Statistical concerns such as confounding and multiplicity, for which solutions have existed for years, are common in biomarker validation studies; however, published validation studies may not address these issues. By not only raising the issues but also describing possible solutions, this discussion may help decrease false discovery and enhance the reproducibility of validation study findings.
Biomarker validation, like any other confirmatory process based on statistical methodology, must discern associations that occur by chance from those reflecting true biological relationships. Validity of a biomarker is established by authenticating its correlation with clinical outcome. Validated biomarkers can lead to targeted therapy, improve clinical diagnosis, and serve as useful prognostic and predictive factors of clinical outcome. Statistical concerns such as confounding and multiplicity are common in biomarker validation studies. This article discusses four major areas of concern in the biomarker validation process and some of the proposed solutions. Because present-day statistical packages enable the researcher to address these common concerns, the purpose of this discussion is to raise awareness of these statistical issues in the hope of improving the reproducibility of validation study findings.
Biomarker; Selection bias; Confounding factors; Validation studies
To study the pharmacokinetic profile after hepatic arterial embolization with superabsorbent microspheres (QuadraSpheres) loaded with doxorubicin.
Materials and Methods
Rabbits with hepatic VX2 tumors were treated with intra-arterial administration of QuadraSpheres loaded with doxorubicin, or transarterial chemoembolization (TACE) using doxorubicin, Lipiodol and Embospheres, or hepatic arterial infusion (HAI) of doxorubicin. Tumor specimens were evaluated by fluorescence microscopy, and plasma and tumor concentrations of doxorubicin were measured.
The peak plasma concentration of doxorubicin was lower in the QuadraSphere group (309.9 ng/ml) than in the HAI (673.4 ng/ml) or TACE (360.5 ng/ml) groups, suggesting higher tumor retention in the QuadraSphere group. Intratumoral doxorubicin levels declined to negligible levels at 1 and 3 days after treatment, respectively in the HAI and TACE groups. In the QuadraSphere groups, intratumoral doxorubicin level declined after day 1, but was still detectable at 14 days after treatment and was higher than that in the other groups at 1, 3, and 7 days. Intratumoral doxorubicin fluorescence was detected at all time points in the QuadraSphere group, but only at 1 day after treatment in the TACE group.
Hepatic arterial administration of doxorubicin-loaded QuadraSpheres enables the sustained release of doxorubicin to hepatic tumors.
Hepatic artery embolization; Drug-eluting microspheres; Pharmacokinetics
The VX2 rabbit model of liver cancer is commonly used to evaluate the efficacy of locoregional anticancer therapy and knowledge of the hepatic arterial anatomy in the rabbit is important for catheter-directed experiments.
To describe the normal anatomy and anatomic variations of the celiac axis and hepatic artery in the rabbit.
Material and Methods
Angiograms of 222 rabbits were retrospectively reviewed. The branching pattern of the celiac axis was classified and the diameters of the major branches were measured. Paired t-tests were used to compare the difference between the average sizes of arteries.
Variant celiac axis or hepatic artery anatomy was noted in 25.9% of angiograms, with the gastric branches arising from the proper hepatic artery in 23.3% of cases. The celiac axis could be successfully classified into one of five distinct branching patterns in 193 (86.9%) cases. The mean diameters of the right and left hepatic arteries were 0.67 mm (95% CI [0.64, 0.7]) and 1.25 mm (95% CI [1.19, 1.31]), respectively. The mean diameters of the medial and lateral branches of the left hepatic artery were 0.63 mm (95% CI [0.6, 0.67]) and 0.91 mm (95% CI [0.86, 0.96]), respectively. The right hepatic artery was significantly smaller than the left hepatic artery and the lateral branch of the left hepatic artery (all p-values <0.0001).
Arterial variants in the rabbit are not uncommon. The proper hepatic artery often gives origin to gastric artery branches. To facilitate superselective intra-arterial intervention, the left lateral lobe of the liver should be targeted for tumor implantation because of the significant size difference between the right and left hepatic arteries.
rabbit; liver angiography; arterial variants; VX2 tumor; liver-directed therapy
This study was designed to investigate the intratumoral uptake of hollow gold nanospheres (HAuNS) after hepatic intra-arterial (IA) and intravenous (IV) injection in a liver tumor model.
