Analytical performance of the FDA-cleared Parsortix® PC1 system.

Introduction: The Parsortix® PC1 system, Food and Drug Administration (FDA) cleared for use in metastatic breast cancer (MBC) patients, is an epitope-independent microfluidic device for the capture and harvest of circulating tumor cells from whole blood based on cell size and deformability. This report details the analytical characterization of linearity, detection limit, precision, and reproducibility for this device. Methods: System performance was determined using K2-EDTA blood samples collected from self-declared healthy female volunteers (HVs) and MBC patients spiked with prelabeled cultured breast cancer cell lines (SKBR3, MCF7, or Hs578T). Samples were processed on Parsortix® PC1 systems and captured cells were harvested and enumerated. Results: The system captured and harvested live SKBR3, MCF7, and Hs578T cells and fixed SKBR3 cells linearly between 2 and ~100 cells, with average harvest rates of 69%, 73%, 79%, and 90%, respectively. To harvest ≥1 cell ≥95% of the time, the system required 3, 5 or 4 live SKBR3, MCF7 or Hs578T cells, respectively. Average harvest rates from precision studies using 5, 10, and ~50 live cells spiked into blood for each cell line ranged from 63.5% to 76.2%, with repeatability and reproducibility percent coefficient of variation (%CV) estimates ranging from 12.3% to 32.4% and 13.3% to 34.1%, respectively. Average harvest rates using ~20 fixed SKBR3 cells spiked into HV and MBC patient blood samples were 75.0% ± 16.1% (%CV = 22.3%) and 68.4% ± 14.3% (%CV = 21.1%), respectively. Conclusions: These evaluations demonstrate the Parsortix® PC1 system linearly and reproducibly harvests tumor cells from blood over a range of 1 to ~100 cells.

HE4 and CA125 serum biomarker monitoring in women with epithelial ovarian cancer.

BACKGROUND: CA125 is the gold standard serum biomarker for monitoring patients with epithelial ovarian cancer (EOC). Human epididymal protein 4 (HE4) is a novel serum biomarker for EOC patients. OBJECTIVE: The objective of this trial was to examine the utility of measuring serum HE4 levels for monitoring EOC patients and to compare HE4 performance parameters to serum CA125. METHODS: A retrospective trial using residual longitudinal serum samples drawn during treatment and monitoring from EOC patients. Serum CA125 and HE4 levels were analyzed at each time point, and a velocity of change was calculated and correlated with clinical status. The null hypothesis was that HE4 is inferior to CA125, and this was tested using concordance and two-sided Fisher's exact testing. McNemar's test was used to assess the overall agreement of the two assays with the clinical status. RESULTS: A total of 129 patients with 272 separate clinical periods and 1739 events (serum samples) were evaluated. Using a 25% change in serum biomarker levels to indicate change in disease status, the accuracy and NPV determined for HE4 versus CA125 were 81.8% versus 82.6% (p = 0.846) and 87.4% versus 89.7% (p = 0.082), respectively. Concordance comparison of HE4 accuracy / CA125 accuracy was 0.990, indicating HE4 was not inferior to CA125 (McNemar's test p-value = 0.522). Performing a velocity of change analysis, the accuracy and NPV determined for HE4 versus CA125 were 78.3% versus 78.6% (p = 0.995) and 74.9% versus 76.3% (p = 0.815), respectively. Concordance comparison of HE4 velocity accuracy / CA125 velocity accuracy was 0.996, again indicating HE4 was not inferior to CA125 (McNemar's test p-value = 0.884). The combination of HE4 and CA125 velocity changes showed a similar accuracy of 81.3% (p = 0.797 compared to HE4 and CA125 alone) and NPV of 81.1% (p≥0.172 compared to HE4 and CA125 alone), and an increased sensitivity of 70.5% (p≤0.070 compared to HE4 and CA125 alone). CONCLUSION: HE4 is equivalent to CA125 for monitoring of EOC patients. The combination of CA125 and HE4 velocities is superior to either marker alone.

Analysis of serum HE4 levels in various histologic subtypes of epithelial ovarian cancer and other malignant tumors.

