Molecular Profiling of Circulating Tumour Cells and Circulating Tumour DNA: Complementary Insights from a Single Blood Sample Utilising the Parsortix® System.

The study of molecular drivers of cancer is an area of rapid growth and has led to the development of targeted treatments, significantly improving patient outcomes in many cancer types. The identification of actionable mutations informing targeted treatment strategies are now considered essential to the management of cancer. Traditionally, this information has been obtained through biomarker assessment of a tissue biopsy which is costly and can be associated with clinical complications and adverse events. In the last decade, blood-based liquid biopsy has emerged as a minimally invasive, fast, and cost-effective alternative, which is better suited to the requirement for longitudinal monitoring. Liquid biopsies allow for the concurrent study of multiple analytes, such as circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA), from a single blood sample. Although ctDNA assays are commercially more advanced, there is an increasing awareness of the clinical significance of the transcriptome and proteome which can be analysed using CTCs. Herein, we review the literature in which the microfluidic, label-free Parsortix® system is utilised for CTC capture, harvest and analysis, alongside the analysis of ctDNA from a single blood sample. This detailed summary of the literature demonstrates how these two analytes can provide complementary disease information.

Serum liquid biopsy for brain tumours: a scoping analysis of practicable approaches in low- and middle-income countries.

Approaches to brain tumour diagnosis and detecting recurrence after treatment are costly and significantly invasive. Developing peripheral-sample liquid biopsy tools is the key to enhancing our ability to prognosticate brain tumour subtypes and molecular heterogeneity. The present scoping review was designed to discuss current updates in liquid biopsy tools for diagnosis and guiding clinical management of brain tumours; we evaluated the literature within the context of low-and-middle-income country challenges. Circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), cell-free DNA (cfDNA), extracellular vesicle-associated biomarkers, protein biomarkers, microRNAs, and serum metabolites are discussed with the collation of current data supporting their utility in liquid biopsy. Further challenges to implanting liquid biopsy tools at a systematic level are highlighted.

Heterogeneous circulating tumor cells correlate with responses to neoadjuvant chemotherapy and prognosis in patients with locally advanced breast cancer.

Neoadjuvant chemotherapy (NCT) is the standard treatment for patients with locally advanced breast cancer (LABC). The predictive value of heterogeneous circulating tumor cells (CTCs) in NCT response has not been determined. All patients were staged as LABC, and blood samples were collected at the time of biopsy, and after the first and eighth NCT courses. Patients were divided into High responders (High-R) and Low responders (Low-R) according to Miller-Payne system and changes in Ki-67 levels after NCT treatment. A novel SE-i·FISH strategy was applied to detect CTCs. Heterogeneities were successfully analyzed in patients undergoing NCT. Total CTCs increased continuously and were higher in Low-R group, while in High-R group, CTCs increased slightly during NCT before returning to baseline levels. Triploid and tetraploid chromosome 8 increased in Low-R but not High-R group. The number of small CTCs in Low-R group increased significantly until the last sample, however, remained constant in High-R group. The patients with more CTCs had shorter PFS and OS than those with less CTCs after the eighth course of NCT. Total CTCs following NCT could predict patients' responses. More detailed characterizations of CTC blood profiles may improve predictive capacity and treatments of LABC.

Different Patterns of Vascularization in Preinvasive States and at the Initial Stages of Invasive Growth of the Neoplasms.

