Clinical significance of circulating tumor cells in colorectal cancer with peritoneal metastases: a prospective cohort study using a novel method for monitoring treatment response, and assessing minimal residual disease.

BACKGROUND: The treatment for patients with colorectal cancer with metastases to the peritoneum is complex and may involve both surgery and chemotherapy. Circulating tumor cells (CTCs) have been poorly investigated in peritoneal metastatic colorectal cancer. The aim of the study is to examine the role of circulating tumor cells (CTCs) as a biomarker for monitoring disease progression, treatment response and residual disease using CellMate® - a new promising in vitro diagnostic platform technology. MATERIALS AND METHODS: We prospectively followed clinical outcomes of 46 patients treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) for colorectal cancer with peritoneal metastases and examined whether CTCs were present the week of surgery. The CTC measurements were made with the CellMate® technology, which is a platform technology to detect CTCs based on the difference in biomechanical properties compared to blood resident cells. The study was registered online (ClinicalTrials.gov). RESULTS: CTCs were detected in 17 (37%) patients. The presence of CTCs was associated with shorter recurrence-free survival and overall survival after CRS and HIPEC. Both recurrence free survival (HR 4.00, 95%CI 1.15-13.9; P=0.029) and overall survival (HR 5.91; 95% CI 1.18-29.7; P=0.03) were significantly worse if CTCs were detected after neoadjuvant treatment. In the subgroup of patients with CTCs detected, adjuvant therapy tended to improve the prognosis while in CTC negative patients it did not. CONCLUSIONS: Pending a prospective multi-center trial to validate these findings, CTCs may in the future be used as a dynamic personalized biomarker for prognostication, predicting response to therapy, and for monitoring disease progression in colorectal cancer with metastases to the peritoneum.

Clinical Significance of Circulating Tumor Cells in Epithelial Appendiceal Neoplasms with Peritoneal Metastases.

Appendiceal tumors are uncommon and, at times, discovered incidentally during histological examination. The histopathological classification of the disease is complex and has generated some controversy. The analysis of circulating tumor cells can be used for the early detection of metastatic potential. The aim of the present study was to examine the prognostic value of circulating tumor cells in patients with appendiceal tumors and peritoneal metastases. To our knowledge, this is the first study to examine CTCs in appendiceal tumors. We performed a prospective cohort study of consecutive patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy between 2015 and 2019 at a HIPEC referral center. In total, 31 patients were included in the analysis, and circulating tumor cells were detected in 15 patients (48%). CTC positivity was not associated with overall or recurrence-free survival, nor was it correlated with PCI score or histopathological grading. Surprisingly, however, CTCs were found in almost half the patients. The presence or quantities of these cells did not, on their own, predict systemic metastatic potential during the observed time, and they did not appear to significantly correlate with the oncological outcomes recorded.

Higher Human Cytomegalovirus (HCMV) Specific IgG Antibody Levels in Plasma Samples from Patients with Metastatic Brain Tumors Are Associated with Longer Survival.

Background: Human cytomegalovirus (HCMV) has been detected in tissue samples from patients with glioblastoma but little is known about the systemic immunological response to HCMV in these patients. Objectives: To investigate the presence and clinical significance of HCMV antibodies levels in plasma samples obtained from patients with brain tumors. Materials and Methods: HCMV-specific IgG and IgM antibody levels were determined in 59 plasma samples collected from brain tumor patients included in a prospective study and in 114 healthy individuals. We examined if the levels of HCMV specific antibodies varied in patients with different brain tumor diagnoses compared to healthy individuals, and if antibody levels were predictive for survival time. Results: HCMV specific IgG antibodies were detected by ELISA in 80% and 89% of patients with GBM and astrocytoma grades II-III, respectively, in all samples (100%) from patients with secondary GBM and brain metastases, as well as in 80% of healthy donors (n = 114). All plasma samples were negative for HCMV-IgM. Patients with brain metastases who had higher plasma HCMV-IgG titers had longer survival times (p = 0.03). Conclusions: HCMV specific IgG titers were higher among all brain tumor patient groups compared with healthy donors, except for patients with secondary GBM. Higher HCMV specific IgG levels in patients with brain metastases but not in patients with primary brain tumors were associated with prolonged survival time.