Materials and Methods
Fifteen VX2 tumor-bearing rabbits were randomized into five groups (N=3 in each group) that received either IV 64Cu-labeled PEG-HAuNS (IV-PEG-HAuNS), IA 64Cu-labeled PEG-HAuNS (IA-PEG-HAuNS), IV cyclic peptide (RGD)-conjugated 64Cu-labeled PEG-HAuNS (IV-RGD-PEG-HAuNS), IA RGD-conjugated 64Cu-labeled PEG-HAuNS (IA-RGD-PEG-HAuNS), or IA 64Cu-labeled PEG-HAuNS with lipiodol (IA-PEG-HAuNS-lipiodol). The animals underwent PET/CT 1 hour after injection, and uptake expressed as percentage of injected dose per gram of tissue (%ID/g) was measured in tumor and major organs. The animals were euthanized 24 hours after injection, and tissues were evaluated for radioactivity.
At 1 hour after injection, animals in the IA-PEG-HAuNS-lipiodol group showed significantly higher tumor uptake (P < 0.001) and higher ratios of tumor-to-normal liver uptake (P < 0.001) than those in all other groups. The biodistribution of radioactivity 24 hours after injection showed that IA delivery of PEG-HAuNS with lipiodol resulted in the highest tumor uptake (0.33 %ID/g; P < 0.001) and tumor-to-normal liver ratio (P < 0.001) among all delivery methods. At 24 hours, the IA-RGD-PEG-HAuNS group showed higher tumor uptake than the IA-PEG-HAuNS group (0.20 %ID/g vs. 0.099 %ID/g; P < 0.001).
Adding iodized oil to IA-PEG-HAuNS maximizes nanoparticle delivery to hepatic tumors and therefore may be useful in targeted chemotherapy and photoablative therapy. PET/CT can be used to noninvasively monitor the biodistribution of radiolabeled HAuNS after IV or IA injection.
Hollow gold nanospheres; liver tumor; intraarterial injection; PET/CT; copper-64; lipiodol
Demographic, behavioral and environmental factors have been associated with increased risk of colorectal cancer (CRC). We reviewed the published evidence and explored associations between risk factors and CRC incidence.
We identified 12 established non-screening CRC risk factors and performed a comprehensive review and meta-analyses to quantify each factor’s impact on CRC risk. We used random effects models of the logarithms of risks across studies: inverse variance weighted averages for dichotomous factors and generalized least squares for dose-response for multi-level factors.
Significant risk factors include inflammatory bowel disease (RR = 2.93, 95% CI: 1.79–4.81); CRC history in first-degree relative (RR = 1.79, 95% CI: 1.60–2.02); body mass index (BMI) to overall population (RR = 1.10 per 8 kg/m2 increase, 95% CI: 1.08–1.12); physical activity (RR = 0.88, 95% CI: 0.86–0.91 for 2 standard deviations increased physical activity score); cigarette smoking (RR = 1.06, 95% CI: 1.03–1.08 for 5 pack-years), and consumption of red meat (RR = 1.13, 95% CI: 1.09–1.16 for 5 servings/week), fruit (RR = 0.85, 95% CI: 0.75–0.96 for 3 servings/day), and vegetables (RR = 0.86, 95% CI: 0.78–0.94 for 5 servings/day).
We developed a comprehensive risk modeling strategy that incorporates multiple effects to predict an individual’s risk of developing colorectal cancer. Inflammatory bowel disease and history of CRC in first-degree relatives are associated with much higher risk of CRC. Increased BMI, red meat intake, cigarette smoking, low physical activity, low vegetable consumption, and low fruit consumption were associated with moderately increased risk of CRC.
Colorectal cancer; colon neoplasms; colonic neoplasms and colorectal neoplasms; colorectal risk factors; and colorectal cancer prevention; meta-analysis
Obesity increases the risk of cancer death among postmenopausal women with estrogen receptor–positive (ER+) breast cancer, but the direct evidence for the mechanisms is lacking. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating this epidemiologic phenomenon.
We analyzed transcriptomic profiles of pretreatment biopsies from a prospective cohort of 137 ER+ breast cancer patients. We generated transgenic (MMTV-TGFα;A
/a) and orthotopic/syngeneic (A
/a) obese mouse models to investigate the effect of obesity on tumorigenesis and tumor progression and to determine biological mechanisms using whole-genome transcriptome microarrays and protein analyses. We used a coculture system to examine the impact of adipocytes/adipokines on breast cancer cell proliferation. All statistical tests were two-sided.
Functional transcriptomic analysis of patients revealed the association of obesity with 59 biological functional changes (P < .05) linked to cancer hallmarks. Gene enrichment analysis revealed enrichment of AKT-target genes (P = .04) and epithelial–mesenchymal transition genes (P = .03) in patients. Our obese mouse models demonstrated activation of the AKT/mTOR pathway in obesity-accelerated mammary tumor growth (3.7- to 7.0-fold; P < .001; n = 6–7 mice per group). Metformin or everolimus can suppress obesity-induced secretion of adipokines and breast tumor formation and growth (0.5-fold, P = .04; 0.3-fold, P < .001, respectively; n = 6–8 mice per group). The coculture model revealed that adipocyte-secreted adipokines (eg, TIMP-1) regulate adipocyte-induced breast cancer cell proliferation and invasion. Metformin suppress adipocyte-induced cell proliferation and adipocyte-secreted adipokines in vitro.
Adipokine secretion and AKT/mTOR activation play important roles in obesity-accelerated breast cancer aggressiveness in addition to hyperinsulinemia, estrogen signaling, and inflammation. Metformin and everolimus have potential for therapeutic interventions of ER+ breast cancer patients with obesity.
Hyperglycemia during hyper-CVAD chemotherapy is associated with poor outcomes of acute lymphoblastic leukemia (ALL).
To examine whether an intensive insulin regimen could improve outcomes compared with conventional anti-diabetic pharmacotherapy, a randomized trial was conducted comparing glargine plus aspart versus conventional therapy (control). Between 4/2004 and 7/2008, 52 newly diagnosed ALL, Burkitt’s lymphoma, or lymphoblastic lymphoma patients on hyper-CVAD in the inpatient setting and had random serum glucose >180 mg/dL in ≥ 2 occasions during chemotherapy were enrolled.
The trial was terminated early due to futility regarding ALL clinical outcomes despite improved glycemic control. Secondary analysis revealed that molar insulin-to-C-peptide ratio (I/C) >0.175 (a surrogate measure of exogenous insulin usage) was associated with decreased overall survival, complete remission duration and progression-free survival (PFS) while metformin and/or thiazolidinedione usage were associated with increased PFS. In multivariate analyses, factors which significantly predicted short overall survival included age≥60 years (P=0.0002), I/C≥0.175 (P=0.0016) and average glucose≥180 mg/dL (P=0.0236). Factors that significantly predicted short progression-free survival included age≥60 years (P=0.0008), I/C≥0.175 (P=0.0002), high systemic risk (P=0.0173) and average glucose≥180 mg/dL (P=0.0249). I/C ≥ 0.175 was the only significant (P=0.0042) factor that predicted short complete remission duration.
A glargine-plus-aspart intensive insulin regimen did not improve ALL outcomes in hyperglycemic patients. Exogenous insulin may be associated with poor outcomes while metformin and thiazolidinediones may be associated with improved outcomes. These results suggest that the choice of anti-diabetic pharmacotherapy may influence ALL outcomes.
Diabetes; intensive insulin regimen; secretagogues; metformin; thiazolidinediones
As therapy for non-small cell lung cancer (NSCLC) patients becomes more personalized, additional tissue in the form of core needle biopsies (CNBs) for biomarker analysis is increasingly required for determining appropriate treatment and for enrollment into clinical trials. We report our experience with small-caliber percutaneous transthoracic (PT) CNBs for the evaluation of multiple molecular biomarkers in BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination), a personalized, targeted therapy NSCLC clinical trial.
The medical records of patients who underwent PTCNB for consideration of enrollment in BATTLE, were reviewed for diagnostic yield of 11 predetermined molecular markers, and procedural complications. Univariate and multivariate analyses of factors related to patient and lesion characteristics were performed to determine possible influences on diagnostic yield.
One hundred and seventy PTCNBs were performed using 20-gauge biopsy needles in 151 NSCLC patients screened for the trial. 82.9% of the biopsy specimens were found to have adequate tumor tissue for analysis of the required biomarkers. On multivariate analysis, metastatic lesions were 5.4 times more likely to yield diagnostic tissue as compared to primary tumors (p = 0.0079). Pneumothorax and chest tube insertion rates were 15.3% and 9.4%, respectively.
Image-guided 20-gauge PTCNB is safe and provides adequate tissue for analysis of multiple biomarkers in the majority of patients being considered for enrollment into a personalized, targeted therapy NSCLC clinical trial. Metastatic lesions are more likely to yield diagnostic tissue as compared to primary tumors.
research biopsy; biomarker analysis; percutaneous transthoracic biopsy
Inhibition of mTOR with everolimus may result improve efficacy of taxanes. Everolimus and docetaxel are both metabolized by CYP3A4, which could result in a pharmacokinetic (PK) interaction.
Patients and Methods
15 patients with metastatic breast cancer were treated with docetaxel (doses of 40-75 mg/m2 IV on day 1 of a 21 day cycle) in combination with everolimus (doses ranging from 20-50 mg po on days 1 and 8 of a 21 day cycle) in a phase I trial using the continuous reassessment method (CRM) to determine maximum tolerated dose (MTD). The first two patients developed DLT (neutropenic infection), prompting a mandatory dose reduction and PK evaluation of both everolimus and docetaxel for patients enrolled in subsequent dosing cohorts.
15 patients were treated. Dose limiting toxicity included grade 3 mucositis (n=1), prolonged grade 4 neutropenia (n=1), and grade 3 infection/febrile neutropenia (n=3). Day 8 of everolimus was commonly held for neutropenia despite a dose reduction in docetaxel to 40mg/m2. Eleven patients underwent complete PK evaluation for everolimus and 9 patients underwent complete PK evaluation for both everolimus and docetaxel. Widely variable changes in clearance were seen for both drugs and the study was terminated due to lack of efficacy and concerns regarding toxicity seen with the combination.
Weekly everolimus in combination with Q 3-week docetaxel was associated with excessive neutropenia and variable clearance of both drugs making combination therapy unpredictable, even at low doses of both drugs.
Phase I; everolimus; docetaxel
Obese women with breast cancer have worse prognosis than women with normal body mass index. Endocrine therapy resistance is in part mediated by insulin resistance in obese women with breast cancer. We investigated the tolerability and pharmacokinetics of exemestane in combination with metformin and rosiglitazone in non-diabetic overweight and obese postmenopausal women with hormone receptor positive metastatic breast cancer.
Patients had previously received chemotherapy and endocrine therapy for breast cancer. Exemestane was given as 25 mg orally per day. Metformin (M) and rosiglitazone (R) were given twice daily. Dose level 1 consisted of M 1500 mg/day and R 6 mg/day. Dose level 2 consisted of M 2000 mg/day and R 8 mg/day. Plasma concentrations of exemestane were measured on days 1, 8, and 15.
Twenty patients were enrolled. Fourteen patients received exemestane, metformin and rosiglitazone. Six patients received exemestane with metformin only (2000 mg/day). Both regimens were well tolerated at the highest doses tested, and there were no notable changes in plasma exemestane levels. Six patients (30%) had stable disease for 6 months or longer.
Oral daily administration of exemestane (25 mg) and metformin (2000 mg), with and without rosiglitazone (8 mg) daily was well tolerated. Exemestane pharmacokinetics were not altered by metformin and rosiglitazone.
Breast neoplasm; exemestane; metformin; rosiglitazone; phase I trial
Patients with ovarian serous tumors of low malignant potential (OSLMP) who have peritoneal implants, especially invasive implants, are at increased risk of developing tumor recurrence. The ability of peritoneal washing (PW) cytology, to detect the presence and type of peritoneal implants has not been adequately investigated, and its prognostic significance is unknown.