Tissue samples obtained during surgery from 90 patients with malignant neoplasms of various localizations were studied (the presence of precancerous dysplastic alterations and their transition to invasive cancer in the slides were obligatory condition). In addition to traditional histological methods, immunohistochemical reactions for detection of HIF-1α, GLUT1, CAIX, and CD31 were performed. At the precancerous stage including cancer in situ, progressive signs of reduced blood vessel density and hypoxia were observed. At the earliest stages of invasion, hypoxia was compensated by abundant vascularization of the stroma, which was confirmed by disappearance of hypoxia markers in tumor cells and their persistence in the deep layers of the tumor far from blood vessels. At the same time, the ischemic phenotype was preserved in tumor cells even in abundantly vascularized stroma, which can attest to deep metabolic changes in some tumor cells similar to the Warburg effect. Thus, the initial stages of carcinogenesis are associated with reduction of the vascular network up to the complete absence of blood vessels in the cancer in situ. After migration to the vascularized subepithelial stroma, e.g., having started invasion that compensated for hypoxia, the tumor cells no longer expressed markers of hypoxia, except the cells located far from blood vessels. In parallel, neoplastic cell clones that presumably have changed their phenotype and transformed their metabolism similar to the Warburg effect were detected. In the deep layers of the tumors, these cells coexist in different proportions. Analysis of the content of these cells, their alternation, and mutual transformation will be very valuable for estimating the sensitivity of tumor cells to therapeutic measures.

Circulating tumor cells may serve as a supplement to RECIST in neoadjuvant chemotherapy of patients with locally advanced breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) have been shown to be associated with the response to neoadjuvant chemotherapy (NCT) and the prognosis of locally advanced breast cancer (LABC) patients. Our study aimed to investigate whether the change of CTC status during NCT could serve as a supplement to the Response Evaluation Criteria in Solid Tumors (RECIST) in the treatment and evaluation of LABC patients. METHODS: 6 ml of blood samples were collected before NCT, after the first cycle of NCT and after the completion of NCT, respectively. According to the change of CTC number during NCT, the patients were divided into "CTC low-response (low-R)" group and "CTC high-response (high-R)" group. Survival data of each group of patients were obtained through long-term follow-up. RESULTS: A total of 35 patients diagnosed with LABC were enrolled. The median follow-up for distant metastasis was 27 months (range 7-36 months). There was no significant difference in distant metastasis-free survival (DMFS) between PR/CR group and PD/SD group (P = 0.0914), while CTC low-R group had a worse DMFS than CTC high-R group (P = 0.0199). In PR/CR subgroup, patients with CTC low-R showed a lower DMFS compared with those with CTC high-R (P = 0.0159). However, in PD/SD subgroup, there was no significant difference in DMFS between CTC low-R and CTC high-R group (P = 0.7521). In terms of assessing response to NCT, CTC change or RECIST classification alone had an AUC of 0.533 (95% CI 0.277-0.790) and 0.700 (95% CI 0.611-0.789), respectively. When combining the two, the AUC slightly increased to 0.713 (95% CI 0.532-0.895). CONCLUSION: The change of CTC number during NCT has a potential to serve as a supplement to RECIST in the assessment of NCT efficacy and the prognosis of LABC patients.

Folate receptor-positive circulating tumor cells in the preoperative diagnosis of indeterminate pulmonary nodules.

BACKGROUND: The use of FR + CTC to distinguish lung cancer from benign lung disease has been well studied. However, the effective method to differentiate precursor glandular lesions from benign/malignant pulmonary diseases is rare. METHODS: 380 patients with indeterminate pulmonary nodules were prospectively recruited. Peripheral blood samples were collected from all participants before surgery for analyzing FR + CTC levels. The performance of FR + CTC to identify lung precursor lesions were analyzed by receiver operating characteristic (ROC) curve. RESULTS: FR + CTC can effectively differentiate precursor from benign pulmonary diseases in all included patients (cutoff: 9.22 FU/3 ml, AUC = 0.807, (p < 0.0001, sensitivity: 69.17%, specificity: 82.46%) and patients with single pulmonary lesion (cutoff: 9.03 FU/3 ml, AUC = 0.842, p = 0.0001, sensitivity: 75.20%, specificity: 83.00%). However, FR + CTC cannot differentiate precursor from benign pulmonary diseases in multiple lesions patients (p = 0.110). FR + CTC neither differentiate precursor from malignant pulmonary lesions in all included patients (p = 0.715), single nor multiple lesions patients (p = 0.867, p = 0.692, respectively). Total number of pulmonary nodules, MTD, location (lower vs upper) were independent risk factors for malignancy (AOR, 95% CI: 3.104 (1.525, 6.316), 3.148 (1.722, 5.754), 2.098 (1.132, 3.888), respectively. CONCLUSION: Preoperative FR + CTC can be identified in precursor glandular lesions and utilized to differentiate from benign pulmonary diseases. Total number of pulmonary nodules, MTD, location (lower vs upper) were independent risk factors for malignancy.