BACKGROUND: The measurement of serum HE4 levels has emerged as a sensitive and specific biomarker for epithelial ovarian cancers (EOCs). However, serum levels in women diagnosed with various histologic subtypes of EOC and in women with metastatic non-ovarian primary malignancies have not been widely reported. OBJECTIVE: The goal of this study was to identify how serum HE4 levels vary in women diagnosed with different histologic subtypes of EOC and non-ovarian malignancies. METHODS: Data from six prospective pelvic mass clinical trials was combined and an evaluation of serum HE4 levels in women diagnosed with a malignancy was performed. For all patients, serum was obtained prior to surgery and final pathology, including primary tumor site, histologic subtype, grade and stage, were recorded. The mean, median, standard deviation, maximum, and minimum HE4 levels were determined for each group. RESULTS: A total of 984 patients were included in this study, with the average patient age being 60 years old. There were 230 premenopausal and 754 postmenopausal patients. Serum HE4 levels were elevated (≥70.0 pMol) in 85%of EOCs, 40%of LMP tumors, 21%of non-EOCs (germ cell tumors), 25%of cervical cancers, and 47%of non-gynecologic metastatic cancers. Analysis of histologic subtypes revealed 90%(n = 391) of serous, 85%(n = 73) of endometrioid, 45%(n = 42) of mucinous, 86%(n = 51) of mixed tumors, and 69%(n = 36) of clear cell tumors had elevated serum HE4 levels. CONCLUSIONS: Serum HE4 levels are most often elevated in women with high grade serous and endometrioid EOCs, and though serum elevations are seen more often with advanced stage disease, HE4 is also often elevated in early stage disease and lower grade tumors.

Characterization of RNA-Binding Motif 3 (RBM3) Protein Levels and Nuclear Architecture Changes in Aggressive and Recurrent Prostate Cancer.

Background: The RNA-binding motif protein 3 (RBM3) has been shown to be up-regulated in several types of cancer, including prostate cancer (PCa), compared to normal tissues. Increased RBM3 nuclear expression has been linked to improved clinical outcomes. Aims: Given that RBM3 has been hypothesized to play a role in critical nuclear functions such as chromatin remodeling, DNA damage response, and other post-transcriptional processes, we sought to: (1) quantify RBM3 protein levels in archival PCa samples; (2) develop a nuclear morphometric model to determine if measures of RBM3 protein levels and nuclear features could be used to predict disease aggressiveness and biochemical recurrence. Methods & Results: This study utilized two tissue microarrays (TMAs) stained for RBM3 that included 80 total cases of PCa stratified by Gleason score. A software-mediated image processing algorithm identified RBM3-positive cancerous nuclei in the TMA samples and calculated twenty-two features quantifying RBM3 expression and nuclear architecture. Multivariate logistic regression (MLR) modeling was performed to determine if RBM3 levels and nuclear structural changes could predict PCa aggressiveness and biochemical recurrence (BCR). Leave-one-out cross validation (LOOCV) was used to provide insight on how the predictive capabilities of the feature set might behave with respect to an independent patient cohort to address issues such as model overfitting. RBM3 expression was found to be significantly downregulated in highly aggressive GS ≥ 8 PCa samples compared to other Gleason scores (P < 0.0001) and significantly down-regulated in recurrent PCa samples compared to non-recurrent samples (P = 0.0377). An eleven-feature nuclear morphometric MLR model accurately identified aggressive PCa, yielding a receiver operating characteristic area under the curve (ROC-AUC) of 0.90 (P < 0.0001) in the raw data set and 0.77 (95% CI: 0.83-0.97) for LOOCV testing. The same eleven-feature model was then used to predict recurrence, yielding a ROC-AUC of 0.92 (P = 0.0004) in the raw data set and 0.76 (95% CI: 0.64-0.87) for LOOCV testing. Conclusions: The RBM3 biomarker alone is a strong prognostic marker for the prediction of aggressive PCa and biochemical recurrence. Further, RBM3 appears to be down-regulated in aggressive and recurrent tumors.

Tumour-derived extracellular vesicles in blood of metastatic cancer patients associate with overall survival.