We retrospectively reviewed records and PW specimens of 101 patients diagnosed with and treated for OSLMP between 1996 and 2010 at The University of Texas MD Anderson Cancer Center. We compared patients’ staging biopsy findings with those of our review of the PWs. Follow-up data were also analyzed.
Of the 96 patients with staging biopsy results available, 26 (27%) had peritoneal implants (17 noninvasive, 9 invasive), 19 (20%) had endosalpingiosis, and 51 (53%) had negative findings. The PW specimens of 18 of the 26 patients (69%) with peritoneal implants were positive for serous neoplasm, and a correlation was found between cytologic and histologic findings (p < 0.0001). The sensitivity, specificity, positive predictive, and negative predictive values were 69%, 84%, 62% and 88%, respectively. Four of 101 patients had disease recurrence, 3 of these patients had invasive implants and 1 patient had noninvasive implants. None of the patients who had negative staging biopsy findings or endosalpingiosis but PW specimens positive for serous neoplasm developed disease recurrence.
PW cytology detects the presence of peritoneal implants with moderate accuracy. Long-term studies are needed to determine whether positive PW cytologic findings are an independent predictor of tumor recurrence.
To evaluate the effects of near-infrared (NIR) laser irradiation of microspheres (MS) containing hollow gold nanospheres (HAuNS) and paclitaxel (PTX) administered intra-arterially in an animal model.
Materials and Methods
For the ex-vivo experiments, VX2 tumor-bearing rabbits underwent hepatic artery (HA) administration of MS-HAuNS or MS. The animals were killed, the liver tumors were subjected to NIR irradiation, and temperature changes were estimated with magnetic resonance imaging. For the in-vivo study, VX2 tumor-bearing rabbits were randomized to 3 groups: MS-HAuNS-PTX-plus-NIR, MS-HAuNS-PTX, and saline-plus-NIR. Laser irradiation was delivered at 1 hour and at 3 days after HA administration of saline or MS-HAuNS-PTX. Animals were euthanized and tumors were analyzed for necrosis and apoptosis. Plasma samples were collected from the MS-HAuNS-PTX-plus-NIR animals for PTX analysis.
Ex-vivo experiments showed intratumoral heating in animals that received MS-HAuNS but no temperature change in animals that received MS. Animals treated with MS-HAuNS-PTX-plus-NIR showed a transient increase in plasma PTX levels after each NIR irradiation and significantly greater tumor necrosis than did those that received MS-HAuNS-PTX or saline-plus-NIR (44.9% vs. 13.8% or 23.7%, respectively; P < .0001). The mean apoptotic index in the MS-HAuNS-PTX-NIR group (5.01 ± 1.66) was significantly higher than that in the MS-HAuNS-PTX (2.99 ± 0.97) or saline-plus-NIR (1.96 ± 0.40) groups (P = .0013).
NIR laser irradiation after MS-HAuNS-PTX administration results in intratumoral heating and increases the efficacy of treatment. Further studies are required to evaluate the optimal laser settings to maximize therapeutic efficacy.
Human acellular dermal matrix (HADM) is used for ventral hernia repair, as it resists infection and remodels via surrounding tissue. However, the tissue source and impact of basement membrane (BM) on cell and vessel infiltration have not been determined. We hypothesized that musculofascia would be the primary tissue source of cells and vessels infiltrating into HADM and the BM would inhibit infiltration.
Fifty-six guinea pigs underwent inlay HADM ventral hernia repair with the BM oriented toward or away from the peritoneum. At postoperative weeks 1, 2, or 4, repair sites were completely excised. Histologic and immunohistochemical analyses were performed to quantify cell and vessel density within repair-site zones, including interface (lateral, beneath musculofascia) and center (beneath subcutaneous fat) zones. Cell and vessel quantities were compared as functions of zone, BM orientation, and time.
Cellular and vascular infiltration increased over time universally. The interface demonstrated greater mean cell density than the center (weeks 1 and 2, p=0.01, p<0.0001). Cell density was greater with the BM oriented toward the peritoneum at week 4 (p=0.02). The interface zone had greater mean vessel density than the center zone at week 4 (p<0.0001). Orienting the BM toward the peritoneum increased vessel density at week 4 (p=0.0004).
