Radiomics analysis to predict pulmonary nodule malignancy using machine learning approaches.

BACKGROUND: Low-dose CT screening can reduce lung cancer-related mortality. However, most screen-detected pulmonary abnormalities do not develop into cancer and it often remains challenging to identify malignant nodules, particularly among indeterminate nodules. We aimed to develop and assess prediction models based on radiological features to discriminate between benign and malignant pulmonary lesions detected on a baseline screen. METHODS: Using four international lung cancer screening studies, we extracted 2060 radiomic features for each of 16 797 nodules (513 malignant) among 6865 participants. After filtering out low-quality radiomic features, 642 radiomic and 9 epidemiological features remained for model development. We used cross-validation and grid search to assess three machine learning (ML) models (eXtreme Gradient Boosted Trees, random forest, least absolute shrinkage and selection operator (LASSO)) for their ability to accurately predict risk of malignancy for pulmonary nodules. We report model performance based on the area under the curve (AUC) and calibration metrics in the held-out test set. RESULTS: The LASSO model yielded the best predictive performance in cross-validation and was fit in the full training set based on optimised hyperparameters. Our radiomics model had a test-set AUC of 0.93 (95% CI 0.90 to 0.96) and outperformed the established Pan-Canadian Early Detection of Lung Cancer model (AUC 0.87, 95% CI 0.85 to 0.89) for nodule assessment. Our model performed well among both solid (AUC 0.93, 95% CI 0.89 to 0.97) and subsolid nodules (AUC 0.91, 95% CI 0.85 to 0.95). CONCLUSIONS: We developed highly accurate ML models based on radiomic and epidemiological features from four international lung cancer screening studies that may be suitable for assessing indeterminate screen-detected pulmonary nodules for risk of malignancy.

Circulating proteome for pulmonary nodule malignancy.

BACKGROUND: Although lung cancer screening with low-dose computed tomography is rolling out in many areas of the world, differentiating indeterminate pulmonary nodules remains a major challenge. We conducted one of the first systematic investigations of circulating protein markers to differentiate malignant from benign screen-detected pulmonary nodules. METHODS: Based on 4 international low-dose computed tomography screening studies, we assayed 1078 protein markers using prediagnostic blood samples from 1253 participants based on a nested case-control design. Protein markers were measured using proximity extension assays, and data were analyzed using multivariable logistic regression, random forest, and penalized regressions. Protein burden scores (PBSs) for overall nodule malignancy and imminent tumors were estimated. RESULTS: We identified 36 potentially informative circulating protein markers differentiating malignant from benign nodules, representing a tightly connected biological network. Ten markers were found to be particularly relevant for imminent lung cancer diagnoses within 1 year. Increases in PBSs for overall nodule malignancy and imminent tumors by 1 standard deviation were associated with odds ratios of 2.29 (95% confidence interval: 1.95 to 2.72) and 2.81 (95% confidence interval: 2.27 to 3.54) for nodule malignancy overall and within 1 year of diagnosis, respectively. Both PBSs for overall nodule malignancy and for imminent tumors were substantially higher for those with malignant nodules than for those with benign nodules, even when limited to Lung Computed Tomography Screening Reporting and Data System (LungRADS) category 4 (P < .001). CONCLUSIONS: Circulating protein markers can help differentiate malignant from benign pulmonary nodules. Validation with an independent computed tomographic screening study will be required before clinical implementation.

Using Recurrent Neural Networks to Extract High-Quality Information From Lung Cancer Screening Computerized Tomography Reports for Inter-Radiologist Audit and Feedback Quality Improvement.

