Integrating clinical research in an operative screening and diagnostic breast imaging department: First experience, results and perspectives using microwave imaging.

Rationale and objectives: Clinical research is crucial for evaluating new medical procedures and devices. It is important for healthcare units and hospitals to minimize the disruptions caused by conducting clinical studies; however, complex clinical pathways require dedicated recruitment and study designs.This work presents the effective introduction of novel microwave breast imaging (MBI), via MammoWave apparatus, into the clinical routine of an operative screening and diagnostic breast imaging department for conducting a multicentric clinical study. Materials and methods: Microwave breast imaging, using MammoWave apparatus, was performed on volunteers coming from different clinical pathways. Clinical data, comprising demographics and conventional radiologic reports (used as reference standard), was collected; a satisfaction questionnaire was filled by every volunteer. Microwave images were analyzed by an automatic clinical decision support system, which quantified their corresponding features to discriminate between breasts with no relevant radiological findings (NF) and breasts with described findings (WF). Results: Conventional breast imaging (DBT, US, MRI) and MBI were performed and adapted to assure best clinical practices and optimum pathways. 180 volunteers, both symptomatic and asymptomatic, were enrolled in the study. After microwave images' quality assessment, 48 NF (15 dense) and 169 WF (88 dense) breasts were used for the prospective study; 48 (18 dense) breasts suffered from a histology-confirmed carcinoma. An overall sensitivity of 85.8 % in breasts lesions' detection was achieved by the microwave imaging apparatus. Conclusion: An optimum recruitment strategy was implemented to assess MBI. Future trials may show the clinical usefulness of microwave imaging, which may play an important role in breast screening.

Dielectric Characterization of Breast Biopsied Tissues as Pre-Pathological Aid in Early Cancer Detection: A Blinded Feasibility Study.

Dielectric characterization has significant potential in several medical applications, providing valuable insights into the electromagnetic properties of biological tissues for disease diagnosis, treatment planning, and monitoring of therapeutic interventions. This work presents the use of a custom-designed electromagnetic characterization system, based on an open-ended coaxial probe, for discriminating between benign and malignant breast tissues in a clinical setting. The probe's development involved a well-balanced compromise between physical feasibility and its combined use with a reconstruction algorithm known as the virtual transmission line model (VTLM). Immediately following the biopsy procedure, the dielectric properties of the breast tissues were reconstructed, enabling tissue discrimination based on a rule-of-thumb using the obtained dielectric parameters. A comparative analysis was then performed by analyzing the outcomes of the dielectric investigation with respect to conventional histological results. The experimental procedure took place at Complejo Hospitalario Universitario de Toledo-Hospital Virgen de la Salud, Spain, where excised breast tissues were collected and subsequently analyzed using the dielectric characterization system. A comprehensive statistical evaluation of the probe's performance was carried out, obtaining a sensitivity, specificity, and accuracy of 81.6%, 61.5%, and 73.4%, respectively, compared to conventional histological assessment, considered as the gold standard in this investigation.

Breast cancer mortality after eight years of an improved screening program using digital breast tomosynthesis.