Analysis of circulating cell-free DNA after endoscopic ultrasound-guided fine needle aspiration in pancreatic ductal adenocarcinoma.

BACKGROUND/OBJECTIVES: Recently, increase in cell-free DNA (cfDNA) concentration or newly detected KRAS mutation after endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) biopsy were reported to be related to the occurrence of new distant metastasis. In this study, we investigated whether cfDNA concentration increased with the release of tumor components into the blood after EUS-FNA and whether its increase was related to prognosis. METHODS: Sixty-eight patients underwent EUS-FNA and were pathologically confirmed as having pancreatic ductal adenocarcinoma (PDAC). We measured plasma cfDNA concentration and the copy number of KRAS mutation in 68 patients and circulating tumor cells in 8 before and after EUS-FNA. RESULTS: The average cfDNA concentration after EUS-FNA (672.5 ± 919.6 ng/mL) was significantly higher than that before EUS-FNA (527.7 ± 827.3 ng/mL) (P < 0.001). KRAS mutation in plasma was detected in 8 patients (11.8%), however a significant increase in cfDNA concentration after EUS-FNA was not related to the change in KRAS-mutant copy number. Minimal increase in circulating tumor cells was observed in 3 of 8 patients. New distant metastasis was observed within 286 days to initial metastasis detection in 6 of 12 patients with ≥2-fold increase in cfDNA concentration and 26 of 56 patients with <2-fold increase within 185 days. In 32 patients who underwent surgery, ≥2-fold increase in cfDNA did not affect early recurrence. CONCLUSIONS: The increase in cfDNA concentration after EUS-FNA was not caused by tumor cell components released into blood vessels. Hence, the risk of seeding via the blood stream after EUS-FNA may need not be considered.

CIRCULATING TUMOR CELLS MAY KAM WELL BE A PROGNOSTIC MARKER IN THE CLINICAL MANAGEMENT OF HEAD AND NECK SQUAMOUS CELL CARCINOMA PATIENTS.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: Xun Y, Cao Q, Zhang J, Guan B, Wang M. Clinicopathological and prognostic significance of circulating tumor cells in head and neck squamous cell carcinoma: A systematic review and meta-analysis. Oral Oncol. 2020;104:104,638. doi:10.1016/j.oraloncology.2020.104638 SOURCE OF FUNDING: Information not available TYPE OF STUDY/DESIGN: Systematic review with meta-analysis of data.

Association between Postoperative Detection of Circulating Tumor Cells and Recurrence in Patients with Prostate Cancer.

PURPOSE: The clinical implications of postoperative detection of circulating tumor cells in prostate cancer are largely unknown. We investigated the association between postoperative circulating tumor cell detection after radical prostatectomy and disease recurrence in prospectively enrolled patients with prostate cancer. MATERIALS AND METHODS: A total of 203 patients with an undetectable prostate specific antigen who had undergone radical prostatectomy for prostate cancer were prospectively enrolled. Circulating tumor cell sampling was performed at a median of 4.5 months after surgery. The primary end point was biochemical recurrence-free survival. Detection of circulating tumor cells in the blood of patients was performed using a novel approach with a replication-competent adenovirus controlled by prostate specific antigen/prostate specific membrane antigen transcription regulatory elements (Ad5/35E1aPSESE4). RESULTS: Circulating tumor cells were detected in 73 (36.0%) patients with undetectable prostate specific antigen concentrations after surgery. The 3-year biochemical recurrence-free survival rate from the time of surgery was significantly higher in circulating tumor cell-negative than in circulating tumor cell-positive cases (81.6% vs 48.9%, log rank p <0.001). Multivariable analysis showed that postoperative circulating tumor cell detection was independently associated with an increased risk of biochemical recurrence (HR 5.42, 95% CI 3.24-9.06, p <0.001). C-index was increased in combinations of multivariable model and postoperative circulating tumor cell detection compared with the multivariable model alone. CONCLUSIONS: Circulating tumor cells in the blood were frequently detected in patients with undetectable prostate specific antigen levels after radical prostatectomy for localized prostate cancer. Furthermore, circulating tumor cell detection was associated with an increased risk of biochemical recurrence, suggesting that circulating tumor cell detection precedes prostate specific antigen rise after surgery in cases of prostate cancer recurrence. Large-scale validation is needed in the future.