PURPOSE: Lung cancer screening programs generate a high volume of low-dose computed tomography (LDCT) reports that contain valuable information, typically in a free-text format. High-performance named-entity recognition (NER) models can extract relevant information from these reports automatically for inter-radiologist quality control. METHODS: Using LDCT report data from a longitudinal lung cancer screening program (8,305 reports; 3,124 participants; 2006-2019), we trained a rule-based model and two bidirectional long short-term memory (Bi-LSTM) NER neural network models to detect clinically relevant information from LDCT reports. Model performance was tested using F1 scores and compared with a published open-source radiology NER model (Stanza) in an independent evaluation set of 150 reports. The top performing model was applied to a data set of 6,948 reports for an inter-radiologist quality control assessment. RESULTS: The best performing model, a Bi-LSTM NER recurrent neural network model, had an overall F1 score of 0.950, which outperformed Stanza (F1 score = 0.872) and a rule-based NER model (F1 score = 0.809). Recall (sensitivity) for the best Bi-LSTM model ranged from 0.916 to 0.991 for different entity types; precision (positive predictive value) ranged from 0.892 to 0.997. Test performance remained stable across time periods. There was an average of a 2.86-fold difference in the number of identified entities between the most and the least detailed radiologists. CONCLUSION: We built an open-source Bi-LSTM NER model that outperformed other open-source or rule-based radiology NER models. This model can efficiently extract clinically relevant information from lung cancer screening computerized tomography reports with high accuracy, enabling efficient audit and feedback to improve quality of patient care.

Real-World Treatment Sequencing, Toxicities, Health Utilities, and Survival Outcomes in Patients with Advanced ALK-Rearranged Non-Small-Cell Lung Cancer.

OBJECTIVES: This real-world analysis describes treatment patterns, sequencing and clinical effectiveness, toxicities, and health utility outcomes in advanced-stage, incurable ALK-positive NSCLC patients across five different ALK-TKIs. MATERIALS AND METHODS: Clinicodemographic, treatment, and toxicity data were collected retrospectively in patients with advanced-stage ALK-positive NSCLC at Princess Margaret Cancer Centre. Patient-reported symptoms, toxicities, and health utilities were collected prospectively. RESULTS: Of 148 ALK-positive NSCLC patients seen July 2009-May 2021, median age was 58.9 years; 84 (57%) were female; 112 (76%) never-smokers; 54 (47%) Asian and 40 (35%) white; 139 (94%) received at least one ALK-TKI: crizotinib (n = 74; 54%) and alectinib (n = 61; 44%) were administered mainly as first-line ALK-TKI, ceritinib, brigatinib and lorlatinib were administered primarily after previous ALK-TKI failure. Median overall survival (OS) was 54.0 months; 31 (21%) patients died within two years of advanced-stage diagnosis. Treatment modifications were observed in 35 (47%) patients with crizotinib, 19 (61%) with ceritinib, 41 (39%) with alectinib, 9 (41%) with brigatinib and 8 (30%) with lorlatinib. Prevalence of dose modifications and self-reported toxicities were higher with early versus later generation ALK-TKIs (P<.05). The presence of early treatment modification was not negatively associated with progression-free survival (PFS) and OS analyses. CONCLUSION: Serial ALK-TKI sequencing approaches are viable therapeutic options that can extend quality of life and quantity-of-life, though a fifth of patients died within two years. No best single sequencing approach could be determined. Clinically relevant toxicities occurred across all ALK-TKIs. Treatment modifications due to toxicity may not necessarily compromise outcomes, allowing multiple approaches to deal with ALK-TKI toxicities.

EGFR mutation prevalence, real-world treatment patterns, and outcomes among patients with resected, early-stage, non-small cell lung cancer in Canada.

OBJECTIVES: The ADAURA trial demonstrated the benefit of adjuvant osimertinib among patients with resected, early-stage, epidermal growth factor receptor-mutated (EGFRm) non-small cell lung cancer (NSCLC). To understand the potential population impact, it is critical to deduce the prevalence, management, and outcomes of this patient population in the real-world setting before use of adjuvant osimertinib. MATERIALS AND METHODS: Using PALEOS (Pan-cAnadian Lung cancEr Observational Study) data (2012-2019), a retrospective, multi-center, observational cohort study was conducted among patients with early-stage (IB-IIIA) resected NSCLC who had not received neoadjuvant therapy. Study outcomes included EGFRm prevalence, treatment patterns, recurrence outcomes, and overall and disease-free survival (OS/DFS). RESULTS: Among patients undergoing reflexive EGFRm testing by a pathologist at time of diagnosis irrespective of disease stage (N = 535), 23 % were EGFRm-positive; 15.9 % had common mutations and 5.6 % had uncommon mutations. Within the EGFRm-positive cohort (N = 156), mean age at diagnosis was 68 years, 65 % of patients were female, and 35 % were of Asian descent. At diagnosis, 48 %, 31 %, and 21 % had stage IB, II, or IIIA disease, respectively; 46 % received adjuvant therapy after resection. Half of patients experienced disease recurrence, typically involving distant sites; central nervous system metastasis varied from 12 % to 15.0 % across disease stages. EGFR tyrosine kinase inhibitors were the most commonly received therapy after first metastatic recurrence. Median OS (DFS) was not reached, 71.2 (22.8) months, and 50.1 (18.0) months among stage IB, II, and IIIA patients. Patients with uncommon EGFRm had a lower probability of survival than those with common EGFRm (2 years: 87 % vs 91 %-94 %; 4 years: 56 % vs 73 %-82 %). CONCLUSION: Approximately-one-quarter of patients with resected, early-stage NSCLC were EGFRm-positive in this study. These patients had high recurrence rates and suboptimal long-term survival after treatment with current therapies. New adjuvant treatments are warranted.

Field evaluation of a prototype tuberculosis lipoarabinomannan lateral flow assay on HIV-positive and HIV-negative patients.

Detection of tuberculosis at the point-of-care (POC) is limited by the low sensitivity of current commercially available tests. We describe a diagnostic accuracy field evaluation of a prototype urine Tuberculosis Lipoarabinomannan Lateral Flow Assay (TB-LAM LFA) in both HIV-positive and HIV-negative patients using fresh samples with sensitivity and specificity as the measures of accuracy. This prototype combines a proprietary concentration system with a sensitive LFA. In a prospective study of 292 patients with suspected pulmonary tuberculosis in Uganda, the clinical sensitivity and specificity was compared against a microbiological reference standard including sputum Xpert MTB/RIF Ultra and solid and liquid culture. TB-LAM LFA had an overall sensitivity of 60% (95%CI 51-69%) and specificity of 80% (95%CI 73-85%). When comparing HIV-positive (N = 86) and HIV-negative (N = 206) patients, there was no significant difference in sensitivity (sensitivity difference 8%, 95%CI -11% to +24%, p = 0.4351) or specificity (specificity difference -9%, 95%CI -24% to +4%, p = 0.2051). Compared to the commercially available Alere Determine TB-LAM Ag test, the TB-LAM LFA prototype had improved sensitivity in both HIV-negative (difference 49%, 95%CI 37% to 59%, p<0.0001) and HIV-positive patients with CD4+ T-cell counts >200cells/µL (difference 59%, 95%CI 32% to 75%, p = 0.0009). This report is the first to show improved performance of a urine TB LAM test for HIV-negative patients in a high TB burden setting. We also offer potential assay refinement solutions that may further improve sensitivity and specificity.

A Non-Canonical Calmodulin Target Motif Comprising a Polybasic Region and Lipidated Terminal Residue Regulates Localization.

