A phase 1 'window-of-opportunity' trial testing evofosfamide (TH-302), a tumour-selective hypoxia-activated cytotoxic prodrug, with preoperative chemoradiotherapy in oesophageal adenocarcinoma patients.

BACKGROUND: Neo-adjuvant chemoradiotherapy followed by surgery is the standard treatment with curative intent for oesophageal cancer patients, with 5-year overall survival rates up to 50 %. However, patients' quality of life is severely compromised by oesophagectomy, and eventually many patients die due to metastatic disease. Most solid tumours, including oesophageal cancer, contain hypoxic regions that are more resistant to chemoradiotherapy. The hypoxia-activated prodrug evofosfamide works as a DNA-alkylating agent under these hypoxic conditions, which directly kills hypoxic cancer cells and potentially minimizes resistance to conventional therapy. This drug has shown promising results in several clinical studies when combined with chemotherapy. Therefore, in this phase I study we investigate the safety of evofosfamide added to the chemoradiotherapy treatment of oesophageal cancer. METHODS/DESIGN: A phase I, non-randomized, single-centre, open-label, 3 + 3 trial with repeated hypoxia PET imaging, will test the safety of evofosfamide in combination with neo-adjuvant chemoradiotherapy in potentially resectable oesophageal adenocarcinoma patients. Investigated dose levels range from 120 mg/m2 to 340 mg/m2. Evofosfamide will be administered one week before the start of chemoradiotherapy (CROSS-regimen) and repeated weekly up to a total of six doses. PET/CT acquisitions with hypoxia tracer (18)F-HX4 will be made before and after the first administration of evofosfamide, allowing early assessment of changes in hypoxia, accompanied with blood sampling to measure hypoxia blood biomarkers. Oesophagectomy will be performed according to standard clinical practice. Higher grade and uncommon non-haematological, haematological, and post-operative toxicities are the primary endpoints according to the CTCAEv4.0 and Clavien-Dindo classifications. Secondary endpoints are reduction in hypoxic fraction based on (18)F-HX4 imaging, pathological complete response, histopathological negative circumferential resection margin (R0) rate, local and distant recurrence rate, and progression free and overall survival. DISCUSSION: This is the first clinical trial testing evofosfamide in combination with chemoradiotherapy. The primary objective is to determine the dose limiting toxicity of this combined treatment and herewith to define the maximum tolerated dose and recommended phase 2 dose for future clinical studies. The addition of non-invasive repeated hypoxia imaging ('window-of-opportunity') enables us to identify the biologically effective dose. We believe this approach could also be used for other hypoxia targeted drugs. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02598687 .

Network Consumption and Storage Needs when Working in a Full-Time Routine Digital Environment in a Large Nonacademic Training Hospital.

OBJECTIVE: Digital pathology is indisputably connected with high demands on data traffic and storage. As a consequence, control of the logistic process and insight into the management of both traffic and storage is essential. METHOD: We monitored data traffic from scanners to server and server to workstation and registered storage needs for diagnostic images and additional projects. RESULTS: The results showed that data traffic inside the hospital network (1 Gbps) never exceeded 80 Mbps for scanner-to-server activity, and activity from the server to the workstation took at most 5 Mbps. Data storage per image increased from 300 MB to an average of 600 MB as a result of camera and software updates, and, due to the increased scanning speed, the scanning time was reduced with almost 8 h/day. Introduction of a storage policy of only 12 months for diagnostic images and rescanning if needed resulted in a manageable storage window of 45 TB for the period of 1 year. CONCLUSION: Using simple registration tools allowed the transition of digital pathology into a concise package that allows planning and control. Incorporating retrieval of such information from scanning and storage devices will reduce the fear of losing control by the management when introducing digital pathology in daily routine.

Expertise in clinical pathology: combining the visual and cognitive perspective.