OBJECTIVES: To assess screening quality metrics and to describe mortality rates eight years after redesign of breast cancer screening and diagnosis pathways, and the introduction of digital breast tomosynthesis. SETTING: Breast Unit of the Toledo Health Area in the region of Castilla-La Mancha (Spain). METHODS: We recorded screening metrics and mortality data following the introduction of digital breast tomosynthesis in 2011 for screening and diagnosis pathways. We then compared the mortality between Toledo Health Area and the rest of Castilla-La Mancha, where digital breast tomosynthesis is not available. RESULTS: All screening quality metrics improved following the introduction of digital breast tomosynthesis. The cancer detection rate significantly increased from 2.3 (95% confidence interval (CI): 1.9-3.6) to 4.5 per 1000 women (95% CI: 3.2-5.2) on average between the periods 2005-2009 and 2015-2018, while the recall rate significantly decreased from 7.0% (95% CI: 6.8%-8.2%) to 2.6% (95% CI: 2.0%-3.6%). Comparing breast cancer mortality rates for 2014-2018 in the Toledo Health Area with the rest of Castilla-La Mancha, which had similar cancer treatment access and management protocols but without digital breast tomosynthesis, the crude mortality rate was 17.79 (95% CI: 15.38 -20.19) vs. 24.76 per 100,000 (95% CI: 26.12-23.39), respectively. The cumulative risk of death was also significantly lower for the Toledo Health Area than for Castilla-La Mancha. CONCLUSION: The introduction of digital breast tomosynthesis improved screening quality indicators. Breast cancer mortality simultaneously decreased with respect to the rest of Castilla-La Mancha. Further research is needed to assess the long-term results, and the role that the redesign may have played in reducing mortality.

Diagnostic value of halo sign in young women (aged 45 to 49 years) in a breast screening programme with synthesized 2D mammography.

OBJECTIVE:: To assess the clinical performance of the halo sign in tomosynthesis and synthesized 2D mammography, and to identify age groups where its diagnostic value may be greater. METHODS:: 183 patients with nodules were recalled from the breast screening programme (with tomosynthesis and 2D synthesized mammograms). The patients were separated into two groups, 45-49 years and 50-69 years, and depending on the presence or not of halo sign. We calculated the predictive values for the different age groups. RESULTS:: In 45-49 years group, 86 nodular lesions were recalled, 66 (76.7%) with positive halo sign and 20 (23.3%) with negative halo sign. In positive halo sign group, biopsy was considered in 23 (34.8%), with histological features of benignity. In 50-69 years group, 98 nodular lesions from 97 patients were recalled, 51 (52%) with positive halo sign and 47 (48%) with negative halo sign. In positive halo sign group, biopsy was considered in 13 (25.5%); four (30.8%) were malignant and nine (69.2%) were benign. CONCLUSION:: Halo sign could be considered as a marker of benign lesion in females < 50 years. In females ≥ 50 years, other breast imaging techniques should be considered, with or without histological studies, to rule out malignancy. ADVANCES IN KNOWLEDGE:: The trend of a positive halo sign to act as a marker of benign lesion could be improve the recall rate and positive predictive values in the breast screening programme with tomosynthesis and synthesized 2D mammography, especially in young females.

Sarcopenic obesity and myosteatosis are associated with higher mortality in patients with cirrhosis.

BACKGROUND AND AIMS: Obesity is frequently associated with cirrhosis, and cirrhotic patients may develop simultaneous loss of skeletal muscle and gain of adipose tissue, culminating in the condition of sarcopenic obesity. Additionally, muscle depletion is characterized by both a reduction in muscle size and increased proportion of muscular fat, termed myosteatosis. In this study, we aimed to establish the frequency and clinical significance of sarcopenia, sarcopenic obesity and myosteatosis in cirrhotic patients. METHODS: We analysed 678 patients with cirrhosis. Sarcopenia, sarcopenic obesity and myosteatosis were analysed by CT scan using the third lumbar vertebrae skeletal muscle and attenuation indexes, using previously validated gender-and body mass index-specific cutoffs. RESULTS: Patients were predominately men (n = 457, 67%), and cirrhosis aetiology was hepatitis C virus in 269 patients (40%), alcohol in 153 (23%), non-alcoholic steatohepatitis/cryptogenic in 96 (14%), autoimmune liver disease in 55 (8%), hepatitis B virus in 43 (6%), and others in 5 patients (1%). Sarcopenia was present in 292 (43%), 135 had sarcopenic obesity (20%) and 353 had myosteatosis (52%). Patients with sarcopenia (22 ± 3 vs. 95 ± 22 months, P < 0.001), sarcopenic obesity (22 ± 3 vs. 95 ± 22 months, P < 0.001), and myosteatosis (28 ± 5 vs. 95 ± 22 months, P < 0.001) had worse median survival than patients without muscular abnormalities. By multivariate Cox regression analysis, both sarcopenia [hazard ratio (HR) 2.00, 95% confidence interval (CI) 1.44-2.77, P < 0.001], and myosteatosis (HR 1.42, 95% CI 1.02-1.07, P = 0.04) were associated with mortality. CONCLUSIONS: Sarcopenia, sarcopenic obesity and myosteatosis are often present in patients with cirrhosis, and sarcopenia and myosteatosis are independently associated with a higher long-term mortality in cirrhosis.

