Qing H. Meng

Epithelial–Mesenchymal Transitioned Circulating Tumor Cells Capture for Detecting Tumor Progression

Purpose: This study aimed to detect cell-surface vimentin (CSV) on the surface of epithelial–mesenchymal transitioned (EMT) circulating tumor cells (CTC) from blood of patients with epithelial cancers. Experimental Design: In this study, 101 patients undergoing postsurgery adjuvant chemotherapy for metastatic colon cancer were recruited. EMT CTCs were detected from blood of patients using the 84-1 monoclonal antibody against CSV as a marker. EMT CTCs isolated were characterized further using EMT-specific markers, fluorescent in situ hybridization, and single-cell mutation analysis. Results: Using the 84-1 antibody, we detected CSV exclusively on EMT CTCs from a variety of tumor types but not in the surrounding normal cells in the blood. The antibody exhibited very high specificity and sensitivity toward different epithelial cancer cells. With this antibody, we detected and enumerated EMT CTCs from patients. From our observations, we defined a cutoff of <5 or ≥5 EMT CTCs as the optimal threshold with respect to therapeutic response using ROC curves. Using this defined threshold, the presence of ≥5 EMT CTCs was associated with progressive disease, whereas patients with <5 EMT CTCs showed therapeutic response. Conclusion: Taken together, the number of EMT CTCs detected correlated with the therapeutic outcome of the disease. These results establish CSV as a universal marker for EMT CTCs from a wide variety of tumor types and thus provide the foundation for emerging CTC detection technologies and for studying the molecular regulation of these EMT CTCs. Clin Cancer Res; 21(4); 899–906. ©2014 AACR.

Circulating Tumor Cell Enumeration with a Combination of Epithelial Cell Adhesion Molecule– and Cell-Surface Vimentin–Based Methods for Monitoring Breast Cancer Therapeutic Response

BACKGROUND Detection, isolation, and enumeration of circulating tumor cells (CTCs) from cancer patients has become an important modality in clinical management of patients with breast cancer. Although CellSearch, an epithelial cell adhesion molecule (EpCAM)-based method that is used to isolate epithelial CTCs, has gained prominence, its inability to detect mesenchymal CTCs from breast cancer patients raises concerns regarding its utility in clinical management. METHODS To address this gap in technology, we recently discovered the utility of cell-surface vimentin (CSV) as a marker for detecting mesenchymal CTCs from sarcoma tumors. In the present study, we tested the sensitivity and specificity of detecting CTCs from blood collected at a random time during therapy from each of 58 patients with metastatic breast cancer by use of 84-1 (a monoclonal antibody against CSV to detect epithelial/mesenchymal-transition CTCs) and CellSearch methods. Additionally, we tested the possibility of improving the sensitivity and specificity of detection by use of additional parameters including nuclear EpCAM localization and epithelial mesenchymal ratios. RESULTS CTC counts with CSV were significant (P = 0.0053) in differentiating populations responsive and nonresponsive to treatment compared with CTC counts with CellSearch (P = 0.0564). The specificity of CTC detection was found to be highest when the sum of CTC counts from the 2 methods was above a threshold of 8 CTCs/7.5 mL. CONCLUSIONS The sum of CTC counts from the CellSearch and CSV methods appears to provide new insights for assessment of therapeutic response and thus provides a new approach to personalized medicine in breast cancer patients.

Prognostic significance of pretreatment serum levels of albumin, LDH and total bilirubin in patients with non-metastatic breast cancer