Expertise studies in the medical domain often focus on either visual or cognitive aspects of expertise. As a result, characteristics of expert behaviour are often described as either cognitive or visual abilities. This study focuses on both aspects of expertise and analyses them along three overarching constructs: (1) encapsulations, (2) efficiency, and (3) hypothesis testing. This study was carried out among clinical pathologists performing an authentic task: diagnosing microscopic slides. Participants were 13 clinical pathologists (experts), 12 residents in pathology (intermediates), and 13 medical students (novices). They all diagnosed seven cases in a virtual microscope and gave post hoc explanations for their diagnoses. The collected data included eye movements, microscope navigation, and verbal protocols. Results showed that experts used lower magnifications and verbalized their findings as diagnoses. Also, their diagnostic paths were more efficient, including fewer microscope movements and shorter reasoning chains. Experts entered relevant areas later in their diagnostic process, and visited fewer of them. Intermediates used relatively high magnifications and based their diagnoses on specific abnormalities. Also, they took longer to reach their diagnosis and checked more relevant areas. Novices searched in detail, described findings by their appearances, and uttered long reasoning chains. These results indicate that overarching constructs can justly be identified: encapsulations and efficiency are apparent in both visual and cognitive aspects of expertise.

Expertise under the microscope: processing histopathological slides.

OBJECTIVES: Although the obvious goal of training in clinical pathology is to bring forth capable diagnosticians, developmental stages and their characteristics are unknown. This study therefore aims to find expertise-related differences in the processing of histopathological slides using a combination of eye tracking data and verbal data. METHODS: Participants in this study were 13 clinical pathologists (experts), 12 pathology residents (intermediates) and 13 medical students (novices). They diagnosed 10 microscopic images of colon tissue for 2 seconds. Eye movements, the given diagnoses, and the vocabulary used in post hoc verbal explanations were registered. Eye movements were analysed according to changes over trial time and the processing of diagnostically relevant areas. The content analysis of verbal data was based on a categorisation system developed from the literature. RESULTS: Although experts and intermediates showed equal levels of diagnostic accuracy, their visual and cognitive processing differed. Whereas experts relied on their first findings and checked the image further for other abnormalities, intermediates tended to double-check their first findings. In their explanations, experts focused on the typicality of the tissue, whereas intermediates mainly mentioned many specific pathologies. Novices looked less often at the relevant areas and were incomplete, incorrect and inconclusive in their explanations. Their diagnostic accuracy was correspondingly poor. CONCLUSIONS: This study indicates that in the case of intermediates and experts, different visual and cognitive strategies can result in equal levels of diagnostic accuracy. Lessons for training underline the relevance of the distinction between normal and abnormal tissue for novices, especially when the mental rotation of 2-D images is required. Intermediates need to be trained to see deviations in abnormalities. Feedback and an educational design that is specific to these developmental stages might improve training.

The evaluation of colon biopsies using virtual microscopy is reliable.

AIMS: Virtual microscopy offers major advantages for pathology practice, separating slide evaluation from slide production. The aim of this study was to investigate the reliability of using whole slide images as compared with routine glass slides for diagnostic purposes. METHODS AND RESULTS: Colon biopsies (n = 295) were assessed using both glass slides and whole slide images by four pathologists and two residents. Two pathologists scored the digital images of biopsies in a primary diagnostic setting. For each case, the consensus diagnosis was defined as the majority diagnosis on the study's glass slides. All diagnoses were grouped into seven main diagnostic categories, and further divided into subgroups. The overall concordance rates were 89.6% for whole slide images and 91.6% for light microscopy. The concordance rates of the subgroups 'adenoma' and 'adenocarcinoma' between whole slide images and conventional microscopy showed only small variability. The intraobserver (whole slide images versus glass slide) agreement, including subgroups, was substantial, with a mean κ-value of 0.78, and was higher than the interobserver agreement for glass slides (interobserver κ-value of 0.69). CONCLUSIONS: This study shows good diagnostic accuracy and reproducibility for virtual microscopy, indicating that this technology can reliably be used for pathological evaluation of colon biopsies in a primary clinical setting.

PET/CT-based metabolic tumour volume for response prediction of neoadjuvant chemoradiotherapy in oesophageal carcinoma.