Fibrosis in nonalcoholic Fatty liver disease: mechanisms and clinical implications.

Nonalcoholic fatty liver disease (NAFLD) is tightly associated with obesity and the metabolic syndrome in the United States and other Western countries. It is also the liver disease most rapidly increasing in prevalence in the United States, and has become a major indication for liver transplantation worldwide. Compelling evidence shows that the degree of liver fibrosis dictates liver prognosis in NAFLD. This review focuses on fibrosis based on clinical and basic perspectives. The authors summarize the physiopathology of fibrosis development and progression in NAFLD, highlighting its molecular mechanisms, clinical consequences of fibrosis, the diagnostic approach and management strategies.

Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Histologic analysis of liver biopsy specimens allows for grading and staging of nonalcoholic fatty liver disease (NAFLD). We performed a longitudinal study to investigate the long-term prognostic relevance of histologic features for patients with NAFLD. METHODS: We performed a retrospective analysis of 619 patients diagnosed with NAFLD from 1975 through 2005 at medical centers in the United States, Europe, and Thailand. Patients underwent laboratory and biopsy analyses, and were examined every 3-12 months after their diagnosis. Outcomes analyzed were overall mortality, liver transplantation, and liver-related events. Cumulative outcomes were compared by log-rank analysis. Cox proportional-hazards regression was used to estimate adjusted hazard ratios (HRs). Time at risk was determined from the date of liver biopsy to the date of outcome or last follow-up examination. RESULTS: Over a median follow-up period of 12.6 years (range, 0.3-35.1 y), 193 of the patients (33.2%) died or underwent liver transplantation. Features of liver biopsies significantly associated with death or liver transplantation included fibrosis stage 1 (HR, 1.88; 95% confidence interval [CI], 1.28-2.77), stage 2 (HR, 2.89; 95% CI, 1.93-4.33), stage 3 (HR, 3.76; 95% CI, 2.40-5.89), and stage 4 (HR, 10.9; 95% CI, 6.06-19.62) compared with stage 0, as well as age (HR, 1.07; 95% CI, 1.05-1.08), diabetes (HR, 1.61; 95% CI, 1.13-2.30), current smoking (HR, 2.62; 95% CI, 1.67-4.10), and statin use (HR, 0.32; 95% CI, 0.14-0.70). Twenty-six patients (4.2%) developed liver-related events; fibrosis stage 3 (HR, 14.2; 95% CI, 3.38-59.68) and stage 4 (HR, 51.5; 95% CI, 9.87-269.2) compared with stage 0, were associated significantly with the events. Patients with fibrosis, regardless of steatohepatitis or NAFLD activity score, had shorter survival times than patients without fibrosis. CONCLUSIONS: In a longitudinal study of patients with NAFLD, fibrosis stage, but no other histologic features of steatohepatitis, were associated independently with long-term overall mortality, liver transplantation, and liver-related events.

Nonalcoholic fatty liver disease: a precursor of the metabolic syndrome.