Calmodulin (CaM) is a Ca2+-sensor that regulates a wide variety of target proteins, many of which interact through short basic helical motifs bearing two hydrophobic 'anchor' residues. CaM comprises two globular lobes, each containing a pair of EF-hand Ca2+-binding motifs that form a Ca2+-induced hydrophobic pocket that binds an anchor residue. A central flexible linker allows CaM to accommodate diverse targets. Several reported CaM interactors lack these anchors but contain Lys/Arg-rich polybasic sequences adjacent to a lipidated N- or C-terminus. Ca2+-CaM binds the myristoylated N-terminus of CAP23/NAP22 with intimate interactions between the lipid and a surface comprised of the hydrophobic pockets of both lobes, while the basic residues make electrostatic interactions with the negatively charged surface of CaM. Ca2+-CaM binds farnesylcysteine, derived from the farnesylated polybasic C-terminus of KRAS4b, with the lipid inserted into the C-terminal lobe hydrophobic pocket. CaM sequestration of the KRAS4b farnesyl moiety disrupts KRAS4b membrane association and downstream signaling. Phosphorylation of basic regions of N-/C-terminal lipidated CaM targets can reduce affinity for both CaM and the membrane. Since both N-terminal myristoylated and C-terminal prenylated proteins use a Singly Lipidated Polybasic Terminus (SLIPT) for CaM binding, we propose these polybasic lipopeptide elements comprise a non-canonical CaM-binding motif.

Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety.

KRAS4b is a small guanosine triphosphatase (GTPase) protein that regulates several signal transduction pathways that underlie cell proliferation, differentiation, and survival. KRAS4b function requires prenylation of its C terminus and recruitment to the plasma membrane, where KRAS4b activates effector proteins including the RAF family of kinases. The Ca2+-sensing protein calmodulin (CaM) has been suggested to regulate the localization of KRAS4b through direct, Ca2+-dependent interaction, but how CaM and KRAS4b functionally interact is controversial. Here, we determined a crystal structure, which was supported by solution nuclear magnetic resonance (NMR), that revealed the sequestration of the prenyl moiety of KRAS4b in the hydrophobic pocket of the C-terminal lobe of Ca2+-bound CaM. Our engineered fluorescence resonance energy transfer (FRET)-based biosensor probes (CaMeRAS) showed that, upon stimulation of Ca2+ influx by extracellular ligands, KRAS4b reversibly translocated in a Ca2+-CaM-dependent manner from the plasma membrane to the cytoplasm in live HeLa and HEK293 cells. These results reveal a mechanism underlying the inhibition of KRAS4b activity by Ca2+ signaling pathways.

Noncompetitive inhibitors of TNFR1 probe conformational activation states.

Tumor necrosis factor receptor 1 (TNFR1) is a central mediator of the inflammatory pathway and is associated with several autoimmune diseases such as rheumatoid arthritis. A revision to the canonical model of TNFR1 activation suggests that activation involves conformational rearrangements of preassembled receptor dimers. Here, we identified small-molecule allosteric inhibitors of TNFR1 activation and probed receptor dimerization and function. Specifically, we used a fluorescence lifetime-based high-throughput screen and biochemical, biophysical, and cellular assays to identify small molecules that noncompetitively inhibited the receptor without reducing ligand affinity or disrupting receptor dimerization. We also found that residues in the ligand-binding loop that are critical to the dynamic coupling between the extracellular and the transmembrane domains played a key gatekeeper role in the conformational dynamics associated with signal propagation. Last, using a simple structure-activity relationship analysis, we demonstrated that these newly found molecules could be further optimized for improved potency and specificity. Together, these data solidify and deepen the new model for TNFR1 activation.

A mobile-phone based high-resolution microendoscope to image cervical precancer.

Nearly 90% of cervical cancer cases and deaths occur in low- and middle-income countries that lack comprehensive national HPV immunization and cervical cancer screening programs. In these settings, it is difficult to implement screening programs due to a lack of infrastructure and shortage of trained personnel. Screening programs based on visual inspection with acetic acid (VIA) have been successfully implemented in some low-resource settings. However, VIA has poor specificity and up to 90% of patients receiving treatment based on a positive VIA exam are over-treated. A number of studies have suggested that high-resolution cervical imaging to visualize nuclear morphology in vivo can improve specificity by better distinguishing precancerous and benign lesions. To enable high-resolution imaging in low-resource settings, we developed a portable, low-cost, high-resolution microendoscope that uses a mobile phone to detect and display images of cervical epithelium in vivo with subcellular resolution. The device was fabricated for less than $2,000 using commercially available optical components including filters, an LED and triplet lenses assembled in a 3D-printed opto-mechanical mount. We show that the mobile high-resolution microendoscope achieves similar resolution and signal-to-background ratio as previously reported high-resolution microendoscope systems using traditional cameras and computers to detect and display images. Finally, we demonstrate the ability of the mobile high-resolution microendoscope to image normal and precancerous squamous epithelium of the cervix in vivo in a gynecological referral clinic in Barretos, Brazil.

Expression and Purification of Calmodulin for NMR and Other Biophysical Applications.

Calmodulin (CaM) is a ubiquitous calcium-sensing protein that has one of the most highly conserved sequences among eukaryotes. CaM has been a useful tool for biologists studying calcium signaling for decades. In recent years, CaM has also been implicated in numerous cancer-associated pathways, and rare CaM mutations have been identified as a cause of human cardiac arrhythmias. Here, we present a collection of our most recent and effective protocols for the expression and purification of recombinant CaM from Escherichia coli, including various isotopic labeling schemes, primarily for nuclear magnetic resonance (NMR) spectroscopy and other biophysical applications.

Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation.

Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled 'dark state'.

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin-myosin structure and function.

We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin-myosin structure and function. Actin-myosin interactions play crucial roles in the generation of cellular force and movement. Numerous mutations and post-translational modifications of actin or myosin disrupt muscle function and cause life-threatening syndromes. Here, we used a FRET biosensor to identify modulators that bind to the actin-myosin interface and alter the structural dynamics of this complex. We attached a fluorescent donor to actin at Cys-374 and a nonfluorescent acceptor to a peptide containing the 12 N-terminal amino acids of the long isoform of skeletal muscle myosin's essential light chain. The binding site on actin of this acceptor-labeled peptide (ANT) overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-ANT complex as in the actin-myosin complex and (b) a significant decrease in actin-ANT FRET upon binding myosin. A high-throughput FRET screen of a small-molecule library (NCC, 727 compounds), using a unique fluorescence lifetime readout with unprecedented speed and precision, showed that FRET is significantly affected by 10 compounds in the micromolar range. Most of these "hits" alter actin-activated myosin ATPase and affect the microsecond dynamics of actin detected by transient phosphorescence anisotropy. We conclude that the actin-ANT TR-FRET assay enables detection of pharmacologically active compounds that affect actin structural dynamics and actomyosin function. This assay establishes feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

Gestational hypoxia disrupts the neonatal leptin surge and programs hyperphagia and obesity in male offspring in the Sprague-Dawley rat.

The effect of gestational hypoxia on the neonatal leptin surge, development of hypothalamic arcuate nuclei (ARH) projections and appetite that could contribute to the programming of offspring obesity is lacking. We examined the effect of 12% O2 from gestational days 15-19 in the Sprague-Dawley rat on post-weaning appetite, fat deposition by MRI, adipose tissue cytokine expression, the neonatal leptin surge, ARH response to exogenous leptin, and αMSH projections to the paraventricular nucleus (PVN) in response to a high fat (HFD) or control diet (CD) in male offspring. Normoxia (NMX) and Hypoxia (HPX) offspring exhibited increased food intake when fed a HFD from 5-8 weeks post-birth; HPX offspring on the CD had increased food intake from weeks 5-7 vs. NMX offspring on a CD. HPX offspring on a HFD remained hyperphagic through 23 weeks. Body weight were the same between offspring from HPX vs. NMX dams from 4-12 weeks of age fed a CD or HFD. By 14-23 weeks of age, HPX offspring fed the CD or HFD as well as male NMX offspring fed the HFD were heavier vs. NMX offspring fed the CD. HPX offspring fed a CD exhibited increased abdominal adiposity (MRI) that was amplified by a HFD. HPX offspring fed a HFD exhibited the highest abdominal fat cytokine expression. HPX male offspring had higher plasma leptin from postnatal day (PN) 6 through 14 vs. NMX pups. HPX offspring exhibited increased basal c-Fos labeled cells in the ARH vs. NMX pups on PN16. Leptin increased c-Fos staining in the ARH in NMX but not HPX offspring at PN16. HPX offspring had fewer αMSH fibers in the PVN vs. NMX offspring on PN16. In conclusion, gestational hypoxia impacts the developing ARH resulting in hyperphagia contributing to adult obesity on a control diet and exacerbated by a HFD.

Soluble Extracellular Domain of Death Receptor 5 Inhibits TRAIL-Induced Apoptosis by Disrupting Receptor-Receptor Interactions.

Dysregulation of tumor necrosis factor (TNF) receptor signaling is a key feature of various inflammatory disorders. Current treatments for TNF-related diseases function either by sequestering ligand or blocking ligand-receptor interactions, which can cause dangerous side effects by inhibiting the receptors that are not involved in the disease condition. Thus, alternate strategies that target receptor-receptor interactions are needed. We hypothesized that the soluble extracellular domain (ECD) of long isoform of death receptor 5 (DR5) could block endogenous receptor assembly, mimicking the biological effect of decoy receptors that lack the death domain to trigger apoptosis. Using live-cell fluorescence resonance energy transfer studies, we demonstrated that soluble ECD disrupts endogenous DR5-DR5 interactions. Cell viability assays were used to demonstrate the complete inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the ECD, although TRAIL is still able to bind to the receptor. Importantly, we used mutagenesis to prove that the inhibition of TRAIL-induced apoptosis by the ECD predominantly comes from the disruption of DR5 oligomerization and not ligand sequestration. Inhibition of death receptor activation should have important therapeutic applications in diseases such as nonalcoholic fatty liver disease. More generally, this approach should be generalized to enable the inhibition of other TNF receptor signaling mechanisms that are associated in a wide range of clinical conditions.

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor-receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

High-resolution microendoscopy: a point-of-care diagnostic for cervical dysplasia in low-resource settings.

Cervical cancer is the third leading cause of cancer-related death among women in low-to-middle income countries. Pap testing and pathological services are difficult to implement under these settings. Alternative techniques for the diagnosis of cervical precancer in these settings are needed to reduce the burden of the disease. The objective of this study was to evaluate the diagnostic accuracy of a low-cost, high-resolution microendoscope imaging system in identifying precancerous lesions of the cervix in vivo. A retrospective study of 59 patients undergoing colposcopy for an abnormal Pap test was performed at Hospital de Câncer de Barretos in Brazil. All patients underwent colposcopy as per standard of care, and acetowhite lesions were recorded. High-resolution microendoscopy (HRME) images were obtained from one colposcopically normal region and from all lesions observed on colposcopy. Biopsies of abnormal areas were obtained and reviewed by three independent, blinded pathologists and compared with HRME findings. The mean nuclear area and the median nuclear eccentricity were calculated from HRME images acquired from each site. A diagnostic algorithm to distinguish histopathologically diagnosed cervical intraepithelial neoplasias of grade 2 or more severe lesions (high grade) from less severe lesions (low grade) was developed using these parameters. A test of trend was used to analyze the relationship between HRME positivity and severity of histopathogical diagnosis. Fisher's exact test was used to analyze differences in HRME positivity between high-grade and low-grade lesions. Evaluable images were obtained from 108 of 143 discrete sites. Of these, 71 sites were colposcopically normal or low grade according to histopathology and 37 were diagnosed as high grade on the basis of histopathology. Using the mean nuclear area and the median nuclear eccentricity, HRME images from 59 colposcopically abnormal sites were classified as high grade or low grade with 92% sensitivity and 77% specificity compared with histopathological findings. Increasing HRME positivity showed a significant trend with increasing severity of diagnosis (Ptrend<0.001). We found a strong association (P<0.001) between HRME positivity and a histopathological diagnosis of cervical intraepithelial neoplasia of grade 2 or higher. HRME demonstrated an accurate in-situ diagnosis of high-grade dysplasia. In low-resource settings in which colposcopy and histopathology services are severely limited or unavailable, HRME may provide a low-cost, accurate method for diagnosis of cervical precancer without the need for biopsy, allowing for a single 'screen-and-treat' approach.

A paper-based immunoassay to determine HPV vaccination status at the point-of-care.

OBJECTIVE: To develop and evaluate a paper-based point-of-care HPV serology test to determine if an individual has received two or more HPV immunizations. METHODS: The paper-based immunoassay was constructed using a nitrocellulose lateral flow strip with adsorbed HPV16 virus-like particles serving as the capturing moiety. Three capture zones containing virus-like particles were placed in series to allow for visual discrimination between high and low HPV16 plasma antibody concentrations. A plasma separation membrane was used to allow whole blood to be applied directly to the assay. All reagents were dried on glass fiber pads during device fabrication and were rehydrated with buffer at the time of use. A pilot study consisting of 35 subjects with a history of zero, one, two or three HPV vaccines was conducted to evaluate the immunoassay. The completed paper-based immunoassays were scanned for visual interpretation by three researchers who were blinded to the true results and separately evaluated quantitatively using MATLAB. RESULTS: For the 28 tests valid for analysis, fifteen subjects reported receiving two or more HPV vaccines, three reported receiving one, and ten reported having no HPV vaccinations. The paper-based immunoassays for all fifteen subjects who reported having received two or more HPV vaccines were judged positive by all researchers. Twelve of the thirteen tests from individuals reporting one or zero vaccinations were deemed negative by all observers. One test from an unvaccinated individual was judged positive by two out of three reviewers. Quantitatively, all tests were correctly separated between the two groups. CONCLUSIONS: We successfully designed and tested a HPV serology test amenable to the point-of-care. The device showed promising results in a pilot study for discriminating between those who received two or more HPV vaccinations and those who did not. Furthermore, this device offers a platform for producing other semi-quantitative point-of-care serological tests.

Synchronous primary anorectal melanoma and sigmoid adenocarcinoma: a case report.

Synchronous primary anorectal melanoma and colorectal adenocarcinoma is extremely rare, with only 5 cases reported in the literature. Here, a case is reported and the currently available literature is summarized. A 72-year-old white male presented with changes in his bowel habits and unintentional weight loss. Colonoscopy revealed a polypoid mass in the distal rectum extending to the anal verge anteriorly and a circumferential polypoid mass in the distal sigmoid colon. Biopsies were taken, which revealed poorly differentiated melanoma of the anorectal mass and moderately differentiated adenocarcinoma of the sigmoid mass with nodal involvement. Computed tomography of the abdomen showed liver metastasis. An extended abdominoperineal resection was undertaken for palliation, relief of symptoms, and definitive histology to guide further management. Consequently, a resection of the hepatic metastasis was attempted; however, macroscopic deposits were discovered on 7 of 8 liver segments perioperatively. He was subsequently referred to medical oncology for palliative chemotherapy. Synchronous primary anorectal melanoma and colorectal adenocarcinoma is rare, this being the sixth report found in the literature. In summary of the available cases, all synchronous cancers were located in the rectosigmoid and had very similar presentations. Most presented relatively late and were generally treated with abdominoperineal resection, which appears to be the best treatment option. Overall, prognosis appears to be dismal. General and colorectal surgeons should always be aware of the possibilities of simultaneous primary cancers because this can affect treatment modalities and prognosis for the patient.

High-resolution microendoscope for the detection of cervical neoplasia.

Cervical cancer causes 275,000 deaths each year with 85 % of these deaths occurring in the developing world. One of the primary reasons for the concentration of deaths in developing countries is a lack of effective screening methods suited for the infrastructure of these countries. In order to address this need, we have developed a high-resolution microendoscope (HRME). The HRME is a fiber-based fluorescence microscope with subcellular resolution. Using the vital stain proflavine, we are able to image cell nuclei in vivo and evaluate metrics such as nuclear-to-cytoplasmic ratio, critical to identifying precancerous epithelial regions. In this chapter, we detail the materials and methods necessary to build this system from commercially available parts.