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Cannabidiol (CBD)-containing supplements are used by children with cerebral palsy (CP), but the prevalence and efficacy of their use have not been studied. We sought to describe CBD use patterns and perceived efficacy in the pediatric population with CP, evaluating any association between CBD use and health-related quality of life. Patients with CP were prospectively enrolled, and caregivers were offered the Caregiver Priorities and Child Health Index of Life with Disabilities (CPCHILD) Questionnaire and a survey assessing CBD use. Of 119 participants, 20 (16.8%) endorsed CBD use (CBD+) and 99 (83.2%) denied it (CBD−). Participants in the CBD+ group had worse functional status (85% Gross Motor Function Classification System level IV–V for CBD+ vs 37.4% for CBD−, P P =.001). Spasticity was the rationale most cited for CBD use (29%), followed by pain and anxiety (both 22.6%). CBD was perceived to be most effective for improving emotional health, spasticity, and pain. Fifty percent of the patients in the CBD+ group underwent surgery in the previous 2 years and most endorsed a general benefit in the postoperative setting. The most common side effects noted were fatigue and increased appetite (both 12%). Most participants endorsed no side effects (60%). CBD may serve as a useful adjunct for some children with CP, especially those with worse disease severity. Caregivers perceive CBD as offering some benefits, particularly in the domains of emotional health, spasticity, and pain. We found no evidence of severe adverse events in our small cohort. [ Orthopedics . 202x;4x(x):xx–xx.]

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Methods for quantitative immunofluorescence and IFN gamma treatment of PDXs

Supplementary Methods on Tuba-seq analysis

In lung adenocarcinoma, oncogenic EGFR mutations co-occur with many tumor suppressor gene alterations; however, the extent to which these contribute to tumor growth and response to therapy in vivo remains largely unknown. By quantifying the effects of inactivating 10 putative tumor suppressor genes in a mouse model of EGFR-driven Trp53-deficient lung adenocarcinoma, we found that Apc, Rb1, or Rbm10 inactivation strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2—the strongest drivers of growth in a KRAS-driven model—reduced EGFR-driven tumor growth. These results are consistent with mutational frequencies in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, KEAP1 inactivation reduced the sensitivity of EGFR-driven tumors to the EGFR inhibitor osimertinib, and mutations in genes in the KEAP1 pathway were associated with decreased time on tyrosine kinase inhibitor treatment in patients. Our study highlights how the impact of genetic alterations differs across oncogenic contexts and that the fitness landscape shifts upon treatment.Significance:By modeling complex genotypes in vivo, this study reveals key tumor suppressors that constrain the growth of EGFR-mutant tumors. Furthermore, we uncovered that KEAP1 inactivation reduces the sensitivity of these tumors to tyrosine kinase inhibitors. Thus, our approach identifies genotypes of biological and therapeutic importance in this disease.This article is highlighted in the In This Issue feature, p. 1601

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In lung adenocarcinoma, oncogenic EGFR mutations co-occur with many tumor suppressor gene alterations; however, the extent to which these contribute to tumor growth and response to therapy in vivo remains largely unknown. By quantifying the effects of inactivating 10 putative tumor suppressor genes in a mouse model of EGFR-driven Trp53-deficient lung adenocarcinoma, we found that Apc, Rb1, or Rbm10 inactivation strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2—the strongest drivers of growth in a KRAS-driven model—reduced EGFR-driven tumor growth. These results are consistent with mutational frequencies in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, KEAP1 inactivation reduced the sensitivity of EGFR-driven tumors to the EGFR inhibitor osimertinib, and mutations in genes in the KEAP1 pathway were associated with decreased time on tyrosine kinase inhibitor treatment in patients. Our study highlights how the impact of genetic alterations differs across oncogenic contexts and that the fitness landscape shifts upon treatment. Significance: By modeling complex genotypes in vivo, this study reveals key tumor suppressors that constrain the growth of EGFR-mutant tumors. Furthermore, we uncovered that KEAP1 inactivation reduces the sensitivity of these tumors to tyrosine kinase inhibitors. Thus, our approach identifies genotypes of biological and therapeutic importance in this disease. This article is highlighted in the In This Issue feature, p. 1601

