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Advanced Non-Small Cell Lung Carcinoma (NSCLC) patients with ROS1 gene rearrangement have shown significant therapeutic responses to tyrosine kinase inhibitors approved by the US Food and Drug Administration, with approximately 40 fusion partners documented in the existing literature. Our report highlights a novel fusion partner of ROS1 that has demonstrated a conclusive response to the current standard of treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: This systematic review and meta-analysis aimed to evaluate the neurocognitive adverse events (NAEs) related to lorlatinib in patients with ALK/ROS1-positive non-small cell lung cancer (NSCLC). A random-effects model was used for the meta-analysis and the proportions were pooled with 95% confidence intervals (CIs). In our pooled analysis, cognitive and mood effects were the most important NAEs, with 14% and 11% overall rates, respectively. Speech changes and psychotic effects had an overall frequency of 7% and 5%, respectively. In a subanalysis, mood effects were significantly more frequent in clinical trials than in real-world studies. Therefore, this study showed that lorlatinib-related NAEs are emerging important toxicities in clinical practice. Hence, increasing awareness about NAEs is paramount to improving their recognition and reporting, and thus enhancing patients' quality of life. Lorlatinib has been FDA-approved as a systemic therapy for ALK/ROS1-positive non-small cell lung cancer (NSCLC) patients. However, it has been associated with an increased frequency of neurocognitive adverse events (NAEs). Therefore, we conducted a systematic review and meta-analysis to assess the NAEs related to lorlatinib therapy in NSCLC patients. PubMed, Scopus, the Cochrane Library, and prominent conference proceedings were searched for eligible studies of lorlatinib in NSCLC patients. NAEs included cognitive, mood, speech, and psychotic effects. A total of 1147 patients from 12 studies were included; 62% had brain metastases. A pooled analysis of NAEs showed frequencies of cognitive effects of 14.57% (95% CI, 8.37 to 24.14, I2 = 84%), mood effects of 11.17% (95% CI, 5.93 to 20.07, I2 = 84%), speech effects of 7.24% (95% CI, 3.39 to 15.20, I2 = 72%), and psychotic effects of 4.97% (95% CI, 3.27 to 7.49, I2 = 21%). Clinical trials reported a significantly higher frequency of mood effects than was indicated by real-world data. These results highlight the importance of educating patients and healthcare professionals about lorlatinib-related NAEs for early detection and management to improve NSCLC patients' quality of life. [ABSTRACT FROM AUTHOR]

Purpose: Lung cancer is the leading cause of cancer-related deaths worldwide. However, with the optimization of screening strategies and advances in treatment, mortality has been decreasing in recent years. In this study, we describe non-small cell lung cancer patients diagnosed between 2021 and 2022 at a high-complexity hospital in Latin America, as well as the immunohistochemistry techniques used to screen for ROS1 rearrangements, in the context of the recent approval of crizotinib for the treatment of ROS1 rearrangements in non-small cell lung cancer in Colombia. Methods: A descriptive cross-sectional study was conducted. Sociodemographic, clinical, and molecular pathology information from non-small cell lung cancer individuals who underwent immunohistochemistry to detect ROS1 rearrangements between 2021 and 2022 at Fundación Valle del Lili (Cali, Colombia) was recorded. The clinical outcomes of confirmed ROS1 rearrangements in non-small cell lung cancer patients were reported. Results: One hundred and thirty-six patients with non-small cell lung cancer were included. The median age at diagnosis was 69.8 years (interquartile range 61.9– 77.7). At diagnosis, 69.8% (n = 95) were at stage IV. ROS1 immunohistochemistry was performed using the monoclonal D4D6 antibody clone in 54.4% (n = 74) of the cases, while 45.6% (n = 62) were done with the monoclonal SP384 antibody clone. Two patients were confirmed to have ROS1 rearrangements in non-small cell lung cancer using next-generation sequencing and received crizotinib. On follow-up at months 5.3 and 7.0, one patient had a partial response, and the other had oligo-progression, respectively. Conclusion: Screening for ROS1 rearrangements in non-small cell lung cancer is imperative, as multiple prospective studies have shown improved clinical outcomes with tyrosine kinase inhibitors. Given the recent approval of crizotinib in Colombia, public health policies must be oriented toward early detection of driver mutations and prompt treatment. Additionally, future approvals of newly tested tyrosine kinase inhibitors should be anticipated. [ABSTRACT FROM AUTHOR]

Background: Radiomics, an evolving paradigm in medical imaging, involves the quantitative analysis of tumor features and demonstrates promise in predicting treatment responses and outcomes. This study aims to investigate the predictive capacity of radiomics for genetic alterations in non-small cell lung cancer (NSCLC). Methods: This exploratory, observational study integrated radiomic perspectives using computed tomography (CT) and genomic perspectives through next-generation sequencing (NGS) applied to liquid biopsies. Associations between radiomic features and genetic mutations were established using the Area Under the Receiver Operating Characteristic curve (AUC-ROC). Machine learning techniques, including Support Vector Machine (SVM) classification, aim to predict genetic mutations based on radiomic features. The prognostic impact of selected gene variants was assessed using Kaplan–Meier curves and Log-rank tests. Results: Sixty-six patients underwent screening, with fifty-seven being comprehensively characterized radiomically and genomically. Predominantly males (68.4%), adenocarcinoma was the prevalent histological type (73.7%). Disease staging is distributed across I/II (38.6%), III (31.6%), and IV (29.8%). Significant correlations were identified with mutations of ROS1 p.Thr145Pro (shape_Sphericity), ROS1 p.Arg167Gln (glszm_ZoneEntropy, firstorder_TotalEnergy), ROS1 p.Asp2213Asn (glszm_GrayLevelVariance, firstorder_RootMeanSquared), and ALK p.Asp1529Glu (glcm_Imc1). Patients with the ROS1 p.Thr145Pro variant demonstrated markedly shorter median survival compared to the wild-type group (9.7 months vs. not reached, p = 0.0143; HR: 5.35; 95% CI: 1.39–20.48). Conclusions: The exploration of the intersection between radiomics and cancer genetics in NSCLC is not only feasible but also holds the potential to improve genetic predictions and enhance prognostic accuracy. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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). Results: Of the 82 advanced ROS1-rearranged NSCLC patients, 38 exhibited PDL1 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). Conclusions: 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. [ABSTRACT FROM AUTHOR]

OBJECTIVE: 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. MATERIAL AND METHODS: The histopathological and molecular data of all NSCLC cases from our department between May 2019 and April 2022 were evaluated. RESULTS: 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). CONCLUSION: 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. [ABSTRACT FROM AUTHOR]

This letter, published in the Journal of Translational Medicine, discusses the association between ROS1 mutation and cancer immunity, as well as its impact on the efficacy of pan-cancer immunotherapy. The authors conducted a comprehensive analysis using bioinformatics and clinical data, finding that ROS1 mutation was associated with longer overall survival and increased response to immune checkpoint inhibitors. They also explored the immune characteristics of ROS1-mutant tumors, including higher mutation loads, increased expression of immune-related molecules, and enhanced immune cell infiltration. Overall, the study suggests that ROS1 mutation may serve as a favorable biomarker for outcomes in pan-cancer immunotherapy. [Extracted from the article]

Background: Inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal soft tissue sarcomas that often present diagnostic challenges due to their wide and varied morphology. A subset of IMTs have fusions involving ALK or ROS1. The role of next‐generation sequencing (NGS) for classification of unselected sarcomas remains controversial. Methods and Results: We report a case of a metastatic sarcoma in a 34‐year‐old female originally diagnosed as an unclassified spindle cell sarcoma with myofibroblastic differentiation and later reclassified as IMT after NGS revealed a TFG‐ROS1 rearrangement. Histologically, the neoplasm had spindle cell morphology with a lobulated to focally infiltrative growth pattern with scant inflammatory cell infiltrate. Immunohistochemistry demonstrated focal desmin and variable smooth muscle actin staining but was negative for SOX10, S100, and CD34. Fluorescence in situ hybridization was negative for USP6 or ALK gene rearrangements. NGS revealed a TFG‐ROS1 rearrangement and the patient was treated with crizotinib with clinical benefit. Conclusions: We discuss the role of NGS as well as its potential benefit in patients with unresectable, ALK‐negative metastatic disease. Considering this case and previous literature, we support the use of NGS for patients requiring systemic treatment. [ABSTRACT FROM AUTHOR]

Repotrectinib, licensed in November 2023, is a novel ROS1 tyrosine kinase inhibitor (TKI) with activity against G2032R, the most common resistance mutation to prior generations of ROS1 TKIs. Here, we report a case of a patient who was heavily pretreated, with advanced L1951R and L2026M mutated ROS1-rearranged NSCLC, who initially responded to repotrectinib but later developed further on-target resistance with the emergence of an L2086F mutation. The disease then responded to cabozantinib, a separate class of ROS1 TKI with preclinical activity against L2086F.

In recent years, particular interest has developed in molecular biology applied to the field of dermatopathology, with a focus on nevi of the Spitz spectrum. From 2014 onwards, an increasing number of papers have been published to classify, stratify, and correctly frame molecular alterations, including kinase fusions. In this paper, we try to synthesize the knowledge gained in this area so far. In December 2023, we searched Medline and Scopus for case reports and case series, narrative and systematic reviews, meta-analyses, observational studies—either longitudinal or historical, case series, and case reports published in English in the last 15 years using the keywords spitzoid neoplasms, kinase fusions, ALK, ROS1, NTRK (1-2-3), MET, RET, MAP3K8, and RAF1. ALK-rearranged Spitz tumors and ROS-1-rearranged tumors are among the most studied and characterized entities in the literature, in an attempt (although not always successful) to correlate histopathological features with the probable molecular driver alteration. NTRK-, RET-, and MET-rearranged Spitz tumors present another studied and characterized entity, with several rearrangements described but as of yet incomplete information about their prognostic significance. Furthermore, although rarer, rearrangements of serine–threonine kinases such as BRAF, RAF1, and MAP3K8 have also been described, but more cases with more detailed information about possible histopathological alterations, mechanisms of etiopathogenesis, and also prognosis are needed. The knowledge of molecular drivers is of great interest in the field of melanocytic diagnostics, and it is important to consider that in addition to immunohistochemistry, molecular techniques such as FISH, PCR, and/or NGS are essential to confirm and classify the different patterns of mutation. Future studies with large case series and molecular sequencing techniques are needed to allow for a more complete and comprehensive understanding of the role of fusion kinases in the spitzoid tumor family.

Abstract The maturation of spermatozoa is a regulated process, influenced by genes expressing essential secreted proteins in the proximal epididymis. Recent genetic studies in rodents have identified the non-sex steroidal molecular signals that regulate gene expression in the proximal epididymis. Germ cells in the testis secrete ligand proteins into the seminiferous tubule lumen The ligand proteins travel through the male reproductive tract lumen to the epididymis, where they bind to receptors, triggering the differentiation of the luminal epithelium for sperm maturation. It is, however, not fully unveiled if such a testis-epididymis trans-luminal signaling mechanism exists in other species, especially humans. In the present study, the rodent-type testis-epididymis trans-luminal signaling in the human male reproductive tract was evaluated in a step-by-step manner by analyzing testis and epididymis gene expression and signaling mediator protein function. There was a significant correlation between the epididymal expressions of mouse genes upregulated by the trans-luminal signaling and those of their human orthologs, as evaluated by the correlation coefficient of 0.604. The transcript expression of NELL2 and NICOL encoding putative ligand proteins was also observed in human testicular cells. In vitro experiments demonstrated that purified recombinant human NELL2 and NICOL formed a molecular complex with similar properties to rodent proteins, which was evaluated by a dissociation equilibrium constant of 110 nM. Recombinant human NELL2 also specifically bound to its putative receptor human ROS1 in vitro. Collectively, these findings suggest that the rodent-type testis-epididymis secreted signaling mechanism is also possible in the human male reproductive tract.

Abstract Background Triple-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. Methods We 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. Results The 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

Abstract 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.

Background: Driver oncogene mutations, such as c-ros oncogene 1 (ROS1) and epidermal growth factor receptor (EGFR) were previously believed to be mutually exclusive in non-small cell lung cancer (NSCLC). Only sporadic cases of ROS1 and EGFR co-mutations have been reported. Hence, appropriate treatment options for these patients are still controversial. Case presentation: A 48-year-old female patient presented at our hospital complaining of a persistent cough that had been ongoing for a month. A chest computed tomography showed a mass in the left lung along with hilar and mediastinal lymphadenopathy. Pathological analysis of bronchoscopic biopsy and lung mass puncture confirmed the presence of lung adenocarcinoma. The patient was diagnosed with stage IIIC left lung adenocarcinoma with a clinical stage of cT2N3M0. Next-generation sequencing analysis conducted at both puncture sites revealed an EFGR 19 deletion mutation combined with ROS1 rearrangement. The lung mass exhibited a higher mutation abundance. Treatment with a combination of third-generation EGFR tyrosine kinase inhibitors (TKIs) and crizotinib yielded satisfactory results. During the follow-up period, the mass significantly reduced and almost disappeared. Conclusion: The co-mutation of EGFR and ROS1 is a rare phenomenon. Nevertheless, the combination of EGFR-TKI and crizotinib treatment appears to hold promise in providing positive results for patients, with manageable side effects. This therapeutic approach has the potential to enhance patients' overall prognosis. [ABSTRACT FROM AUTHOR]

In recent years, particular interest has developed in molecular biology applied to the field of dermatopathology, with a focus on nevi of the Spitz spectrum. From 2014 onwards, an increasing number of papers have been published to classify, stratify, and correctly frame molecular alterations, including kinase fusions. In this paper, we try to synthesize the knowledge gained in this area so far. In December 2023, we searched Medline and Scopus for case reports and case series, narrative and systematic reviews, meta-analyses, observational studies—either longitudinal or historical, case series, and case reports published in English in the last 15 years using the keywords spitzoid neoplasms, kinase fusions, ALK, ROS1, NTRK (1-2-3), MET, RET, MAP3K8, and RAF1. ALK-rearranged Spitz tumors and ROS-1-rearranged tumors are among the most studied and characterized entities in the literature, in an attempt (although not always successful) to correlate histopathological features with the probable molecular driver alteration. NTRK-, RET-, and MET-rearranged Spitz tumors present another studied and characterized entity, with several rearrangements described but as of yet incomplete information about their prognostic significance. Furthermore, although rarer, rearrangements of serine–threonine kinases such as BRAF, RAF1, and MAP3K8 have also been described, but more cases with more detailed information about possible histopathological alterations, mechanisms of etiopathogenesis, and also prognosis are needed. The knowledge of molecular drivers is of great interest in the field of melanocytic diagnostics, and it is important to consider that in addition to immunohistochemistry, molecular techniques such as FISH, PCR, and/or NGS are essential to confirm and classify the different patterns of mutation. Future studies with large case series and molecular sequencing techniques are needed to allow for a more complete and comprehensive understanding of the role of fusion kinases in the spitzoid tumor family. [ABSTRACT FROM AUTHOR]

Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5–7%, 1–2%, and 1–2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability. [ABSTRACT FROM AUTHOR]

Summary: Dynamic DNA methylation regulatory networks are involved in many biological processes. However, how DNA methylation patterns change during flower senescence and their relevance with gene expression and related molecular mechanism remain largely unknown.Here, we used whole genome bisulfite sequencing to reveal a significant increase of DNA methylation in the promoter region of genes during natural and ethylene‐induced flower senescence in carnation (Dianthus caryophyllus L.), which was correlated with decreased expression of DNA demethylase gene DcROS1. Silencing of DcROS1 accelerated while overexpression of DcROS1 delayed carnation flower senescence.Moreover, among the hypermethylated differentially expressed genes during flower senescence, we identified two amino acid biosynthesis genes, DcCARA and DcDHAD, with increased DNA methylation and reduced expression in DcROS1 silenced petals, and decreased DNA methylation and increased expression in DcROS1 overexpression petals, accompanied by decreased or increased amino acids content. Silencing of DcCARA and DcDHAD accelerates carnation flower senescence. We further showed that adding corresponding amino acids could largely rescue the senescence phenotype of DcROS1, DcCARA and DcDHAD silenced plants.Our study not only demonstrates an essential role of DcROS1‐mediated remodeling of DNA methylation in flower senescence but also unravels a novel epigenetic regulatory mechanism underlying DNA methylation and amino acid biosynthesis during flower senescence. [ABSTRACT FROM AUTHOR]

Simple Summary: Crizotinib, an oral tyrosine kinase inhibitor that targets ALK, MET, and ROS1 kinase, has emerged as an effective treatment for ROS1-rearranged NSCLC. In South Korea's real-world setting, characterized by a higher elderly population and increased brain/central nervous system metastasis rates, crizotinib remains a cornerstone in treating ROS1-rearranged NSCLC, providing lasting clinical benefits with a favorable safety profile. Liquid biopsies, utilizing biomarkers, are instrumental in detecting targetable mutations, monitoring the disease burden, and identifying resistance mechanisms. In our study, next-generation sequencing using cell-free total nucleic acids enables the detection of ROS1 fusions and the identification of resistance mechanisms during disease progression. Real-world data on the use and outcomes of crizotinib in ROS1-rearranged non-small-cell lung cancer (NSCLC) are limited. This study aims to analyze the real-world efficacy of crizotinib in South Korea and explore the utilization of liquid biopsies that implement next-generation sequencing (NGS) using cell-free total nucleic acids. In this prospective multicenter cohort study, 40 patients with ROS1-rearranged NSCLC, either starting or already on crizotinib, were enrolled. Patients had a median age of 61 years, with 32.5% presenting brain/central nervous system (CNS) metastases at treatment initiation. At the data cutoff, 48.0% were still in treatment; four continued with it even after disease progression due to the clinical benefits. The objective response rate was 70.0%, with a median duration of response of 27.8 months. The median progression-free survival was 24.1 months, while the median overall survival was not reached. Adverse events occurred in 90.0% of patients, primarily with elevated transaminases, yet these were mostly manageable. The NGS assay detected a CD74–ROS1 fusion in 2 of the 14 patients at treatment initiation and identified emerging mutations, such as ROS1 G2032R, ROS1 D2033N, and KRAS G12D, during disease progression. These findings confirm crizotinib's sustained clinical efficacy and safety in a real-world context, which was characterized by a higher elderly population and higher rates of brain/CNS metastases. The study highlights the clinical relevance of liquid biopsy for detecting resistance mechanisms, suggesting its value in personalized treatment strategies. [ABSTRACT FROM AUTHOR]

Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Triple-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. Methods: We 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. Results: The 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. Conclusion: Our 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. [ABSTRACT FROM AUTHOR]

Introduction: ROS1 as a driver mutation is observed in approximately 1%-2% of all nonsmall cell lung cancer (NSCLC). Given its rarity, we share our experience regarding ROS1-positive NSCLC including the access to ROS1 tyrosine kinase inhibitors (TKIs) in a lowmiddle income country like India. Methods: It is a retrospective analysis of ROS1-positive NSCLC patients registered between January 2015 to December 2021 for demographics, treatment patterns and outcomes i.e., overall survival (OS) and progression free survival (PFS). Results: Baseline characteristics were available for 70 patients of 78 patients positive for ROS1 by fluorescent in situ hybridisation. Median age at presentation was 52 years, 39 (55.7%) were males, most (51, 72.86%) were non-smokers and ten patients (14.3%) had poor Eastern Cooperative Oncology Group (ECOG) performance status (PS) i.e., PS >2 at presentation. A total of 67 patients receiving cancer directed therapy were analysed for survival. The first line (1L) therapies included - ROS1 TKIs in 38, chemotherapy in 20, epidermal growth factor receptor TKI in eight and chemotherapy-bevacizumab in one only. ROS1 TKI was provided to 20 patients as part of an assistance programme. The median OS for patients who received ROS1 TKI was not attained (95% CI 37.85-NA), while it was 8.11 (95% CI 6.31-NA) months for those who did not (HR-0.1673). The median PFS for the 1L ROS1 TKI compared to the no-TKI group was 27.07 (95% CI 24.28-NA) months versus 5.78 (95% CI 3.42-12) months (HR: 0.2047). Poor ECOG PS at presentation was the only independent prognosticator for survival. Conclusion: Using ROS1 TKI improves clinical outcomes in all-comers though statistically not significant. To further improve outcomes, future trials should pay special attention to patients with poor PS and find a way to increase the current limited access to TKI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The maturation of spermatozoa is a regulated process, influenced by genes expressing essential secreted proteins in the proximal epididymis. Recent genetic studies in rodents have identified the non-sex steroidal molecular signals that regulate gene expression in the proximal epididymis. Germ cells in the testis secrete ligand proteins into the seminiferous tubule lumen The ligand proteins travel through the male reproductive tract lumen to the epididymis, where they bind to receptors, triggering the differentiation of the luminal epithelium for sperm maturation. It is, however, not fully unveiled if such a testis-epididymis trans-luminal signaling mechanism exists in other species, especially humans. In the present study, the rodent-type testis-epididymis trans-luminal signaling in the human male reproductive tract was evaluated in a step-by-step manner by analyzing testis and epididymis gene expression and signaling mediator protein function. There was a significant correlation between the epididymal expressions of mouse genes upregulated by the trans-luminal signaling and those of their human orthologs, as evaluated by the correlation coefficient of 0.604. The transcript expression of NELL2 and NICOL encoding putative ligand proteins was also observed in human testicular cells. In vitro experiments demonstrated that purified recombinant human NELL2 and NICOL formed a molecular complex with similar properties to rodent proteins, which was evaluated by a dissociation equilibrium constant of 110 nM. Recombinant human NELL2 also specifically bound to its putative receptor human ROS1 in vitro. Collectively, these findings suggest that the rodent-type testis-epididymis secreted signaling mechanism is also possible in the human male reproductive tract. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal soft tissue sarcomas that often present diagnostic challenges due to their wide and varied morphology. A subset of IMTs have fusions involving ALK or ROS1. The role of next‐generation sequencing (NGS) for classification of unselected sarcomas remains controversial. Methods and Results We report a case of a metastatic sarcoma in a 34‐year‐old female originally diagnosed as an unclassified spindle cell sarcoma with myofibroblastic differentiation and later reclassified as IMT after NGS revealed a TFG‐ROS1 rearrangement. Histologically, the neoplasm had spindle cell morphology with a lobulated to focally infiltrative growth pattern with scant inflammatory cell infiltrate. Immunohistochemistry demonstrated focal desmin and variable smooth muscle actin staining but was negative for SOX10, S100, and CD34. Fluorescence in situ hybridization was negative for USP6 or ALK gene rearrangements. NGS revealed a TFG‐ROS1 rearrangement and the patient was treated with crizotinib with clinical benefit. Conclusions We discuss the role of NGS as well as its potential benefit in patients with unresectable, ALK‐negative metastatic disease. Considering this case and previous literature, we support the use of NGS for patients requiring systemic treatment.

Background: ROS1 rearrangements (ROS1+) define a distinct molecular subset of lung adenocarcinomas. ROS1 + tumors are known to occur more in never-smokers, but the frequency and outcome of ROS1 positivity by sex and smoking intensity are not clearly documented. Patients and methods: This patient cohort study included all never- (

Introduction: C-ros oncogene 1 (ROS1) translocation is an oncogenic driver-mutation identified in 1–2% of non-small-cell lung cancer (NSCLC) cases. Although crizotinib, a tyrosine kinase inhibitor (TKI) against ALK/ROS1, is known to be effective against ROS1-fusion-positive NSCLC, such cases sometimes progress with brain metastases. The most frequently reported crizotinib-resistance mutation is ROS1 G2032R, and some studies have found that even newly developed ROS1 TKIs, such as entrectinib and lorlatinib, show a decreased efficacy against it. The optimal therapies for ROS1-fusion-positive NSCLC and how such cases can be sequenced have not yet been established. Case Presentation: We herein report a patient with ROS1-fusion-positive NSCLC diagnosed at 34 years old. Crizotinib was started at the diagnosis and switched after 25 months to cisplatin/pemetrexed/bevacizumab once the disease progressed with multiple brain metastases that were resistant to stereotactic radiation therapy. The cytotoxic chemotherapy stabilized the patient’s condition for 17 months until he developed leptomeningeal metastasis (LM). He underwent lumboperitoneal shunting and whole-brain radiotherapy, followed by crizotinib re-administration. Despite crizotinib treatment, his neurological symptoms, such as double vision, headache, weakness in the legs, and walking difficulties, progressed. Eventually, subsequent entrectinib treatment was initiated, which resolved all of the symptoms mentioned above. Regrettably, liquid next-generation sequencing had failed to detect the resistance mechanism due to minimal ctDNA in this case. Conclusion: These findings imply that sequential entrectinib administration may be effective in patients with disease progression limited to central nervous system metastases during crizotinib administration.

Abstract Background The vast majority of patients with ROS1 positive non‐small cell lung cancer (NSCLC) derive clinical benefit from currently approved ROS1 therapies, including crizotinib and entrectinib. However, a small proportion of patients treated with ROS1 inhibitors fail to derive any clinical benefit and demonstrate rapid disease progression. The biological mechanisms underpinning intrinsic resistance remain poorly understood for oncogene‐driven cancers. Methods We generated a patient‐derived cell line, CUTO33, from a ROS1 therapy naive patient with CD74‐ROS1+ NSCLC, who ultimately did not respond to a ROS1 inhibitor. We evaluated a panel of ROS1+ patient‐derived NSCLC cell lines and used cell‐based assays to determine the mechanism of intrinsic resistance to ROS1 therapy. Results The CUTO33 cell line expressed the CD74‐ROS1 gene fusion at the RNA and protein level. The ROS1 fusion protein was phosphorylated at baseline consistent with the known intrinsic activity of this oncogene. ROS1 phosphorylation could be inhibited using a wide array of ROS1 inhibitors, however these inhibitors did not block cell proliferation, confirming intrinsic resistance in this model and consistent with the patient's lack of response to a ROS1 inhibitor. CUTO33 expressed high levels of AXL, which has been associated with drug resistance. Combination of an AXL inhibitor or AXL knockdown with a ROS1 inhibitor partially reversed resistance. Conclusions In summary, we demonstrate that AXL overexpression is a mechanism of intrinsic resistance to ROS1 inhibitors.

ROS proto‐oncogene 1, receptor tyrosine kinase (ROS1) rearrangements are a crucial therapeutic target in non‐small cell lung cancer (NSCLC). However, there is limited comprehensive analysis of the molecular patterns of ROS1 fusions. This study aimed to address this gap by analysing 135 ROS1 fusions from 134 Chinese NSCLC patients using next‐generation sequencing (NGS). The fusions were categorized into common and uncommon based on their incidence. Our study revealed, for the first time, a unique distribution preference of breakpoints within ROS1, with common fusions occurring in introns 31–33 and uncommon fusions occurring in introns 34 and 35. Additionally, we identified previously unknown breakpoints within intron 28 of ROS1. Furthermore, we identified a close association between the distribution patterns of fusion partners and breakpoints on ROS1, providing important insights into the molecular landscape of ROS1 fusions. We also confirmed the presence of inconsistent breakpoints in ROS1 fusions between DNA‐based NGS and RNA‐based NGS through rigorous validation methods. These inconsistencies were attributed to alternative splicing resulting in out‐of‐frame or exonic ROS1 fusions. These findings significantly contribute to our understanding of the molecular characteristics of ROS1 fusions, which have implications for panel design and the treatment of NSCLC patients with ROS1 rearrangements.

Lorlatinib has been FDA-approved as a systemic therapy for ALK/ROS1-positive non-small cell lung cancer (NSCLC) patients. However, it has been associated with an increased frequency of neurocognitive adverse events (NAEs). Therefore, we conducted a systematic review and meta-analysis to assess the NAEs related to lorlatinib therapy in NSCLC patients. PubMed, Scopus, the Cochrane Library, and prominent conference proceedings were searched for eligible studies of lorlatinib in NSCLC patients. NAEs included cognitive, mood, speech, and psychotic effects. A total of 1147 patients from 12 studies were included; 62% had brain metastases. A pooled analysis of NAEs showed frequencies of cognitive effects of 14.57% (95% CI, 8.37 to 24.14, I2 = 84%), mood effects of 11.17% (95% CI, 5.93 to 20.07, I2 = 84%), speech effects of 7.24% (95% CI, 3.39 to 15.20, I2 = 72%), and psychotic effects of 4.97% (95% CI, 3.27 to 7.49, I2 = 21%). Clinical trials reported a significantly higher frequency of mood effects than was indicated by real-world data. These results highlight the importance of educating patients and healthcare professionals about lorlatinib-related NAEs for early detection and management to improve NSCLC patients’ quality of life.

Aim: Crizotinib, approved in Japan (2017) for ROS1-positive NSCLC, has limited real-world data. Materials & methods: Crizotinib monotherapy real-world effectiveness and treatment status were analyzed from claims data (June 2017–March 2021; Japanese Medical Data Vision; 58 patients tested for ROS1-NSCLC). Results: Median duration of treatment ([DoT]; primary end point), any line: 12.9 months; 22 patients on crizotinib, 23 discontinued, 13 receiving post-crizotinib treatment. 1L (n = 27) median DoT: 13.0 months (95% CI, 4.4–32.0 months); 13 patients on crizotinib; seven discontinued; seven receiving post-crizotinib treatment. 2L (n = 13) median DoT: 14.0 months (95% CI, 4.6–22.2 months); 2L+ (n = 31): nine patients on crizotinib; 16 discontinued; six receiving post-crizotinib treatment. Post-crizotinib treatments (chemotherapy, cancer immunotherapy, anti–VEGF/R) did not affect crizotinib DoT. Conclusion: Data supplement crizotinib's effectiveness in ROS1-positive NSCLC previously seen in clinical trials/real-world. Non-small-cell lung cancer (NSCLC) is a common type of cancer in the lung that is often caused by mutations in specific genes in the DNA. One type of NSCLC occurs when you have mutations in a gene called ROS1, whose normal function is not well understood. Crizotinib, an oral medicine, was approved in Japan for the treatment of NSCLC with mutations in ROS1 in 2017; however, this was based upon data from controlled clinical trials. This study was looking at crizotinib use in Japan based upon claims data from the Japanese Medical Data Vision database, which captures all use of medications provided in Japan. Data was collected from June 2017 to March 2021 for 58 Japanese patients who had NSCLC, tested positive for ROS1 mutations, and received crizotinib. Patients took crizotinib for a median of 13.0 months as a first treatment option and 14.0 months as a second treatment option for their NSCLC. The type of and duration of anticancer treatments given before crizotinib did not have an effect on the length of time crizotinib was used. Other treatments outside of crizotinib were given before or after crizotinib and include chemotherapy, therapy that modifies the immune system to treat cancer, or treatments that inhibit the growth of blood vessels that help the cancer grow/spread. Together, these real-world data provide evidence supporting the use of crizotinib in the treatment of patients with NSCLC and ROS1 mutations. For ROS1-positive NSCLC patients, duration of treatment (DOT) with crizotinib in real world was identified by using claims database in Japan. DOT of crizotinib for any line was 391 days (N=58). Details are here: (link of manuscript) #ROS1, #NSCLC, #RealWorldData [ABSTRACT FROM AUTHOR]

Background: The vast majority of patients with ROS1 positive non-small cell lung cancer (NSCLC) derive clinical benefit from currently approved ROS1 therapies, including crizotinib and entrectinib. However, a small proportion of patients treated with ROS1 inhibitors fail to derive any clinical benefit and demonstrate rapid disease progression. The biological mechanisms underpinning intrinsic resistance remain poorly understood for oncogene-driven cancers. Methods: We generated a patient-derived cell line, CUTO33, from a ROS1 therapy naive patient with CD74-ROS1+ NSCLC, who ultimately did not respond to a ROS1 inhibitor. We evaluated a panel of ROS1+ patient-derived NSCLC cell lines and used cell-based assays to determine the mechanism of intrinsic resistance to ROS1 therapy. Results: The CUTO33 cell line expressed the CD74-ROS1 gene fusion at the RNA and protein level. The ROS1 fusion protein was phosphorylated at baseline consistent with the known intrinsic activity of this oncogene. ROS1 phosphorylation could be inhibited using a wide array of ROS1 inhibitors, however these inhibitors did not block cell proliferation, confirming intrinsic resistance in this model and consistent with the patient’s lack of response to a ROS1 inhibitor. CUTO33 expressed high levels of AXL, which has been associated with drug resistance. Combination of an AXL inhibitor or AXL knockdown with a ROS1 inhibitor partially reversed resistance. Conclusions: In summary, we demonstrate that AXL overexpression is a mechanism of intrinsic resistance to ROS1 inhibitors. [ABSTRACT FROM AUTHOR]

ROS1 is the largest receptor tyrosine kinase in the human genome. Rearrangements of the ROS1 gene result in oncogenic ROS1 kinase fusion proteins that are currently the only validated biomarkers for targeted therapy with ROS1 TKIs in patients. While numerous somatic missense mutations in ROS1 exist in the cancer genome, their impact on catalytic activity and pathogenic potential is unknown. We interrogated the AACR Genie database and identified 34 missense mutations in the ROS1 tyrosine kinase domain for further analysis. Our experiments revealed that these mutations have varying effects on ROS1 kinase function, ranging from complete loss to significantly increased catalytic activity. Notably, Asn and Gly substitutions at Asp2113 in the ROS1 kinase domain were found to be TKI‐sensitive oncogenic variants in cell‐based model systems. In vivo experiments showed that ROS1 D2113N induced tumor formation that was sensitive to crizotinib and lorlatinib, FDA‐approved ROS1‐TKIs. Collectively, these findings highlight the tumorigenic potential of specific point mutations within the ROS1 kinase domain and their potential as therapeutic targets with FDA‐approved ROS1‐TKIs. Synopsis: The current understanding of ROS1 oncogenes is predominantly focused on ROS1 kinase fusion proteins, limiting clinical interventions with tyrosine kinase inhibitors (TKIs). Here, activating point mutations in ROS1 were investigated.D2113N and D2113G were identified as activating point mutations through functional screenings.ROS1 D2113N mutation was suggested to exert a major dynamic effect within the kinase domain by increasing the flexibility of the A‐loop in the active (DFG‐in) conformation.ROS1 D2113N and D2113G substitutions induced ROS1 TKI sensitive cell transformation in vitro, and ROS1 D2113N drove crizotinib and lorlatinib‐sensitive tumor formation in vivo.D2113N and D2113G downstream signalling effects overlapped with those of ROS1 fusion oncogenes, but were not identical.Pharmacological inhibition of signalling effectors such as SHP2 and MEK1/2 robustly inhibited ROS1 mutation driven cell proliferation. [ABSTRACT FROM AUTHOR]

ROS proto‐oncogene 1, receptor tyrosine kinase (ROS1) rearrangements are a crucial therapeutic target in non‐small cell lung cancer (NSCLC). However, there is limited comprehensive analysis of the molecular patterns of ROS1 fusions. This study aimed to address this gap by analysing 135 ROS1 fusions from 134 Chinese NSCLC patients using next‐generation sequencing (NGS). The fusions were categorized into common and uncommon based on their incidence. Our study revealed, for the first time, a unique distribution preference of breakpoints within ROS1, with common fusions occurring in introns 31–33 and uncommon fusions occurring in introns 34 and 35. Additionally, we identified previously unknown breakpoints within intron 28 of ROS1. Furthermore, we identified a close association between the distribution patterns of fusion partners and breakpoints on ROS1, providing important insights into the molecular landscape of ROS1 fusions. We also confirmed the presence of inconsistent breakpoints in ROS1 fusions between DNA‐based NGS and RNA‐based NGS through rigorous validation methods. These inconsistencies were attributed to alternative splicing resulting in out‐of‐frame or exonic ROS1 fusions. These findings significantly contribute to our understanding of the molecular characteristics of ROS1 fusions, which have implications for panel design and the treatment of NSCLC patients with ROS1 rearrangements. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammatory myofibroblastic tumor (IMT) is a distinctive tumor composed of spindle cells accompanied by mixed inflammatory cells, and immunohistochemical positivity for ALK (anaplastic lymphoma kinase protein) can be detected in half of IMTs. The diagnosis of ALK-negative IMT could be a challenge. Recently, the fusions of some kinase genes, such as RET, NTRK1, ROS1, etc., are revealed in ALK-negative IMT. Case presentation A 19-year-old woman presented with swelling of the left upper arm. Magnetic resonance imaging (MRI) scan revealed a tumor in the left postbrachium extended to the left axillary, serratus anterior muscle, and latissimus dorsi muscle. Histopathologically, the irregular-circumscribed tumor was composed of dense spindle-shaped cells with eosinophilic abundant cytoplasm and hyalinized mesenchyme in an inflammatory background. Immunohistochemically (IHC), tumor cells were positive for SMA, MDM2, and p16; the cells were negative for desmin, MyoD1, Myogenin, pan-cytokeratin, S100, SOX10, HMB45, Malen-A, CD34, CD31, CD99, and ALK. By RNA-based NGS, a novel fusion between TPD52L2 3’ end of exon 1–4 and ROS1 5’ end of exon 36–43 was revealed. ROS1 IHC staining was negative. The final diagnosis of IMT with TPD52L2-ROS1 fusion was made. Subsequently, the patient experienced a good clinical response to Crizotinib, and clinical follow-up showed stable disease after 9 months. Conclusion This report expands the spectrum of ROS1 gene rearrangements in the IMT and highlights the importance of molecular analysis of IMT for getting a diagnostic clue and determining potential therapeutic strategies.

Background: Radiomics, an evolving paradigm in medical imaging, involves the quantitative analysis of tumor features and demonstrates promise in predicting treatment responses and outcomes. This study aims to investigate the predictive capacity of radiomics for genetic alterations in non-small cell lung cancer (NSCLC). Methods: This exploratory, observational study integrated radiomic perspectives using computed tomography (CT) and genomic perspectives through next-generation sequencing (NGS) applied to liquid biopsies. Associations between radiomic features and genetic mutations were established using the Area Under the Receiver Operating Characteristic curve (AUC-ROC). Machine learning techniques, including Support Vector Machine (SVM) classification, aim to predict genetic mutations based on radiomic features. The prognostic impact of selected gene variants was assessed using Kaplan–Meier curves and Log-rank tests. Results: Sixty-six patients underwent screening, with fifty-seven being comprehensively characterized radiomically and genomically. Predominantly males (68.4%), adenocarcinoma was the prevalent histological type (73.7%). Disease staging is distributed across I/II (38.6%), III (31.6%), and IV (29.8%). Significant correlations were identified with mutations of ROS1 p.Thr145Pro (shape_Sphericity), ROS1 p.Arg167Gln (glszm_ZoneEntropy, firstorder_TotalEnergy), ROS1 p.Asp2213Asn (glszm_GrayLevelVariance, firstorder_RootMeanSquared), and ALK p.Asp1529Glu (glcm_Imc1). Patients with the ROS1 p.Thr145Pro variant demonstrated markedly shorter median survival compared to the wild-type group (9.7 months vs. not reached, p = 0.0143; HR: 5.35; 95% CI: 1.39–20.48). Conclusions: The exploration of the intersection between radiomics and cancer genetics in NSCLC is not only feasible but also holds the potential to improve genetic predictions and enhance prognostic accuracy.

Abstract Background ROS1 fusion is an infrequent, but attractive target for therapy in patients with metastatic non- small-cell lung cancer. In studies on mainly late-stage disease, the prevalence of ROS1 fusions is about 1–3%. In early-stage lung cancer ROS1 might also provide a fruitful target for neoadjuvant or adjuvant therapy. In the present study, we investigated the prevalence of ROS1 fusion in a Norwegian cohort of early-stage lung cancer. We also explored whether positive ROS1 immunohistochemical (IHC) stain was associated with certain mutations, clinical characteristics and outcomes. Methods The study was performed using biobank material from 921 lung cancer patients including 542 patients with adenocarcinoma surgically resected during 2006–2018. Initially, we screened the samples with two different IHC clones (D4D6 and SP384) targeting ROS1. All samples that showed more than weak or focal staining, as well as a subgroup of negative samples, were analyzed with ROS1 fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) with a comprehensive NGS DNA and RNA panel. Positive ROS1-fusion was defined as those samples positive in at least two of the three methods (IHC, FISH, NGS). Results Fifty cases were IHC positive. Of these, three samples were both NGS and FISH-positive and considered positive for ROS1 fusion. Two more samples were FISH positive only, and whilst IHC and NGS were negative. These were also negative with Reverse Transcription quantitative real time Polymerase Chain Reaction (RT-qPCR). The prevalence of ROS1 fusion in adenocarcinomas was 0.6%. All cases with ROS1 fusion had TP53 mutations. IHC-positivity was associated with adenocarcinoma. Among SP384-IHC positive cases we also found an association with never smoking status. There was no association between positive IHC and overall survival, time to relapse, age, stage, sex or pack-year of smoking. Conclusions ROS1 seems to be less frequent in early-stage disease than in advanced stages. IHC is a sensitive, but less specific method and the results need to be confirmed with another method like FISH or NGS.

Background: Inflammatory myofibroblastic tumor (IMT) is a distinctive tumor composed of spindle cells accompanied by mixed inflammatory cells, and immunohistochemical positivity for ALK (anaplastic lymphoma kinase protein) can be detected in half of IMTs. The diagnosis of ALK-negative IMT could be a challenge. Recently, the fusions of some kinase genes, such as RET, NTRK1, ROS1, etc., are revealed in ALK-negative IMT. Case presentation: A 19-year-old woman presented with swelling of the left upper arm. Magnetic resonance imaging (MRI) scan revealed a tumor in the left postbrachium extended to the left axillary, serratus anterior muscle, and latissimus dorsi muscle. Histopathologically, the irregular-circumscribed tumor was composed of dense spindle-shaped cells with eosinophilic abundant cytoplasm and hyalinized mesenchyme in an inflammatory background. Immunohistochemically (IHC), tumor cells were positive for SMA, MDM2, and p16; the cells were negative for desmin, MyoD1, Myogenin, pan-cytokeratin, S100, SOX10, HMB45, Malen-A, CD34, CD31, CD99, and ALK. By RNA-based NGS, a novel fusion between TPD52L2 3' end of exon 1–4 and ROS1 5' end of exon 36–43 was revealed. ROS1 IHC staining was negative. The final diagnosis of IMT with TPD52L2-ROS1 fusion was made. Subsequently, the patient experienced a good clinical response to Crizotinib, and clinical follow-up showed stable disease after 9 months. Conclusion: This report expands the spectrum of ROS1 gene rearrangements in the IMT and highlights the importance of molecular analysis of IMT for getting a diagnostic clue and determining potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

Abstract ROS1 is the largest receptor tyrosine kinase in the human genome. Rearrangements of the ROS1 gene result in oncogenic ROS1 kinase fusion proteins that are currently the only validated biomarkers for targeted therapy with ROS1 TKIs in patients. While numerous somatic missense mutations in ROS1 exist in the cancer genome, their impact on catalytic activity and pathogenic potential is unknown. We interrogated the AACR Genie database and identified 34 missense mutations in the ROS1 tyrosine kinase domain for further analysis. Our experiments revealed that these mutations have varying effects on ROS1 kinase function, ranging from complete loss to significantly increased catalytic activity. Notably, Asn and Gly substitutions at Asp2113 in the ROS1 kinase domain were found to be TKI‐sensitive oncogenic variants in cell‐based model systems. In vivo experiments showed that ROS1 D2113N induced tumor formation that was sensitive to crizotinib and lorlatinib, FDA‐approved ROS1‐TKIs. Collectively, these findings highlight the tumorigenic potential of specific point mutations within the ROS1 kinase domain and their potential as therapeutic targets with FDA‐approved ROS1‐TKIs.

ROS proto-oncogene 1 (ROS1) rearrangements occur in 0.9–2.6% of patients with non small cell lung cancer (NSCLC), conferring sensitivity to treatment with specific tyrosine-kinase inhibitors (TKI). Crizotinib, a first-generation TKI, was the first target-therapy approved for the first-line treatment of ROS1-positive NSCLC. Recently, entrectinib, a multitarget inhibitor with an anti-ROS1 activity 40 times more potent than crizotinib and better activity on the central nervous system (CNS), received approval for treatment-naive patients. After a median time-to-progression of 5.5–20 months, resistance mechanisms can occur, leading to tumor progression. Therefore, newer generation TKI with greater potency and brain penetration have been developed and are currently under investigation. This review summarizes the current knowledge on clinicopathological characteristics of ROS1-positive NSCLC and its therapeutic options. [ABSTRACT FROM AUTHOR]

Abstract Background Entrectinib is an effective drug for treating solid tumors with NTRK gene rearrangement and non‐small cell lung cancer (NSCLC) with ROS1 gene rearrangement. However, its efficacy is limited by tolerance and acquired resistance, the mechanisms of which are not fully understood. The growth factors produced by the tumor microenvironment, including hepatocyte growth factor (HGF) produced by tumor‐associated fibroblasts, critically affect the sensitivity to targeted drugs. Methods We investigated whether growth factors that can be produced by the microenvironment affect sensitivity of NTRK1‐rearranged colon cancer KM12SM cells and ROS1‐rearranged NSCLC HCC78 cells to entrectinib both in vitro and in vivo. Results Among the growth factors assessed, HGF most potently induced entrectinib resistance in KM12SM and HCC78 cells by activating its receptor MET. HGF‐induced entrectinib resistance was reversed by the active‐HGF‐specific macrocyclic peptide HiP‐8 and the MET kinase inhibitor capmatinib in vitro. In addition, HGF‐producing fibroblasts promoted entrectinib resistance in vitro (culture model) and in vivo (subcutaneous tumor model). The use of capmatinib circumvented entrectinib resistance in a subcutaneous tumor model inoculated with KM12SM and HGF‐producing fibroblasts. Conclusion Our findings suggest that growth factors in the tumor microenvironment, such as HGF, may induce resistance to entrectinib in tumors with NTRK1 or ROS1 rearrangements. Our results further suggest that optimally co‐administering inhibitors of resistance‐inducing growth factors may maximize the therapeutic efficacy of entrectinib.

Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5–7%, 1–2%, and 1–2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability.

Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies.

Real-world data on the use and outcomes of crizotinib in ROS1-rearranged non-small-cell lung cancer (NSCLC) are limited. This study aims to analyze the real-world efficacy of crizotinib in South Korea and explore the utilization of liquid biopsies that implement next-generation sequencing (NGS) using cell-free total nucleic acids. In this prospective multicenter cohort study, 40 patients with ROS1-rearranged NSCLC, either starting or already on crizotinib, were enrolled. Patients had a median age of 61 years, with 32.5% presenting brain/central nervous system (CNS) metastases at treatment initiation. At the data cutoff, 48.0% were still in treatment; four continued with it even after disease progression due to the clinical benefits. The objective response rate was 70.0%, with a median duration of response of 27.8 months. The median progression-free survival was 24.1 months, while the median overall survival was not reached. Adverse events occurred in 90.0% of patients, primarily with elevated transaminases, yet these were mostly manageable. The NGS assay detected a CD74–ROS1 fusion in 2 of the 14 patients at treatment initiation and identified emerging mutations, such as ROS1 G2032R, ROS1 D2033N, and KRAS G12D, during disease progression. These findings confirm crizotinib’s sustained clinical efficacy and safety in a real-world context, which was characterized by a higher elderly population and higher rates of brain/CNS metastases. The study highlights the clinical relevance of liquid biopsy for detecting resistance mechanisms, suggesting its value in personalized treatment strategies.

Introduction: Treatment with lorlatinib for patients with advanced ALK- and ROS1-rearranged NSCLC (ALK+ and ROS1+ NSCLC) is associated with a unique set of adverse events (AEs) often requiring dose reduction. However, the impact of dose reductions on outcomes remains unclear and is mainly limited to analyses from prospective studies of lorlatinib in the first-line setting. Methods: We reviewed the course of 144 patients with advanced ALK- or ROS1-rearranged NSCLC treated with lorlatinib in the second-line or later setting to assess the frequency of dose reductions resulting from treatment-related AEs (TRAEs) and the association between dose reductions and progression-free survival (PFS) and overall survival (OS). Results: A total of 58 patients (40%) had TRAE-related dose reductions, most (59%) owing to neurocognitive AEs or neuropathy. Among all patients, the median PFS was 8.1 months (95% confidence interval [CI]: 6.4–11.8); the median OS was 20.7 months (95% CI: 16.3–30.5). Among patients who were started on lorlatinib 100 mg/d (n = 122), a Cox regression model with the occurrence of a dose reduction as a time-dependent covariate indicated no association between dose reduction and PFS (hazard ratio = 0.86, 95% CI: 0.54–1.39) or OS (hazard ratio = 0.78, 95% CI: 0.47–1.30). Conclusions: Lorlatinib dose reductions were not associated with inferior clinical outcomes in this multicenter analysis. Prompt identification of lorlatinib TRAEs and implementation of dose reductions may help maximize tolerability without compromising outcomes.

Introduction: Translocations of the ROS1 gene were found to drive tumorigenesis in 1% to 2% of lung adenocarcinoma. In clinical practice, ROS1 rearrangements are often screened by immunohistochemistry (IHC) before confirmation with either fluorescence in situ hybridization or molecular techniques. This screening test leads to a non-negligible number of cases that have equivocal or positive ROS1 IHC, without ROS1 translocation. Methods: In this study, we have analyzed retrospectively 1021 cases of nonsquamous NSCLC having both ROS1 IHC and molecular analysis using next-generation sequencing. Results: ROS1 IHC was negative in 938 cases (91.9%), equivocal in 65 cases (6.4%), and positive in 18 cases (1.7%). Among these 83 equivocal or positive cases, only two were ROS1 rearranged, leading to a low predictive positive value of the IHC test (2%). ROS1-positive IHC was correlated with an increased mRNA ROS1 transcripts. Moreover, we have found a mean statistically significant relationship between ROS1 expression and EGFR gene mutations, suggesting a crosstalk mechanism between these oncogenic driver molecules. Conclusion: This study demonstrates that ROS1 IHC represents true ROS1 mRNA expression, and raises the question of a potential benefit of combined targeted therapy in EGFR-mutated NSCLC.

Background: ROS1 fusion is an infrequent, but attractive target for therapy in patients with metastatic non- small-cell lung cancer. In studies on mainly late-stage disease, the prevalence of ROS1 fusions is about 1–3%. In early-stage lung cancer ROS1 might also provide a fruitful target for neoadjuvant or adjuvant therapy. In the present study, we investigated the prevalence of ROS1 fusion in a Norwegian cohort of early-stage lung cancer. We also explored whether positive ROS1 immunohistochemical (IHC) stain was associated with certain mutations, clinical characteristics and outcomes. Methods: The study was performed using biobank material from 921 lung cancer patients including 542 patients with adenocarcinoma surgically resected during 2006–2018. Initially, we screened the samples with two different IHC clones (D4D6 and SP384) targeting ROS1. All samples that showed more than weak or focal staining, as well as a subgroup of negative samples, were analyzed with ROS1 fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) with a comprehensive NGS DNA and RNA panel. Positive ROS1-fusion was defined as those samples positive in at least two of the three methods (IHC, FISH, NGS). Results: Fifty cases were IHC positive. Of these, three samples were both NGS and FISH-positive and considered positive for ROS1 fusion. Two more samples were FISH positive only, and whilst IHC and NGS were negative. These were also negative with Reverse Transcription quantitative real time Polymerase Chain Reaction (RT-qPCR). The prevalence of ROS1 fusion in adenocarcinomas was 0.6%. All cases with ROS1 fusion had TP53 mutations. IHC-positivity was associated with adenocarcinoma. Among SP384-IHC positive cases we also found an association with never smoking status. There was no association between positive IHC and overall survival, time to relapse, age, stage, sex or pack-year of smoking. Conclusions: ROS1 seems to be less frequent in early-stage disease than in advanced stages. IHC is a sensitive, but less specific method and the results need to be confirmed with another method like FISH or NGS. [ABSTRACT FROM AUTHOR]

Key Clinical Message: In conclusion author highlights the tumor cell genetic testing or molecular pathological diagnosis plays a key role in the individualized treatment of PSC, which could benefit patients with advanced PSC. An uncommon form of non‐small‐cell lung cancer (NSCLC) with a poor prognosis is pulmonary sarcomatoid carcinoma (PSC). Surgical resection is currently the preferred treatment, but guidelines for adjuvant chemotherapy have not yet been established, especially for the advanced stage. The development of molecular subgroups in the field of tumors may be advantageous to advanced PSC patients with the ongoing progress of genomics and immunology. A 54‐year‐old man presented to Xishan People's Hospital of Wuxi City with recurrent intermittent dry cough with fever for 1 month. Further examinations suggested the diagnosis of PSC occupying almost the entire right interlobar fissure area combined with malignant pleural effusion (Stage IVa). Pathological examination confirmed the diagnosis of PSC with ROS1 overexpressing via genetic testing. However, after three cycles of chemo‐, antiangiogenetic‐ and immunochemical therapy, the lesion was localized, and pleural effusion disappeared, the patient subsequently received an operation which was performed as R0 resection. Unfortunately, the patient became deteriorated quickly followed by extensive metastatic nodules in the thoracic cavity. Although the patient continued to receive chemo‐ and immunochemical‐therapy, it did not limit the progress of the tumor, leading to widespread metastasis, and eventually died of multiple organ failure. For PSC patients with Stage IVa, chemo‐, antiangiogenetic‐ and immunochemical‐therapy performs well in clinical efficacy, and comprehensive panel‐based genetic testing may offer PSC patients a somewhat better prognosis. However, blindly implementing surgical treatment may bring harm to the patient and affect long‐term survival. It's essential to know the surgical indications precisely by NSCLC guidelines. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) presents the greatest number of identified therapeutic targets, some of which have therapeutic utility. Currently, detecting EGFR, BRAF, KRAS and MET mutations, ALK, ROS1, NTRK and RET translocations, and PD-L1 expression in these patients is considered essential. The use of next-generation sequencing facilitates precise molecular diagnosis and allows the detection of other emerging mutations, such as the HER2 mutation and predictive biomarkers for immunotherapy responses. In this consensus, a group of experts in the diagnosis and treatment of NSCLC selected by the Spanish Society of Pathology and the Spanish Society of Medical Oncology have evaluated currently available information and propose a series of recommendations to optimize the detection and use of biomarkers in daily clinical practice. [ABSTRACT FROM AUTHOR]