Noninvasive Detection of Clinically Significant Prostate Cancer Using Circulating Tumor Cells.

PURPOSE: Prostate specific antigen testing results in unnecessary biopsy and over diagnosis with consequent overtreatment. Tissue biopsy is an invasive procedure associated with significant morbidity. More accurate noninvasive or minimally invasive diagnostic approaches should be developed to avoid unnecessary prostate biopsy and over diagnosis. We investigated the potential of using circulating tumor cell analysis in cancer diagnosis, particularly to predict clinically significant prostate cancer in prebiopsy cases. MATERIALS AND METHODS: We enrolled 155 treatment naïve patients with prostate cancer and 98 before biopsy for circulating tumor cell enumeration. RNA was extracted from circulating tumor cells of 184 patients for gene expression analysis. The Kruskal-Wallis and Spearman rank tests, multivariate logistic regression and the random forest method were applied to assess the association of circulating tumor cells with aggressive prostate cancer. RESULTS: Of patients with localized prostate cancer 54% were scored as having positive circulating tumor cells, which was associated with a higher Gleason score (p=0.0003), risk group (p <0.0001) and clinically significant prostate cancer (p <0.0001). In the prebiopsy group a positive circulating tumor cell score combined with prostate specific antigen predicted clinically significant prostate cancer (AUC 0.869). A 12-gene panel prognostic for clinically significant prostate cancer was also identified. When combining the prostate specific antigen level, the circulating tumor cell score and the 12-gene panel, the AUC of clinically significant prostate cancer prediction was 0.927. Adding those data to cases with available multiparametric magnetic resonance imaging data significantly increased prediction accuracy (AUC 0.936 vs 0.629). CONCLUSIONS: Circulating tumor cell analysis has the potential to significantly improve patient stratification by prostate specific antigen and/or multiparametric magnetic resonance imaging for biopsy and treatment.

Identification and Characterization of Circulating Tumor Cells in Men Who have Undergone Prostatectomy for Clinically Localized, High Risk Prostate Cancer.

PURPOSE: Approximately 15% of men with newly diagnosed prostate cancer have high risk features which increase the risk of recurrence and metastasis. Better predictive biomarkers could allow for earlier detection of biochemical recurrence and change surveillance and adjuvant treatment paradigms. Circulating tumor cells are thought to represent the earliest form of metastases. However, their role as biomarkers in men with high risk, localized prostate cancer is not well defined. MATERIALS AND METHODS: Two to 5 months after prostatectomy we obtained blood samples from 37 patients with high risk, localized prostate cancer, defined as stage T3a or higher, Gleason score 8 or greater, or prostate specific antigen 20 ng/ml or greater. Circulating tumor cells were enumerated using a commercial platform. Matched tumor and single circulating tumor cell sequencing was performed. RESULTS: Circulating tumor cells were detected in 30 of 37 samples (81.1%) with a median of 2.4 circulating tumor cells per ml (range 0 to 22.9). Patients with detectable circulating tumor cells showed a trend toward shorter recurrence time (p=0.12). All patients with biochemical recurrence had detectable circulating tumor cells. Androgen receptor over expression was detected in 7 of 37 patients (18.9%). Patients with biochemical recurrence had more circulating tumor cell copy number aberrations (p=0.027). Matched tumor tissue and single circulating tumor cell sequencing revealed heterogeneity. CONCLUSIONS: We noted a high incidence of circulating tumor cell detection after radical prostatectomy and shorter time to biochemical recurrence in men with a higher circulating tumor cell burden and more circulating tumor cell copy number aberrations. Genomic alterations consistent with established copy number aberrations in prostate cancer were detectable in circulating tumor cells but often discordant with cells analyzed in bulk from primary lesions. With further testing in appropriately powered cohorts early circulating tumor cell detection could be an informative biomarker to assist with adjuvant treatment decisions.

