diameter (P<0.001), pleural invasion (P<0.001), lymphovascular invasion (P<0.001), nerve invasion (P=0.013), lymph node metastasis (P<0.001), clinical stage (P<0.001) and histological type (P<0.001). There was a statistical correlation between STAS and ALK protein expression (P=0.001). Multivariate analysis showed that STAS positive was correlated with pleural invasion (P=0.001), vascular invasion (P<0.001), lymph node metastasis (P=0.005)and ALK protein expression (P=0.032). STAS is associated with highly aggressive biological behavior of lung adenocarcinoma, suggesting a poor prognosis.

Targeted Therapies in Early-Stage Resectable Non-Small-Cell Lung Cancer: New Kids on the Block.

With increased adoption of next-generation sequencing, tailored therapy on the basis of molecular status is being delivered for patients with early-stage resectable non-small-cell lung cancer (NSCLC). The purpose of this narrative review was to focus on recent developments of targeted therapies in the adjuvant and neoadjuvant/adjuvant setting for early-stage disease. A systematic search of the MEDLINE/PubMed database was performed, focusing on studies published within the past 10 years. Our search queried "early-stage NSCLC (AND) tyrosine kinase inhibitor (TKI; OR) epidermal growth factor receptor (EGFR; OR) anaplastic lymphoma kinase (ALK)" and was limited only to prospective and ongoing studies. Most studies examining the benefit of targeted therapies in early-stage resectable NSCLC have been for EGFR-TKIs in the adjuvant setting. Currently, only one study, the ADAURA trial of adjuvant osimertinib, has demonstrated an overall survival benefit with the use of an EGFR-TKI in the adjuvant setting. Future work to build on the success of the ADAURA trial is focused on determining the optimal duration of targeted therapies and using biomarkers, such as circulating tumor DNA, to risk-stratify patients and guide maintenance targeted therapy duration. The results of several ongoing studies are eagerly awaited regarding the use of targeted therapies in the neoadjuvant/adjuvant setting and for more uncommon or rare mutations such as ALK, ROS proto-oncogene 1, rearranged during transfection, mesenchymal-epithelial transition factor, and B-Raf proto-oncogene V600E. The treatment landscape for early-stage NSCLC harboring actionable mutations is likely to shift dramatically in the upcoming decade.