The association between Annexin A2 and epithelial cell adhesion molecule in breast cancer cells.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane and glycosylated protein, which is overexpressed in many neoplasms. However, EpCAM has no known ligand partners and the mechanisms by which it functions are not fully understood. AIM: This study was performed to discover novel partners of EpCAM, which may provide a better understanding of its functions. METHODS: The membrane fraction of the ERα+ noninvasive breast cancer cell line ZR-75-1 and MCF-7 was extracted and followed by co-immunoprecipitation of EpCAM using C-10, a mouse monoclonal antibody raised against amino acids 24-93 of the EpCAM molecule. As a negative control, MDA-MB-231 and Hs578T were used since they express a negligible amount of EpCAM and are known as EpCAM-/low ERα-/low invasive and tumorigenic breast cancer cell lines. RESULTS: Annexin A2 (ANXA2) was found to be selectively and differentially co-immunoprecipitated with EpCAM in the ERα+ breast cancer cells MCF-7 and ZR-75-1. ANXA2 is a multifunctional protein and known to act as a co-receptor for tissue plasminogen activator (tPA) on the surface of endothelial and cancer cells, thereby affecting fibrinolytic activity and neoangiogenesis as well as invasive and metastatic properties. In this study, the association between EpCAM and ANXA2 was found to affect the activity of tPA. CONCLUSION: This study concludes that ANXA2 co-localizes with EpCAM at the plasma membrane, and the co-localization may have functional implications. Data suggest that EpCAM supports ANXA2 to function as a co-receptor for the tPA, and that EpCAM has a regulatory function on the expression and subcellular localization of ANXA2.

Screening Circulating Tumor Cells as a Noninvasive Cancer Test in 3388 Individuals from High-Risk Groups (ICELLATE2).

Cancer is known to spread up to 12 years before clinical symptoms occur, but few screening tests exist. Early detection would give the opportunity for early treatment, potentially improving prognosis. To this end, 3388 subjectively healthy individuals of age 45 to 80 who had been exposed to cancer risk factors were screened for the occurrence of circulating tumor cells in their blood. Presence of circulating tumor cells is a suspicious finding indicative of spreading cancer, since cancer metastasizes by way of the blood and offers the opportunities to (a) follow up the individual clinically based on established guidelines for early detection of cancer and (b) evaluate the cells further analytically. 107 individuals showed one or more circulating tumor cells in a 7.5 ml blood sample, which constitutes a positive circulating tumor cell test, based on the iCellate IsoPic™ laboratory test. That number compares favorably with the cancer incidence per 100,000 people/year that is 157.1 in Peru, given that a high-risk group of individuals was screened and that the screening results would be expected to correspond to an accumulated incidence of up to 12 years. The present findings therefore identify screening for circulating tumor cells as a promising new test.

Prostate biopsy sampling causes hematogenous dissemination of epithelial cellular material.

The extent of epithelial cellular material (ECM) occurring in venous blood samples after diagnostic core needle biopsy (CNB) was studied in 23 patients with CNB diagnosed prostate cancer without provable metastases and 15 patients without cancer. The data show a significant increase of ECM in the peripheral blood sampled 20 seconds or 30 minutes after the last of 10 CNB procedures compared to the number of ECM detectable in the blood samples taken before the performance of CNB. The data indicate that diagnostic CNB of prostate cancer causes an extensive tissue trauma with a potential risk of cancer cell dissemination.

EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells.

Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell "marker" and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM(+). In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.

Activation of neural and pluripotent stem cell signatures correlates with increased malignancy in human glioma.

The presence of stem cell characteristics in glioma cells raises the possibility that mechanisms promoting the maintenance and self-renewal of tissue specific stem cells have a similar function in tumor cells. Here we characterized human gliomas of various malignancy grades for the expression of stem cell regulatory proteins. We show that cells in high grade glioma co-express an array of markers defining neural stem cells (NSCs) and that these proteins can fulfill similar functions in tumor cells as in NSCs. However, in contrast to NSCs glioma cells co-express neural proteins together with pluripotent stem cell markers, including the transcription factors Oct4, Sox2, Nanog and Klf4. In line with this finding, in high grade gliomas mesodermal- and endodermal-specific transcription factors were detected together with neural proteins, a combination of lineage markers not normally present in the central nervous system. Persistent presence of pluripotent stem cell traits could only be detected in solid tumors, and observations based on in vitro studies and xenograft transplantations in mice imply that this presence is dependent on the combined activity of intrinsic and extrinsic regulatory cues. Together these results demonstrate a general deregulated expression of neural and pluripotent stem cell traits in malignant human gliomas, and indicate that stem cell regulatory factors may provide significant targets for therapeutic strategies.

Uncoupling of the ERα regulated morphological phenotype from the cancer stem cell phenotype in human breast cancer cell lines.

The CD24(low/-)CD44(+)EpCAM(+) phenotype is associated with breast cancer initiating cells. To investigate if these putative breast cancer stem cell markers are regulated by estrogen receptor alpha (ERα) we have determined the expression levels of EpCAM, CD44 and CD24 in several well characterized breast cancer cell lines. The expression levels of the three adhesion proteins were quantitatively different in the cell lines but the composite CD24(low/-)CD44(+)EpCAM(+) breast cancer stem cell phenotype was shown to exist as a small fraction, between 0.1% and 1.2%, in all breast cancer cell lines tested. Experimental silencing of ERα resulted in a reduced epithelial appearance and partial reduction of CD24 mRNA, while levels of CD44 and EpCAM were unaltered. Moreover, knockdown of ERα led to a change in the morphology of the cells similar to the epithelial to mesenchymal transition phenotype and was associated with decreased E-cadherin expression. Our findings offer new insights into the regulation of the breast cancer stem cell phenotype by ERα and suggest that treatments targeting the breast cancer stem cell adhesion molecules and the ERα pathway may be complementary.

Protein extraction from solid tissue.

Maximal extraction and solubilization of protein from diseased or healthy tissue is important to make the whole protein complement available for proteomic analysis. It also helps to maximize reproducibility and to minimize waste. Minimal degradation of the protein amino acid backbone or dephosphorylation is essential to preserve the analytical utility of the extract. Containment of the sample is important to minimize the risk of contamination to and from the sample. The proposed standard protocol for protein extraction and solubilization can result in 98% solubilization of brain tissue, corresponding to about 100 µg protein per mg tissue wet weight, by a frozen disintegration/SDS-based solubilization method: Tissue is crushed in the frozen state in a cryotube by shaking with a sterile steel ball. The crushing is followed by the extraction and solubilization in 2% SDS for 10 min, at 70°C, in a volume corresponding to ten times the tissue wet weight, with shaking. The containment in a cryotube helps to prevent contamination. The treatment with SDS sample buffer can inhibit protease and phosphatase activity. The resulting protein extracts can be used for SDS PAGE, 2-D PAGE, Western blotting, ESI-MS, and ELISA. The proposed standard protocol has the potential to find wide application where protein extraction, solubilization, identification, and quantitation from cryopreserved clinical samples are desirable.

Handling of solid brain tumor tissue for protein analysis.

Optimal protein analysis requires unfixed tissue samples. We suggest handling the brain tumor tissue sterilely and coldly (on ice) for as short time as possible prior to processing, but for no more than 8 h. This simple protocol results in apparently intact morphology, immunoreactivity, protein integrity, and protein phosphorylation with the criteria we apply. Sample handling for Pathological Anatomical Diagnosis (PAD) and for protein analysis can be one and the same.

Blood plasma handling for protein analysis.

Blood handling routines have been worked out that result in consistent protein analytic results in clinical practice. It would seem reasonable to build on this experience when devising handling routines for new protein biomarker discovery. Consequently, normal blood sample handling precautions apply to blood sample handling for new biomarker discovery. The blood sample handling protocol mentioned below describes room temperature, or 4°C, platelet poor EDTA plasma collected within 90 min of venipuncture, handled, and screened to eliminate hemolysis. DNA can be isolated from the "buffy coat" that results as blood cells are sedimented to isolate the plasma.

Targeting the insulin-like growth factor-1 receptor by picropodophyllin as a treatment option for glioblastoma.

Glioblastoma (GB) is the most common malignant brain tumor in adults. It has limited treatment opportunities and is almost exclusively fatal. Owing to the central role the insulin-like growth factor-1 receptor (IGF-1R) plays in malignant cells, it has been suggested as a target for anticancer therapy including GB. The cyclolignan picropodophyllin (PPP) inhibits IGF-1R without affecting the highly homologous insulin receptor. Here, we show that PPP inhibits growth of human GB cell lines along with reduced phosphorylation of IGF-1R and AKT. In vivo, PPP-treatment causes dramatic tumor regression not only in subcutaneous xenografts but also in intracerebral xenografts, indicating passage of PPP across the blood-brain barrier.

Optimized protein extraction from cryopreserved brain tissue samples.

Optimal standard conditions for protein extraction and solubilization from frozen tissue samples have been examined. Quantitative differences in specific protein amounts or post-translational modifications underlie many, if not all, disease states. Maximal and standardized extraction and solubilization of protein from diseased or healthy tissue is important to make the whole protein complement available for proteomic analysis, and to make the best use of a precious resource. Minimal degradation of the protein amino acid backbone, or of phosphorylated amino acid side chains, during sample preparation is essential to preserve the analytical utility of the extract. We have investigated parameters of brain tissue disintegration, and of extraction/solubilization temperature, time and volume and have reached 98% extraction of brain tissue, corresponding to about 100 microg protein per mg tissue wet weight, by an SDS-based method: Tissue disintegration in the frozen state, by ball mill grinding followed by extraction and solubilization in 2% SDS for 10 min, at 70 degrees C, in a volume corresponding to ten times the tissue wet weight, with shaking. The treatment with SDS sample buffer can inhibit protease and phosphatase activity. Moreover, endogenous enzymes can be inhibited by incubation at high pH. The resulting protein extracts can be used for both one-dimensional SDS gel-electrophoresis and for two-dimensional isoelectric focusing/SDS electrophoresis. The proposed standard protocol has the potential to find wide application where protein extraction, solubilization, identification and quantitation from cryopreserved clinical samples are desirable.

Frozen tissue biobanks. Tissue handling, cryopreservation, extraction, and use for proteomic analysis.

The potential and the limitations of protein analysis of tissue samples are surveyed. The complexity, concentration range and dynamics of the human proteome are reviewed, as is the effect of handling and cryopreservation. Protein extraction, solubilization, resolution and detection are discussed, in relation to the properties of the human proteome.

Cloning of Schistosoma mansoni Seven in Absentia (SmSINA)(+) homologue cDNA, a gene involved in ubiquitination of SmRXR1 and SmRXR2.

Drosophila (SINA) and human Seven in Absentia (SIAH-1 and SIAH-2) have been implicated in ubiquitin-mediated proteolysis of different target proteins. Using the Schistosoma mansoni nuclear receptor SmRXR2 as bait in a yeast two-hybrid system, we identified a DNA fragment that encodes part of the schistosome homologue of the Seven in Absentia protein (SmSINA). Screening of S. mansoni cDNA expression library resulted in the isolation of a cDNA containing the full-length coding region of SmSINA. SmSINA contains the characteristic structural features of other SINA proteins including a conserved N-terminal RING finger domain and a cysteine-rich C-terminus. We demonstrate that SmSINA associates with SmRXR2 and SmRXR1 both in vivo and in vitro, and define the binding domains in SmRXR2 and SmRXR1 that mediate their interaction. Schistosome SINA co-localizes with SmRXR2 and SmRXR1 in vitelline cells. In addition, we show that SmSINA stimulates the ubiquination of both SmRXR2 and SmRXR1 in vitro. Our findings suggest that SmSINA regulates ubiquitination and ubiquitin-induced degradation of schistosome nuclear receptors (RXR1 and RXR2) via the ubiquitin-proteasome pathway.

Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster.

A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (NPFR76F, also called GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62-66% similarity and 32-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila.