A Nasal Swab Classifier to Evaluate the Probability of Lung Cancer in Patients With Pulmonary Nodules.

BACKGROUND: Accurate assessment of the probability of lung cancer (pCA) is critical in patients with pulmonary nodules (PNs) to help guide decision-making. We sought to validate a clinical-genomic classifier developed using whole-transcriptome sequencing of nasal epithelial cells from patients with a PN ≤ 30 mm who smoke or have previously smoked. RESEARCH QUESTION: Can the pCA in individuals with a PN and a history of smoking be predicted by a classifier that uses clinical factors and genomic data from nasal epithelial cells obtained by cytologic brushing? STUDY DESIGN AND METHODS: Machine learning was used to train a classifier using genomic and clinical features on 1,120 patients with PNs labeled as benign or malignant established by a final diagnosis or a minimum of 12 months of radiographic surveillance. The classifier was designed to yield low-, intermediate-, and high-risk categories. The classifier was validated in an independent set of 312 patients, including 63 patients with a prior history of cancer (other than lung cancer), comparing the classifier prediction with the known clinical outcome. RESULTS: In the primary validation set, sensitivity and specificity for low-risk classification were 96% and 42%, whereas sensitivity and specificity for high-risk classification was 58% and 90%, respectively. Sensitivity was similar across stages of non-small cell lung cancer, independent of subtype. Performance compared favorably with clinical-only risk models. Analysis of 63 patients with prior cancer showed similar performance as did subanalyses of patients with light vs heavy smoking burden and those eligible for lung cancer screening vs those who were not. INTERPRETATION: The nasal classifier provides an accurate assessment of pCA in individuals with a PN ≤ 30 mm who smoke or have previously smoked. Classifier-guided decision-making could lead to fewer diagnostic procedures in patients without cancer and more timely treatment in patients with lung cancer.

Interstitial lung abnormalities in a large clinical lung cancer screening cohort: association with mortality and ILD diagnosis.

BACKGROUND: Interstitial lung abnormalities (ILA) are CT findings suggestive of interstitial lung disease in individuals without a prior diagnosis or suspicion of ILD. Previous studies have demonstrated that ILA are associated with clinically significant outcomes including mortality. The aim of this study was to determine the prevalence of ILA in a large CT lung cancer screening program and the association with clinically significant outcomes including mortality, hospitalizations, cancer and ILD diagnosis. METHODS: This was a retrospective study of individuals enrolled in a CT lung cancer screening program from 2012 to 2014. Baseline and longitudinal CT scans were scored for ILA per Fleischner Society guidelines. The primary analyses examined the association between baseline ILA and mortality, all-cause hospitalization, and incidence of lung cancer. Kaplan-Meier plots were generated to visualize the associations between ILA and lung cancer and all-cause mortality. Cox regression proportional hazards models were used to test for this association in both univariate and multivariable models. RESULTS: 1699 subjects met inclusion criteria. 41 (2.4%) had ILA and 101 (5.9%) had indeterminate ILA on baseline CTs. ILD was diagnosed in 10 (24.4%) of 41 with ILA on baseline CT with a mean time from baseline CT to diagnosis of 4.47 ± 2.72 years. On multivariable modeling, the presence of ILA remained a significant predictor of death, HR 3.87 (2.07, 7.21; p < 0.001) when adjusted for age, sex, BMI, pack years and active smoking, but not of lung cancer and all-cause hospital admission. Approximately 50% with baseline ILA had progression on the longitudinal scan. CONCLUSIONS: ILA identified on baseline lung cancer screening exams are associated with all-cause mortality. In addition, a significant proportion of patients with ILA are subsequently diagnosed with ILD and have CT progression on longitudinal scans. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov; No.: NCT04503044.

Vascular Invasion Predicts Recurrence in Stage IA2-IB Lung Adenocarcinoma but not Squamous Cell Carcinoma.

BACKGROUND: Lymphovascular invasion (LVI) is an adverse prognostic feature in resected stage I non-small cell lung cancer (NSCLC); however, it is unclear if the prognostic significance applies to both lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). MATERIALS AND METHODS: A retrospective review of H&E-stained slides from surgically resected AJCC 8th ed. stage IA2-IB LUAD (n = 344) and LUSC (n = 102) from two institutions was performed. LVI was defined as either lymphatic (LI) or vascular (VI) invasion. Outcomes were assessed by 5-year recurrence-free survival (RFS) estimates using the Kaplan-Meier method. RESULTS: The cohorts of LUAD and LUSC showed no significant differences in 5-year RFS (81% each), stage, age, race, or surgical procedure. The presence of LVI, VI, and LI was predictive of 5-year RFS for LUAD (LVI + 71% vs. LVI - 92%, P < 0.001; VI + 64% vs. VI - 90%, P < 0.001; LI + 75% vs. LI - 84%, P = 0.030) but not LUSC (LVI + 84% vs. LVI - 79%, P = 0.740; VI + 83% vs. VI- 80%, P = 0.852; LI + 84% vs. LI - 81%, P = 0.757). Among LUAD with LVI, VI was a stronger predictor of 5-year RFS than the remaining subset of VI-LI + tumors (64% vs. 87%, P = 004). Subset analysis of LI among LUAD stratified by VI showed no significant prognostic advantage to adding LI for risk stratification (VI-LI + 87% vs. VI-LI - 92%, P = 0.347 & VI+LI + 62% vs. VI + LI- 66%, P = 0.422). VI was present in 36% of LUAD. CONCLUSION: Vascular invasion is a strong predictor of recurrence in stage IA2-IB LUAD but not in LUSC. Adjuvant therapy trials should be directed at this subgroup.

Vascular invasion predicts the subgroup of lung adenocarcinomas ≤2.0 cm at risk of poor outcome treated by wedge resection compared to lobectomy.

Background: Recent randomized control trials (JCOG0802 and CALGB140503) have shown sublobar resection to be noninferior to lobectomy for non-small cell lung cancer (NSCLC) ≤2.0 cm. We have previously proposed histologic criteria stratifying lung adenocarcinoma into indolent low malignant potential (LMP) and aggressive angioinvasive adenocarcinomas, resulting in better prognostication than provided by World Health Organization grade. Here we determine whether pathologic classification is reproducible and whether subsets of adenocarcinomas predict worse outcomes when treated by wedge resection compared to lobectomy. Methods: A retrospective cohort of 108 recipients of wedge resection and 187 recipients of lobectomy for stage I/0 lung adenocarcinomas ≤2.0 cm was assembled from 2 institutions. All tumors were classified by a single pathologist, and interobserver reproducibility was assessed in a subset (n = 92) by 5 pathologists. Results: Angioinvasive adenocarcinoma (21%-27% of cases) was associated with worse outcomes when treated with wedge resection compared to lobectomy (5-year recurrence-free survival, 57% vs 85% [P = .007]; 5-year disease-free survival [DSS], 70% vs 90% [P = .043]; 7-year overall survival, 37% vs 58% [P = .143]). Adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and LMP exhibited 100% 5-year DSS regardless of the surgical approach. Multivariable analysis showed that angioinvasion, tumor size, margin status, and extent of nodal sampling were significantly associated with recurrence but not with surgical procedure. There was substantial interobserver reproducibility among the pathologists for the diagnosis of angioinvasive adenocarcinoma (κ = 0.71) and the combined indolent AIS/MIA/LMP group (κ = 0.74). Conclusions: The majority (∼75%) of lung adenocarcinomas ≤2 cm are adequately managed with wedge resection; however, angioinvasive adenocarcinomas (∼25%) treated by wedge resection with suboptimal nodal sampling exhibit poor outcomes, with a 40% to 45% rate of recurrence within 5 years and 60% to 65% overall mortality at 7 years.

MicroRNA Associated with the Invasive Phenotype in Clear Cell Renal Cell Carcinoma: Let-7c-5p Inhibits Proliferation, Migration, and Invasion by Targeting Insulin-like Growth Factor 1 Receptor.

Differential microRNA (miRNA) expression can portend clear cell renal cell carcinoma (ccRCC) progression. In a previous study, we identified a subset of dysregulated miRNA in small renal masses, pT1 ccRCC (≤5 cm) that are associated with an aggressive phenotype. The present study investigated miRNA expression in clinical stage I (cT1) tumors (≤5 cm), comparing pathologic stage I (pT1) tumors to those upstaged to pathologic stage 3 (pT3) after surgery following identification of renal vein invasion or invasion into adjacent fat tissue within Gerota's fascia. Twenty cT1 tumors were examined in an miRNA screening, 10 pT1 and 10 pT3 tumors. The ccRCC cell lines 786-O and Caki-1 were used to assess the impact of let-7c-5p and its protein target insulin-like growth factor 1 receptor (IGF1R). Cells were transfected with pre-let-7c-5p and assessed through cell proliferation, migration, and invasion assays. IGF1R expression was evaluated through Simple Western, and interaction between let-7c-5p and IGF1R was confirmed via luciferase reporter assay. Screening identified 20 miRNA, including let-7c-5p, that were dysregulated between pT1 and pT3 upstaged tumors. This miRNA was also downregulated in our previous study of pT1 tumors that progressed to metastatic disease. Transfection of ccRCC cells with pre-let-7c-5p significantly inhibited proliferation, migration, invasion, and IGF1R expression. These findings suggest that miRNA dysregulation is involved in ccRCC progression, specifically through invasion, and that let-7c-5p downregulation contributes to the aggressiveness of small ccRCC tumors, in part, through its regulation of IGF1R.

Smoking modulates different secretory subpopulations expressing SARS-CoV-2 entry genes in the nasal and bronchial airways.

SARS-CoV-2 infection and disease severity are influenced by viral entry (VE) gene expression patterns in the airway epithelium. The similarities and differences of VE gene expression (ACE2, TMPRSS2, and CTSL) across nasal and bronchial compartments have not been fully characterized using matched samples from large cohorts. Gene expression data from 793 nasal and 1673 bronchial brushes obtained from individuals participating in lung cancer screening or diagnostic workup revealed that smoking status (current versus former) was the only clinical factor significantly and reproducibly associated with VE gene expression. The expression of ACE2 and TMPRSS2 was higher in smokers in the bronchus but not in the nose. scRNA-seq of nasal brushings indicated that ACE2 co-expressed genes were highly expressed in club and C15orf48+ secretory cells while TMPRSS2 co-expressed genes were highly expressed in keratinizing epithelial cells. In contrast, these ACE2 and TMPRSS2 modules were highly expressed in goblet cells in scRNA-seq from bronchial brushings. Cell-type deconvolution of the gene expression data confirmed that smoking increased the abundance of several secretory cell populations in the bronchus, but only goblet cells in the nose. The association of ACE2 and TMPRSS2 with smoking in the bronchus is due to their high expression in goblet cells which increase in abundance in current smoker airways. In contrast, in the nose, these genes are not predominantly expressed in cell populations modulated by smoking. In individuals with elevated lung cancer risk, smoking-induced VE gene expression changes in the nose likely have minimal impact on SARS-CoV-2 infection, but in the bronchus, smoking may lead to higher viral loads and more severe disease.

Vascular invasion identifies the most aggressive histologic subset of stage I lung adenocarcinoma: Implications for adjuvant therapy.

OBJECTIVES: Approximately 15% of stage I lung adenocarcinomas will recur despite adequate surgical therapy. Adjuvant therapy may benefit specific high-risk subsets; however, it is unclear which patients are sufficiently predisposed to recurrence to warrant intensified therapy. MATERIALS AND METHODS: 517 AJCC 8th edition stage I/0 lung adenocarcinomas ≤ 4 cm total size were graded (WHO-2015 and WHO-2021) and compared to stage subgroupings using 7-year recurrence free (RFS), disease specific (DSS), and overall survival (OS). Low malignant potential (LMP) adenocarcinoma was assigned as previously defined. Univariate/multivariate analysis was performed to assess risk factors associated with aggressive behavior. RESULTS: Vascular invasion was the most significant histologic feature on multivariate analysis for both RFS (HR = 4.68, p < 0.001) and DSS (HR = 3.67, p = 0.001) and nearly reached significance for OS (HR = 1.47, p = 0.060). Angioinvasive adenocarcinomas comprised 26 % of the cohort and exhibited a 7-year 64 % RFS, 73 % DSS, and 50 % OS; in contrast to 20 % WHO-2015-G3 (7-year 71 % RFS, 79 % DSS, & 54 % OS), 44 % WHO-2021-G3 (7-year 79 % RFS, 85 % DSS, & 56 % OS), and 21 % stage IB (7-year 72 % RFS, 79 % DSS, and 50 % OS) adenocarcinomas. The majority (>50 %) of overall mortality was disease specific for angioinvasive adenocarcinoma whereas ≤25 % of overall mortality was disease specific for the remaining tumors. Angioinvasive adenocarcinomas were proportionally more common among those still smoking at diagnosis (49 %), male sex (49 %), and black race (16 %) than other subtypes. CONCLUSION: Patients with AJCC 8th ed. stage I angioinvasive lung adenocarcinomas are at high-risk of cancer-specific mortality and should be considered for clinical trials evaluating benefit of adjuvant therapy.

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.

MiRNA-424-5p Suppresses Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma and Attenuates Expression of O-GlcNAc-Transferase.

MicroRNAs (miRNAs) are non-coding post-transcriptional regulators of gene expression that are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in tumor progression. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors, including miR-424-5p, that are associated with an aggressive phenotype. We investigate the impact of this dysregulated miRNA and its protein target O-GlcNAc-transferase (OGT) to better understand the mechanisms behind aggressive stage I ccRCC. The ccRCC cell lines 786-O and Caki-1 were used to assess the impact of miR-424-5p and OGT. Cells were transfected with pre-miR-424-5p, a lentiviral anti-OGT shRNA, or were treated with the demethylating agent 5-Aza-2'-deoxycytidine. Cell proliferation was measured via MT cell viability assay. Cell migration and invasion were analyzed using Transwell assays. The expression of miR-424-5p was determined through qRT-PCR, while OGT protein expression was evaluated through Western blotting. The interaction between miR-424-5p and OGT was confirmed via luciferase reporter assay. The transfection of ccRCC cells with pre-miR-424-5p or anti-OGT shRNA significantly inhibited cell proliferation, migration, and OGT expression, while miR-424-5p also attenuated cell invasion. Addition of the demethylating agent significantly reduced cell proliferation, migration, invasion, and OGT expression, while significantly increasing the expression of miR-424-5p. Altogether, these findings suggest that epigenetic downregulation of miR-424-5p, which in turn augments OGT expression, contributes to the creation of aggressive forms of stage I ccRCC.

Qualitative coronary artery calcification scores and risk of all cause, COPD and pneumonia hospital admission in a large CT lung cancer screening cohort.

BACKGROUND: Patients at high-risk for lung cancer and qualified for CT lung cancer screening (CTLS) are at risk for numerous cardio-pulmonary comorbidities. We sought to examine if qualitatively assessed coronary artery calcifications (CAC) on CTLS exams could identify patients at increased risk for non-cardiovascular events such as all cause, COPD and pneumonia related hospitalization and to verify previously reported associations between CAC and mortality and cardiovascular events. STUDY DESIGN AND METHODS: Patients (n = 4673) from Lahey Hospital and Medical Center who underwent CTLS from January 12, 2012 through September 30, 2017 were included with clinical follow-up through September 30, 2019. CTLS exams were qualitatively scored for the presence and severity of CAC at the time of exam interpretation using a four point scale: none, mild, moderate, and marked. Multivariable Cox regression models were used to evaluate the association between CT qualitative CAC and all-cause, COPD-related, and pneumonia-related hospital admissions. RESULTS: 3631 (78%) of individuals undergoing CTLS had some degree of CAC on their baseline exam: 1308 (28.0%), 1128 (24.1%), and 1195 (25.6%) had mild, moderate and marked coronary calcification, respectively. Marked CAC was associated with all-cause hospital admission and pneumonia related admissions HR 1.48; 95% CI 1.23-1.78 and HR 2.19; 95% 1.30-3.71, respectively. Mild, moderate and marked CAC were associated with COPD-related admission HR 2.30; 95% CI 1.31-4.03, HR 2.17; 95% CI 1.20-3.91 and HR 2.27; 95% CI 1.24-4.15. CONCLUSION: Qualitative CAC on CTLS exams identifies individuals at elevated risk for all cause, pneumonia and COPD-related hospital admissions.

Qualitative emphysema and risk of COPD hospitalization in a multicenter CT lung cancer screening cohort study.

BACKGROUND: In the United States, 9 to 10 million Americans are estimated to be eligible for computed tomographic lung cancer screening (CTLS). Those meeting criteria for CTLS are at high-risk for numerous cardio-pulmonary co-morbidities. The objective of this study was to determine the association between qualitative emphysema identified on screening CTs and risk for hospital admission. STUDY DESIGN AND METHODS: We conducted a retrospective multicenter study from two CTLS cohorts: Lahey Hospital and Medical Center (LHMC) CTLS program, Burlington, MA and Mount Auburn Hospital (MAH) CTLS program, Cambridge, MA. CTLS exams were qualitatively scored by radiologists at time of screening for presence of emphysema. Multivariable Cox regression models were used to evaluate the association between CT qualitative emphysema and all-cause, COPD-related, and pneumonia-related hospital admission. RESULTS: We included 4673 participants from the LHMC cohort and 915 from the MAH cohort. 57% and 51.9% of the LHMC and MAH cohorts had presence of CT emphysema, respectively. In the LHMC cohort, the presence of emphysema was associated with all-cause hospital admission (HR 1.15, CI 1.07-1.23; p < 0.001) and COPD-related admission (HR 1.64; 95% CI 1.14-2.36; p = 0.007), but not with pneumonia-related admission (HR 1.52; 95% CI 1.27-1.83; p < 0.001). In the MAH cohort, the presence of emphysema was only associated with COPD-related admission (HR 2.05; 95% CI 1.07-3.95; p = 0.031). CONCLUSION: Qualitative CT assessment of emphysema is associated with COPD-related hospital admission in a CTLS population. Identification of emphysema on CLTS exams may provide an opportunity for prevention and early intervention to reduce admission risk.

MicroRNAs MiR-15a and MiR-26a cooperatively regulate O-GlcNAc-transferase to control proliferation in clear cell renal cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs), a group of non-coding post-transcriptional regulators of gene expression, are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in carcinogenesis. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors associated with progression to metastatic disease. OBJECTIVE: To investigate the impact of two of these dysregulated miRNA, miR-15a-5p and -26a-5p, in an effort to elucidate the mechanisms underpinning aggressive forms of stage I ccRCC. METHODS: The ccRCC cell line 786-O was transfected with pre-miRs-15a-5p and -26a-5p to rescue expression. Cell proliferation was measured via MT Cell Viability Assay. O-GlcNAc-transferase (OGT), a known protein in ccRCC proliferation, was identified by bioinformatics analysis as a target of both miRNA and validated via luciferase reporter assay to confirm binding of each miR to the 3' untranslated region (UTR). OGT protein expression was evaluated via western blotting. RESULTS: Luciferase assay confirmed specificity of miR-15a-5p and -26a-5p for the OGT UTR. Western blot analysis for OGT showed reduced expression following co-transfection of both miRNAs compared to negative control or individual transfection. Co-transfection of these miRNAs greatly reduced proliferation when compared to negative control or the individual transfections. CONCLUSION: Our results indicate that the dysregulation of miR-15a-5p and -26a-5p contribute cooperatively to the proliferation of ccRCC through their regulation of OGT. These results give insight into the pathogenesis of aggressive early stage ccRCC and suggest potential therapeutic targets for future research.

Pulmonary Adenocarcinomas of Low Malignant Potential: Proposed Criteria to Expand the Spectrum Beyond Adenocarcinoma In Situ and Minimally Invasive Adenocarcinoma.

Lung cancer screening has improved mortality among high-risk smokers but has coincidentally detected a fraction of nonprogressive adenocarcinoma historically classified as bronchoalveolar carcinoma (BAC). In the National Lung Screening Trial (NLST) the majority of BAC-comprising 29% of computed tomography-detected stage I lung adenocarcinoma-were considered overdiagnosis after extended follow-up comparison with the control arm. In the current classification, adenocarcinoma in situ and minimally invasive adenocarcinoma have replaced BAC but together comprise only ∼5% of stage I lung adenocarcinoma. Lepidic and subsets of papillary and acinar adenocarcinoma also infrequently recur. We, therefore, propose criteria for low malignant potential (LMP) adenocarcinoma among nonmucinous adenocarcinoma measuring ≤3 cm in total, exhibiting ≥15% lepidic growth, and lacking nonpredominant high-grade patterns (≥10% cribriform, ≥5% micropapillary, ≥5% solid), >1 mitosis per 2 mm2, angiolymphatic or visceral pleural invasion, spread through air spaces or necrosis. We tested these criteria in a multi-institutional cohort of 328 invasive stage I (eighth edition) and in situ adenocarcinomas and observed 16% LMP and 7% adenocarcinoma in situ/minimally invasive adenocarcinoma which together (23%) approximated the frequency of overdiagnosed stage I BAC in the NLST. The LMP group had 100% disease-specific survival. The proposed LMP criteria, incorporating multiple histologic parameters, may be a clinically useful "low-grade" prognostic group. Validation of these criteria in additional retrospective cohorts and prospective screen-detected cohorts should be considered.

Protein Expression Profiles among Lichen Sclerosus Urethral Strictures-Can Urethroplasty Success be Predicted?

PURPOSE: Urethroplasty of lichen sclerosus strictures has a significantly higher failure rate than strictures due to other causes. We sought to determine predictors of urethroplasty failure in men with lichen sclerosus urethral stricture disease by evaluating protein expression profiles. MATERIALS AND METHODS: Urethral tissue was excised from patients with lichen sclerosus who were undergoing urethroplasty of urethral stricture disease at a single institution. A tissue microarray was created with cores from each sample. Immunohistochemistry was performed to compare protein expression related to inflammation, cell cycle disruption, oxidative stress, hormone receptor status and infection. Stricture recurrence was defined by the need for a subsequent unanticipated procedure for urethral stricture disease. RESULTS: We evaluated 50 men with lichen sclerosus urethral stricture disease, including 31 with successful reconstruction and 19 with recurrent stricture. Recurrent strictures expressed lower levels of several inflammatory markers and had a lower Ki-67 mitotic index and significantly higher vascular endothelial growth factor levels than nonrecurrent strictures. CONCLUSIONS: To our knowledge this is the first study to use tissue protein expression to identify risk factors for urethroplasty failure among men with lichen sclerosus urethral stricture disease. Our findings suggest that recurrent lichen sclerosus strictures demonstrate a suppressed inflammatory response, a decreased cell turnover rate, and poor oxygenation and nutrient delivery. Prospective studies are needed to clarify the role of these pathways in the pathophysiology of lichen sclerosus urethral stricture disease, determine whether preoperative biopsy can predict urethroplasty success, help counsel patients and develop future treatments.

Two-photon images reveal unique texture features for label-free identification of ovarian cancer peritoneal metastases.

For cancer patients, treatment selection fundamentally relies on staging, with "under-staging" considered a common problem. Imaging modalities that can complement conventional white-light laparoscopy are needed to detect more accurately small metastatic lesions in patients undergoing operative cancer care. Biopsies from healthy parietal peritoneum and ovarian peritoneal metastases obtained from 8 patients were imaged employing a two-photon laser scanning microscope to generate collagen-second harmonic generation (SHG) and fluorescence images at 755 nm and 900 nm excitation and 460 ± 20 nm and 525 ± 25 nm emission. Forty-one images were analyzed by automated image processing algorithms and statistical textural analysis techniques, namely gray level co-occurrence matrices. Two textural features (contrast and correlation) were employed to describe the spatial intensity variations within the captured images and outcomes were used for discriminant analysis. We found that healthy tissues displayed large variations in contrast and correlation features as a function of distance, corresponding to repetitive, increased local intensity fluctuations. Metastatic tissue images exhibited decreased contrast and correlation related values, representing more uniform intensity patterns and smaller fibers, indicating the destruction of the healthy stroma by the cancerous infiltration. The textural outcomes resulted in high classification accuracy as evaluated quantitatively by discriminant analysis.

Insights into the Pathophysiology of Urethral Stricture Disease due to Lichen Sclerosus: Comparison of Pathological Markers in Lichen Sclerosus Induced Strictures vs Nonlichen Sclerosus Induced Strictures.

PURPOSE: We evaluated the pathophysiology of lichen sclerosus and nonlichen sclerosus urethral stricture disease by comparing protein expression related to inflammation, cell cycle disruption, oxidative stress, hormone receptor status and infection. MATERIALS AND METHODS: Tissue samples were collected from the urethral strictures of 81 patients undergoing urethroplasty. Clinical and demographic data were obtained by chart review. After identifying areas pathognomonic for lichen sclerosus a tissue microarray was created with cores from each sample and immunohistochemistry was performed. RESULTS: Patients had similar baseline demographics and comorbidities. Of the 81 strictures 58 were and 23 were not due to lichen sclerosus. Lichen sclerosus strictures were significantly longer and showed higher levels of inflammation. The proportion of T cells which stained positive for CD8 was significantly higher in strictures due to lichen sclerosus (50% vs 13%, p = 0.004). CCL-4 was expressed significantly more in strictures due to lichen sclerosus (76% vs 42%, p = 0.01). Several other inflammatory markers were only found in strictures due to lichen sclerosus. Block-like p16, a surrogate for high risk human papillomavirus infection, and varicella zoster virus were found only in lichen sclerosus urethral stricture disease samples, although both were rare. Epstein-Barr virus RNA was found in significantly more lichen sclerosus samples (37% vs 10%, p = 0.024). CONCLUSIONS: To our knowledge this is the first study to evaluate protein expression in lichen sclerosus urethral stricture disease. These strictures demonstrate increased inflammation compared to nonlichen sclerosus urethral strictures. Markers of oxidative stress, cell cycle dysregulation and the androgen receptor do not appear to be uniquely associated with lichen sclerosus urethral stricture disease. Positive staining for several viruses in samples of lichen sclerosus urethral stricture disease suggests a possible infectious etiology.

Clinical Proof-of-concept of a Novel Platform Utilizing Biopsy-derived Live Single Cells, Phenotypic Biomarkers, and Machine Learning Toward a Precision Risk Stratification Test for Prostate Cancer Grade Groups 1 and 2 (Gleason 3 + 3 and 3 + 4).

OBJECTIVE: To examine the ability of a novel live primary-cell phenotypic (LPCP) test to predict postsurgical adverse pathology (P-SAP) features and risk stratify patients based on SAP features in a blinded study utilizing radical prostatectomy (RP) surgical specimens. METHODS: Two hundred fifty-one men undergoing RP were enrolled in a prospective, multicenter (10), and proof-of-concept study in the United States. Fresh prostate samples were taken from known areas of cancer in the operating room immediately after RP. Samples were shipped and tested at a central laboratory. Utilizing the LPCP test, a suite of phenotypic biomarkers was analyzed and quantified using objective machine vision software. Biomarkers were objectively ranked via machine learning-derived statistical algorithms (MLDSA) to predict postsurgical adverse pathological features. Sensitivity and specificity were determined by comparing blinded predictions and unblinded RP surgical pathology reports, training MLDSAs on 70% of biopsy cells and testing MLDSAs on the remaining 30% of biopsy cells across the tested patient population. RESULTS: The LPCP test predicted adverse pathologies post-RP with area under the curve (AUC) via receiver operating characteristics analysis of greater than 0.80 and distinguished between Prostate Cancer Grade Groups 1, 2, and 3/Gleason Scores 3 + 3, 3 + 4, and 4 + 3. Further, LPCP derived-biomarker scores predicted Gleason pattern, stage, and adverse pathology with high precision-AUCs>0.80. CONCLUSION: Using MLDSA-derived phenotypic biomarker scores, the LPCP test successfully risk stratified Prostate Cancer Grade Groups 1, 2, and 3 (Gleason 3 + 3 and 7) into distinct subgroups predicted to have surgical adverse pathologies or not with high performance (>0.85 AUC).

Spatiotemporal microRNA profile in peripheral nerve regeneration: miR-138 targets vimentin and inhibits Schwann cell migration and proliferation.

While the peripheral nervous system has regenerative ability, restoration of sufficient function remains a challenge. Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration, including myelination, neuroplasticity, kinase signaling in nerve axoplasm, and cell migration; however, the mechanisms regulating its expression within Schwann cell (SC) remain unexplored. The aim of this study was to profile the spatial and temporal expression profile of microRNA (miRNA) in a regenerating rat sciatic nerve after transection, and explore the potential role of miR-138-5p targeting vimentin in SC proliferation and migration. A rat sciatic nerve transection model, utilizing a polyethylene nerve guide, was used to investigate miRNA expression at 7, 14, 30, 60, and 90 days during nerve regeneration. Relative levels of miRNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction. In vitro assays were conducted with cultured Schwann cells transfected with miRNA mimics and assessed for migratory and proliferative potential. The top seven dysregulated miRNAs reported in this study have been implicated in cell migration elsewhere, and GO and KEGG analyses predicted activities essential to wound healing. Transfection of one of these, miRNA-138-5p, into SCs reduced cell migration and proliferation. miR-138-5p has been shown to directly target vimentin in cancer cells, and the luciferase assay performed here in rat Schwann cells confirmed it. These results detail a role of miR-138-5p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.

Profiling microRNA from nephrectomy and biopsy specimens: predictors of progression and survival in clear cell renal cell carcinoma.

OBJECTIVE: To identify microRNA (miRNA) characteristic of metastatic clear cell renal cell carcinoma (ccRCC) and those indicative of cancer-specific survival (CSS) in nephrectomy and biopsy specimens. We also sought to determine if a miRNA panel could differentiate benign from ccRCC tissue. MATERIALS AND METHODS: RNA was isolated from nephrectomy and kidney biopsy specimens (n = 156 and n = 46, respectively). Samples were grouped: benign, non-progressive, and progressive ccRCC. MiRNAs were profiled by microarray and validated by quantitative reverse transcription-polymerase chain reaction. Biomarker signatures were developed to predict cancer status in nephrectomy and biopsy specimens. CSS was examined using Kaplan-Meier and Cox proportional hazards analyses. RESULTS: Microarray analysis revealed 20 differentially expressed miRNAs comparing non-progressive with progressive tumours. A biomarker signature validated in nephrectomy specimens had a sensitivity of 86.7% and a specificity of 92.9% for differentiating benign and ccRCC specimens. A second signature differentiated non-progressive vs progressive ccRCC with a sensitivity of 93.8% and a specificity of 83.3%. These biomarkers also discriminated cancer status in biopsy specimens. Levels of miR-10a-5p, -10b-5p, and -223-3p were associated with CSS. CONCLUSION: This study identified miRNAs differentially expressed in ccRCC samples; as well as those correlating with CSS. Biomarkers identified in this study have the potential to identify patients who are likely to have progressive ccRCC, and although preliminary, these results may aid in differentiating aggressive and indolent ccRCC based on biopsy specimens.

MicroRNA Expression Profile Identifies High Grade, Non-Muscle-Invasive Bladder Tumors at Elevated Risk to Progress to an Invasive Phenotype.

The objective of this study was to identify a panel of microRNAs (miRNAs) differentially expressed in high-grade non-muscle invasive (NMI; TaG3-T1G3) urothelial carcinoma that progress to muscle-invasive disease compared to those that remain non-muscle invasive, whether recurrence happens or not. Eighty-nine high-grade NMI urothelial carcinoma lesions were identified and total RNA was extracted from paraffin-embedded tissue. Patients were categorized as either having a non-muscle invasive lesion with no evidence of progression over a 3-year period or as having a similar lesion showing progression to muscle invasion over the same period. In addition, comparison of miRNA expression levels between patients with and without prior intravesical therapy was performed. Total RNA was pooled for microarray analysis in each group (non-progressors and progressors), and qRT-PCR of individual samples validated differential expression between non-progressive and progressive lesions. MiR-32-5p, -224-5p, and -412-3p were associated with cancer-specific survival. Downregulation of miR-203a-3p and miR-205-5p were significantly linked to progression in non-muscle invasive bladder tumors. These miRNAs include those implicated in epithelial mesenchymal transition, previously identified as members of a panel characterizing transition from the non-invasive to invasive phenotype in bladder tumors. Furthermore, we were able to identify specific miRNAs that are linked to postoperative outcome in patients with high grade NMI urothelial carcinoma of the bladder (UCB) that progressed to muscle-invasive (MI) disease.