Background Although EGFR mutant tumors exhibit low response rates to immune checkpoint blockade overall, some EGFR mutant tumors do respond to these therapies; however, there is a lack of understanding of the characteristics of EGFR mutant lung tumors responsive to immune checkpoint blockade. Patients and methods We retrospectively analyzed de-identified clinical and molecular data on 171 cases of EGFR mutant lung tumors treated with immune checkpoint inhibitors from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, University of California Los Angeles, and Dana Farber Cancer Institute. A separate cohort of 383 EGFR mutant lung cancer cases with sequencing data available from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, and The Cancer Genome Atlas was compiled to assess the relationship between tumor mutation burden and specific EGFR alterations. Results Compared with 212 EGFR wild-type lung cancers, outcomes with programmed cell death 1 or programmed death-ligand 1 (PD-(L)1) blockade were worse in patients with lung tumors harboring alterations in exon 19 of EGFR (EGFRΔ19) but similar for EGFRL858R lung tumors. EGFRT790M status and PD-L1 expression did not impact response or survival outcomes to immune checkpoint blockade. PD-L1 expression was similar across EGFR alleles. Lung tumors with EGFRΔ19 alterations harbored a lower tumor mutation burden compared with EGFRL858R lung tumors despite similar smoking history. Conclusions EGFR mutant tumors have generally low response to immune checkpoint inhibitors, but outcomes vary by allele. Understanding the heterogeneity of EGFR mutant tumors may be informative for establishing the benefits and uses of PD-(L)1 therapies for patients with this disease.

Cancer genome sequencing has uncovered substantial complexity in the mutational landscape of tumors. Given this complexity, experimental approaches are necessary to establish the impact of combinations of genetic alterations on tumor biology and to uncover genotype-dependent effects on drug sensitivity. In lung adenocarcinoma, EGFR mutations co-occur with many putative tumor suppressor gene alterations, however the extent to which these alterations contribute to tumor growth and their response to therapy in vivo has not been explored experimentally. By integrating a novel mouse model of oncogenic EGFR-driven Trp53-deficient lung adenocarcinoma with multiplexed CRISPR–Cas9-mediated genome editing and tumor barcode sequencing, we quantified the effects of inactivation of ten putative tumor suppressor genes. Inactivation of Apc, Rb1, or Rbm10 most strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2 – which are the strongest drivers of tumor growth in an oncogenic Kras-driven model – reduced EGFR-driven tumor growth. These results are consistent with the relative frequency of these tumor suppressor gene alterations in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, Keap1 inactivation reduces the sensitivity of EGFR-driven Trp53-deficient tumors to the EGFR inhibitor osimertinib. Importantly, in human EGFR/TP53 mutant lung adenocarcinomas, mutations in the KEAP1 pathway correlated with decreased time on tyrosine kinase inhibitor treatment. Our study highlights how genetic alterations can have dramatically different biological consequences depending on the oncogenic context and that the fitness landscape can shift upon drug treatment.

Background: The Female Athlete Triad (Triad) is the interrelationship of low energy availability, reproductive irregularities, and low bone mineral density. It is theorized that mental health illness can both contribute to and result from low energy availability. Limited data exists that describes the relationship between the Female Athlete Triad and mental health symptoms. Hypothesis/Purpose: The purpose of this study was to investigate the relationship between the Triad and mental health illness, specifically depression and anxiety symptoms, in high school athletes. Methods: This was a cross-sectional survey study of high school athletes distributed via social media with responses accepted from 12/30/20 – 2/15/21. Data was collected including age, sex, training history, menstrual history, history of bone stress injuries, and eating behaviors. The Female Athlete Triad Coalition Consensus Statement Cumulative Risk Assessment Tool was used to score athletes (range: 0-10 points) and to classify them as low-, moderate-, or high-risk for Female Athlete Triad. Mental health was assessed using the Patient-Reported Outcomes Measurement Information System (PROMIS) Emotional Distress Depression and Anxiety short form questionnaires. Mental health scores were scored as none, mild, moderate, or severe self-reported depressive or anxious symptoms. Ordinal logistic regressions were performed. Results: There were 78 self-reported female high school participants (mean age = 16). Depression symptoms were reported in 74% of participants (19% mild, 40% moderate, 15% severe). Anxiety symptoms were reported in 64% of participants (18% mild, 26% moderate, 21% severe). Triad risk assessment responses categorized participants as 45% low risk, 41% moderate risk, and 14% high risk of developing the Triad. Depression symptoms were significantly associated with increasing Triad risk assessment score: Odds Ratio (per 1 point Triad risk score) = 1.245 (1.004 – 1.544) ( p = .0462). Anxiety symptoms were also significantly associated with increasing Triad risk assessment score: Odds Ratio (per 1 point Triad risk score) = 1.264 (1.020 – 1.567) ( p = .0319). Conclusion: Anxiety and depression symptoms were both significantly associated with increasing Triad risk scores in this preliminary cross-sectional study. Notably, this study is limited by sampling bias. Further research is needed to explore the relationship between mental health illness and the Female Athlete Triad.

Mechanisms of acquired resistance to immune checkpoint inhibitors (ICI) are poorly understood. We leveraged a collection of 14 ICI-resistant lung cancer samples to investigate whether alterations in genes encoding HLA Class I antigen processing and presentation machinery (APM) components or interferon signaling play a role in acquired resistance to PD-1 or PD-L1 antagonistic antibodies. Recurrent mutations or copy-number changes were not detected in our cohort. In one case, we found acquired homozygous loss of B2M that caused lack of cell-surface HLA Class I expression in the tumor and a matched patient-derived xenograft (PDX). Downregulation of B2M was also found in two additional PDXs established from ICI-resistant tumors. CRISPR-mediated knockout of B2m in an immunocompetent lung cancer mouse model conferred resistance to PD-1 blockade in vivo, proving its role in resistance to ICIs. These results indicate that HLA Class I APM disruption can mediate escape from ICIs in lung cancer. Significance: As programmed death 1 axis inhibitors are becoming more established in standard treatment algorithms for diverse malignancies, acquired resistance to these therapies is increasingly being encountered. Here, we found that defective antigen processing and presentation can serve as a mechanism of such resistance in lung cancer. Cancer Discov; 7(12); 1420–35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1355

Immune checkpoint inhibitors (ICIs) that block negative regulators of T cell function can generate lasting responses in lung cancer patients, yet only a minority of tumors respond to therapy and ~50% of patients whose tumors initially respond eventually develop acquired resistance. Mechanisms of acquired resistance to ICIs in lung cancer are not fully understood and strategies to overcome resistance are lacking. To address this challenge, we developed a new mouse model of acquired resistance to ICIs by knocking out the DNA mismatch repair gene Msh2 in Kras mutant mouse lung adenocarcinoma cells, leading to a higher mutation burden in these cells. Unlike Msh2 WT controls, Msh2 KO tumors transplanted into syngeneic mice are sensitive to ICIs. Importantly, chronic treatment with αPD-1 or αPD-1 + αCTLA-4 led to Msh2 KO tumors that initially responded to therapy but then developed acquired resistance. Tumors remained resistant to ICIs upon transplantation to new hosts, suggesting that tumor-intrinsic programs drive resistance. Gene set enrichment analysis (GSEA) of single cell RNA sequencing data revealed that tumors with acquired resistance to ICIs downregulated antigen processing and presentation gene signatures compared to tumors that were regressing on therapy. Specifically, tumor cells with acquired resistance downregulated expression of MHC class II, which was confirmed by flow cytometry, and this was accompanied by a decrease in expression of Stat1, Irf1, and Ciita, which drive MHC-II expression in response to IFNγ. Depletion of CD4+ T cells during αPD-1 + αCTLA-4 treatment abrogated the response to therapy, suggesting a functional role for tumor cell MHC-II expression. GSEA also revealed that tumors with acquired resistance to ICIs upregulated hypoxia and glycolysis gene signatures compared to tumors responding to therapy. Consistent with these results, resistant tumor cells had increased hypoxia, detected by pimonidazole staining, compared to tumor cells responding to therapy. Tumor infiltrating lymphocytes have been shown to be excluded from hypoxic tumor regions, suggesting that hypoxia in the tumor microenvironment may contribute to acquired resistance to ICIs by excluding T cells from these regions. Moreover, we found that Msh2 KO tumor cells upregulated MHC-II in response to IFNγ under normoxic in vitro conditions, but had lower MHC-II expression under hypoxia. Our data suggest that tumor hypoxia may contribute to both tumor-cell intrinsic transcriptional programs, such as alterations in antigen processing and presentation machinery, as well as tumor-cell extrinsic effects, such as exclusion of T cells from the tumor microenvironment, which together may impact the development of acquired resistance to immune checkpoint blockade. Our results can inform strategies to overcome acquired resistance, addressing a critical unmet need in cancer therapy. Citation Format: Camila Robles-Oteiza, Katherine Hastings, Susan M. Kaech, Katerina Politi. A hypoxia-driven transcriptional program characterizes acquired resistance to immune checkpoint inhibitors in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1613.

Oncogenic alterations in the Epidermal Growth Factor Receptor (EGFR) gene are found in ~15% of lung adenocarcinomas (LUADs). EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and these drugs are approved for the first-line treatment of patients with EGFR mutant lung cancer. Response rates to TKIs are high, however, there is variability in the duration and depth of response between patients and acquired resistance to TKIs inevitably emerges. EGFR mutant tumors harbor numerous additional co-occurring alterations in addition to the driver mutations that are likely to contribute to the malignant phenotype of these tumors and the variability in therapeutic response. TP53 is the most common tumor suppressor gene (TSG) co-mutated with EGFR, and alterations in TP53 correlate with worse outcomes upon TKI treatment. However, the functional contribution of loss of other LUAD-associated TSGs to tumor progression and drug resistance in EGFR mutant tumors remains poorly understood. To study the role of 10 putative tumor suppressor genes–that are frequently altered in human LUADs–for EGFR mutant tumors, we developed a conditional lung cancer model based on the inducible expression of EGFRL858R and deletion of Tp53 in the lungs via lentiviral delivery of Cre recombinase. We coupled this EGFRL858R;p53flox/flox mouse model with multiplexed CRISPR-Cas9-mediated genome editing and performed an in vivo screen to quantify the effects of inactivation of these 10 TSGs on tumor growth and TKI sensitivity. We found that inactivation of Apc, Rb1, and Rbm10 each strongly promoted growth while loss of Lkb1 or Setd2 reduced tumor growth. In contrast, in a KrasG12D;p53flox/flox model inactivation of Lkb1 or Setd2 were the strongest drivers of tumor growth. To establish the relevance of the functional data from mouse models for human tumors, we examined the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE) database. Consistent with the mouse data, of the 10 TSGs screened, APC, RB1 and RBM10 were the most frequently mutated in EGFR/TP53 mutant LUADs. We also tested whether inactivation of any of the 10 TSGs was associated with a reduced response to TKIs, by treating mice infected with the lentiviral pool targeting the TSGs with the 3rd generation TKI osimertinib and found that loss of Keap1 was associated with a diminished response to therapy. Collectively, our data have: i) uncovered striking specificity of TSG function in EGFR mutant LUADs, ii) discovered that inactivation of some TSGs can have diametrically opposite effects depending on the nature of the initiating oncogene and iii) established a causal link between certain tumor genotypes and differential responses to EGFR inhibition. Ongoing studies are aimed at investigating the mechanistic underpinnings of these findings. Citation Format: Giorgia Foggetti, Chuan Li, Hongchen Cai, Wen-Yang Lin, Deborah Ayeni, Katherine Hastings, Laura Andrejka, Dylan Maghini, Robert Homer, Dmitri A. Petrov, Monte M. Winslow, Katerina Politi. Genetic determinants of EGFR-driven lung cancer growth and therapeutic response in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1094.

Immune-checkpoint inhibitors (ICI), particularly inhibitors of the PD-1 axis, have altered the management of non–small cell lung cancer (NSCLC) over the last 10 years. First demonstrated to improve outcomes in second-line or later therapy of advanced disease, ICIs were shown to improve overall survival compared with chemotherapy in first-line therapy for patients whose tumors express PD-L1 on at least 50% of cells. More recently, combining ICIs with chemotherapy has been shown to improve survival in patients with both squamous and nonsquamous NSCLC, regardless of PD-L1 expression. However, PD-L1 and, more recently, tumor mutational burden have not proven to be straightforward indicative biomarkers. We describe the advances to date in utilizing these biomarkers, as well as novel markers of tumor inflammation, to ascertain which patients are most likely to benefit from ICIs. Ongoing translational work promises to improve the proportion of patients who benefit from these agents.

Immune checkpoint inhibitors (ICIs) including programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) antagonist antibodies for lung tumors mark a new era of cancer therapeutics harnessing a patient’s immune system to induce durable antitumor responses. Despite their impressive activity in some patients, challenges remain as most tumors exhibit primary or acquired resistance (AR) to ICIs, and the biologic mechanisms for resistance are poorly understood. Using a collection of 14 ICI-resistant lung cancer samples, we investigated whether alterations in genes encoding components of the HLA Class I antigen processing and presentation machinery or interferon signaling pathways play a role in AR to PD-1 or PD-L1 antagonistic antibodies. Although we did not detect any recurrent mutations or copy number changes in our AR cohort, we noted one case of an acquired homozygous loss of B2M that resulted in lack of HLA class I expression on the cell surface in both the patient’s tumor sample and the corresponding patient-derived xenograft (PDX). Downregulation of B2M was also found in two additional PDXs established from ICI-resistant tumors. To test if B2m expression confers sensitivity to ICIs in vivo, we used a CRISPR-mediated approach to knockout B2m in an ICI-sensitive, murine lung cancer cell line and transplanted the cells into syngeneic, immunocompetent mice. We found that the B2m knockout tumors were resistant to ICI therapy in contrast to the wild type tumors. Furthermore, RNA-seq analysis of a subset of the samples in the acquired resistance to ICI cohort revealed an inflammatory tumor microenvironment with significant upregulation of the inhibitory receptors LAG-3 and PD-1 in tumor-infiltrating T cells at ICI resistance. Overall, these findings provide a novel system for the evaluation and screening of candidate genes for their ability to mediate AR to ICIs in vivo. Moreover, they also provide evidence for the disruption of HLA Class I antigen processing and presentation as a mechanism for escape from ICIs in lung cancer and provide information on the immune microenvironment in ICI-resistant tumors. Citation Format: Katherine Hastings, Scott Gettinger, Choi Jungmin, Anna Truini, Ila Datar, Ryan Sowell, Anna Wurtz, Weilai Dong, Guoping Cai, Mary Ann Melnick, Joseph Schlessinger, Sarah Goldberg, Anne Chiang, Ignacio Melero, Jackeline Agorreta, Luis Montuenga, Richard Lifton, Soldano Ferrone, Paula Kavathas, David Rimm, Susan Kaech, Kurt Schalper, Roy Herbst, Katerina Politi. Impaired HLA Class I antigen processing and presentation as a mechanism of acquired Rrsistance to immune checkpoint inhibitors in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A09.

In this paper, we present and discuss issues related to volume estimation of various brain components using MR brain images. We discuss pre-processing techniques to remove elements such as skull, blood vessels and fats from the MR images since these are non-essential to the volume calculations. The volume estimation is based on image segmentation. A challenge in MR brain image segmentation is to distinguish central gray matter from surrounding matters. This paper provides a frame work from pre-processing stage through volume estimation for white matter, gray matter and cerebrospinal fluid (CSF). The main contribution of this paper contains two parts. First, it provides a software-based method for interactive image pre-processing; second, it introduces a software-based, supervised interactive image segmentation method to deal with the segmentation of different matters, especially the central gray matter. Experiments with real data demonstrate the efficiency of our frame work.