Identification of a gene expression signature of vascular invasion and recurrence in stage I lung adenocarcinoma via bulk and spatial transcriptomics.

Microscopic vascular invasion (VI) is predictive of recurrence and benefit from lobectomy in stage I lung adenocarcinoma (LUAD) but is difficult to assess in resection specimens and cannot be accurately predicted prior to surgery. Thus, new biomarkers are needed to identify this aggressive subset of stage I LUAD tumors. To assess molecular and microenvironment features associated with angioinvasive LUAD we profiled 162 resected stage I tumors with and without VI by RNA-seq and explored spatial patterns of gene expression in a subset of 15 samples by high-resolution spatial transcriptomics (stRNA-seq). Despite the small size of invaded blood vessels, we identified a gene expression signature of VI from the bulk RNA-seq discovery cohort (n=103) and found that it was associated with VI foci, desmoplastic stroma, and high-grade patterns in our stRNA-seq data. We observed a stronger association with high-grade patterns from VI+ compared with VI- tumors. Using the discovery cohort, we developed a transcriptomic predictor of VI, that in an independent validation cohort (n=60) was associated with VI (AUROC=0.86; p=5.42×10-6) and predictive of recurrence-free survival (HR=1.98; p=0.024), even in VI- LUAD (HR=2.76; p=0.003). To determine our VI predictor's robustness to intra-tumor heterogeneity we used RNA-seq data from multi-region sampling of stage I LUAD cases in TRACERx, where the predictor scores showed high correlation (R=0.87, p<2.2×10-16) between two randomly sampled regions of the same tumor. Our study suggests that VI-associated gene expression changes are detectable beyond the site of intravasation and can be used to predict the presence of VI. This may enable the prediction of angioinvasive LUAD from biopsy specimens, allowing for more tailored medical and surgical management of stage I LUAD.

Elevated Serum Androstenedione Level in a Patient With Ectopic Adrenocorticotropic Hormone Syndrome.

Background/Objective: Ectopic Cushing syndrome can be challenging to diagnose when its presentation is atypical. Herein, we highlight features of ectopic adrenocorticotropic hormone (ACTH) syndrome in a patient with worsening hypertension, hypokalemia, ACTH-dependent hypercortisolism, and disproportionate elevation in serum androstenedione levels. Case Report: A 59-year-old woman presented with rapidly progressing hypertension, severe hypokalemia, confusion, and weakness. Her medical history included well-controlled hypertension receiving amlodipine 5 mg/day, which worsened 3 months prior to admission requiring losartan and spironolactone therapy, with twice daily potassium supplementation. Physical examination was notable for bruising, muscle wasting, thin extremities, facial fullness, and abdominal adiposity despite body mass index 17 kg/m2. Laboratory evaluation showed potassium 2.6 mEq/L (3.5-5.3), morning cortisol >50 mcg/dL (8-25), 24-hour urine cortisol 8369 mcg/day (<50), ACTH 308 pg/mL (<46), androstenedione 398 ng/dL (20-75), dehydroepiandrosterone sulfate 48 mcg/dL (≤430), and testosterone 11 ng/dL (≤4.5) levels. A 3.8-cm carcinoid right lung tumor was identified, and resection was performed with clean margins. Cortisol, androstenedione, and potassium levels rapidly normalized postoperatively and blood pressure returned to baseline, well-controlled on amlodipine. Discussion: Our case illustrates disproportionate elevation in androstenedione levels despite normal dehydroepiandrosterone sulfate and testosterone in a woman with ectopic ACTH syndrome. Limited reports have observed similar discordance in androgen profiles in ectopic versus pituitary ACTH hypersecretion, potentially attributable to differential activation of androgen biosynthesis. Conclusion: Adrenal androgen assessment may help differentiate pituitary versus ectopic ACTH secretion in which androstenedione is elevated, but studies are needed to determine whether disproportionate androstenedione elevation reliably predicts the origin of ACTH excess.

Pathologic and gene expression comparison of CT- screen detected and routinely detected stage I/0 lung adenocarcinoma in NCCN risk-matched cohorts.

INTRODUCTION: Although three randomized control trials have proven mortality benefit of CT lung cancer screening (CTLS), <5% of eligible US smokers are screened. Some attribute this to fear of harm conveyed at shared decision visits, including the harm of overdiagnosis/overtreatment of indolent BAC-like adenocarcinoma. METHODS: Since the frequency of indolent cancers has not been compared between CTLS and routinely detected cohorts, we compare pathology and RNA expression of 86 NCCN high-risk CTLS subjects to 83 high-risk (HR-R) and 51 low-risk (LR-R) routinely detected patients. Indolent adenocarcinoma was defined as previously described for low malignant potential (LMP) adenocarcinoma along with AIS/MIA. Exome RNA sequencing was performed on a subset of high-risk (CTLS and HR-R) FFPE tumor samples. RESULTS: Indolent adenocarcinoma (AIS, MIA, and LMP) showed 100% disease-specific survival (DSS) with similar frequency in CTLS (18%) and HR-R (20%) which were comparatively lower than LR-R (33%). Despite this observation, CTLS exhibited intermediate DSS between HR-R and LR-R (5-year DSS: 88% CTLS, 82% HR-R, & 95% LR-R, p = 0.047), possibly reflecting a 0.4 cm smaller median tumor size and lower frequency of tumor necrosis compared to HR-R. WGCNA gene modules derived from TCGA lung adenocarcinoma correlated with aggressive histologic patterns, mitotic activity, and tumor invasive features, but no significant differential expression between CTLS and HR-R was observed. CONCLUSION: CTLS subjects are at no greater risk of overdiagnosis from indolent adenocarcinoma (AIS, MIA, and LMP) than risk-matched patients whose cancers are discovered in routine clinical practice. Improved outcomes likely reflect detection and treatment at smaller size.

Elevated T cell repertoire diversity is associated with progression of lung squamous cell premalignant lesions.

OBJECTIVE: The immune response to invasive carcinoma has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological, and molecular features. METHODS: Endobronchial biopsies (n=295) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via autofluorescence bronchoscopy and CT, were profiled by RNA-seq. We applied the TCR Repertoire Utilities for Solid Tissue/Tumor tool to the RNA-seq data to identify TCR CDR3 sequences across all samples. In the biopsies, we measured the correlation of TCR diversity with previously derived immune-associated PML transcriptional signatures and PML outcome. We also quantified the spatial and temporal distribution of shared and clonally expanded TCRs. Using the biopsies and brushes, the ratio of private (ie, found in one patient only) and public (ie, found in two or more patients) TCRs was quantified, and the CDR3 sequences were compared with those found in curated databases with known antigen specificities. RESULTS: We detected 39,303 unique TCR sequences across all samples. In PML biopsies, TCR diversity was negatively associated with a transcriptional signature of T cell mediated immune activation (p=4e-4) associated with PML outcome. Additionally, in lesions of the proliferative molecular subtype, TCR diversity was decreased in regressive versus progressive/persistent PMLs (p=0.045). Within each patient, TCRs were more likely to be shared between biopsies sampled at the same timepoint than biopsies sampled at the same anatomic location at different times. Clonally expanded TCRs, within a biopsied lesion, were more likely to be expanded at future time points than non-expanded clones. The majority of TCR sequences were found in a single sample, with only 3396 (8.6%) found in more than one sample and 1057 (2.7%) found in two or more patients (ie, public); however, when compared with a public database of CDR3 sequences, 4543 (11.6%) of TCRs were identified as public. TCRs with known antigen specificities were enriched among public TCRs (p<0.001). CONCLUSIONS: Decreased TCR diversity may reflect nascent immune responses that contribute to PML elimination. Further studies are needed to explore the potential for immunoprevention of PMLs.

Substratum stiffness tunes membrane voltage in mammary epithelial cells.

Membrane voltage (Vm) plays a critical role in the regulation of several cellular behaviors, including proliferation, apoptosis and phenotypic plasticity. Many of these behaviors are affected by the stiffness of the underlying extracellular matrix, but the connections between Vm and the mechanical properties of the microenvironment are unclear. Here, we investigated the relationship between matrix stiffness and Vm by culturing mammary epithelial cells on synthetic substrata, the stiffnesses of which mimicked those of the normal mammary gland and breast tumors. Although proliferation is associated with depolarization, we surprisingly observed that cells are hyperpolarized when cultured on stiff substrata, a microenvironmental condition that enhances proliferation. Accordingly, we found that Vm becomes depolarized as stiffness decreases, in a manner dependent on intracellular Ca2+. Furthermore, inhibiting Ca2+-gated Cl- currents attenuates the effects of substratum stiffness on Vm. Specifically, we uncovered a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of Vm by substratum stiffness. Taken together, these results suggest a novel role for CFTR and membrane voltage in the response of mammary epithelial cells to their mechanical microenvironment.

Substratum stiffness signals through integrin-linked kinase and ß1-integrin to regulate midbody proteins and abscission during EMT.

Abscission is the final stage of cytokinesis during which the parent cell physically separates to yield two identical daughters. Failure of abscission results in multinucleation (MNC), a sign of genomic instability and a precursor to aneuploidy, enabling characteristics of neoplastic progression. Induction of epithelial-mesenchymal transition (EMT) causes MNC in mammary epithelial cells cultured on stiff microenvironments that have mechanical properties similar to those found in breast tumors, but not on soft microenvironments reminiscent of the normal mammary gland. Here we report that on stiff microenvironments, EMT signaling through Snail up-regulates the midbody-associated proteins septin-6, Mklp1, and anillin, leading to abscission failure and MNC. To uncover the mechanism by which stiff microenvironments promote MNC in cells undergoing EMT, we investigated the role of cell-matrix adhesion through ß1-integrin and integrin-linked kinase (ILK). We found that ILK expression, but not kinase activity, is required for EMT-associated MNC in cells on stiff microenvironments. Conversely, increasing focal adhesions by expressing an autoclustering mutant of ß1-integrin promotes MNC in cells on soft microenvironments. Our data suggest that signaling through focal adhesions causes failure of cytokinesis in cells actively undergoing EMT. These results highlight the importance of tissue mechanics and adhesion in regulating the cellular response to EMT inducers.

Matrix degradation and cell proliferation are coupled to promote invasion and escape from an engineered human breast microtumor.

Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype.

Combination chemotherapy plus dasatinib leads to comparable overall survival and relapse-free survival rates as allogeneic hematopoietic stem cell transplantation in Philadelphia positive acute lymphoblastic leukemia.

BACKGROUND: The Philadelphia chromosome is associated with a poor prognosis in acute lymphoblastic leukemia (ALL). While hematopoietic stem cell transplantation (HSCT) has been regarded as a favorable treatment option in adult Philadelphia-positive (Ph+) ALL, its benefit is less clear in the era of newer generation tyrosine kinase inhibitors (TKIs) like dasatinib. METHODS: This was a retrospective study that analyzed the outcomes of adult patients with Ph+ ALL treated with either combination chemotherapy plus dasatinib or combination chemotherapy plus dasatinib followed by allogeneic HSCT. RESULTS: A total of 70 patients were included; 30 (42.9%) underwent allogeneic HSCT while 40 (57.1%) received only chemotherapy plus dasatinib. In comparing overall survival (OS) rates, results between the 2 groups were similar with a 1-year OS of 93.3% versus 100% (P = 0.20), 2-year OS of 89.8% versus 86.2% (P = 0.72), and 3-year OS of 76% versus 71.3% (P = 0.56) in the transplant versus nontransplant groups, respectively. The 3-year relapse-free survival (RFS) rates were also similar at 70.5% in the transplant group and 80.1% in the nontransplant group (P = 0.94). Subgroup analyses were performed for patients with specific poor prognostic factors (higher white blood count, older age, positive minimal residual disease status), but results again showed no significant survival difference between transplant and nontransplant patients. CONCLUSIONS: While HSCT has historically led to a survival advantage in Ph+ ALL, the results of our study demonstrate that it may have a less beneficial role in the era of newer generation TKIs such as dasatinib.

Molecular subtyping reveals immune alterations associated with progression of bronchial premalignant lesions.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.

A Soft Microenvironment Protects from Failure of Midbody Abscission and Multinucleation Downstream of the EMT-Promoting Transcription Factor Snail.

Multinucleation is found in more than one third of tumors and is linked to increased tolerance for mutation, resistance to chemotherapy, and invasive potential. The integrity of the genome depends on proper execution of the cell cycle, which can be altered through mechanotransduction pathways as the tumor microenvironment stiffens during tumorigenesis. Here, we show that signaling downstream of matrix metalloproteinase-3 (MMP3) or TGFß, known inducers of epithelial-mesenchymal transition (EMT), also promotes multinucleation in stiff microenvironments through Snail-dependent expression of the filament-forming protein septin-6, resulting in midbody persistence, abscission failure, and multinucleation. Consistently, we observed elevated expression of Snail and septin-6 as well as multinucleation in a human patient sample of metaplastic carcinoma of the breast, a rare classification characterized by deposition of collagen fibers and active EMT. In contrast, a soft microenvironment protected mammary epithelial cells from becoming multinucleated by preventing Snail-induced upregulation of septin-6. Our data suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression.Significance: These findings reveal tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Cancer Res; 78(9); 2277-89. ©2018 AACR.

Integrated Genomics Reveals Convergent Transcriptomic Networks Underlying Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis.

RATIONALE: Despite shared environmental exposures, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease are usually studied in isolation, and the presence of shared molecular mechanisms is unknown. OBJECTIVES: We applied an integrative genomic approach to identify convergent transcriptomic pathways in emphysema and IPF. METHODS: We defined the transcriptional repertoire of chronic obstructive pulmonary disease, IPF, or normal histology lungs using RNA-seq (n = 87). MEASUREMENTS AND MAIN RESULTS: Genes increased in both emphysema and IPF relative to control were enriched for the p53/hypoxia pathway, a finding confirmed in an independent cohort using both gene expression arrays and the nCounter Analysis System (n = 193). Immunohistochemistry confirmed overexpression of HIF1A, MDM2, and NFKBIB members of this pathway in tissues from patients with emphysema or IPF. Using reads aligned across splice junctions, we determined that alternative splicing of p53/hypoxia pathway-associated molecules NUMB and PDGFA occurred more frequently in IPF or emphysema compared with control and validated these findings by quantitative polymerase chain reaction and the nCounter Analysis System on an independent sample set (n = 193). Finally, by integrating parallel microRNA and mRNA-Seq data on the same samples, we identified MIR96 as a key novel regulatory hub in the p53/hypoxia gene-expression network and confirmed that modulation of MIR96 in vitro recapitulates the disease-associated gene-expression network. CONCLUSIONS: Our results suggest convergent transcriptional regulatory hubs in diseases as varied phenotypically as chronic obstructive pulmonary disease and IPF and suggest that these hubs may represent shared key responses of the lung to environmental stresses.

Therapeutic benefits of a component of coffee in a rat model of Alzheimer's disease.

A minor component of coffee unrelated to caffeine, eicosanoyl-5-hydroxytryptamide (EHT), provides protection in a rat model for Alzheimer's disease (AD). In this model, viral expression of the phosphoprotein phosphatase 2A (PP2A) endogenous inhibitor, the I2(PP2A), or SET protein in the brains of rats leads to several characteristic features of AD including cognitive impairment, tau hyperphosphorylation, and elevated levels of cytoplasmic amyloid-ß protein. Dietary supplementation with EHT for 6-12 months resulted in substantial amelioration of all these defects. The beneficial effects of EHT could be associated with its ability to increase PP2A activity by inhibiting the demethylation of its catalytic subunit PP2Ac. These findings raise the possibility that EHT may make a substantial contribution to the apparent neuroprotective benefits associated with coffee consumption as evidenced by numerous epidemiologic studies indicating that coffee drinkers have substantially lowered risk of developing AD.