BACKGROUND: Circulating tumour cells (CTCs) in blood associate with overall survival (OS) of cancer patients, but they are detected in extremely low numbers. Large tumour-derived extracellular vesicles (tdEVs) in castration-resistant prostate cancer (CRPC) patients are present at around 20 times higher frequencies than CTCs and have equivalent prognostic power. In this study, we explored the presence of tdEVs in other cancers and their association with OS. METHODS: The open-source ACCEPT software was used to automatically enumerate tdEVs in digitally stored CellSearch® images obtained from previously reported CTC studies evaluating OS in 190 CRPC, 450 metastatic colorectal cancer (mCRC), 179 metastatic breast cancer (MBC) and 137 non-small cell lung cancer (NSCLC) patients before the initiation of a new treatment. RESULTS: Presence of unfavourable CTCs and tdEVs is predictive of OS, with respective hazard ratios (HRs) of 2.4 and 2.2 in CRPC, 2.7 and 2.2 in MBC, 2.3 and 1.9 in mCRC and 2.0 and 2.4 in NSCLC, respectively. CONCLUSIONS: tdEVs have equivalent prognostic value as CTCs in the investigated metastatic cancers. CRPC, mCRC, and MBC (but not NSCLC) patients with favourable CTC counts can be further prognostically stratified using tdEVs. Our data suggest that tdEVs could be used in clinical decision-making.

Multiple biomarker algorithms to predict epithelial ovarian cancer in women with a pelvic mass: Can additional makers improve performance?

INTRODUCTION: Management of a woman with a pelvic mass is complicated by difficulty in discriminating malignant from benign disease. Many serum biomarkers have been examined to determine their sensitivity for detecting malignancy. This study was designed to evaluate if the addition of biomarkers to HE4 and CA125, as used in the Risk of Malignancy Algorithm (ROMA), can improve the detection of EOC. METHODS: This was an IRB approved, prospective clinical trial examining serum obtained from women diagnosed with a pelvic mass who subsequently underwent surgery. Serum biomarker levels for CA125, HE4, YKL-40, transthyretin, ApoA1, Beta-2-microglobulin, transferrin, and LPA were measured. Logistic regression analysis was performed for various marker combinations, ROC curves were generated, and the area under the curves (AUCs) were determined. RESULTS: A total of 184 patients met inclusion criteria with a median age of 56 years (Range 20-91). Final pathology revealed there were 103 (56.0%) benign tumors, 4 (2.2%) LMP tumors, 61 EOC (33.1%), 2 (1.1%) non-EOC ovarian cancers, 6 (3.3%) gynecologic cancers with metastasis to the ovary and 8 (4.3%) non-gynecologic cancers with metastasis to the ovary. The combination of HE4 and CA125 (i.e. ROMA) achieved an AUC of 91.2% (95% CI: 86.0-96.4) for the detection of EOC vs benign disease. The combination of CA125, HE4, YKL-40, transthyretin, ApoA1, Beta 2 microglobulin, transferrin, LPA and menopausal status achieved the highest AUC of 94.6% (95% CI: 90.1-99.2) but this combination was not significantly better than the HE4 and CA125 combination alone (p = 0.078). CONCLUSIONS: The addition of select further serum biomarkers to HE4 and CA125 does not add to the performance of the dual marker combination for the detection of ovarian cancer.

The Parsortix™ Cell Separation System-A versatile liquid biopsy platform.

Cancer cells from solid tumors can enter the circulatory system and survive to subsequently form distant metastases. The CellSearch® system (Menarini-Silicon Biosystems, Huntingdon Valley, PA) was the first, FDA-cleared system that provided a reliable tool for the investigation of circulating tumor cells (CTCs), which have been shown to be strongly associated with poor survival and therapy failure. Since that time, a number of new technologies have been introduced to improve CTC detection and/or isolation for further characterization. The continued and growing interest in the "liquid biopsy" field has spurred the development of numerous different CTC technologies. However, selecting the most appropriate CTC platform for individual applications can be challenging. No consensus has yet been reached in the community regarding which liquid biopsy technology is optimal. Here, we introduce the Parsortix™ Cell Separation System (ANGLE North America, Inc., King of Prussia, PA), a microfluidic based technology that captures rare cells based on size and deformability, offers reproducibly high capture efficiency, and produces highly enriched, viable (viability dependent on preservative used) CTCs that are amenable to a multitude of downstream analyses, including the isolation and interrogation of single cells. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

PBOV1 as a potential biomarker for more advanced prostate cancer based on protein and digital histomorphometric analysis.