The conventional paradigm of nonalcoholic fatty liver disease representing the "hepatic manifestation of the metabolic syndrome" is outdated. We identified and summarized longitudinal studies that, supporting the association of nonalcoholic fatty liver disease with either type 2 diabetes mellitus or metabolic syndrome, suggest that nonalcoholic fatty liver disease precedes the development of both conditions. Online Medical databases were searched, relevant articles were identified, their references were further assessed and tabulated data were checked. Although several cross-sectional studies linked nonalcoholic fatty liver disease to either diabetes and other components of the metabolic syndrome, we focused on 28 longitudinal studies which provided evidence for nonalcoholic fatty liver disease as a risk factor for the future development of diabetes. Moreover, additional 19 longitudinal reported that nonalcoholic fatty liver disease precedes and is a risk factor for the future development of the metabolic syndrome. Finally, molecular and genetic studies are discussed supporting the view that aetiology of steatosis and lipid intra-hepatocytic compartmentation are a major determinant of whether fatty liver is/is not associated with insulin resistance and metabolic syndrome. Data support the novel paradigm of nonalcoholic fatty liver disease as a strong determinant for the development of the metabolic syndrome, which has potentially relevant clinical implications for diagnosing, preventing and treating metabolic syndrome.

An extension of STARD statements for reporting diagnostic accuracy studies on liver fibrosis tests: the Liver-FibroSTARD standards.

BACKGROUND & AIMS: Chronic liver diseases are highly prevalent and require an accurate evaluation of liver fibrosis to determine patient management. Over the last decade, great effort has been made to develop non-invasive liver fibrosis tests. The ensuing increase of literature is, however, impaired by extensive heterogeneity in the quality of published reports. The Standards for Reporting of Diagnostic Accuracy Studies (STARD), first published in 2003, were developed to improve the quality of research reports on diagnostic studies. We aimed to evaluate STARD statements in the setting of diagnostic studies on non-invasive liver fibrosis tests, and to propose an extended version developed specifically for those studies. METHODS: Eight French experts evaluated STARD statement adequacy in 10 studies on non-invasive liver fibrosis tests and then developed an extended version with a glossary. The new checklist and glossary were independently evaluated by seven international experts. RESULTS: Fourteen of the 25 STARD items were considered only partially adequate for the evaluation of diagnostic studies on non-invasive liver fibrosis tests. Inter-expert agreement was at least very good for 8 STARD items (32%), moderate for 9 (36%), and poor or very poor for 8 (32%). The experts' proposals were developed into the new Liver-FibroSTARD standards including a checklist with 62 items/sub-items and a corresponding comprehensive glossary. New proposals were inserted in the 25 STARD items as a complementary module. Independent evaluation of the Liver-FibroSTARD checklist showed at least very good inter-expert agreement for 39 items/sub-items (63%), moderate agreement for 11 (18%), and poor or very poor agreement for only 12 (19%). CONCLUSIONS: As a supplement of the STARD statements, the Liver-FibroSTARD checklist and its glossary are new tools specifically designed for the evaluation of diagnostic studies about non-invasive liver fibrosis tests.

The evaluation of hepatic fibrosis scores in children with nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in children and can progress to liver cirrhosis during childhood. Patients with more advanced fibrosis on biopsy tend to have more liver complications. Noninvasive hepatic fibrosis scores have been developed for adult patients with NAFLD; however, these scores have not been validated in children. The aim of our study was to evaluate some of these scores in assessing the presence of fibrosis in children with biopsy-proven NAFLD. METHODS: Our study consisted of 92 biopsy-proven NAFLD children from five major US centers. Fibrosis was determined by an experienced pathologist (F0-4). Clinically significant fibrosis was defined as fibrosis stage ≥ 2, and advanced fibrosis was defined as F3-4. The following fibrosis scores were calculated for each child: AST/ALT ratio, AST/platelet ratio index (APRI), NAFLD fibrosis score (NFS), and FIB-4 index. ROC was performed to assess the performance of different scores for prediction of presence of any, significant, or advanced fibrosis. A p value < 0.05 was considered statistically significant. RESULTS: Mean age was 13.3 ± 3 years, and 33 % were females. Eleven (12 %) subjects had no fibrosis, 35 (38 %) had fibrosis score of 1, 26 (28 %) had fibrosis score of 2, and 20 (22 %) had a score of 3. APRI had a fair diagnostic accuracy for the presence of any fibrosis (AUC of 0.80) and poor diagnostic accuracy for significant or advanced fibrosis. AST/ALT, NFS, and FIB-4 index all either had poor diagnostic accuracy or failed to diagnose the presence of any, significant, or advanced fibrosis. CONCLUSION: Noninvasive hepatic fibrosis scores developed in adults had poor performance in diagnosing significant fibrosis in children with NAFLD. Our results highlight the urgent need to develop a reliable pediatric fibrosis score.

Valoración de las categorías BI-RADS ® de la densidad mamaria entre radiólogos y respecto a un método cuantitativo de medición / Evaluation of the BI-RADS® categories between readers and with regard to a quantitative method

Objetivo. Valorar la concordancia al clasificar las densidades mamarias empleando el sistema BI-RADS® y la variabilidad entre este método empleado por un lector experto en comparación con los valores obtenidos mediante un sistema de medición automática (Quantra®). Pacientes y métodos. Estudio descriptivo en 2 fases. Primero se evaluó la clasificación BI-RADS® de 5 lectores de una muestra seleccionada de forma aleatoria y estratificada por grupos (516 pacientes), para medir la variabilidad intraobservador e interobservador (índice kappa). Finalmente se analizó la concordancia entre el radiólogo con más experiencia y el software Quantra® que permite la valoración volumétrica de la densidad. Resultados. La concordancia por parejas varió entre débil a moderada (kappa 0,35-0,67), el grupo presentó un valor moderado de reproducibilidad (0,58), siendo mayor en las densidades extremas y en los grupos de edad límite. La variabilidad intraobservador fue de moderada a buena (0,52-0,79). Respecto a la clasificación de la densidad por categorías entre el lector experto y la aplicación, no se evidenció concordancia (kappa = 0,0737). Conclusiones. Los valores de concordancia obtenidos corroboran la evidencia descrita en la literatura, considerando que la lectura la realizaron radiólogos con experiencia y dedicación exclusiva en mama. No se apreció concordancia en la clasificación lector versus Quantra® (esta pondera las densidades en menor porcentaje respecto al análisis visual), y esto podría deberse al punto de corte por categoría (AU)

Objective. To assess agreement in breast density classification using the BI-RADS® system and the concordance between this measurement method compared with a dedicated software program (Quantra®). Patients and methods. A descriptive study was carried out in 2 phases. First, we analyzed the BI-RADS® breast density classification assessed by 5 readers in a sample of 516 patients randomly selected and stratified into groups. Intraobserver and interobserver reproducibility were calculated (kappa index). Second, we evaluated the agreement between the most experienced reader and a software package that calculates the volumetric fraction of fibroglandular tissue (Quantra®). Results. The agreement between pairs ranged from fair to moderate (kappa 0.35-0.67). The reading group showed moderate reproducibility (0.58) with the highest values in high breast densities and in the youngest and oldest patients. Intraobserver agreement ranged from moderate to substantial (0.52-0.79). In density classification, there was no evidence of agreement between the expert reader and the software application (kappa = 0.0737). Conclusions. The agreement values obtained corroborate the evidence described in the literature on reading performed by experienced radiologists working exclusively with mammograms. No agreement was found in reader classification versus Quantra® (which classifies densities with a lesser percentage than visual analysis), which could be due to the cut off by category (AU)

Noninvasive prediction of hepatic steatosis.

The diagnosis of hepatic steatosis requires demonstration of fat infiltration of the liver in imaging studies or liver biopsy. Noninvasive scores composed of clinical and laboratory variables routinely measured in clinical practice can be used to predict hepatic steatosis without imaging or liver biopsy. Using two large and well-defined populations, Meffert et al. externally validated two of those noninvasive scoring systems, named Fatty Liver Index and the Hepatic Steatosis Index, and created and validated a new score named the SHIP score. Although the three scores had mixed accuracies, they perform relatively well in predicting the presence of hepatic steatosis.

