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Biomarkers for the early diagnosis of hepatocellular carcinoma (HCC) are needed to decrease mortality from this cancer. However, as new biomarkers have been slow to be brought to clinical practice, we have developed a diagnostic algorithm that utilizes commonly used clinical measurements in those at risk of developing HCC. Briefly, as α-fetoprotein (AFP) is routinely used, an algorithm that incorporated AFP values along with four other clinical factors was developed. Discovery analysis was performed on electronic data from patients who had liver disease (cirrhosis) alone or HCC in the background of cirrhosis. The discovery set consisted of 360 patients from two independent locations. A logistic regression algorithm was developed that incorporated log-transformed AFP values with age, gender, alkaline phosphatase, and alanine aminotransferase levels. We define this as the Doylestown algorithm. In the discovery set, the Doylestown algorithm improved the overall performance of AFP by 10%. In subsequent external validation in over 2,700 patients from three independent sites, the Doylestown algorithm improved detection of HCC as compared with AFP alone by 4% to 20%. In addition, at a fixed specificity of 95%, the Doylestown algorithm improved the detection of HCC as compared with AFP alone by 2% to 20%. In conclusion, the Doylestown algorithm consolidates clinical laboratory values, with age and gender, which are each individually associated with HCC risk, into a single value that can be used for HCC risk assessment. As such, it should be applicable and useful to the medical community that manages those at risk for developing HCC. Cancer Prev Res; 9(2); 172–9. ©2015 AACR.

Data description and detailed statistical information regarding model development. Supplementary Table 1. Patient information on samples from the University of Michigan. Supplementary Table 2. Patient information on samples from HALT-C. Supplementary Table 3. Patient information on samples from EDRN. Supplementary Table 4. Patient information on samples from Thomas Jefferson University. Supplementary Table 5. Patient information on samples from the University of Texas Southwestern Medical Center. Supplementary Table 6. Odds ratios for each predictor in univariate and multivariate logistic regressions. Supplementary Table 7. Indices of goodness-of-fit and apparent validation of candidate logistic regression models. Supplementary Table 8. Performance of leave one out cross validation (LOOCV). Supplementary Table 9. Cross Validation of bootstrap method. Supplementary Table 10. AUCs and IDIs from 3-fold Cross-Validation. Supplementary Table 11. Summary statistics of Doylestown model. Supplementary Table 12. Comparison of logistic regression, classification and regression tree (CART) and conditional inference tree (CTREE). Supplementary Figure 1. The distribution of AFP in each data set by cases and controls: Supplementary Figure 2. Quartiles for AFP in the individual patient sets. Supplementary Figure 3. Histogram of predictions and observe occurrence proportion of HCC for the top 4 models.

In a previous study of smoking cessation in veterans with lung cancer, we noted as an incidental finding that current smokers were much younger than former smokers at diagnosis. To confirm and extend this observation, we analyzed the association of smoking status with age at diagnosis and survival of lung cancer patients. The Jefferson Cancer Registry collects information on all cancer patients registered at this hospital. Information on smoking status has been recorded since 1995. We determined age at diagnosis and survival of current and former smokers with lung cancer. 5111 lung cancer cases were identified in the registry from 1995 to 2011 inclusive. Smoking status was recorded in 4687 cases (91.7%). Of these, 1859 (39.7%) were current, 2423 (51.7%) were former, and 405 (8.6%) were never smokers. There was a 6-year difference in median age at lung cancer diagnosis between the current (63 years) and former smokers (69 years) (P

Background: Use of chemotherapy in stage II colorectal cancer (CRC) is controversial because it improves survival only in some patients. We aimed to develop a statistical model using routine and readily available blood tests to predict the prognosis of patients with stage II CRC and to identify which patients are likely to benefit from chemotherapy. Methods: We divided 422 patients with stage II CRC into a training and a testing set. The association of routine laboratory variables and disease-free survival (DFS) was analyzed. A prognostic model was developed incorporating clinically relevant laboratory variables with demographic and tumor characteristics. A prognostic score was derived by calculating the sum of each variable weighted by its regression coefficient in the model. Model performance was evaluated by constructing receiver operating characteristic curves and calculating the area under the curve (AUC). Results: Significant associations were seen between 5 laboratory variables and patient DFS in univariate analyses. After stepwise selection, 3 variables (carcinoembryonic antigen, hemoglobin, creatinine) were retained in the multivariate model with an AUC of 0.75. Compared with patients with a low prognostic score, those with a medium and high prognostic score had a 1.99- and 4.78-fold increased risk of recurrence, respectively. The results from the training set were validated in the testing set. Moreover, chemotherapy significantly improved DFS in high-risk patients, but not in low- and medium-risk patients. Conclusions: A routine laboratory variable-based model may help predict DFS of patients with stage II CRC and identify high-risk patients more likely to benefit from chemotherapy.

Purpose/objective(s) Definitive chemoradiation is essential in non-operative thoracic esophageal cancer. Concerns have posed onto the possible radiation-induced lymphopenia and correlation with different survival outcomes. Neutrophil to lymphocyte ratio (NLR) is a simple way to surrogate the inflammation-immunity status and predict survival. We aim to evaluate the clinical variables and radiation dosimetric parameters associated with hematologic variables changes. Materials/methods Between 2010 and 2015, we identified 93 newly diagnosed thoracic esophageal squamous-cell cancer patients who had completed definitive concurrent chemoradiotherapy (CCRT). Patients' clinical, dosimetric, and hematological data, including absolute neutrophil count, absolute lymphocyte count, and NLR were analyzed at baseline and during CCRT. Cox regression model and Kaplan-Meier analyses were used to validate different survival outcomes. The possible relationships between clinical, hematologic, and dosimetric variables were tested using Spearman's rank or Pearson correlation coefficients, and a further multivariable logistic regression model was created to verify these significant variables. Results The participant group (mean age = 58.6 y) predominantly comprised males (94%) with 27% stage II (n = 25) and 73% stage III (n = 68). Median overall survival (OS) was 13 months (95% confidence interval: 10.304-15.696). We confirmed that baseline NLR (NLR-b) and highest NLR during CCRT (NLR-h) significantly predicted OS, progression-free survival, disease-specific survival, and freedom from distant metastasis. Dichotomized NLR-b with > 3.68 or ≤3.68 also predicted the survival outcomes. Primary esophageal tumor length (Spearman's correlation coefficient r = 0.324, P = 0.011) and baseline body weight (Spearman's r = -0.251, P = 0.019) were significantly correlated with baseline NLR > 3.68. In multivariable logistic regression, primary esophageal tumor length (OR = 1.345, P = 0.021) remained associated with a higher NLR-b. For NLR-h, lung V5 (Pearson r = 0.254, P = 0.014) and V10 (Pearson r = 0.317, P = 0.002) were significantly correlated. In the percentage of ALC nadir decreased during CCRT, lung V5 (Pearson r = 0.299, P = 0.005) and heart V10 (Pearson r = 0.273, P = 0.011) were significantly correlated. Conclusion Inflammation predicts survival outcomes and is correlated with tumor size. Low-dose thoracic irradiation affects inflammation-immunity dynamics because lung V5 and V10 are related to NLR-h; lung V5 and heart V10 with extent of lymphocyte decrease. A novel approach to decrease these unnecessary exposures may further improve survival outcomes in esophageal cancer treated with CCRT.

Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin-free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment with heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined Dulbecco's modified Eagle's medium/F-12-based medium on either Matrigel or defined matrices like vitronectin and Synthemax. One of heparin's main functions was to act as a Wnt modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost-effective method for cardiomyocyte derivation from hPSCs that can be used for potential large-scale drug screening, disease modeling, and future cellular therapies.

Current clinical guidelines state that the use of erythropoiesis-stimulating agents (ESAs) may be considered to treat chemotherapy-induced anemia in the non-curative setting to alleviate anemia-related symptoms. However, no convincing survival benefit has been demonstrated to support the use of ESAs in these patients. Using the comprehensive data collected in the National Cancer Institute (NCI)-surveillance epidemiology and end results (SEER) and Medicare-linked database, we analyzed the effect of ESA use on the short-term (18-month) and long-term (60-month) survival rates of chemotherapy-treated metastatic breast cancer patients. Confounding variables were adjusted using a propensity score approach. We also analyzed the effects of ESA on the survival of patients receiving trastuzumab, a commonly prescribed targeted therapy agent in treating HER2-positive tumors. Metastatic breast cancer patients who received ESA treatment exhibited similar 60-month survival rate to those without ESA treatment (22.8 vs. 24.9%, p = 0.8). ESA-treated patients had a trend toward better 18-month survival [crude hazard ratio (HR) 0.86, 95% confidence intervals (CI) 0.68-1.09, p = 0.21]. This protective effect during the first 18 months of chemotherapy became marginally significant after adjusting for the propensity of receiving ESAs (HR 0.80, 95% CI 0.63-1.01, p = 0.070). An interaction effect between ESA and trastuzumab on patient survival was noticeable but not statistically significant. ESAs did not negatively affect the long-term survival of metastatic breast cancer patients. Moreover, ESAs improved patients' survival during the first 18 months of chemotherapy treatment. These findings endorse the current clinical guideline. Given the short survival of these patients, the potential short-term beneficial effects of ESAs are clinically meaningful.

ARID2 (ARID2), CTNNB1 (β catenin), tumor protein 53 (p53), and PIK3CA (p110α) mutations are implicated in hepatocellular carcinoma (HCC); and previous work has contributed to thorough molecular characterization of these events. However, studies that assess the impact of these mutations on downstream protein expression, especially those that evaluate all 4 cancer markers simultaneously, are relatively lacking. Hence, the present study uses immunohistochemistry to assess protein expression patterns of ARID2, β-catenin, p53, and p110α in HCCs and adjacent nonneoplastic cirrhotic tissues from 58 explanted livers. Notably, this study is the first to our knowledge to investigate ARID2 protein expression in the liver. The frequency of ARID2 mutations detected using our immunohistochemistry method was similar to that reported in previous molecular studies. Furthermore, we found that loss of ARID2 protein expression may be associated with recurrence, although further studies must be done to validate these findings in a larger population. We found that expression patterns of the 4 cancer markers were independent of each other, suggesting separate pathways of hepatocarcinogenesis. We also did not observe an association between viral etiology and protein expression. Consistent with previous studies, overexpression of p53 correlated with poor differentiation. Lastly, 17.5% of HCCs paradoxically had diffuse loss of the oncoprotein p110α compared with strong expression in background cirrhotic liver. The exact mechanism is unclear, but enigmatic loss of oncoprotein function has been described in other carcinomas and could potentially have significant implications for the use of mechanistic target of rapamycin (mTOR) drug therapies.

AT-rich interactive domain 2 (ARID2), catenin (cadherin-associated protein), beta 1, 88kDa (β-catenin), tumor protein 53 (p53), and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (p110α) mutations are implicated in hepatocellular carcinoma (HCC); and previous work has contributed to thorough molecular characterization of these events. However, studies that assess the impact of these mutations on downstream protein expression, especially those that evaluate all 4 cancer markers simultaneously, are relatively lacking. Hence, the present study uses immunohistochemistry to assess protein expression patterns of ARID2, β-catenin, p53, and p110α in HCCs and adjacent nonneoplastic cirrhotic tissues from 58 explanted livers. Notably, this study is the first to our knowledge to investigate ARID2 protein expression in the liver. The frequency of ARID2 mutations detected using our immunohistochemistry method was similar to that reported in previous molecular studies. Furthermore, we found that loss of ARID2 protein expression may be associated with recurrence, although further studies must be done to validate these findings in a larger population. We found that expression patterns of the 4 cancer markers were independent of each other, suggesting separate pathways of hepatocarcinogenesis. We also did not observe an association between viral etiology and protein expression. Consistent with previous studies, overexpression of p53 correlated with poor differentiation. Lastly, 17.5% of HCCs paradoxically had diffuse loss of the oncoprotein p110α compared with strong expression in background cirrhotic liver. The exact mechanism is unclear, but enigmatic loss of oncoprotein function has been described in other carcinomas and could potentially have significant implications for the use of targeted mechanistic target of rapamycin (serine/threonine kinase) drug therapies.

Background and Aim APRI (aspartate aminotransferase [AST] to platelet ratio index) is widely used to assess fibrosis and cirrhosis risk, especially in hepatitis C virus (HCV)-infected patients. Few studies have evaluated APRI and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) risk. Prospective evidence is needed to assess whether APRI predicts HCC risk in HBV patients. Method In a prospectively enrolled clinical cohort of 855 HBV patients with a 1-year exclusion window (followed for > 1 year and did not develop HCC within 1 year), the predictive value of APRI in HCC risk was evaluated by Cox proportional hazards model using univariate and multivariate analyses and longitudinal analysis. Results Higher APRI prospectively conferred a significantly increased risk of HCC in univariate analysis (quartile analysis, P trend = 2.9 × 10−7). This effect remained highly significant after adjusting for common host characteristics but not cirrhosis (P trend = 7.1 × 10−5), and attenuated when cirrhosis is adjusted (P trend = 0.021). The effect remained prominent when the analysis was restricted to patients with a more stringent 2-year exclusion window (P trend = 0.008 in quartile analysis adjusting all characteristics including cirrhosis), indicating that the association was unlikely due to including undetected HCC patients in the cohort, thus minimizing the reverse-causation limitation in most retrospective studies. Longitudinal comparison demonstrated a persistently higher APRI value in HBV patients who developed HCC during follow-up than those remaining cancer free. Conclusion APRI might be a marker of HCC risk in HBV patients in cirrhosis-dependent and -independent manners. Further studies are warranted to validate this finding and test its clinical applicability in HCC prevention.

The use of erythropoiesis stimulating agents (ESAs) to treat anemia in breast cancer patients who are treated with chemotherapy is a matter of ongoing debate. Several recent randomized trials challenged conventional wisdom, which holds that ESAs are contraindicated for breast cancer patients undergoing curative treatment. We aimed to perform the first large national population-based study to analyze the association between ESA use and breast cancer patient outcomes. Cytotoxic chemotherapy-treated invasive breast cancer patients were identified from the Surveillance, Epidemiology, and End Results (SEER)-Medicare database. Non-ESA users were sequentially 1:1 matched to 2,000 randomly sampled ESA users on demographics (age, diagnosis year, race, marital status, and socioeconomic status), tumor presentation (stage, grade, and status of hormone receptors), and treatments (surgery, radiation, and sub-types of chemotherapy) using a minimum distant strategy. Breast cancer-specific survival of ESA and matched non-ESA users was compared using Fine and Gray competing risk model. Compared to ESA users, non-ESA users exhibited dramatically different baseline characteristics such as less advanced tumor, and fewer co-morbidities. Non-ESA users had a significantly more favorable breast cancer-specific survival (subdistribution hazard ratio [sHR] = 0.75, p < 0.0001). This survival disparity was progressively diminished in the sequential matching of demographics (sHR = 0.74, p = 0.0004), presentation (sHR = 0.86, p = 0.06), and treatment (sHR = 0.89, p = 0.17) variables. Stratified analyses identified subgroups of patients whose breast cancer-specific survival were not different between ESA and non-ESA users. In the SEER-Medicare database, ESA usage does not seem to be associated with unfavorable breast cancer-specific survival in breast cancer patients receiving cytotoxic chemotherapy. The ESA-breast cancer prognosis association is complex and requires more intensive investigations.

Background and Aim Hyperphosphatemia has been implicated in the development and treatment of various cancers. However, whether it can be used as a direct prognostic marker of colorectal cancer (CRC) has remained unexplored. Given new insights into the importance of hyperphosphatemia in CRC, we sought to evaluate the association of hyperphosphatemia with the clinical outcomes of this disease. Methods In a retrospective analysis of a well-characterized clinic-based cohort with 1241 CRC patients, we assessed the association of postoperative hyperphosphatemia with patient overall survival. Results Postoperative hyperphosphatemia measured within the first month after surgery was significantly associated with CRC survival. Compared to patients with a normal phosphate level, those with hyperphosphatemia exhibited a significant unfavorable overall survival with a hazard ratio (HR) of 1.84 (95% confidence interval [CI] 1.49–2.29, P = 2.6 × 10−8 (log-rank P = 1.2 × 10−7). Stratified analyses indicated the association was more pronounced in patients with colon (HR = 2.00, 95% CI 1.57–2.56, P = 3.17 × 10−8) but not rectal cancer (HR = 0.96, 95% CI 0.58–1.59, P = 0.889) (P interaction = 0.023), as well as in those not receiving chemotherapy (HR = 2.15, 95% CI 1.59–2.90, P = 6.2 × 10−7) but not in those receiving chemotherapy (HR = 1.30, 95% CI 0.92–1.82, P = 0.136) (P interaction = 0.012). Flexible parametric survival model demonstrated that the increased risk for death conferred by postoperative hyperphosphatemia persisted over 150 months after surgery. Conclusion Our data indicated that postoperative hyperphosphatemia might be used as a prognostic marker of CRC patients after surgery. Since phosphate level is routinely tested in clinics, it may be incorporated into clinical models to predict CRC survival.

Prospective and longitudinal epidemiological evidence is needed to assess the association between telomere length and risk of hepatocellular carcinoma (HCC). In 323 cancer-free Korean-American HBV patients with 1-year exclusion window (followed for >1 year and did not develop HCC within 1 year), we measured the relative telomere length (RTL) in baseline serum DNAs and conducted extensive prospective and longitudinal analyses to assess RTL-HCC relationship. We found that long baseline RTL conferred an increased HCC risk compared to short RTL [hazard ratio (HR) = 4.93, P = 0.0005). The association remained prominent when the analysis was restricted to patients with a more stringent 5-year exclusion window (HR = 7.51, P = 0.012), indicating that the association was unlikely due to including undetected HCC patients in the cohort, thus minimizing the reverse-causation limitation in most retrospective studies. Adding baseline RTL to demographic variables increased the discrimination accuracy of the time-dependent receiver operating characteristic analysis from 0.769 to 0.868 (P = 1.0 × 10-5). In a nested longitudinal subcohort of 16 matched cases-control pairs, using a mixed effects model, we observed a trend of increased RTL in cases and decreased RTL in controls along 5 years of follow-up, with a significant interaction of case/control status with time (P for interaction=0.002) and confirmed the association between long RTL and HCC risk [odds ratio [OR] = 3.63, P = 0.016]. In summary, serum DNA RTL may be a novel non-invasive prospective marker of HBV-related HCC. Independent studies are necessary to validate and generalize this finding in diverse populations and assess the clinical applicability of RTL in HCC prediction.

Many studies recently focus on complicated and expensive genomic tests, but the prognostic values of biochemical markers which are easily obtained in clinics are largely overlooked and without further exploration. This study assesses the association of neutrophil–lymphocyte-ratio (NLR) with prognosis of lung cancer patients. In 1032 patients with histologically confirmed lung cancer, the association of pretreatment NLR values with overall survival (OS) was evaluated using a Cox proportional hazards model and the temporal relationship of longitudinal NLR was assessed using a mixed effects model. Compared to the patients with a low pretreatment NLR value, those with elevated NLR exhibited a statistically significant worse OS with a hazard ratio (HR) of 1.50 (P

Recent studies have demonstrated a potential link between circulating cell-free mitochondrial DNA (mtDNA) content and cancers. However, there is no study evaluating the association between circulating mtDNA as a non-invasive marker of hepatocellular carcinoma (HCC) risk. We conducted a nested case-control study to determine circulating mtDNA content in serum samples from 116 HBV-related HCC cases and 232 frequency-matched cancer-free HBV controls, and evaluate the retrospective association between mtDNA content and HCC risk using logistic regression and their temporal relationship using a mixed effects model. HCC cases had significantly lower circulating mtDNA content than controls (1.06 versus 2.47, P = 1.7 × 10−5). Compared to HBV patients with higher mtDNA content, those with lower mtDNA content had a significantly increased risk of HCC with an odds ratio (OR) of 2.19 (95% confidence interval [CI] 1.28–3.72, P = 0.004). Quartile analyses revealed a significant dose-dependent effect (Ptrend = 0.001) for this association. In a pilot longitudinal sub-cohort of 14 matched cases-control pairs, we observed a trend of dramatically decreased mtDNA content in cases and slightly decreased mtDNA content in controls, with a significant interaction of case-control status with time (Pinteraction = 0.049). Our findings suggest that circulating mtDNA is a potential novel non-invasive biomarker of HCC risk in HBV patients.

In this study, we have generated a high-throughput screening (HTS)-compatible 3D cell culture platform by chemically "welding" polystyrene scaffolds into standard 2D polystyrene 96-well plates. The variability of scaffolds was minimized by introducing automation into the fabrication process. The fabricated 3D cell culture plates were compared with several commercially available 3D cell culture platforms with light and scanning electron microscopy. Voltage-gated calcium channel functionality was used to access the Z' factors of all plates, including a 2D standard plate control. It was found that with the No-Wash Fluo-4 calcium assay and neural progenitor cells, all plates display acceptable Z' factors for use in HTS. The plates with "welded" polystyrene scaffolds have several advantages, such as being versatile and economical, and are ready to use off the shelf. These characteristics are especially desired in HTS preclinical drug discovery applications.

Many whole cell-based assays in use today rely on flat, two-dimensional (2D) glass or plastic substrates that may not produce results characteristic of in vivo conditions. In this study, a three-dimensional (3D) cell-based assay platform was established by integrating 3D synthetic polymer scaffolds with standard cell culture dishes and multi-well plates. This technology can be used to feasibly modify any traditional 2D cell-based assay vessels for 3D cell-based assay with currently used high throughput screening (HTS) systems. We examined neural stem (NS) cells' growth profile, morphology, cell-matrix interaction, gene expression and voltage gated calcium channel (VGCC) functionality of this novel 3D assay platform. Our results showed that unlike the NS cells cultured on traditional 2D planar surfaces, cells in 3D scaffolds are more physiologically relevant with respect to in vivo characteristics exhibited by in-vivo surrogates such as neural spheres. This new biomimetic cell-based assay platform may provide a broadly applicable 3D cell-based system for use in drug discovery programs and other research fields.

We evaluated differences in treatment of black vs white patients with colon cancer and assessed their effects on survival, based on cancer stage.We collected data from the Surveillance, Epidemiology, and End Results-Medicare database and identified 6190 black and 61,951 white patients with colon cancer diagnosed from 1998 through 2009 and followed up through 2011. Three sets of 6190 white patients were matched sequentially, using a minimum distance strategy, to the same set of 6190 black patients based on demographic (age; sex; diagnosis year; and Surveillance, Epidemiology, and End Results registry), tumor presentation (demographic plus comorbidities, tumor stage, grade, and size), and treatment (presentation plus therapies) variables. We conducted sensitivity analyses to explore the effects of socioeconomic status in a subcohort that included 2000 randomly selected black patients. Racial differences in treatment were assessed using a logistic regression model; their effects on racial survival disparity were evaluated using the Kaplan-Meier method and the Cox proportional hazards model.After patients were matched for demographic variables, the absolute 5-year difference in survival between black and white patients was 8.3% (white, 59.2% 5-y survival; blacks, 50.9% 5-y survival) (P.0001); this value decreased significantly, to 5.0% (P .0001), after patients were matched for tumor presentation, and decreased to 4.9% (P.0001) when patients were matched for treatment. Differences in treatment therefore accounted for 0.1% of the 8.3% difference in survival between black and white patients. After patients were matched for tumor presentation, racial disparities were observed in almost all types of treatment; the disparities were most prominent for patients with advanced-stage cancer (stages III or IV, up to an 11.1% difference) vs early stage cancer (stages I or II, up to a 4.3% difference). After patients were matched for treatment, there was a greater reduction in disparity for black vs white patients with advanced-stage compared with early-stage cancer. In sensitivity analyses, the 5-year racial survival disparity was 7.7% after demographic match, which was less than the 8.3% observed in the complete cohort. This reduction likely was owing to the differences between the subcohort and the complete cohort in those variables that were not included in the demographic match. This value was reduced to 6.5% (P = .0001) after socioeconomic status was included in the demographic match. The difference decreased significantly to 2.8% (P = .090) after tumor presentation match, but was not reduced further after treatment match.We observed significant disparities in treatment and survival of black vs white patients with colon cancer. The disparity in survival appears to have been affected more strongly by tumor presentation at diagnosis than treatment. The effects of treatment differences on disparities in survival were greater for patients with advanced-stage vs early-stage cancer.

Biomarkers for the early diagnosis of hepatocellular carcinoma (HCC) are needed to decrease mortality from this cancer. However, as new biomarkers have been slow to be brought to clinical practice, we have developed a diagnostic algorithm that utilizes commonly used clinical measurements in those at risk of developing HCC. Briefly, as α-fetoprotein (AFP) is routinely used, an algorithm that incorporated AFP values along with four other clinical factors was developed. Discovery analysis was performed on electronic data from patients who had liver disease (cirrhosis) alone or HCC in the background of cirrhosis. The discovery set consisted of 360 patients from two independent locations. A logistic regression algorithm was developed that incorporated log-transformed AFP values with age, gender, alkaline phosphatase, and alanine aminotransferase levels. We define this as the Doylestown algorithm. In the discovery set, the Doylestown algorithm improved the overall performance of AFP by 10%. In subsequent external validation in over 2,700 patients from three independent sites, the Doylestown algorithm improved detection of HCC as compared with AFP alone by 4% to 20%. In addition, at a fixed specificity of 95%, the Doylestown algorithm improved the detection of HCC as compared with AFP alone by 2% to 20%. In conclusion, the Doylestown algorithm consolidates clinical laboratory values, with age and gender, which are each individually associated with HCC risk, into a single value that can be used for HCC risk assessment. As such, it should be applicable and useful to the medical community that manages those at risk for developing HCC. Cancer Prev Res; 9(2); 172–9. ©2015 AACR.

Mitochondrial dysfunction has been implicated in the pathophysiology of Alzheimer's disease (AD) brains. To unravel the mechanism(s) underlying this dysfunction, we demonstrate that phospholipases A2(PLA2s), namely the cytosolic and the calcium-independent PLA2s (cPLA2and iPLA2), are key enzymes mediating oligomeric amyloid-β peptide (Aβ1–42)-induced loss of mitochondrial membrane potential and increase in production of reactive oxygen species from mitochondria in astrocytes. Whereas the action of iPLA2is immediate, the action of cPLA2requires a lag time of ∼12–15 min, probably the time needed for initiating signaling pathways for the phosphorylation and translocation of cPLA2to mitochondria. Western blot analysis indicated the ability of oligomeric Aβ1–42to increase phosphorylation of cPLA2in astrocytes through the NADPH oxidase and mitogen-activated protein kinase pathways. The involvement of PLA2in Aβ1–42-mediated perturbations of mitochondrial function provides new insights to the decline in mitochondrial function, leading to impairment in ATP production and increase in oxidative stress in AD brains.

A survival disparity of black versus white breast cancer patients has been extensively documented but not adequately explained. Blacks and whites also have significant differences in hematologic traits including hemoglobin (HGB). However, a link between survival disparity and hematologic differences has not been reported. We aimed to explore the effect of pre-treatment hematologic variables on this survival disparity. We sequentially matched 443 black patients, using a minimum distance approach, to four different sets of 443 whites on demographics (age, year of diagnosis, smoking, and drinking status), tumor presentation (all demographic variables plus tumor stage, grade, and hormone receptor status), treatment (all presentation variables plus surgery, chemotherapy, radiation therapy, and hormone therapy), and presentation plus pre-treatment hematologic variables. Racial survival for each matched dataset was analyzed by Cox proportional hazards model. We found that white patients matched on demographic characteristics had more favorable survival than blacks [hazard ratio (HR) 0.57, 95 % confidence interval (CI) 0.42–0.77, p log-rank = 0.0002]. Presentation match diminished this disparity [HR 0.72 (0.54–0.95), p log-rank = 0.0199], which was not further reduced in treatment match [HR 0.73 (0.55–0.96), p log-rank = 0.0249]. However, the survival disparity was largely reduced when pre-treatment level of HGB or red blood cell distribution width was further matched in addition to presentation match [HR 0.83 (0.64–1.09), p log-rank = 0.1819 and HR 0.83 (0.64–1.09), p log-rank = 0.1760, respectively]. We found that in our patient population, differences in tumor presentation and certain pre-treatment hematologic traits, but not treatment, were associated with the survival disparity between black and white breast cancer patients.

Cancer patients undergo routine clinical monitoring with an array of blood tests that may carry long-term prognostic information. We aimed to develop a new prognostic model predicting survival for patients with advanced non-small cell lung cancer (NSCLC), based on laboratory tests commonly performed in clinical practice. A cohort of 1,161 stage IIIB or IV NSCLC patients was divided into training (n = 773) and testing (n = 388) cohorts. We analyzed the associations of 32 commonly tested laboratory variables with patient survival in the training cohort. We developed a model based on those significant laboratory variables, together with important clinical variables. The model was then evaluated in the testing cohort. Five variables, including albumin, total protein, alkaline phosphatase, blood urea nitrogen and international normalized ratio, were significantly associated with patient survival after stepwise selection. A model incorporating these variables classified patients into low-, medium- and high-risk groups with median survival of 16.9, 7.2 and 2.1 months, respectively (p0.0001). Compared with low-risk group, patients in the medium- and high-risk groups had a significantly higher risk of death at 1 year, with hazard ratio (HR) of 1.95 (95% CI 1.62-2.36) and 5.22 (4.30-6.34), respectively. These results were validated in the testing cohort. Overall, we developed a prognostic model relying entirely on readily available variables, with similar predictive power to those which depend on more specialized and expensive molecular assays. Further study is necessary to validate and further refine this model, and compare its performance to models based on more specialized and expensive testing.

Hyperphosphatemia has been implicated in the development and treatment of various cancers. However, whether it can be used as a direct prognostic marker of colorectal cancer (CRC) has remained unexplored. Given new insights into the importance of hyperphosphatemia in CRC, we sought to evaluate the association of hyperphosphatemia with the clinical outcomes of this disease.In a retrospective analysis of a well-characterized clinic-based cohort with 1241 CRC patients, we assessed the association of postoperative hyperphosphatemia with patient overall survival.Postoperative hyperphosphatemia measured within the first month after surgery was significantly associated with CRC survival. Compared to patients with a normal phosphate level, those with hyperphosphatemia exhibited a significant unfavorable overall survival with a hazard ratio (HR) of 1.84 (95% confidence interval [CI] 1.49-2.29, P = 2.6 × 10(-8) (log-rank P = 1.2 × 10(-7) ). Stratified analyses indicated the association was more pronounced in patients with colon (HR = 2.00, 95% CI 1.57-2.56, P = 3.17 × 10(-8) ) but not rectal cancer (HR = 0.96, 95% CI 0.58-1.59, P = 0.889) (P interaction = 0.023), as well as in those not receiving chemotherapy (HR = 2.15, 95% CI 1.59-2.90, P = 6.2 × 10(-7) ) but not in those receiving chemotherapy (HR = 1.30, 95% CI 0.92-1.82, P = 0.136) (P interaction = 0.012). Flexible parametric survival model demonstrated that the increased risk for death conferred by postoperative hyperphosphatemia persisted over 150 months after surgery.Our data indicated that postoperative hyperphosphatemia might be used as a prognostic marker of CRC patients after surgery. Since phosphate level is routinely tested in clinics, it may be incorporated into clinical models to predict CRC survival.

It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC) functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG) were cultured on two dimensional (2D) flat surfaces and in three dimensional (3D) synthetic poly-L-lactic acid (PLLA) and polystyrene (PS) polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K(+) depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells' functionality, transcriptase expression and related membrane protein distributions (caveolin-1) were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns.

Cells cultured in three dimensional (3D) scaffolds as opposed to traditional two-dimensional (2D) substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate). Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue.

In this review, we discuss the microenvironmental cues that modulate the status of cells to yield physiologically more relevant three-dimensional (3D) cell-based high throughput drug screening (HTS) platforms for drug discovery. Evidence is provided to support the view that simplifying 3D cell culture platforms for HTS applications calls for identifying and validating ubiquitous three-dimensionality biomarkers. Published results from avascular tumorigenesis and early stages of inflammatory wound healing, where cells transition from a two-dimensional (2D) to 3D microenvironment, conclusively report regulation by cytokines, providing the physiological basis for focusing on cytokines as potential three-dimensionality biomarkers. We discuss additional support for cytokines that comes from numerous 2D and 3D comparative transcriptomic and proteomic studies, which generally report upregulation of cytokines in 3D compared with 2D culture counterparts.

Inflammatory breast cancer (IBC) is an extremely aggressive form of locally advanced breast cancer that affects about 5% of breast cancer patients. The prognosis of IBC patients is remarkably poor, with a three-year survival rate of approximately 30% compared to 60% for patients with non-IBC breast cancers. These facts highlight the importance of accurate characterization, early detection, and timely treatment of IBC patients. Thus, it is important to develop novel and clinically applicable non-invasive biomarkers to characterize the unique presentation of IBC. In this study, we searched for somatic mutations in the circulating tumor DNAs (ctDNAs) that could be used to non-invasively characterize IBC patients and inform their clinical management. Using ctDNAs extracted from plasma of 10 pairs of IBC and non-IBC patients that were matched on major demographic and clinical variables, we conducted a high-depth target next-generation sequencing study that interrogated a comprehensive panel of 127 TCGA (The Cancer Genome Atlas)-reported cancer-related genes with >7000 uniquely designed and validated probes. Overall, we obtained >500x coverage in >80% of the interrogated regions, and >100x coverage in >97% of the regions. We found that C>T mutations predominated in well-reported mutated genes such as TP53, PIK3CA, EGFR, and CDH1. Compared to non-IBC patients, IBC patients appeared to have a higher percentage of mutations in PIK3CA but a lower percentage in TP53. Interestingly, about 78% of mutated genes that were only detected in IBC patients encode zinc finger-related proteins, a family of transcriptional factors that have been implicated in IBC development. In comparison, about 43% of genes that were detected only in non-IBC patients encode proteins important to cell division regulation. Furthermore, network-based stratification (NBS) analysis of the mutation profile revealed clusters of IBC relative to non-IBC samples, indicating the potential of mutation profiling in identifying molecularly distinct subtypes of IBC patients. Preliminary longitudinal analysis of ctDNAs from three patients with multiple plasma samples indicated that de novo mutations in important genes including PIK3CA, RB1, and KRAS appeared in patient blood after chemotherapy and/or targeted therapy treatments. Moreover, the emergence of some of these mutations was temporally correlated with the responses of patients to the treatments they received. Overall, this study provides novel evidence that ctDNA mutation status may help to non-invasively characterize IBC tumors, and might also serve as a novel non-invasive marker to monitor treatment efficacy and prognosis of breast cancer patients. Future studies with larger sample sizes are warranted to confirm our findings and identify additional clinically useful markers for the characterization and management of IBC and non-IBC patients. Citation Format: Hushan Yang, Xue Zhong, Qiang Wei, Zhaomei Mu, Zhong Ye, Yinzhi Lai, Huei-Wen Lin, Rebecca Jaslow, Tiffany Avery, Laura Austin, Zhaohui Sun, Shengrong Lin, Grace Zhao, Ling Fang Tang, Ronald E. Myers, Juan P. Palazzo, Laura Biederman, Bingshan Li, Massimo Cristofanilli. Comprehensive high-depth target sequencing in circulating tumor DNAs of patients with inflammatory and non-inflammation breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2788. doi:10.1158/1538-7445.AM2015-2788

Background: In a recent retrospective case-control study, we showed that patients with HBV-related hepatocellular carcinoma (HCC) had significantly longer telomeres in circulating serum DNA than cancer-free HBV patients, indicating that circulating DNA telomere length may be a novel HCC marker. Prospective and longitudinal evidence is needed to confirm this finding. Method: In a cohort of 323 cancer-free HBV patients who had been followed for >1 year and did not develop HCC within 1 year, we determined the relative telomere length (RTL) in baseline serum DNA samples and conducted prospective and longitudinal analyses to assess the relationships between RTL and HCC risk. Results: In a median follow-up of 4.5 years (range, 1.1-21.1), 37 patients developed HCC. Using the median baseline RTL as cut-off, long RTL conferred a significantly increased HCC risk compared to short RTL (HR=4.93, 95% CI 2.00-12.13, P=0.0005, Plog-rank=1.7x10-6). This association remained significant when the analysis was more stringently restricted to patients who had been followed for >5 years and did not develop HCC within 5 years (HR=7.51, 95% CI 1.56-36.18, P=0.012, Plog-rank=0.0024), indicating that the RTL-HCC association was unlikely due to the inclusion of undiagnosed HCC patients in the cancer-free HBV cohort, thus further minimizing the effect of the reverse-causation limitation that is inherent in most previous telomere length-related retrospective studies. The area under the curve (AUC) in the time-dependent ROC analysis increased from 0.769 (demographic variables only) to 0.868 (demographic variables and baseline RTL), with a statistically significant difference (P=1.0x10-5) assessed by 10,000-time bootstrap resampling. Finally, in a nested longitudinal sub-population of 22 matched cases-control pairs with multiple samples for each patient collected at different follow-up time points, we found a significant increase in RTL from baseline to HCC diagnosis in cases (slope=0.032, P=4.0x10-5), in contrast to a RTL decrease from baseline to last follow-up in controls (slope=-0.020, P=0.026). Using a mixed-effects model for longitudinal case-control analysis, we showed that long RTL conferred a significantly increased risk of HCC (OR=4.55, 95% CI 1.91-10.89, P=0.0007). Collectively, the longitudinal data were consistent with the prospective analysis results, indicating that telomere lengthening may be an important driving factor in hepatocarcinogenesis. Conclusions: To our best knowledge, this is one of the first prospective and longitudinal evaluations of RTL in cancer risk prediction. Our data suggests that RTL of circulating serum DNA may be a novel non-invasive prospective marker of HBV-related HCC. Future studies are needed to generalize this finding in broader populations and test its clinical applicability in HCC prevention. Citation Format: Hushan Yang, Hie-won Hann, Shaogui Wan, Yinzhi Lai, Kejin Zhang, Richard Hann, Ronald E. Myers. Prospective and longitudinal evaluations of telomere length in circulating serum DNA as a noninvasive risk factor of hepatocellular carcinoma in HBV patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 110. doi:10.1158/1538-7445.AM2013-110

Oligomeric amyloid-beta peptide (Abeta) is known to induce cytotoxic effects and to damage cell functions in Alzheimer's disease. However, mechanisms underlying the effects of Abeta on cell membranes have yet to be fully elucidated. In this study, Abeta 1-42 (Abeta(42)) was shown to cause a temporal biphasic change in membranes of astrocytic DITNC cells using fluorescence microscopy of Laurdan. Abeta(42) made astrocyte membranes became more molecularly-disordered within the first 30 min to 1 h, but gradually changed to more molecularly-ordered after 3 h. However, Abeta(42) caused artificial membranes of vesicles made of rat whole brain lipid extract to become more disordered only. The trend for more molecularly-ordered membranes in astrocytes induced by Abeta(42) was abrogated by either an NADPH oxidase inhibitor, apocynin, or an inhibitor of cytosolic phospholipase A(2) (cPLA(2)), but not by an inhibitor of calcium-independent PLA(2) (iPLA(2)). Apocynin also suppressed the increased production of superoxide anions (O(2)(-)) and phosphorylation of cPLA(2) induced by Abeta(42). In addition, hydrolyzed products of cPLA(2), arachidonic acid (AA), but not lysophosphatidylcholine (LPC) caused astrocyte membranes to become more molecularly-ordered. These results suggest (1) a direct interaction of Abeta(42) with cell membranes making them more molecularly-disordered, and (2) Abeta(42) also indirectly makes membranes become more molecularly-ordered by triggering the signaling pathway involving NADPH oxidase and cPLA(2) in astrocytes.