Less micrometastatic risk related to circulating tumor cells after endoscopic breast cancer surgery compared to open surgery.

BACKGROUND: Increase of circulating tumor cells (CTCs) has been found after surgery for various carcinomas but not confirmed for breast cancer, and whether endoscopic surgery confers identical effect to CTCs as open surgery did is not clear. The present study aimed to investigate whether CTCs increase after surgery and whether there is a difference between open surgery and endoscopic surgery. METHODS: Pre- and postoperative peripheral blood (5 mL) obtained from 110 female patients with operable breast cancer (53 underwent endoscopic surgery, 57 underwent open radical mastectomy). Quantitative real-time reverse transcription-PCR was done to detect cytokeratin 19 mRNA-positive CTC. CTC detection rate, cell number and the increase after surgery (named micrometastasis) were compared between the two groups. RESULTS: In the open group, CTC positive rate before and after surgery were 22.81 and 33.33%; median CTC number before and after surgery were 0.21 and 0.43 and 17 patients (29.82%) had increased micrometastatic risk. In the endoscopic group, CTC positive rate before and after surgery were 24.53 and 28.30%; median CTC number before and after surgery were 0.27 and 0.36, and 8 patients (15.09%) had increased micrometastatic risk. There was a suggestive higher postoperative CTC detection rate and CTC number and a significant increased postoperation micrometastatic risk was observed in the open group compared to the endoscopic group (OR = 3.19, 95%CI: 1.05-9.65) after adjustment for clinicopathologic characteristics. DISCUSSION: CTC tends to increase in breast cancer patients after surgery, and the micrometastatic risk was higher for open surgery compared to endoscopic surgery. TRIAL REGISTRATION: This study was prospectively registered at Chinese Clinical Trial Register (ChiCTR-OCH-10000859, 24 April 2010).

[Liquid biopsy in colorectal cancer : An overview of ctDNA analysis in tumour diagnostics]. / Liquid Biopsy im kolorektalen Karzinom : Ein Überblick zur ctDNA-Analyse in der Tumordiagnostik.

In current routine diagnostics, the gold standard to determine the genomic profile of colorectal cancers (CRCs) is using biopsy or surgically resected tissues. However, such a tissue sample cannot represent the entire tumour heterogeneity, because it only shows a local and temporal snapshot. As a complement to tumour tissue genotyping, liquid biopsies enable minimally invasive detection of all potential tumour-specific mutations and their dynamic changes for molecular profiling. Furthermore, they can be repeated in certain intervals for monitoring response to treatment, occurrence of drug resistance and detection of relapse. This review focusses on analyzing circulating cell-free tumour DNA (ctDNA), which is mostly released from apoptotic or necrotic tumour cells into the bloodstream or by active secretion of circulating tumour cells (CTCs). Nevertheless, there are some challenges in analyzing ctDNA. First, ctDNA represents only a small fraction of total circulating DNA, because there is an enormous wild-type background of cell-free DNA (cfDNA) released by healthy cells. Second, ctDNA is highly fragmented and the amount of ctDNA in the blood is very low. In this review, we discuss the potential, fields of application as well as challenges and limitations of liquid biopsy approaches. In more detail, we discuss the possibility of using liquid biopsies as a future application for molecular characterization of CRCs, particularly for monitoring CRC patients during anti-EGFR therapy to detect resistance mutations (e.g. KRAS mutations) or further therapy-relevant mutations. In addition, we investigate whether blood-based molecular profiling is a reliable addition to routine diagnostic approaches of tissue-based molecular characterization.

[Clinical significance of recombinant virus-specific detection for viable circulating tumor cells in lung cancer patients].

Objective: To investigate the clinical significance of recombinant virus-specific detection for detecting viable circulating tumor cell (CTC) in lung cancer patients. Methods: From June 2017 to August 2018, 63 lung cancer patients diagnosed by imaging and pathology at the Department of Respiratory and Critical Care Medicine, Beijing Anzhen Hospital were recruited for this study, including 38 cases of adenocarcinoma, 13 squamous cell carcinoma, and 12 small cell carcinoma. The patients consisted of 36 males and 27 females, with an average age of (65±11) years. In addition,21 patients with benign lung diseases were recruited for benign lung disease group, including 11 males and 10 females with an average age of (57±13) years. Another 18 healthy subjects were recruited for the healthy control group, including 10 males and 8 females, with an average age of (32±10) years. The peripheral blood viable CTCs were detected by a CTC in vitro detection kit containing recombinant virus. Fisher's exact test was used to compare the differences between the groups of viable CTC positive rates. Results: Cases with positive viable CTC in the lung cancer group, the benign lung disease group and the healthy control group were 56,4 and 0, respectively. The sensitivity and specificity of this CTC detection system were 89% (56/63) and 90% (35/39) , respectively. The positive rate of CTC detection for lung cancer patients with adenocarcinoma, aquamous cell carcinoma, and small cell carcinoma, were 84%(32/38), 92% (12/13) and 100% (12/12), respectively. The positive cases from adenocarcinoma and squamous cell carcinoma patients with different stages were 2 (n=2) in stageⅠ,6 (n=8) in stage Ⅱ,7(n=8) in stage Ⅲ and 29(n=33) in stage Ⅳ. There were no significant differences in the positive rates of CTC among different histological types or different stages (χ(2)=1.881, 3.419; P>0.05) . Conclusion: This CTC detection system was found to be a simple and non-invasive method for auxiliary diagnosis of lung cancer, with high sensitivity and specificity.

[Circulating tumor cells in pancreatic cancer patients: progression in the detection methods and clinical application].

Circulating tumor cells (CTC) disseminate from primary tumors by undergoing epithelial mesenchymal transition that allow their entry into the circulation to drive metastatic formation in pancreatic cancer patients.Technological advances in detection and characterization of CTC are conducive to the early diagnosis, differential diagnosis, monitoring disease progression and predicating the probability of canceration or the chemotherapeutic efficacy. Nowadays, detection methods of CTC can be based on immunomagnetic beads technique, cell filtration or microfluidic chips technology, but there are great differences in the sample throughput, CTC recovery rate, purity, and CTC viability among them.Owing to the dilemma in detection methods, the intrinsic relevance between the biological characteristics of CTC and clinical manifestations is still not exactly elucidated. By the improved methodology, next generation sequencing technology and exploring the technique for culturing CTC in vitro and establishing xenotransplanted tumor model in nude mice, more and more biological information will be revealed, and finally, individualized treatment is achieved.

Prostate Cancer Disseminated Tumor Cells are Rarely Detected in the Bone Marrow of Patients with Localized Disease Undergoing Radical Prostatectomy across Multiple Rare Cell Detection Platforms.