PURPOSE: Neoadjuvant chemoradiotherapy is increasingly used in oesophageal cancer patients. In general, small tumours are associated with a survival benefit compared to large tumours. Little is known, however, about the relationship between initial tumour volume and response to chemoradiotherapy. Therefore, the aim of this study was to determine whether the pretherapy metabolic tumour volume (MTV) on diagnostic PET/CT in oesophageal cancer patients is correlated with response to chemoradiotherapy in the resection specimen. METHODS: A consecutive series of patients underwent diagnostic PET/CT scanning prior to chemoradiotherapy and oesophagectomy. MTVs were determined on PET/CT and an automated tumour contour was generated using specified standard uptake value thresholds. Response to chemoradiotherapy was determined in the resection specimen according to the scoring system developed by Mandard et al. Patients were divided into different groups according to response to chemoradiotherapy. RESULTS: Between January 2008 and May 2011 a total of 115 patients underwent an oesophagectomy. The MTV determined on diagnostic PET/CT scans was available in 79 patients. Of these 79 patients, 30 (38 %) showed no residual tumour cells at the location of the primary tumour. Three of these patients presented with residual tumour cells in the lymph nodes; 27 patients (34 %) had a complete pathological response. There was a trend towards a better response in patients with a smaller MTV (p = 0.084). CONCLUSION: This study demonstrated a trend towards a correlation between response to chemoradiotherapy in oesophageal cancer patients and smaller MTVs as determined on diagnostic PET/CT prior to neoadjuvant chemoradiotherapy. However, tumour volumes overlapped between groups, indicating the need for multifactorial parameters as predictors. In addition, a complete local tumour response may be accompanied by residual disease in the regional lymph nodes.

Circulating PSA-containing macrophages as a possible target for the detection of prostate cancer: a three-color/five-parameter flow cytometric study on peripheral blood samples.

A major problem of serum prostate-specific antigen (PSA) for predicting prostate cancer risk is diagnostic uncertainty. To detect circulating macrophages with phagocytized fragments (eg, PSA) of prostate tumor cells and determine if the number of circulating PSA-containing macrophages can help differentiate between benign and malignant prostate disease, we collected mononuclear cells from peripheral blood. After labeling the macrophages, phagocytized PSA was detected by incubating the cells with a phycoerythrin-conjugated PSA monoclonal antibody. Flow cytometric analysis was performed. A significant difference was observed in the mean+/-SD percentage of activated macrophages (CD14+/CD16+) between malignant (28.8%+/-13.0%) and benign conditions (17.3%+/-6.8%; P< .0001). A significant increase was detected in the percentage of PSA-containing macrophages in prostate cancer (17.7%+/-12.3%) vs benign disorders (3.6%+/-1.9%; P< .0001) and between localized (10.5%+/-3.4%) and metastasized prostate carcinoma (26.3%+/-14.3%; P= .0002). The new method for detecting circulating PSA-containing macrophages can be suitable for differentiating prostate cancer from benign conditions and, possibly, low-risk from more aggressive prostate cancer.

Why determine only the total prostate-specific antigen, if the free-to-total ratio contains the information?

BACKGROUND: Total prostate-specific antigen (tPSA) is the best available test for the detection of prostate cancer but it lacks specificity. The free-to-total ratio (F/T ratio) is used to increase specificity in the range of tPSA of 4-10 microg/L. MATERIALS AND METHODS: Four hundred and seven biopsy results and quantitative tPSA and F/T ratio data were combined. Using the histological determination, normal/hyperplasia versus malignant as a gold standard, receiver operating characteristic (ROC) curves as well as the areas under the curve (AUC) for tPSA and F/T ratio were determined. The differences between the two AUCs were considered for various tPSA ranges and specificities of F/T ratio and tPSA were calculated. RESULTS: In the total group, there was a gain of specificity of 11% (from 23% to 34%) when the sensitivity was 92% (using a cut-off >0.28 for the F/T ratio and a cut-off >4 microg/L for tPSA). When considering the group of patients for which the F/T ratio is currently used (4-10 microg/L), the gain of specificity was 27% (from 2% to 29%). This implicates that the number of unnecessary biopsies taken will be reduced by 27%. Moreover, the AUC of the F/T ratio was significantly higher at an even broader range of tPSA, i.e. up to 40 microg/L. CONCLUSIONS: This study demonstrates that the F/T ratio has better diagnostic performance than tPSA, not only in the grey zone of tPSA, but also outside the grey zone, i.e. up to 40 microg/L.

Conventional Microscopical versus Digital Whole-Slide Imaging-Based Diagnosis of Thin-Layer Cervical Specimens: A Validation Study.

BACKGROUND: Whole-slide imaging (WSI) has been implemented in many areas of pathology, but primary diagnostics of cytological specimens are lagging behind. One of the objectives of viewing scanned whole-slide images from histological or cytological specimens is remote exchange of knowledge and expertise of professionals to increase diagnostic accuracy. We compared the scoring results of our team obtained in double readings of two different data sets: conventional light microscopy (CLM) versus CLM and CLM versus WSI. We hypothesized that WSI is noninferior to CLM for primary diagnostics of thin-layer cervical slides. MATERIALS AND METHODS: First, we determined the concordance rate at different thresholds of the participating cytotechnicians by double reading with CLM of 500 thin-layer cervical slides (Cohort 1). Next, CLM was compared with WSI examination of another 505 thin-layer cervical slides (Cohort 2) scanned at ×20 in single focus plane. Finally, all major discordant cases of Cohort 1 were evaluated by an external expert in the field of gynecological cytology and of Cohort 2 in the weekly case meetings. RESULTS: The overall concordance rate of Cohort 1 (CLM vs. CLM) was 97.8% (95% confidence interval [CI]: 96.0%-98.7%) and of Cohort 2 was 95.3% (95% CI: 93.0%-96.9%). CONCLUSION: Concordance rates of WSI versus CLM were comparable with those of CLM versus CLM. We have made a step forward paving the road to implementation of WSI also in routine diagnostic cytology.

Early apoptotic responses in transgenic mouse mammary carcinoma for photodynamic therapy.

BACKGROUND: Male transgenic mice expressing the human RAS gene on an FVB strain background develop adenocarcinoma of the breast between 7 and 8 weeks of age. We have utilized this mammary tumour model to investigate apoptotic responses following photodynamic therapy (PDT) with a chlorin-based, water-soluble photosensitizer. METHODS: Detection of apoptosis was accomplished by use of the antibody M30 against a neo-epitope of caspase-cleaved cytokeratin 18 that becomes available at an early stage of the apoptotic cascade. Mice bearing multiple tumours were injected with the photosensitizer intraperitoneally, and following a drug-light interval of 96h, 40J/cm(2) of 652nm laser light was applied to one tumour per animal, while the other tumours were protected from light to serve as host controls. The M30 antibody was used for standard immunohistochemistry of tumour sections and flow cytometric detection of epitope expression coupled to cell cycle analysis in tumour cell populations retrieved from paraffin blocks. RESULTS: M30 staining was significantly increased within 2h following light treatment and persisted until 96h after treatment. Flow cytometric analysis for the S-phase fraction (SPF) of tumour cells post-PDT showed a substantial decrease in SPF at 2h post PDT, and recovery of SPF within 96h. CONCLUSIONS: Cytokeratin 18 cleavage seems to be both an early and ongoing event during the cellular response to PDT. Calculating the M30/SPF ratio at both 2h and 96h suggested distinct cellular dynamics at early and late time points, and we propose the M30/SPF ratio as a tumour dynamic index (TDI) to monitor events post PDT.

Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18.

Cytokeratins are released from carcinoma cells by unclear mechanisms and are commonly used serum tumor markers (TPA, TPS, and CYFRA 21-1). We here report that soluble cytokeratin-18 (CK18) is released from human carcinoma cells during cell death. During necrosis, the cytosolic pool of soluble CK18 was released, whereas apoptosis was associated with significant release of caspase-cleaved CK18 fragments. These results suggested that assessments of different forms of CK18 in patient sera could be used to examine cell death modes. Therefore, CK18 was measured in local venous blood collected during operation of patients with endometrial tumors. In most patient sera, caspase-cleaved fragments constituted a minor fraction of total CK18, suggesting that tumor apoptosis is not the main mechanism for generation of circulating CK18. Monitoring of different CK18 forms in peripheral blood during chemotherapy of prostate cancer patients showed individual differences in the patterns of release. Importantly, several examples were observed where the increase of apoptosis-specific caspase-cleaved CK18 fragments constituted only a minor fraction of the total increase. These results suggest that cell death of epithelially derived tumors can be assessed in patient serum and suggest that tumor apoptosis may not necessarily be the dominating death mode in many tumors in vivo.

Working toward consensus among professionals in the identification of classical cervical cytomorphological characteristics in whole slide images.

INTRODUCTION: Cervical cancer is one of the most common causes of death in women worldwide.([1]) The introduction of cervical cytology in screening programs is an effective way for early detection and treatment of cervical precancerous lesions. Conventional screening of cervical cytology slides is still considered the current "gold standard" for the assessment of proficiency in becoming a cytotechnician, but diagnosis using digital whole slide images (WSI) may offer many advantages. MATERIALS AND METHODS: In this study, we have used a selection of WSI from thin-layer specimens of the most common cervical infections and (pre) neoplastic lesions, and hypothesized that weekly WSI based case-meetings would help to obtain optimal acceptance of the new digital workflow in daily pathology practice. A questionnaire, before and after the test period was used to study the effect of our approach. RESULTS: The participants clearly had to go through a learning curve to get accustomed to viewing WSI. In the beginning, there was a little self-confidence in recognizing classical cervical cytomorphological features in the WSI, and there were complaints about the speed of viewing and insufficient Z-resolution for cell groups. Adjusting the Z-stack settings resulted in better three-dimensional information due to better focusing options. Weekly meetings appeared to be instrumental in the implementation process, as participants had to select and present WSI from thematic cases themselves, and thereby, got used to viewing WSI. Some WSI were replaced by better ones until a final set of 45 representatives WSI remained. Eventually, the consensus was reached among all participants that cytomorphological features in WSI from thin-layers cervical specimens could comparably be appreciated in WSI as by conventional microscopy. The selection of 45 WSI was now used to create a digital WSI based reference atlas to support further studies. CONCLUSION: We have obtained consensus between professionals that WSI from cervical cytology can be used to identify cytomorphological features, necessary for diagnosis. In addition, we observed that active participation of professionals had a positive effect on the overall acceptance of WSI and was important in the change management.

Her2/neu analysis in formalin-fixed, paraffin-embedded breast carcinomas: comparison of immunohistochemistry and multiparameter DNA flow cytometry.

PURPOSE: The determination of Her2/neu over-expression has become necessary for the selection of breast cancer patients for Herceptin therapy. Immunohistochemistry in combination with FISH is currently the most used method for this detection. The objective of our study was to compare the use of multiparameter DNA flow cytometry (MP-FCM) for this assessment with immunohistochemistry (IHC). For this purpose the number of positive cells as well as the intensity of the immunofluorescence signal as determined by MP-FCM were compared with IHC scores. MATERIALS AND METHODS: Single cell suspensions from 110 breast cancers were analysed on a DAKO Galaxy flow cytometer after simultaneous (immuno)labeling for cytokeratin, Her2/neu and DNA (propidium iodide). Next to ploidy and proliferative activity (S-phase fraction), the fraction of Her2/neu-positive epithelial cells was determined. In 25 cases the intensity of the immunofluorescence signal was also determined by MP-FCM in the total cytokeratin+ fraction as well as the cytokeratin+/Her2/neu+fraction. Immunohistochemistry for Her2/neu was done on paraffin sections of all cases using the same polyclonal Her2/neu antibody in a SABC-peroxidase procedure after heat-induced antigen retrieval. RESULTS: Her2/neu overexpression as determined by IHC showed a score of 0 in 30%, 1 in 25%, 2 in 26% and 3 in 19% of the cases. There was a good correlation when comparing the fraction of Her2/neu-positive epithelial cells as determined by MP-FCM and semiquantitative IHC scores (r = 0.89, p < 0.0001). No correlation was found between the IHC-Scores and the intensity of the Her2/neu- staining in the total epithelial compartment as measured by MP-FCM. However, when restricting the intensity-determination to the Her2/neu-positive epithelial cells, a significant correlation was found between MP-FCM and IHC scores (r = 0.72, p < 0.0001). CONCLUSION: The results of the current study show that MP-FCM can be a good alternative for the objective quantification of Her2/neu.

Reproducibility studies for experimental epitope detection in macrophages (EDIM).

INTRODUCTION: We have recently described epitope detection in macrophages (EDIM) by flow cytometry. This is a promising tool for the diagnosis and follow-up of malignancies. However, biological and technical validation is warranted before clinical applicability can be explored. METHODS: The pre-analytic and analytic phases were investigated. Five different aspects were assessed: blood sample stability, intra-individual variability in healthy persons, intra-assay variation, inter-assay variation and assay transferability. The post-analytic phase was already partly standardized and described in an earlier study. RESULTS: The outcomes in the pre-analytic phase showed that samples are stable for 24h after venipuncture. Biological variation over time was similar to that of serum tumor marker assays; each patient has a baseline value. Intra-assay variation showed good reproducibility, while inter-assay variation showed reproducibility similar to that of to established serum tumor marker assays. Furthermore, the assay showed excellent transferability between analyzers. CONCLUSION: Under optimal analytic conditions the EDIM method is technically stable, reproducible and transferable. Biological variation over time needs further assessment in future work.

A travel report of the implementation of virtual whole slide images in routine surgical pathology.

Virtual microscopy is the terminology used to indicate the use of digitized images of whole slides for inspection of cells and tissue sections on computer screens as an add-on or replacement for conventional microscopy using bright field or other types of illumination in combination with a wide variety of microscope brands. Although technically there is no longer a limit in the size and colour composition of the images, the logistics of embedding virtual microscopy in daily routine of a diagnostic process are still a relatively open area where new pitfalls and opportunities can be found. In this article, we described various aspects in the process. None of them had been planned in advance, but mostly originated from observations done during the different steps towards implementation of virtual microscopy in daily routine, for example, the choice between the different scanner types and their (dis)advantages, issues on storing and retrieval and at last, the effect of digitalization on the diagnostic process. This approach resulted in a manuscript that in a way has more the appearance of a story than of a scientific study with strict protocols, with a clear cut question in advance, a research plan and expected outcome. Depending on the purpose of the virtual slides in a given situation, different solutions must be found locally.

Automated colorectal cancer diagnosis for whole-slice histopathology.

In this study, we propose a computational diagnosis system for detecting the colorectal cancer from histopathological slices. The computational analysis was usually performed on patch level where only a small part of the slice is covered. However, slice-based classification is more realistic for histopathological diagnosis. The developed method combines both textural and structural features from patch images and proposes a two level classification scheme. In the first level, the patches in slices are classified into possible classes (adenomatous, inflamed, cancer and normal) and the distribution of the patches into these classes is considered as the information representing the slices. Then the slices are classified using a logistic linear classifier. In patch level, we obtain the correct classification accuracies of 94.36% and 96.34% for the cancer and normal classes, respectively. However, in slice level, the accuracies of the 79.17% and 92.68% are achieved for cancer and normal classes, respectively.

Whole slide images as a platform for initial diagnostics in histopathology in a medium-sized routine laboratory.

INTRODUCTION: Whole slide imaging is the process of digitizing glass slides and the creation of Whole Slide Images (WSI), which enable the examination of pathology samples on a computer screen in a manner comparable to light microscopy. WSI have been used for different applications in pathology but their use for primary diagnostics is still limited. Implementing WSI for primary diagnostics would be a turning point necessitating extensive validation to unravel pitfalls and difficulties that could be encountered within the routine workflow. This article is aimed to describe the gradual integration of WSI into routine pathology diagnostics in a medium-sized routine pathology laboratory. METHODS: This project was started with optimizing the digital work environment including the setting up of validation studies, scanning preferences, storing WSI and the implemented adjustments to the workflow for the laboratory and the pathologist. Afterwards scanning glass slides was initiated in the department of pathology at the Atrium Medical Center, Heerlen, The Netherlands, for performing primary diagnostics of breast biopsies. Later this was extended to other specimen types including resections. RESULTS: The validation studies yielded a high concordance rate between WSI and conventional diagnoses. Routine primary WSI based diagnosis was possible in 82.1% of cases. Failure of digital diagnosis was mainly related to poor image quality and logistic problems. CONCLUSION: The quality of the currently produced WSI is sufficient for primary diagnostics in 82.1% of the cases. Improving image quality, adequate retrieval and controlling scanning error will definitely encourage the wide adaptation in routine diagnostics.

Data-driven compensation for flow cytometry of solid tissues.

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.

FDG-PET provides the best correlation with the tumor specimen compared to MRI and CT in rectal cancer.

PURPOSE: To compare CT-, MR- and PET-CT based tumor length measurements in rectal cancer with pathology. PATIENTS AND METHODS: Twenty-six rectal cancer patients underwent both MR and PET-CT imaging followed by short-course radiotherapy (RT 5×5 Gy) and surgery within 3 days after RT. Tumor length was measured manually and independently by 2 observers on CT, MR and PET. PET-based tumor length measurements were also generated automatically using the signal-to-background-ratio (SBR) method. All measurements were correlated with the tumor length on the pathological specimen. RESULTS: CT-based measurements did not show a valuable correlation with pathology. MR-based measurements correlated only weakly, but still significantly (Pearson correlation=0.55 resp. 0.57; p<0.001). Manual PET measurements reached a good correlation with pathology, but less strong (Pearson correlation 0.72 and 0.76 for the two different observers) than automatic PET-CT based measurements, which provided the best correlation with pathology (Pearson correlation of 0.91 (p<0.001)). Bland-Altman analysis demonstrated in general an overestimation of the tumor diameter using manual measurements, while the agreement of automatic contours and pathology was within acceptable ranges. A direct comparison of the different modalities revealed a significant better precision for PET-based auto-contours as compared to all other measurements. CONCLUSION: Automatically generated PET-CT based contours show the best correlation with the surgical specimen and thus provide a useful and powerful tool to accurately determine the largest tumor dimension in rectal cancer. This could be used as a quick and reliable tool for target delineation in radiotherapy. However, a 3D volume analysis is needed to confirm these results.

CEA in activated macrophages. New diagnostic possibilities for tumor markers in early colorectal cancer.

BACKGROUND: Serum tumor markers show low sensitivity, making them unsuitable for early detection of cancer. Activated macrophages (AM) from peripheral blood can accumulate tumor marker substances and facilitate early detection in prostate cancer. Here it was investigated whether carcinoembryonic antigen (CEA)-containing macrophages (CEACM) can be used to detect colorectal cancer (CRC) at earlier stages than can serum CEA. PATIENTS AND METHODS: Peripheral blood was collected from patients with CRC (n=48), inflammatory colorectal disease (n=5) and from healthy controls (n=18). After separating and labeling AM with CD14-APC/CD16-FITC, AM were intracellularly labeled with anti-CEA antibody and flow cytometrically analyzed. Serum CEA and C-reactive protein (CRP) were measured. RESULTS: The fraction-size of CEACM discriminated between controls and CRC patients, irrespective of AJCC stage (AJCC stage I-IV, p< or =0.0001). Serum CEA values were significantly elevated in AJCC stage II, III and IV (p=0.02, 0.006 and <0.0001, respectively). Combining CEACM with CRP levels separated CRC from inflammatory colorectal disease. CONCLUSION: CEACM combined with CRP appears to have diagnostic potential in early CRC.