Liver function tests (LFTs) have been reported as independent predictors of non-liver disease-related morbidity and mortality in general population and cancer patients. In this study, we evaluated the relationship between pretreatment serum LFTs and overall survival (OS) in non-metastatic Caucasian breast cancer patients. Seven LFTs, including albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase (LDH), total bilirubin and total protein, were measured in pretreatment serum from 2425 female Caucasian patients with newly diagnosed, histologically confirmed non-metastatic invasive breast cancer. Multivariate Cox model was used to estimate hazard ratio (HR) and 95% confidence interval (CI) for the association of individual LFTs with 5-year OS while adjusting for age, smoking status, pathological characteristics and treatment regimen. We found that serum albumin, LDH and total bilirubin were significantly associated with 5-year OS in multivariate Cox analyses. Patients with higher albumin level exhibited 45% reduced risk of death (HR = 0.55, 95% CI: 0.40-0.75) compared with those with lower albumin level. Patients with higher total bilirubin level had a nearly 40% reduction in the risk of death (HR = 0.62, 95% CI: 0.45-0.85) and patients with higher LDH levels had a 1.42-fold increased risk of death (HR = 1.42, 95% CI: 1.08-1.88). Furthermore, cumulative analysis showed a significant dose-response trend of significantly increasing risk of death with increasing number of unfavorable LFT levels. Our result highlighted the potential of using pretreatment serum levels of albumin, LDH and total bilirubin as prognostic factors for OS in patients with non-metastatic breast cancer.

Potential role of nuclear PD-L1 expression in cell-surface vimentin positive circulating tumor cells as a prognostic marker in cancer patients

Although circulating tumor cells (CTCs) have potential as diagnostic biomarkers for cancer, determining their prognostic role in cancer patients undergoing treatment is a challenge. We evaluated the prognostic value of programmed death-ligand 1 (PD-L1) expression in CTCs in colorectal and prostate cancer patients undergoing treatment. Peripheral blood samples were collected from 62 metastatic colorectal cancer patients and 30 metastatic prostate cancer patients. CTCs were isolated from the samples using magnetic separation with the cell-surface vimentin(CSV)-specific 84-1 monoclonal antibody that detects epithelial-mesenchymal transitioned (EMT) CTCs. CTCs were enumerated and analyzed for PD-L1 expression using confocal microscopy. PD-L1 expression was detectable in CTCs and was localized in the membrane and/or cytoplasm and nucleus. CTC detection alone was not associated with poor progression-free or overall survival in colorectal cancer or prostate cancer patients, but nuclear PD-L1 (nPD-L1) expression in these patients was significantly associated with short survival durations. These results demonstrated that nPD-L1 has potential as a clinically relevant prognostic biomarker for colorectal and prostate cancer. Our data thus suggested that use of CTC-based models of cancer for risk assessment can improve the standard cancer staging criteria and supported the incorporation of nPD-L1 expression detection in CTCs detection in such models.

Genetic Variants in the Fibroblast Growth Factor Pathway as Potential Markers of Ovarian Cancer Risk, Therapeutic Response, and Clinical Outcome

BACKGROUND The fibroblast growth factor (FGF) and FGF receptor (FGFR) axis plays a critical role in tumorigenesis, but little is known of its influence in ovarian cancer. We sought to determine the association of genetic variants in the FGF pathway with risk, therapeutic response, and survival of patients with ovarian cancer. METHODS We matched 339 non-Hispanic white ovarian cancer cases with 349 healthy controls and genotyped them for 183 single-nucleotide polymorphisms (SNPs) from 24 FGF (fibroblast growth factor) and FGFR (fibroblast growth factor receptor) genes. Genetic associations for the main effect, gene-gene interactions, and the cumulative effect were determined. RESULTS Multiple SNPs in the FGF-FGFR axis were associated with an increased risk of ovarian cancer. In particular, FGF1 [fibroblast growth factor 1 (acidic)] SNP rs7727832 showed the most significant association with ovarian cancer (odds ratio, 2.27; 95% CI, 1.31-3.95). Ten SNPs were associated with a reduced risk of ovarian cancer. FGF18 (fibroblast growth factor 18) SNP rs3806929, FGF7 (fibroblast growth factor 7) SNP rs9920722, FGF23 (fibroblast growth factor 23) SNP rs12812339, and FGF5 (fibroblast growth factor 5) SNP rs3733336 were significantly associated with a favorable treatment response, with a reduction of risk of nonresponse of 40% to 60%. Eleven SNPs were significantly associated with overall survival. Of these SNPs, FGF23 rs7961824 was the most significantly associated with improved prognosis (hazard ratio, 0.55; 95% CI, 0.39-0.78) and was associated with significantly longer survival durations, compared with individuals with the common genotype at this locus (58.1 months vs. 38.0 months, P = 0.005). Survival tree analysis revealed FGF2 rs167428 as the primary factor contributing to overall survival. CONCLUSIONS Significant associations of genetic variants in the FGF pathway were associated with ovarian cancer risk, therapeutic response, and survival. The discovery of multiple SNPs in the FGF-FGFR pathway provides a molecular approach for risk assessment, monitoring therapeutic response, and prognosis.

Influence of Vitamin D2 Percentage on Accuracy of 4 Commercial Total 25-Hydroxyvitamin D Assays

To the Editor: 25-Hydroxyvitamin D (25-OH-D)1 is the most appropriate marker for monitoring vitamin D storage status. The 2 major forms of 25-OH-D [25-OH-D2 (D2) and 25-OH-D3 (D3)], differ slightly in molecular structures but have essentially the same physiological activity. Therefore, both D2 and D3 should be measured to assess vitamin D status. At our laboratory, 42% of patient samples submitted for vitamin D testing contain D2. Recognizing the ability of an assay to quantify both the D3 and D2 forms equally is therefore crucial. The analytical performance of several total 25-OH-D assays approved by the US Food and Drug Administration is well documented (1); however, information is limited on how D2 affects assay accuracies. Le Goff et al. (2) demonstrated the inconsistency between several assays in their cross-reactivities toward D2. The size of their study was small, however, and the range of D2 percentages (D2%) was unknown. We used a larger sample size than previous studies and a D2% interval of 1.3%–91.2% [3.4–95.1 ng/mL (8.5–238 nmol/L)] and evaluated the influence of D2% on the accuracies of the following assays: DiaSorin Liaison, Abbott Architect, Roche Cobas, and Siemens Centaur. We developed and validated a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay as the reference method. Samples were prepared for the LC-MS/MS assay by mixing with internal standard (D3- d 6/D2- d 6), ZnSO4 solution, and methanol; liquid–liquid …

EMT circulating tumor cells detected by cell-surface vimentin are associated with prostate cancer progression

Recent advances in the field of circulating tumor cells (CTC) have shown promise in this liquid biopsy-based prognosis of patient outcome. However, not all of the circulating cells are tumor cells, as evidenced by a lack of tumor-specific markers. The current FDA standard for capturing CTCs (CellSearch) relies on an epithelial marker and cells captured via CellSearch cannot be considered to have undergone EMT. Therefore, it is difficult to ascertain the presence and relevance of any mesenchymal or EMT-like CTCs. To address this gap in technology, we recently discovered the utility of cell-surface vimentin (CSV) as a marker for detecting mesenchymal CTCs from sarcoma, breast, and colon cancer. Here we studied peripheral blood samples of 48 prostate cancer (PCA) patients including hormone sensitive and castration resistant sub-groups. Blood samples were analyzed for three different properties including our own CSV-based CTC enumeration (using 84-1 mAb against CSV), CellSearch-based epithelial CTC counts, and serum prostate-specific antigen (PSA) quantification. Our data demonstrated that in comparison with CellSearch, the CSV-based method had greater sensitivity and specificity. Further, we observed significantly greater numbers of CTCs in castration resistant patients as measured by our CSV method but not CellSearch. Our data suggests CSV-guided CTC enumeration may hold prognostic value and should be further validated as a possible measurement of PCA progression towards the deadly, androgen-independent form.

Pseudohyperkalemia: A new twist on an old phenomenon

Abstract Severe hyperkalemia is a potentially life-threatening condition requiring immediate medical intervention. Pseudohyperkalemia can be misleading and result in incorrect interpretation and inappropriate patient management. Immediate recognition and appropriate interpretation of pseudohyperkalemia, on the other hand, prevents misdiagnosis and unnecessary intervention. Pseudohyperkalemia is induced by hemolysis and excessive leakage of potassium from cells during or after blood collection. It has been increasingly seen in many hematological disorders such as leukocytosis and thrombocytosis. Reverse pseudohyperkalemia has recently been reported in leukemic patients in whom the plasma potassium levels are greater than the serum potassium levels because of heparin-induced cell membrane damage. Although pseudohyperkalemia has long been recognized and understood, it continues to be misinterpreted. To improve patient care, an algorithm for investigation of pseudohyperkalemia and preventive measures should be established and implemented in the clinical laboratory.

Personalized Prognostic Prediction Models for Breast Cancer Recurrence and Survival Incorporating Multidimensional Data

Background: In this study, we developed integrative, personalized prognostic models for breast cancer recurrence and overall survival (OS) that consider receptor subtypes, epidemiological data, quality of life (QoL), and treatment. Methods: A total of 15 314 women with stage I to III invasive primary breast cancer treated at The University of Texas MD Anderson Cancer Center between 1997 and 2012 were used to generate prognostic models by Cox regression analysis in a two-stage study. Model performance was assessed by calculating the area under the curve (AUC) and calibration analysis and compared with Nottingham Prognostic Index (NPI) and PREDICT. Results: Host characteristics were assessed for 10 809 women as the discovery population (median follow-up = 6.09 years, 1144 recurrence and 1627 deaths) and 4505 women as the validation population (median follow-up = 7.95 years, 684 recurrence and 1095 deaths). In addition to the known clinical/pathological variables, the model for recurrence included alcohol consumption while the model for OS included smoking status and physical component summary score. The AUCs for recurrence and OS were 0.813 and 0.810 in the discovery and 0.807 and 0.803 in the validation, respectively, compared with AUCs of 0.761 and 0.753 in discovery and 0.777 and 0.751 in validation for NPI. Our model further showed better calibration compared with PREDICT. We also developed race-specific and receptor subtype–specific models with comparable AUCs. Racial disparity was evident in the distributions of many risk factors and clinical presentation of the disease. Conclusions: Our integrative prognostic models for breast cancer exhibit high discriminatory accuracy and excellent calibration and are the first to incorporate receptor subtype and epidemiological and QoL data.

Low serum testosterone is associated with tumor aggressiveness and poor prognosis in prostate cancer

Serum testosterone is a potential marker to distinguish between indolent and aggressive prostate cancer (PCa). The present study aimed to investigate whether low levels of total serum testosterone at diagnosis were associated with aggressive PCa and poor clinical outcomes. In total, 762 non-Hispanic Caucasian men with previously untreated PCa were recruited from The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients were categorized into three groups based on their total serum testosterone levels according to clinical guidelines [low (<230 ng/dl), intermediate (230-350 ng/dl) and normal (>350 ng/dl)]. PCa aggressiveness (low-, intermediate- or high-risk, or metastatic) was compared using multinomial logistic regression. Rates of disease progression, mortality from any cause and PCa-specific mortality were compared using the multivariate Cox proportional hazards model. Testosterone levels significantly decreased as PCa aggressiveness increased (P<0.001). Compared with the normal testosterone group, the low testosterone group had 2.9-fold (OR, 2.92; 95% CI, 1.74-4.90; P<0.001), 5.6-fold (OR, 5.63; 95% CI, 3.14-10.12; P<0.001) and 72.4-fold (OR, 72.40; 95% CI, 20.89-250.89; P<0.001) increased risks of having intermediate-risk, high-risk and metastatic PCa, respectively. Furthermore, low levels of testosterone were significantly associated with a 10.7-fold (HR, 10.68; 95% CI, 1.35-84.44; P=0.03) increased risk of PCa-specific mortality. The results of the present study indicate that low levels of total serum testosterone at diagnosis are associated with aggressive PCa and predict poor PCa-specific survival.