PURPOSE: Prostate circulating tumor cells escape into peripheral blood and enter bone marrow as disseminated tumor cells, representing an early step before conventionally detectable metastasis. It is unclear how frequently this occurs in localized disease and existing detection methods rely on epithelial markers with low specificity and sensitivity. We used multiple methodologies of disseminated tumor cell detection in bone marrow harvested at radical prostatectomy. MATERIALS AND METHODS: Bone marrow was harvested from 208 clinically localized cases, 16 controls and 5 metastatic cases with peripheral blood obtained from 37 metastatic cases. Samples were evaluated at 4 centers with 4 distinct platforms using antibody enrichment with the AdnaTest (Qiagen®) or VERSA (versatile exclusion based rare sample analysis), or whole sample interrogation with the RareCyte platform (Seattle, Washington) or HD-SCA (high definition single cell assay) using traditional epithelial markers and prostate specific markers. We investigated the sensitivity and specificity of these markers by evaluating expression levels in control and metastatic cases. RESULTS: EpCAM, NKX3.1 and AR were nonspecifically expressed in controls and in most samples using AdnaTest with no relation to perioperative variables. Only 1 patient with localized disease showed positive results for the prostate specific marker PSA. With the VERSA platform no localized case demonstrated disseminated tumor cells. With the RareCyte and HD-SCA platforms only a single patient had 1 disseminated tumor cell. CONCLUSIONS: Evaluation across multiple platforms revealed that epithelial markers are nonspecific in bone marrow and, thus, not suitable for disseminated tumor cell detection. Using prostate specific markers disseminated tumor cells were typically not detected in patients with localized prostate cancer.

Neurofibromatosis type 1 and optic pathway glioma: Molecular interplay and therapeutic insights.

Children with neurofibromatosis type 1 (NF1) are predisposed to develop central nervous system neoplasms, the most common of which are low-grade gliomas (LGGs). The absence of human NF1 associated LGG-derived cell lines, coupled with an inability to generate patient-derived xenograft models, represents barriers to profile molecularly targeted therapies for these tumors. Thus, genetically engineered mouse models have been identified to evaluate the interplay between Nf1-deficient tumor cells and nonneoplastic stromal cells to evaluate potential therapies for these neoplasms. Future treatments might also consider targeting the nonneoplastic cells in NF1-LGGs to reduce tumor growth and neurologic morbidity in affected children.

Proteasome-Rich PaCS as an Oncofetal UPS Structure Handling Cytosolic Polyubiquitinated Proteins. In Vivo Occurrence, in Vitro Induction, and Biological Role.

In this article, we outline and discuss available information on the cellular site and mechanism of proteasome interaction with cytosolic polyubiquitinated proteins and heat-shock molecules. The particulate cytoplasmic structure (PaCS) formed by barrel-like particles, closely reproducing in vivo the high-resolution structure of 26S proteasome as isolated in vitro, has been detected in a variety of fetal and neoplastic cells, from living tissue or cultured cell lines. Specific trophic factors and interleukins were found to induce PaCS during in vitro differentiation of dendritic, natural killer (NK), or megakaryoblastic cells, apparently through activation of the MAPK-ERK pathway. Direct interaction of CagA bacterial oncoprotein with proteasome was shown inside the PaCSs of a Helicobacter pylori-infected gastric epithelium, a finding suggesting a role for PaCS in CagA-mediated gastric carcinogenesis. PaCS dissolution and autophagy were seen after withdrawal of inducing factors. PaCS-filled cell blebs and ectosomes were found in some cells and may represent a potential intercellular discharge and transport system of polyubiquitinated antigenic proteins. PaCS differs substantially from the inclusion bodies, sequestosomes, and aggresomes reported in proteinopathies like Huntington or Parkinson diseases, which usually lack PaCS. The latter seems more linked to conditions of increased cell proliferation/differentiation, implying an increased functional demand to the ubiquitin⁻proteasome system.

[Circulating tumor cells in pancreatic cancer : Results of morphological and molecular analyses and comparisons with the primary tumor]. / Zirkulierende Tumorzellen beim Pankreaskarzinom : Ergebnisse morphologischer und molekularer Analysen und Vergleiche mit dem Primärtumor.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a disease with a poor prognosis. PDAC shows characteristic mutations within codon 12/13. Circulating tumor cells (CTC) detected in blood samples of patients with cancer are hypothesized as the means of systemic tumor spread. But less is known about morphological/molecular characteristics or the pathophysiological meaning of PDAC CTC. OBJECTIVES: The aim of the study was a cytomorphological and genetic analysis of CTC from patients with PDAC followed by the correlation of the results with those of the corresponding tumor in the pancreas. MATERIAL AND METHODS: Blood samples of 58 patients with PDAC and 10 "normal" control donors were processed through a size-based CTC isolation. KRAS-mutation analyses were performed for CTC and the primary tumor and the results were compared. Furthermore, their potential as a prognostic marker was evaluated. RESULTS: In patients with different UICC stages CTC were detected, but not in normal control patients. There was a trend for a worse median overall survival (OS) for patients with >3 CTC/ml. Patients with a KRASG12V mutation showed a trend for a better median OS compared to those with other KRAS mutations (10 months) or even without KRAS mutation. Fifty-eight percent of the patients presented concordant KRAS mutations in the primary tumor and corresponding CTC, while 42% were discordant. The median OS for both groups was similar. CONCLUSIONS: Detection and characterization of CTC (for example by KRAS mutation analysis) may be useful for prognosis. Furthermore, it expands our knowledge of tumor biology and may detect possible tumor heterogeneity regarding the mutation profile of some cancer types.

[The value of circulating tumor cells detected by chromosome centromere probe identification in diagnosis of non-small cell lung cancer].

Objective: This study explored the value of circulating tumor cells (CTC) detected by chromosome 8 centromere probe in diagnosis of non-small cell lung cancer (NSCLC) as well as correlation between CTC counts and clinical pathological characteristics. Methods: We collected 136 patients with newly diagnosed NSCLC (101 males and 35 females, age ranged from 34 to 79 years), 149 patients with non-malignant pulmonary diseases (103 males and 46 females, age ranged from 24 to 80 years) and 32 healthy volunteers (5 males and 27 females, age ranged from 24 to 42 years). Detection was performed using an epithelial cell adhesion molecule-independent strategy that combined immunocytochemistry staining (ICC) of CD(45) and fluorescence in situ hybridization detection (FISH) with chromosome 8 centromere probe (CEP8). CTC was defined as 4',6-diamidino-2-phenylindole (DAPI) positive, CD(45) negative and CEP8 more than 2 positive points. Quantitative data were reported as x±s and Mann Whitney test was used to compare them. Measurement data were analyzed as contingency tables and Pearson chi-squared test was used. P values of less than 0.05 were considered statistically significant. Results: CTCs were detected in 114 patients (83.8%) with NSCLC, 35 patients (23.5%) with non-malignant pulmonary diseases (P<0.000 1) and 5 volunteers (15.6%). CTC counts in NSCLC patients were (5.98±0.64) per 3.2 ml and (0.60±0.13) per 3.2 ml (P<0.000 1). A receiver operating characteristic curve (ROC) analysis showed similar capability of CTC count, with CEA, in discriminating NSCLC and non-malignant diseases with an area under ROC curve of 0.854 (95% confidence interval: 0.808-0.900, P<0.001). Cutoff of 2 circulating tumor cells per 3.2 ml peripheral blood gave the highest Youden Index of 0.614 in diagnosis of NSCLC with sensitivity of 72.1% (98/136) and specificity of 89.3% (133/149). When patients with CTC≥2/3.2 ml peripheral blood or serum CEA≥5 ng/ml were considered as having NSCLC, sensitivity and specificity of this combined test were 87.5% (119/136) and 85.9% (128/149), with a higher Youden Index of 0.734. No correlation was observed between positive rate of CTC (rate of patients with CTC≥2/3.2 ml peripheral blood) and age, gender, smoking status, pathologic types, and clinical stages. Conclusions: CTC counts detected by CD(45)-FISH is higher in NSCLC patients than in non-malignant disease patients. CTC≥2/3.2 ml peripheral blood is valuable in discriminating NSCLC from nonmalignant diseases, which can be more accurate when combined with CEA.