BACKGROUND: There are few tissue-based biomarkers that can accurately predict prostate cancer (PCa) progression and aggressiveness. We sought to evaluate the clinical utility of prostate and breast overexpressed 1 (PBOV1) as a potential PCa biomarker. METHODS: Patient tumor samples were designated by Grade Groups using the 2014 Gleason grading system. Primary radical prostatectomy tumors were obtained from 48 patients and evaluated for PBOV1 levels using Western blot analysis in matched cancer and benign cancer-adjacent regions. Immunohistochemical evaluation of PBOV1 was subsequently performed in 80 cancer and 80 benign cancer-adjacent patient samples across two tissue microarrays (TMAs) to verify protein levels in epithelial tissue and to assess correlation between PBOV1 proteins and nuclear architectural changes in PCa cells. Digital histomorphometric analysis was used to track 22 parameters that characterized nuclear changes in PBOV1-stained cells. Using a training and test set for validation, multivariate logistic regression (MLR) models were used to identify significant nuclear parameters that distinguish Grade Group 3 and above PCa from Grade Group 1 and 2 PCa regions. RESULTS: PBOV1 protein levels were increased in tumors from Grade Group 3 and above (GS 4 + 3 and ≥ 8) regions versus Grade Groups 1 and 2 (GS 3 + 3 and 3 + 4) regions (P = 0.005) as assessed by densitometry of immunoblots. Additionally, by immunoblotting, PBOV1 protein levels differed significantly between Grade Group 2 (GS 3 + 4) and Grade Group 3 (GS 4 + 3) PCa samples (P = 0.028). In the immunohistochemical analysis, measures of PBOV1 staining intensity strongly correlated with nuclear alterations in cancer cells. An MLR model retaining eight parameters describing PBOV1 staining intensity and nuclear architecture discriminated Grade Group 3 and above PCa from Grade Group 1 and 2 PCa and benign cancer-adjacent regions with a ROC-AUC of 0.90 and 0.80, respectively, in training and test sets. CONCLUSIONS: Our study demonstrates that the PBOV1 protein could be used to discriminate Grade Group 3 and above PCa. Additionally, the PBOV1 protein could be involved in modulating changes to the nuclear architecture of PCa cells. Confirmatory studies are warranted in an independent population for further validation.

Validation of a Commercial Assay and Decision Support Tool for Routine Paclitaxel Therapeutic Drug Monitoring (TDM).

BACKGROUND: The value of therapeutic drug monitoring (TDM) for paclitaxel (PTX) was recently demonstrated in the largest TDM trial ever conducted in oncology. The trial demonstrated significant reduction in neuropathy when using TDM. Dose adjustment for PTX was based on time above a threshold concentration (Tc>0.05). Tc>0.05 must be calculated with a pharmacokinetic model and complex nonlinear mixed-effects software. The use of the software and chromatographic methods to measure PTX requires specialized expertise. User-friendly methods to quantitate PTX and calculate Tc>0.05 could simplify the introduction of TDM into routine clinical practice. METHODS: The immunoassay (MyPaclitaxel) was used to quantitate PTX in samples from the clinical trial; the results were used to calculate Tc>0.05 using a stand-alone computer program with a simple, friendly graphical user interface for nonlinear mixed-effects pharmacokinetic calculations (MyCare Drug Exposure Calculator). The resulting dose recommendations from the calculated Tc>0.05 were compared with those using liquid chromatography-ultraviolet detection and NONMEM to examine the efficacy of the simpler tools for TDM. RESULTS: There was a good agreement between the immunoassay and liquid chromatography-ultraviolet detection: Passing-Bablok regression slope was 1.045 and intercept was -6.00, R was 0.9757, and mean bias was -1.77 ng/mL (-2.07 nmol/L). Dosing recommendations were identical for 70% of the cycles and within 10% for 89% of the samples. All Tc>0.05 values were at the same or adjacent medical decision points. CONCLUSIONS: MyPaclitaxel assay and MyCare Drug Exposure Calculator are convenient, user-friendly tools that may be suitable for routine TDM of PTX in clinical care.

Prospective, Multicenter Study of 5-Fluorouracil Therapeutic Drug Monitoring in Metastatic Colorectal Cancer Treated in Routine Clinical Practice.

BACKGROUND: Studies have demonstrated that body surface area-based dosing of chemotherapy drugs leads to significant individual exposure variability, with a substantial risk of under- or overdosing. The present study was initiated to validate the use of therapeutic drug management (TDM) to personalize 5-fluorouracil (5-FU) dosing in patients with metastatic colorectal cancer treated in routine clinical practice. PATIENTS AND METHODS: A total of 75 patients with metastatic colorectal cancer from 8 German medical centers received ≤ 6 administrations of infusional 5-FU according to the AIO (folinate, 5-FU; n = 16), FOLFOX6 (leucovorin calcium [folinic acid], 5-FU, and oxaliplatin; n = 26), or FUFOX (oxaliplatin plus 5-FU/folinic acid; n = 33) regimen. Initial infusional 5-FU dosing for all patients was determined by the BSA. Individual 5-FU exposure (area under the curve [AUC]) was measured using an immunoassay of a blood sample taken during each infusion. To achieve a target AUC of 20 to 30 mg × h/L, subsequent infusional 5-FU doses were adjusted according to the previous cycle's 5-FU AUC. The primary objective was to confirm that TDM of infusional 5-FU resulted in an increased proportion of patients in the target AUC range at the fourth versus the first administration. The secondary objective was to determine whether 5-FU TDM reduced the treatment-related toxicities compared with the historical data. RESULTS: The average 5-FU AUC at the first administration was 18 ± 6 mg × h/L, with 64%, 33%, and 3% of the patients below, within, or above the target AUC range, respectively. By the fourth administration, the average 5-FU AUC was 25 ± 7 mg × h/L (P < .001), with 54% of patients within the target 5-FU AUC range (P = .0294). The incidence of 5-FU-related grade 3 and 4 diarrhea (4.6%), nausea (3.4%), fatigue (0.0%), and mucositis (0.2%) was reduced compared with the historical data, despite 55% of the patients receiving increased doses. CONCLUSION: Personalization of 5-FU dosing using TDM in routine clinical practice resulted in significantly improved 5-FU exposure and suggested a lower incidence of 5-FU-related toxicities.

A Novel Quantitative Multiplex Tissue Immunoblotting for Biomarkers Predicts a Prostate Cancer Aggressive Phenotype.

BACKGROUND: Early prediction of disease progression in men with very low-risk (VLR) prostate cancer who selected active surveillance (AS) rather than immediate treatment could reduce morbidity associated with overtreatment. METHODS: We evaluated the association of six biomarkers [Periostin, (-5, -7) proPSA, CACNA1D, HER2/neu, EZH2, and Ki-67] with different Gleason scores and biochemical recurrence (BCR) on prostate cancer TMAs of 80 radical prostatectomy (RP) cases. Multiplex tissue immunoblotting (MTI) was used to assess these biomarkers in cancer and adjacent benign areas of 5 µm sections. Multivariate logistic regression (MLR) was applied to model our results. RESULTS: In the RP cases, CACNA1D, HER2/neu, and Periostin expression were significantly correlated with aggressive phenotype in cancer areas. An MLR model in the cancer area yielded a ROC-AUC = 0.98, whereas in cancer-adjacent benign areas, yielded a ROC-AUC = 0.94. CACNA1D and HER2/neu expression combined with Gleason score in a MLR model yielded a ROC-AUC = 0.79 for BCR prediction. In the small biopsies from an AS cohort of 61 VLR cases, an MLR model for prediction of progressors at diagnosis retained (-5, -7) proPSA and CACNA1D, yielding a ROC-AUC of 0.78, which was improved to 0.82 after adding tPSA into the model. CONCLUSIONS: The molecular profile of biomarkers is capable of accurately predicting aggressive prostate cancer on retrospective RP cases and identifying potential aggressive prostate cancer requiring immediate treatment on the AS diagnostic biopsy but limited in BCR prediction. IMPACT: Comprehensive profiling of biomarkers using MTI predicts prostate cancer aggressive phenotype in RP and AS biopsies.

Development of circulating tumor cell-endocrine therapy index in patients with hormone receptor-positive breast cancer.

BACKGROUND: Endocrine therapy (ET) fails to induce a response in one half of patients with hormone receptor (HR)-positive metastatic breast cancer (MBC), and almost all will eventually become refractory to ET. Circulating tumor cells (CTC) are associated with worse prognosis in patients with MBC, but enumeration alone is insufficient to predict the absolute odds of benefit from any therapy, including ET. We developed a multiparameter CTC-Endocrine Therapy Index (CTC-ETI), which we hypothesize may predict resistance to ET in patients with HR-positive MBC. METHODS: The CTC-ETI combines enumeration and CTC expression of four markers: estrogen receptor (ER), B-cell lymphoma 2 (BCL-2), Human Epidermal Growth Factor Receptor 2 (HER2), and Ki67. The CellSearch System and reagents were used to capture CTC and measure protein expression by immunofluorescent staining on CTC. RESULTS: The feasibility of determining CTC-ETI was initially established in vitro and then in a prospective single-institution pilot study in patients with MBC. CTC-ETI was successfully determined in 44 of 50 (88%) patients. Eighteen (41%), 9 (20%), and 17 (39%) patients had low, intermediate, and high CTC-ETI scores, respectively. Interobserver concordance of CTC-ETI determination was from 94% to 95% (Kappa statistic, 0.90-0.91). Inter- and cell-to-cell intrapatient heterogeneity of expression of each of the CTC markers was observed. CTC biomarker expression was discordant from both primary and metastatic tissues. CONCLUSIONS: CTC expression of ER, BCL-2, HER2, and Ki67 can be reproducibly measured with high analytical validity using the CellSearch System. The clinical implications of CTC-ETI, and of the heterogeneity of CTC biomarker expression, are being evaluated in an ongoing prospective trial.

Combining clinical assessment and the Risk of Ovarian Malignancy Algorithm for the prediction of ovarian cancer.

OBJECTIVES: ACOG guidelines for the evaluation of women with a pelvic mass employ a combination of physical exam, imaging, and CA125 to guide physicians in the triage of women to gynecologic oncologists. We studied the use of ROMA with clinical assessment for cancer risk assessment in women with a pelvic mass. METHODS: This was a prospective, multicenter trial evaluating women with a pelvic mass who had an initial clinical risk assessment (ICRA) performed by a generalist. ROMA scores were calculated and sensitivity, specificity, PPV and NPV were determined for ICRA and ICRA+ROMA. RESULTS: A total of 461 women were entered into the study. There were 375 benign tumors, 48 EOC, 18 LMP tumors and 20 non-ovarian malignancies. For detection of ovarian cancer alone, ICRA had a sensitivity of 85.4%, a specificity of 84.3%, and a NPV of 97.8%. Adding ROMA to ICRA produced a significant improvement of 8.4% in sensitivity, achieving a sensitivity of 93.8% with a specificity of 67.2% and a NPV of 98.8%. Examination of all malignancies (ovarian & non-ovarian) provided a sensitivity of 89.7% for ROMA+ICRA in comparison to 77.9% for ICRA alone, a significant increase in sensitivity of 11.8%. The NPV also significantly increased from 95.5% to 97.3%. Overall, ROMA detected 13 additional malignancies missed by ICRA. CONCLUSIONS: Adjunctive use of ROMA with clinical assessment improves the stratification of women with a pelvic mass into low and high risk groups for ovarian cancer. The combination is particularly effective in ruling out malignant disease.

HE4 (WFDC2) gene overexpression promotes ovarian tumor growth.

Selective overexpression of Human epididymal secretory protein E4 (HE4) points to a role in ovarian cancer tumorigenesis but little is known about the role the HE4 gene or the gene product plays. Here we show that elevated HE4 serum levels correlate with chemoresistance and decreased survival rates in EOC patients. HE4 overexpression promoted xenograft tumor growth and chemoresistance against cisplatin in an animal model resulting in reduced survival rates. HE4 displayed responses to tumor microenvironment constituents and presented increased expression as well as nuclear translocation upon EGF, VEGF and Insulin treatment and nucleolar localization with Insulin treatment. HE4 interacts with EGFR, IGF1R, and transcription factor HIF1α. Constructs of antisense phosphorothio-oligonucleotides targeting HE4 arrested tumor growth in nude mice. Collectively these findings implicate increased HE4 expression as a molecular factor in ovarian cancer tumorigenesis. Selective targeting directed towards the HE4 protein demonstrates therapeutic benefits for the treatment of ovarian cancer.