Cellular and vascular infiltration into HADM for ventral hernia repairs was greater from musculofascia than subcutaneous and the BM inhibited cellular and vascular. HADM should be placed adjacent to the best vascularizing tissue to improve fibrovascular incorporation.
Trastuzumab resistance has been linked to activation of the phosphoinositol 3-kinase (PI3K) pathway. Phosphatase and tensin homolog (PTEN) is a dual phosphatase that counteracts the PI3K function; PTEN loss leads to activation of the Akt cascade and the downstream mammalian target of rapamycin (mTOR). Preclinical studies demonstrated that mTOR inhibition sensitized the response to trastuzumab in mice with HER2 overexpressing and PTEN-deficient breast xenografts. Our trial evaluated the safety and efficacy of the combination of everolimus and trastuzumab in women with HER2-overexpressing metastatic breast cancer (MBC) that progressed on trastuzumab-based therapy.
Patients and Methods
This represents a pooled analysis (n = 47), stemming from two trials that occurred concurrently in The University of Texas MD Anderson Cancer Center, Beth Israel Deaconess Medical Center, and Dana-Farber Cancer Institute. Patients with HER2-overexpressing MBC who had progressed on trastuzumab-based therapy received trastuzumab every 3 weeks in combination with daily everolimus.
Among 47 patients, the combination of everolimus and trastuzumab provided partial responses in seven patients (15%) and persistent stable disease (lasting 6 months or longer) in nine patients (19%), resulting in a clinical benefit rate of 34%. The median progression-free survival (PFS) was 4.1 month. Fatigue, infection, and mucositis were the predominant nonhematologic toxicities. Trastuzumab did not have significant influence on the pharmacokinetic profile of everolimus. Patients with PTEN loss demonstrated decreased overall survival (P = .048). However, PFS was not affected by PTEN loss.
Inhibition of mTOR results in clinical benefit and disease response in patients with trastuzumab-resistant HER2-overexpressing MBC.
The ubiquitously expressed 14-3-3 proteins are involved in numerous important cellular functions. The loss of 14-3-3σ is a common event in breast cancer; however, the role of other 14-3-3s in breast cancer is unclear. Recently, we found that 14-3-3ζ overexpression occurs in early stage breast diseases and contributes to transformation of human mammary epithelial cells. Here, we show that 14-3-3ζ overexpression also persisted in invasive ductal carcinoma and contributed to the further progression of breast cancer. To examine the clinical impact of 14-3-3ζ overexpression in advanced stage breast cancer, we performed immunohistochemical analysis of 14-3-3ζ expression in primary breast carcinomas. 14-3-3ζ overexpression occurred in 42% of breast tumors and was determined to be an independent prognostic factor for reduced disease-free survival. 14-3-3ζ overexpression combined with ErbB2 overexpression and positive lymph node status identified a subgroup of patients at high risk for developing distant metastasis. To investigate whether 14-3-3ζ overexpression causally promotes breast cancer progression, we overexpressed 14-3-3ζ by stable transfection or reduced 14-3-3ζ expression by siRNA in cancer cell lines. Increased 14-3-3ζ expression enhanced anchorage independent growth and inhibited stress-induced apoptosis, whereas downregulation of 14-3-3ζ reduced anchorage independent growth and sensitized cells to stress-induced apoptosis via the mitochondrial apoptotic pathway. Transient blockade of 14-3-3ζ expression by siRNA in cancer cells effectively reduced the onset and growth of tumor xenografts in vivo. Therefore, 14-3-3ζ overexpression is a novel molecular marker for disease recurrence in breast cancer patients and may serve as an effective therapeutic target in patients whose tumors overexpress 14-3-3ζ.
14-3-3ζ; breast cancer; apoptosis resistance; disease recurrence; prognostic marker
Healthcare websites that are influential in healthcare decision-making must be evaluated for accuracy, readability and understandability by the average population. Most existing frameworks for designing and evaluating interactive websites focus on the utility and usability of the site. Although these are significant to the design of the basic site, they are not sufficient. We have developed an iterative framework that considers additional attributes.
We outline the methodology used to develop an Internet-based cancer risk
assessment tool and describe factors needed to create and post credible
risk assessment tools or risk calculators.