Hepatic steatosis and steatohepatitis: Are they really two distinct entities?

Non-alcoholic fatty liver disease affects nearly 30% of Americans. A histopathological spectrum exists from simple steatosis to NASH which may progress to cirrhosis and HCC. NASH is currently the third most common indication for liver transplant with increasing incidence. Steatosis can be considered the hepatic manifestation of the metabolic syndrome as insulin resistance is a major risk factor for its development. While liver biopsy is the gold standard for diagnosis, non-invasive methods are currently being developed to appropriately determine who needs histologic evaluation. Management focuses on mitigation of risk factors, since targeted therapies to halt progression of fibrosis have not been validated. Simple steatosis does not affect overall survival, but NASH conveys increased mortality. Because of this, non-invasive strategies to diagnose patients and management algorithms are needed. This review supports the definitions of simple steatosis and NASH as two distinct entities based on pathophysiology, diagnosis, management, and prognosis.

Right upper-quadrant pain in a patient with drug abuse, secondary syphilis and occult hepatitis B virus.

OBJECTIVE: To describe the etiology of hepatitis and identify occult hepatitis B virus (HBV) infection. CLINICAL PRESENTATION AND INTERVENTION: A 40-year-old man presented with severe abdominal pain and jaundice, a history of acute HBV infection that had cleared as well as the use of acetaminophen, methamphetamine, buprenorphine and marijuana. He admitted to having had unprotected sex with multiple partners of both genders. A thorough skin examination revealed papulosquamous lesions on his penis, scrotum, upper and lower extremities and feet. Transaminases and bilirubin were elevated. His rapid plasma reagin was reactive, and hepatitis serologies showed occult HBV. Liver biopsy showed severe hepatitis, but the stains for hepatitis B surface antigen and hepatitis B core antigen were negative. The pathological findings were highly indicative of drug-induced hepatitis without evidence of chronic hepatitis, reactivation of HBV or syphilitic hepatitis. With supportive management and abstinence from drugs, his condition improved. CONCLUSION: This case describes a patient with multiple potential causes for hepatitis and highlights the importance of obtaining a detailed social history. Further, one should consider the presence of occult HBV and recognize the serologic pattern.

Serum ferritin levels lack diagnostic accuracy for liver fibrosis in patients with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Series studies have associated increased serum levels of ferritin with liver fibrosis in patients with nonalcoholic fatty liver disease. We aimed to determine the accuracy with which measurements of serum ferritin determine the presence and severity of liver fibrosis, and whether combining noninvasive scoring systems with serum ferritin analysis increases the accuracy of diagnosis of advanced liver fibrosis. METHODS: We performed a retrospective analysis of data from 1014 patients with liver biopsy-confirmed nonalcoholic fatty liver disease. Three cut points of serum ferritin level, adjusted for sex, were established based on receiver operating characteristic curve analysis: 1.0-, 1.5-, and 2.0-fold the upper limit of normal. Three multiple logistic regression models were created to determine the association of these cutoff values with liver fibrosis, adjusting for age, sex, race, diabetes, body mass index, and level of alanine aminotransferase. RESULTS: A greater proportion of patients with increased serum levels of ferritin had definitive nonalcoholic steatohepatitis and more-advanced fibrosis than patients without increased levels. In all models, serum level of ferritin was significantly associated with the presence and severity of liver fibrosis. However, for all 3 cutoff values, area under the receiver operating characteristic curve values were low (less than 0.60) for the presence of fibrosis or any stage of liver fibrosis; ferritin level identified patients with fibrosis with 16%-41% sensitivity and 70%-92% specificity. The accuracy with which noninvasive scoring systems identified patients with advanced fibrosis did not change with inclusion of serum ferritin values. CONCLUSIONS: Although serum levels of ferritin correlate with more-severe liver fibrosis, based on adjusted multiple logistic regression analysis, serum ferritin levels alone have a low level of diagnostic accuracy for the presence or severity of liver fibrosis in patients with nonalcoholic fatty liver disease.

