Discovery of oncogenic ROS1 missense mutations with sensitivity to tyrosine kinase inhibitors.

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.

Discovery and functional characterization of the oncogenicity and targetability of a novel NOTCH1-ROS1 gene fusion in pediatric angiosarcoma.

Angiosarcomas are rare, malignant soft tissue tumors in children that arise in a wide range of anatomical locations and have limited targeted therapies available. Here, we report a rare case of a pediatric angiosarcoma (pAS) with Li-Fraumeni syndrome (LFS) expressing a novel NOTCH1-ROS1 gene fusion. Although both NOTCH1 and ROS1 are established proto-oncogenes, our study is the first to describe the mechanistic role of NOTCH1-ROS1 fusion arising via intrachromosomal rearrangement. NOTCH1-ROS1 displayed potent neoplastic transformation propensity in vitro, and harbors tumorigenic potential in vivo, where it induced oncogenic activation of the MAPK, PI3K/mTOR, and JAK-STAT signaling pathways in a murine allograft model. We found an unexpected contribution of the NOTCH1 extracellular region in mediating NOTCH1-ROS1 activation and oncogenic function, highlighting the contribution of both NOTCH1 and ROS1 fusion partners in driving tumorigenicity. Interestingly, neither membrane localization nor fusion protein dimerization were found to be essential for NOTCH1-ROS1 fusion oncogenicity. To target NOTCH1-ROS1-driven tumors, we tested both NOTCH1-directed inhibitors and ROS1-targeted tyrosine kinase inhibitors (TKI) in heterologous models (NIH3T3, Ba/F3). Although NOTCH1 inhibitors did not suppress NOTCH1-ROS1-driven oncogenic growth, we found that oral entrectinib treatment effectively suppressed the growth of NOTCH-ROS1-driven tumors. Taken together, we report the first known pAS case with a novel NOTCH1-ROS1 alteration along with a detailed report on the function and therapeutic targeting of NOTCH1-ROS1. Our study highlights the importance of genomic profiling of rare cancers such as pAS to reveal actionable drivers and improve patient outcomes.

MYC Promotes Tyrosine Kinase Inhibitor Resistance in ROS1-Fusion-Positive Lung Cancer.

Targeted therapy of ROS1-fusion-driven non-small cell lung cancer (NSCLC) has achieved notable clinical success. Despite this, resistance to therapy inevitably poses a significant challenge. MYC amplification was present in ∼19% of lorlatinib-resistant ROS1-driven NSCLC. We hypothesized that MYC overexpression drives ROS1-TKI resistance. Using complementary approaches in multiple models, including a MYC-amplified patient-derived cell line and xenograft (LUAD-0006), we established that MYC overexpression induces broad ROS1-TKI resistance. Pharmacologic inhibition of ROS1 combined with MYC knockdown were essential to completely suppress LUAD-0006 cell proliferation compared with either treatment alone. We interrogated cellular signaling in ROS1-TKI-resistant LUAD-0006 and discovered significant differential regulation of targets associated with cell cycle, apoptosis, and mitochondrial function. Combinatorial treatment of mitochondrial inhibitors with crizotinib revealed inhibitory synergism, suggesting increased reliance on glutamine metabolism and fatty-acid synthesis in chronic ROS1-TKI treated LUAD-0006 cells. In vitro experiments further revealed that CDK4/6 and BET bromodomain inhibitors effectively mitigate ROS1-TKI resistance in MYC-overexpressing cells. Notably, in vivo studies demonstrate that tumor control may be regained by combining ROS1-TKI and CDK4/6 inhibition. Our results contribute to the broader understanding of ROS1-TKI resistance in NSCLC. IMPLICATIONS: This study functionally characterizes MYC overexpression as a novel form of therapeutic resistance to ROS1 tyrosine kinase inhibitors in non-small cell lung cancer and proposes rational combination treatment strategies.

Association of Preoperative Hemoglobin A1c and Body Mass Index with Wound Infection Rate in Spinal Surgery.

BACKGROUND: The deleterious effect of diabetes mellitus on surgical outcomes is well documented for joint replacement surgery. We analyzed the large national US Department of Veterans Affairs (VA) database for patients who had undergone elective spinal surgery. METHODS: We retrospectively searched the VA database and identified 174 520 spine cases. RESULTS: There were 7766 (4.5%) wound infections and 49 271 (28%) had hemoglobin A1c (HbA1c) testing (range: 3.0-17.8) prior to surgery. In the preoperative HbA1c-checked group, there were 2941 (6.0% of 49 271) infections and in the without-preoperative HbA1c group, there were 4825 (3.9% of 125 249) infections. The distribution of infections was significantly different (χ2 = 372.577, P < .0001) and suggests a 2.12% increase in the absolute risk of infection based on the presence of preoperative HbA1c testing, regardless of the result. Logistic regression revealed a preoperative HbA1c test was associated with an odds ratio of 1.435 for infection (confidence interval 1.367-1.505, P < .0001). In a separate model based on HbA1c levels, we found that HbA1c is a significant predictor of infection with an odds ratio of 1.042 (confidence interval 1.017-1.068, P = .0009) for each 1% increase in the test result. This analysis differs from using a strict cutoff value of HbA1c of 6.5%. Similar testing for body mass index and age yielded an odds ratio of 1.027 for each increase of 1 kg/m2 and an odds ratio of 1.009 for each 1-year increase in age respectively. CONCLUSIONS: Hemoglobin A1c testing, HgA1c value, body mass index, and age all contribute to the risk of wound infection after elective spine surgery in a large national VA population. These data can be used to estimate surgical risks and to aid in patient counseling about proposed spine surgery. LEVEL OF EVIDENCE: 4.

Trends in Weight and Body Mass Index after Spinal Surgery for Degenerative Disease.

BACKGROUND: Currently, 37% of adults in the United States are obese, and 34% are overweight. Obesity poses a particularly complex challenge in spinal surgery management, whereby risk of adverse surgical outcomes increases with increased body mass index (BMI). When patients are counseled to reduce weight to levels associated with acceptable surgical risks, patients often respond that their spinal problems prohibit the exercise needed to lose the required weight and counter that surgery will allow for increased activity and thereby facilitate weight loss. A retrospective study of a US Veterans Affairs (USVA) nationwide patient database was undertaken. METHODS: A request was made of the USVA Corporate Data Warehouse for data on all patients undergoing elective spine surgery for degenerative conditions over a 10-year period. RESULTS: The mean preoperative age of 65 667 patients identified was 59 years. The mean preoperative weight was 91.8 kg, and BMI was 29.2. Before surgery, 26 772 patients had a BMI of >30. After surgery, 12 564 (46.9%) lost at least 2.3 kg, 9450 (35.3%) gained at least 2.3 kg, and 4758 (17.8%) were unchanged. After surgery, 4853 (18.1%) lost at least 11.3 kg and 1360 (5.1%) lost at least 22.7 kg. At a mean of 1.9 years after index surgery, mean postoperative weight was 92.5 kg, and BMI was 29.4. Of the 65 667 patients, 23 125 (35.2%) patients lost at least 2.3 kg, 27 571 (42.0%) gained at least 2.3 kg, and 14 971 (23.0%) remained within 2.3 kg of their preoperative weight. CONCLUSION: The study results will aid in counseling patients regarding realistic expectations about weight loss after spinal surgery. LEVEL OF EVIDENCE: 4. CLINICAL RELEVANCE: This evidence will allow for realistic patient counseling regarding the likelihood of weight loss after elective spinal surgery.

ROS1-dependent cancers - biology, diagnostics and therapeutics.

The proto-oncogene ROS1 encodes a receptor tyrosine kinase with an unknown physiological role in humans. Somatic chromosomal fusions involving ROS1 produce chimeric oncoproteins that drive a diverse range of cancers in adult and paediatric patients. ROS1-directed tyrosine kinase inhibitors (TKIs) are therapeutically active against these cancers, although only early-generation multikinase inhibitors have been granted regulatory approval, specifically for the treatment of ROS1 fusion-positive non-small-cell lung cancers; histology-agnostic approvals have yet to be granted. Intrinsic or extrinsic mechanisms of resistance to ROS1 TKIs can emerge in patients. Potential factors that influence resistance acquisition include the subcellular localization of the particular ROS1 oncoprotein and the TKI properties such as the preferential kinase conformation engaged and the spectrum of targets beyond ROS1. Importantly, the polyclonal nature of resistance remains underexplored. Higher-affinity next-generation ROS1 TKIs developed to have improved intracranial activity and to mitigate ROS1-intrinsic resistance mechanisms have demonstrated clinical efficacy in these regards, thus highlighting the utility of sequential ROS1 TKI therapy. Selective ROS1 inhibitors have yet to be developed, and thus the specific adverse effects of ROS1 inhibition cannot be deconvoluted from the toxicity profiles of the available multikinase inhibitors. Herein, we discuss the non-malignant and malignant biology of ROS1, the diagnostic challenges that ROS1 fusions present and the strategies to target ROS1 fusion proteins in both treatment-naive and acquired-resistance settings.

Rare but Recurrent ROS1 Fusions Resulting From Chromosome 6q22 Microdeletions are Targetable Oncogenes in Glioma.

PURPOSE: Gliomas, a genetically heterogeneous group of primary central nervous system tumors, continue to pose a significant clinical challenge. Discovery of chromosomal rearrangements involving kinase genes has enabled precision therapy, and improved outcomes in several malignancies. EXPERIMENTAL DESIGN: Positing that similar benefit could be accomplished for patients with brain cancer, we evaluated The Cancer Genome Atlas (TCGA) glioblastoma dataset. Functional validation of the oncogenic potential and inhibitory sensitivity of discovered ROS1 fusions was performed using three independent cell-based model systems, and an in vivo murine xenograft study. RESULTS: In silico analysis revealed previously unreported intrachromosomal 6q22 microdeletions that generate ROS1-fusions from TCGA glioblastoma dataset. ROS1 fusions in primary glioma and ependymoma were independently corroborated from MSK-IMPACT and Foundation Medicine clinical datasets. GOPC-ROS1 is a recurrent ROS1 fusion in primary central nervous system (CNS) tumors. CEP85L-ROS1 and GOPC-ROS1 are transforming oncogenes in cells of astrocytic lineage, and amenable to pharmacologic inhibition with several ROS1 inhibitors even when occurring concurrently with other cancer hotspot aberrations frequently associated with glioblastoma. Oral monotherapy with a brain-permeable ROS1 inhibitor, lorlatinib, significantly prolonged survival in an intracranially xenografted tumor model generated from a ROS1 fusion-positive glioblastoma cell line. CONCLUSIONS: Our findings highlight that CNS tumors should be specifically interrogated for these rare intrachromosomal 6q22 microdeletion events that generate actionable ROS1 fusions. ROS1 fusions in primary brain cancer may be amenable for clinical intervention with kinase inhibitors, and this holds the potential of novel treatment paradigms in these treatment-refractory cancer types, particularly in glioblastoma.

Spinal intradural teratomas: developmental programs gone awry?

Intradural spinal teratomas are rare tumors of the spinal cord that are infrequently encountered in children. Although the mechanistic basis for the formation of these tumors is unclear, several lines of evidence suggest that a dysembryogenic process in the embryo results in their formation. The authors present a case of spinal intradural teratoma in an 18-year-old, previously healthy man and review the literature linking the development of these tumors to defects in neurulation and embryogenesis.