Monitoring apoptosis and Bcl-2 on circulating tumor cells in patients with metastatic breast cancer.

BACKGROUND: Enumeration of circulating tumor cells (CTC) from whole blood permits monitoring of patients with breast carcinoma. Analysis of apoptosis & Bcl-2 expression in CTC might add additional prognostic and predictive information. We estimated the degree of these markers in CTC from patients being treated for metastatic breast cancer. METHODS: Eighty-three evaluable patients initiating a new therapy for metastatic breast cancer were enrolled. Whole blood was collected at baseline, at one of three short term time windows (24, 48, or 72 h) after initiating treatment, and at first follow-up (3-5 weeks). CTC were isolated, enumerated, and expression of M30 and Bcl2 was determined using the CellSearch(®) System. RESULTS: At baseline, window, and 3-5 weeks post-treatment, 41/80 (51%), 40/80 (50%) and 21/75 (28%) patients had ≥5 CTC, respectively. At baseline, the proportion of CTC-apoptosis (M30) was inversely correlated with CTC number, and modestly inversely correlated with CTC-Bcl-2. As expected, higher CTC levels at baseline or first follow-up were associated with worse prognosis. Surprisingly, in patients with elevated CTC, higher levels of CTC-apoptosis were associated with worse prognosis, while higher CTC-Bcl-2 levels correlated with better outcomes. CONCLUSIONS: CTC apoptosis and expression of Bcl-2 can be analytically determined in patients with metastatic breast cancer and may have biological and clinical implications. Characterization of CTC for these and other markers could further increase the utility of CTC monitoring patients in clinical investigations of new anti-neoplastic agents.

Human epididymis protein 4 offers superior specificity in the differentiation of benign and malignant adnexal masses in premenopausal women.

OBJECTIVE: We sought to assess the ability of human epididymis protein 4 (HE4) and CA-125 to distinguish among benign, borderline, and malignant pelvic masses in premenopausal women. STUDY DESIGN: We conducted a subset analysis of data from a prospective clinical trial that enrolled women undergoing surgery for an adnexal mass. Diagnostic performance of CA-125 and HE4 for epithelial ovarian cancer (EOC) detection in premenopausal women was determined. RESULTS: Of 229 premenopausal patients, 195 (85%) had benign masses, 18 (8%) had EOC, and 16 (7%) had borderline ovarian tumor. The sensitivity of CA-125 and HE4 for EOC detection was 83.3% and 88.9%, respectively. The specificity of CA-125 and HE4 was 59.5% and 91.8%, respectively. A normal HE4 level ruled out invasive cancer in 98% of women with an elevated CA-125. CONCLUSION: HE4 offers superior specificity compared to CA-125 for the differentiation of benign and malignant adnexal masses in premenopausal women.

Evaluation of the diagnostic accuracy of the risk of ovarian malignancy algorithm in women with a pelvic mass.

OBJECTIVE: It is often difficult to distinguish a benign pelvic mass from a malignancy and tools to help referring physician are needed. The purpose of this study was to validate the Risk of Ovarian Malignancy Algorithm in women presenting with a pelvic mass. METHODS: This was a prospective, multicenter, blinded clinical trial that included women who presented to a gynecologist, a family practitioner, an internist, or a general surgeon with an adnexal mass. Serum HE4 and CA 125 were determined preoperatively. A Risk of Ovarian Malignancy Algorithm score was calculated and classified patients into high-risk and low-risk groups for having a malignancy. The sensitivity, specificity, negative predictive value, and positive predictive value of the Risk of Ovarian Malignancy Algorithm were estimated. RESULTS: A total of 472 patients were evaluated with 383 women diagnosed with benign disease and 89 women with a malignancy. The incidence of all cancers was 15% and 10% for ovarian cancer. In the postmenopausal group, a sensitivity of 92.3% and a specificity of 76.0% and for the premenopausal group the Risk of Ovarian Malignancy Algorithm had a sensitivity of 100% and specificity of 74.2% for detecting ovarian cancer. When considering all women together, the Risk of Ovarian Malignancy Algorithm had a sensitivity of 93.8%, a specificity of 74.9%, and a negative predictive value of 99.0%. CONCLUSION: The use of the serum biomarkers HE4 and CA 125 with the Risk of Ovarian Malignancy Algorithm has a high sensitivity for the prediction of ovarian cancer in women with a pelvic mass. These findings support the use of the Risk of Ovarian Malignancy Algorithm as a tool for the triage of women with an adnexal mass to gynecologic oncologists. LEVEL OF EVIDENCE: II.

Nuclear roundness variance predicts prostate cancer progression, metastasis, and death: A prospective evaluation with up to 25 years of follow-up after radical prostatectomy.

BACKGROUND: Nuclear structure is often altered in cancer due to spatial rearrangements of chromatin organization via activation of oncogenes and other chromatin remodeling genes. Therefore, we evaluated the prognostic value of nuclear roundness variance (NRV) for prostate cancer (PCa) progression, metastasis and PCa-specific death free survivals in a cohort of 116 men after radical prostatectomy (RP). METHOD: NRV was calculated for each case using the variance of the nuclear roundness from approximately 150 nuclei captured at a magnification of 2,440x for each case in 1992-1993. $${\rm Nuclear}\,{\rm roundness} = {{{\rm Radius}({\rm circumference})} \over {{\rm radius}({\rm area})}} = {R \over r} = {{P/2\pi } \over {\sqrt {A/\pi } }}$$ NRV data were merged with clinical, pathologic, and follow-up data for all patients in 2009. Cox proportional hazards regression and Kaplan-Meier plots were employed to analyze the data. RESULTS: Median follow-up time after RP for all patients was 19 years (range: 1-25 years, mean: 17 years), with approximately 92% (107/116), 71% (82/116), and 47% (55/116) patients having >or=10, 15, and 20 years of follow-up, respectively. NRV was the most significant parameter for prediction of all three outcomes and its concordance-index (C-Index) increased from progression (0.7080) to metastasis (0.7332) to PCa-specific death (0.8090) free survival predictions. Of note, NRV C-Index was significantly higher compared to Gleason Score C-Index for metastasis (0.7332 vs. 0.6046; P = 0.027) and PCa-specific death (0.8090 vs. 0.6336; P = 0.004) free survival predictions. However, the difference between NRV and Gleason Score C-Indexes was not statistically significant for progression free survival prediction (0.7080 vs. 0.6463; P = 0.106). CONCLUSION: NRV is valuable nuclear structural feature that exceeds Gleason score to predict an aggressive phenotype of PCa.

Comparison of a novel multiple marker assay vs the Risk of Malignancy Index for the prediction of epithelial ovarian cancer in patients with a pelvic mass.

OBJECTIVE: We sought to compare the Risk of Malignancy Index (RMI) to the Risk of Ovarian Malignancy Algorithm (ROMA) to predict epithelial ovarian cancer (EOC) in women with a pelvic mass. STUDY DESIGN: In all, 457 women with imaging results from ultrasound, computed tomography, magnetic resonance imaging, and serum HE4 and CA125 determined prior to surgery for pelvic mass were evaluable. RMI values were determined using CA125, imaging score, and menopausal status. ROMA values were determined using HE4, CA125, and menopausal status. RESULTS: At a set specificity of 75%, ROMA had a sensitivity of 94.3% and RMI had a sensitivity of 84.6% for distinguishing benign status from EOC (P = .0029). In patients with stage I and II disease, ROMA achieved a sensitivity of 85.3% compared with 64.7% for RMI (P < .0001). CONCLUSION: The dual marker algorithm utilizing HE4 and CA125 to calculate a ROMA value achieves a significantly higher sensitivity for identifying women with EOC than does RMI.

Significance of Circulating Tumor Cells Detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate Cancer.

The increasing number of treatment options for patients with metastatic carcinomas has created a concomitant need for new methods to monitor their use. Ideally, these modalities would be noninvasive, be independent of treatment, and provide quantitative real-time analysis of tumor activity in a variety of carcinomas. Assessment of circulating tumor cells (CTCs) shed into the blood during metastasis may satisfy this need. We developed the CellSearch System to enumerate CTC from 7.5 mL of venous blood. In this review we compare the outcomes from three prospective multicenter studies investigating the use of CTC to monitor patients undergoing treatment for metastatic breast (MBC), colorectal (MCRC), or prostate cancer (MPC) and review the CTC definition used in these studies. Evaluation of CTC at anytime during the course of disease allows assessment of patient prognosis and is predictive of overall survival.