Noninvasive evaluation of NAFLD.

A common clinical concern in patients with NAFLD is whether they have NASH or simple steatosis and, more importantly, what the stage of fibrosis is and whether the level of fibrosis has increased over time. Such concern is based on the fact that patients with NAFLD with advanced fibrosis are at greatest risk of developing complications of end-stage liver disease. Although it lacks sensitivity, ultrasonography is an accepted tool for steatosis screening. The controlled attenuation parameter or CAP seems a promising screening technique, but requires further validation. Cytokeratin-18 has been extensively validated, but it is an imperfect serum marker of NASH. Ultrasonography-based transient elastography can exclude advanced fibrosis and cirrhosis, but its main limitation is its reduced applicability in patients with NAFLD, which is not completely solved by use of the XL probe. Of the noninvasive serum markers, the NAFLD fibrosis score is the most validated and has appropriate accuracy in distinguishing patients with and without advanced fibrosis. Although noninvasive methods require further validation, they could be useful for selecting those patients with NAFLD who require a liver biopsy. This Review discusses the advantages and limitations of noninvasive methods for the management of adults with NAFLD, including diagnosis and quantification of steatosis, diagnosis of NASH and staging of hepatic fibrosis.

Simple noninvasive systems predict long-term outcomes of patients with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Some patients with nonalcoholic fatty liver disease (NAFLD) develop liver-related complications and have higher mortality than other patients with NAFLD. We determined the accuracy of simple, noninvasive scoring systems in identification of patients at increased risk for liver-related complications or death. METHODS: We performed a retrospective, international, multicenter cohort study of 320 patients diagnosed with NAFLD, based on liver biopsy analysis through 2002 and followed through 2011. Patients were assigned to mild-, intermediate-, or high-risk groups based on cutoff values for 2 of the following: NAFLD fibrosis score, aspartate aminotransferase/platelet ratio index, FIB-4 score, and BARD score. Outcomes included liver-related complications and death or liver transplantation. We used multivariate Cox proportional hazard regression analysis to adjust for relevant variables and calculate adjusted hazard ratios (aHRs). RESULTS: During a median follow-up period of 104.8 months (range, 3-317 months), 14% of patients developed liver-related events and 13% died or underwent liver transplantation. The aHRs for liver-related events in the intermediate-risk and high-risk groups, compared with the low-risk group, were 7.7 (95% confidence interval [CI]: 1.4-42.7) and 34.2 (95% CI: 6.5-180.1), respectively, based on NAFLD fibrosis score; 8.8 (95% CI: 1.1-67.3) and 20.9 (95% CI: 2.6-165.3) based on the aspartate aminotransferase/platelet ratio index; and 6.2 (95% CI: 1.4-27.2) and 6.6 (95% CI: 1.4-31.1) based on the BARD score. The aHRs for death or liver transplantation in the intermediate-risk and high-risk groups compared with the low-risk group were 4.2 (95% CI: 1.3-13.8) and 9.8 (95% CI: 2.7-35.3), respectively, based on the NAFLD fibrosis scores. Based on aspartate aminotransferase/platelet ratio index and FIB-4 score, only the high-risk group had a greater risk of death or liver transplantation (aHR = 3.1; 95% CI: 1.1-8.4 and aHR = 6.6; 95% CI: 2.3-20.4, respectively). CONCLUSIONS: Simple noninvasive scoring systems help identify patients with NAFLD who are at increased risk for liver-related complications or death. NAFLD fibrosis score appears to be the best indicator of patients at risk, based on HRs. The results of this study require external validation.

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.

BACKGROUND: Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). MATERIALS AND METHODS: Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. RESULTS: Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 µM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 µM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). CONCLUSIONS: LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation.