A pilot study to explore circulating tumour cells in pancreatic cancer as a novel biomarker

Background:

Obtaining tissue for pancreatic carcinoma diagnosis and biomarker assessment to aid drug development is challenging. Circulating tumour cells (CTCs) may represent a potential biomarker to address these unmet needs. We compared prospectively the utility of two platforms for CTC enumeration and characterisation in pancreatic cancer patients in a pilot exploratory study.

Patients and methods:

Blood samples were obtained prospectively from 54 consenting patients and analysed by CellSearch and isolation by size of epithelial tumour cells (ISET). CellSearch exploits immunomagnetic capture of CTCs-expressing epithelial markers, whereas ISET is a marker independent, blood filtration device. Circulating tumour cell expression of epithelial and mesenchymal markers was assessed to explore any discrepancy in CTC number between the two platforms.

Results:

ISET detected CTCs in more patients than CellSearch (93% vs 40%) and in higher numbers (median CTCs/7.5 ml, 9 (range 0–240) vs 0 (range 0–144)). Heterogeneity observed for epithelial cell adhesion molecule, pan-cytokeratin (CK), E-Cadherin, Vimentin and CK 7 expression in CTCs may account for discrepancy in CTC number between platforms.

Conclusion:

ISET detects more CTCs than CellSearch and offers flexible CTC characterisation with potential to investigate CTC biology and develop biomarkers for pancreatic cancer patient management.     

Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes

Background:

The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer.

Methods:

CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of ⩾5 CTCs per 7.5 ml blood.

Results:

No significant differences were observed in the absolute CTC counts (P=0.120) or in CTC positivity rates according to ⩾1 and ⩾5 CTCs per 7.5 ml blood detection thresholds (P=0.165 and P=0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as ⩾80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P=0.024). In the total study population, the presence of ⩾5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P<0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B–HER2-negative and TN disease.

Conclusion:

The detection of EpCAM+CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.     

Clinical significance of phenotyping and karyotyping of circulating tumor cells in patients with advanced gastric cancer

BACKGROUND

Karyotyping and phenotyping of circulating tumor cells (CTCs) in therapeutic cancer patients is of particular clinical significance in terms of both identifying chemo-resistant CTC subtypes and understanding CTC evolution.

METHODS

The integrated subtraction enrichment (SET) and immunostaining-fluorescence in situ hybridization (iFISH) platform was applied to detect and characterize CTCs in patients with advanced gastric cancer (AGC). Status of human epidermal growth factor receptor 2 (HER2) expressing and aneuploidy of chromosome 8 in CTCs enriched from the patients was examined by SET-iFISH following clinical chemotherapy or HER2-targeted therapy. CellSearch system was applied as a reference control.

RESULTS

Phenotyping of CTCs in HER2 positive AGC patients demonstrated that HER2⁺ CTCs could be effectively eliminated in response to HER2-targeted therapy. Karyotyping of CTCs indicated that distinct CTCs with different ploidies of chromosomes 8 in AGC patients correlated to either sensitivity or resistance of paclitaxel or cisplatin-based chemotherapy. Examination of the copy number of chromosome 8 in CTCs provides a potential approach for predicting chemotherapeutic efficacy and monitoring chemo-resistance.

CONCLUSIONS

Phenotyping and karyotyping of the enriched CTCs upon ploidy of chromosome 8 or HER2 expression is of clinical potential for monitoring chemo-resistance and evaluating therapeutic efficacy for AGC patients.     

A direct comparison of CellSearch and ISET for circulating tumour-cell detection in patients with metastatic carcinomas

Background:

Circulating tumour cells (CTCs) can provide information on patient prognosis and treatment efficacy. However, there is no universal method to detect CTC currently available. Here, we compared the performance of two CTC detection systems based on the expression of the EpCAM antigen (CellSearch assay) or on cell size (ISET assay).

Methods:

Circulating tumour cells were enumerated in 60 patients with metastatic carcinomas of breast, prostate and lung origins using CellSearch according to the manufacturer''s protocol and ISET by studying cytomorphology and immunolabelling with anti-cytokeratin or lineage-specific antibodies.

Results:

Concordant results were obtained in 55% (11 out of 20) of the patients with breast cancer, in 60% (12 out of 20) of the patients with prostate cancer and in only 20% (4 out of 20) of lung cancer patients.

Conclusion:

Our results highlight important discrepancies between the numbers of CTC enumerated by both techniques. These differences depend mostly on the tumour type. These results suggest that technologies limiting CTC capture to EpCAM-positive cells, may present important limitations, especially in patients with metastatic lung carcinoma.     

Frequency of HER2 expression of circulating tumour cells in patients with metastatic or recurrent gastrointestinal cancer

Background:

The clinical significance of circulating tumour cell (CTC) detection in gastrointestinal (GI) cancer remains controversial and the molecular biological characteristics of CTCs are poorly understood.

Methods:

A total of 87 patients with metastatic or recurrent GI cancer were prospectively enrolled. Circulating tumour cells and their HER2 status were assessed using the CellSearch System.

Results:

Among the 62 CTC-positive cases, we found 22 discordant cases (35.5%). Among the HER2-negative primary tumours, 17 of 54 developed HER2-positive CTCs. Five of eight had HER2-negative CTCs among the HER2-positive primary tumours.

Conclusion:

The findings in the current study suggest that it is critical to evaluate the HER2 status of not only the primary tumour but also the CTCs because the metastasising tumour cells are the primary target of systemic therapy.     

Androgen receptor expression in circulating tumour cells from castration-resistant prostate cancer patients treated with novel endocrine agents

Background:

Abiraterone and enzalutamide are novel endocrine treatments that abrogate androgen receptor (AR) signalling in castration-resistant prostate cancer (CRPC). Here, we developed a circulating tumour cells (CTCs)-based assay to evaluate AR expression in real-time in CRPC and investigated nuclear AR expression in CTCs in patients treated with enzalutamide and abiraterone.

Methods:

CTCs were captured and characterised using the CellSearch system. An automated algorithm to identify CTCs and quantify AR expression was employed. The primary aim was to evaluate the association between CTC AR expression and prior treatment with abiraterone or enzalutamide.

Results:

AR expression in CTCs was evaluated in 94 samples from 48 metastatic CRPC patients. We observed large intra-patient heterogeneity of AR expression in CTCs. Prior exposure to abiraterone or enzalutamide was not associated with a change in CTCs AR expression (median intensity and distribution of AR-positive classes). In support of this, we also confirmed maintained nuclear AR expression in tissue samples collected after progression on abiraterone. AR staining also identified additional AR-positive CD45-negative circulating cells that were CK-negative/weak and therefore missed using standard protocols. The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis.

Conclusions:

We developed a non-invasive method to monitor AR nuclear expression in CTCs. Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments. Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.     

A multimarker QPCR-based platform for the detection of circulating tumour cells in patients with early-stage breast cancer

Background:

The detection of circulating tumour cells (CTCs) has been linked with poor prognosis in advanced breast cancer. Relatively few studies have been undertaken to study the clinical relevance of CTCs in early-stage breast cancer.

Methods:

In a prospective study, we evaluated CTCs in the peripheral blood of 82 early-stage breast cancer patients. Control groups consisted of 16 advanced breast cancer patients and 45 healthy volunteers. The CTC detection was performed using ErbB2/EpCAM immunomagnetic tumour cell enrichment followed by multimarker quantitative PCR (QPCR). The CTC status and common clinicopathological factors were correlated to relapse-free, breast cancer-related and overall survival.

Results:

Circulating tumour cells were detected in 16 of 82 (20%) patients with early-stage breast cancer and in 13 out of 16 (81%) with advanced breast cancer. The specificity was 100%. The median follow-up time was 51 months (range: 17–60). The CTC positivity in early-stage breast cancer patients resulted in significantly poorer relapse-free survival (log rank test: P=0.003) and was an independent predictor of relapse-free survival (multivariate hazard ratio=5.13, P=0.006, 95% CI: 1.62–16.31).

Conclusion:

The detection of CTCs in peripheral blood of early-stage breast cancer patients provided prognostic information for relapse-free survival.     

Detection of circulating tumour cells with a hybrid (epithelial/mesenchymal) phenotype in patients with metastatic non-small cell lung cancer

Background:

Circulating tumour cells (CTC) have a crucial role in metastasis formation and can consistently provide information on patient prognosis. Epithelial-mesenchymal transition (EMT) is considered as an essential process in the metastatic cascade, but there is currently very few data demonstrating directly the existence of the EMT process in CTCs.

Methods:

CTCs were enriched by blood filtration using ISET (isolation by size of epithelial tumour cells), triply labelled with fluorescent anti-vimentin, anti-pan-keratin antibodies and SYTOX orange nuclear dye, and examined by confocal microscopy in six patients with metastatic non-small cell lung cancer (NSCLC). In parallel, CTCs were morphocytologically identified by an experienced cytopathologist.

Results:

Isolated or clusters of dual CTCs strongly co-expressing vimentin and keratin were evidenced in all patients (range 5–88/5 ml). CTCs expressing only vimentin were detected in three patients, but were less frequent (range 3–15/5 ml). No CTC expressing only keratin was detected.

Conclusion:

We showed for the first time the existence of hybrid CTCs with an epithelial/mesenchymal phenotype in patients with NSCLC. Their characterisation should provide further insight on the significance of EMT in CTCs and on the mechanism of metastasis in patients with NSCLC.     

Circulating tumor cells in newly diagnosed inflammatory breast cancer

Introduction

Circulating tumor cells (CTCs) are an independent prognostic factor for progression-free survival (PFS) and overall survival (OS) in patients with metastatic breast cancer. Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer. The prognostic value of a CTC count in newly diagnosed IBC has not been established. The aim of this study was to assess the prognostic value of a baseline CTC count in patients with newly diagnosed IBC.

Methods

This retrospective study included 147 patients with newly diagnosed IBC (77 with locally advanced and 70 with metastatic IBC) treated with neoadjuvant therapy or first-line chemotherapy during the period from January 2004 through December 2012 at The University of Texas MD Anderson Cancer Center. CTCs were detected and enumerated by using the CellSearch system before patients were started with chemotherapy.

Results

The proportion of patients with ≥1 CTC was lower among patients with stage III than among patients with metastatic IBC (54.5% versus 84.3%; P = 0.0002); the proportion of patients with ≥5 CTCs was also lower for stage III than for metastatic IBC (19.5% versus 47.1%; P = 0.0004). Patients with fewer than five CTCs had significantly better progression-free survival (PFS) (hazard ratio (HR) = 0.60; P = 0.02) and overall survival (HR = 0.59; P = 0.03) than patients with five or more CTCs. Among patients with stage III IBC, there was a nonsignificant difference in PFS (HR = 0.66; 95% confidence interval (CI), 0.31 to 1.39; P = 0.29) and OS (HR = 0.54; 95% CI, 0.24 to 1.26; P = 0.48) in patients with no CTCs compared with patients with one or more CTCs. In multivariate analysis, CTC was prognostic for PFS and OS independent of clinical stage.

Conclusions

CTCs can be detected in a large proportion of patients with newly diagnosed IBC and are a strong predictor of worse prognosis in patients with newly diagnosed IBC.     

Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer

Background:

To investigate the clinical relevance of CK-19mRNA-positive circulating tumour cells (CTCs) detected before the initiation of front-line treatment in patients with metastatic breast cancer (MBC).

Methods:

The presence of CTCs was detected in 298 patients with MBC using a real-time PCR (RT-PCR assay. In 44 patients, the detection of CTCs was evaluated by both the CellSearch and the RT-PCR assay. Interaction with known prognostic factors and association of CTCs with clinical outcome were investigated.

Results:

There was a strong correlation between the detection of CTCs by both assays. CK-19mRNA-positive CTCs were detected in 201 (67%) patients and their detection was independent of various patients'' clinico-pathological characteristics. The median progression-free survival (PFS; 9.2 vs 11.9 months (mo), P=0.003) and the overall survival (OS; 29.7 vs 38.9 mo, P=0.016) were significantly shorter in patients with detectable CK-19mRNA-positive CTCs compared with patients without detectable CTCs. Multivariate analysis demonstrated that oestrogen receptor status, performance status and detection of CTCs were emerged as independent prognostic factors associated with decreased PFS and OS.

Conclusion:

The detection of CK-19mRNA-positive CTCs in patients with MBC before front-line therapy could define a subgroup of patients with dismal clinical outcome.     

Dynamic changes of live/apoptotic circulating tumour cells as predictive marker of response to Sunitinib in metastatic renal cancer

Background:

Recently, we developed an apoptotic assay for expanding the monitoring capabilities of the circulating tumour cells (CTC) test during therapy. An automated platform for computing CTCs was integrated with a mAb (M30) targeting a neoepitope disclosed by caspase cleavage at cytokeratin 18 in early apoptosis; we showed that live CTCs were associated with progression, consistent with enhanced cell migration and invasion. The test was first applied here to mRCC.

Methods:

Live/apoptotic CTCs changes were measured in mRCC patients receiving first-line Sunitinib and compared with circulating endothelial cell (CEC) levels.

Results:

The presence of EpCAM-positive, live CTCs predicts progression in individual mRCC patient, being associated with distant metastasis under first-line Sunitinib. Synchronous detection of CTCs and CEC levels discloses for the first time an association between their dynamic changes and outcome: a rapid increase of the CEC number as early as the first cycle of therapy is associated with CTC decrease in non-progressed patients, whereas a delayed response of CECs is related to higher CTC values in the progressed group indicating treatment failure.

Conclusion:

We demonstrated that a delayed response to antiangiogenic treatment indicated by persistent detection of CECs correlates with persistent live CTCs and more aggressive disease.     

Prospective evaluation of serum tissue inhibitor of metalloproteinase 1 and carbonic anhydrase IX in correlation to circulating tumor cells in patients with metastatic breast cancer

Introduction

Circulating tumor cells (CTCs) reflect aggressive tumor behavior by hematogenous tumor cell dissemination. The tissue inhibitor of metalloproteinase 1 (TIMP-1) plays a role in tissue invasion and is also involved in angiogenesis, abrogation of apoptosis and in chemoresistance. Carbonic anhydrase IX (CAIX) is a metalloenzyme involved in cell adhesion, growth and survival of tumor cells. The aim of the study was to investigate whether serum concentrations of TIMP-1 and CAIX are associated with the detection of CTC in metastatic breast cancer.

Methods

Blood was obtained in a prospective multicenter setting from 253 patients with metastatic breast cancer at the time of disease progression. Serum TIMP-1 and CAIX were determined using commercial ELISA-kits (Oncogene Science). CTC were detected with the CellSearch™ system (Veridex).

Results

Five or more CTCs were detected in 122 patients out of 245 evaluable patients (49.8%). Out of 253 metastatic patients 70 (28%) had serum TIMP-1 levels above 454 ng/mL. Serum CAIX was elevated above 506 ng/mL in 90 (35%) patients. Both serum markers had prognostic significance. Median progression free survival (PFS) was 7.2 months with elevated TIMP-1 vs. 11.4 months with non-elevated levels (p < 0.01). OS was 11.5 vs. 19.1 months (p < 0.01). Median PFS was 7.5 months with elevated CAIX vs. 11.7 months with non-elevated levels (p < 0.01), overall survival (OS) was 13.4 months vs. 19.1 months (p < 0.01). In patients with five or more CTCs, serum levels were above the cut-off for CAIX in 47% vs. 25% in those with less than five CTCs (p = 0.01). For TIMP-1, 37% patients with five or more CTCs had elevated serum levels and 17% of patients with less than five CTCs (p = 0.01). Including TIMP-1, CAIX, CTC and established prognostic factors in the multivariate analysis, the presence of CTCs, the therapy line and elevated CAIX remained independent predictors of OS.

Conclusions

Elevated serum levels of the invasion markers TIMP-1 and CAIX in metastatic breast cancer are prognostic markers and are associated with the presence of CTCs. Whether increased secretion of TIMP-1 and/or CAIX might directly contribute to tumor cell dissemination remains to be elucidated in further investigations.

Trial registration

Current Controlled Trials: ISRCTN59722891     

PTEN loss in circulating tumour cells correlates with PTEN loss in fresh tumour tissue from castration-resistant prostate cancer patients

Background:

PTEN gene loss occurs frequently in castration-resistant prostate cancer (CRPC) and may drive progression through activation of the PI3K/AKT pathway. Here, we developed a novel CTC-based assay to determine PTEN status and examined the correlation between PTEN status in CTCs and matched tumour tissue samples.

Methods:

PTEN gene status in CTCs was evaluated on an enrichment-free platform (Epic Sciences) by fluorescence in situ hybridisation (FISH). PTEN status in archival and fresh tumour tissue was evaluated by FISH and immunohistochemistry.

Results:

Peripheral blood was collected from 76 patients. Matched archival and fresh cancer tissue was available for 48 patients. PTEN gene status detected in CTCs was concordant with PTEN status in matched fresh tissues and archival tissue in 32 of 38 patients (84%) and 24 of 39 patients (62%), respectively. CTC counts were prognostic (continuous, P=0.001). PTEN loss in CTCs associated with worse survival in univariate analysis (HR 2.05; 95% CI 1.17–3.62; P=0.01) and with high lactate dehydrogenase (LDH) in metastatic CRPC patients.

Conclusions:

Our results illustrate the potential use of CTCs as a non-invasive, real-time liquid biopsy to determine PTEN gene status. The prognostic and predictive value of PTEN in CTCs warrants investigation in CRPC clinical trials of PI3K/AKT-targeted therapies.     

Genome wide analysis and clinical correlation of chromosomal and transcriptional mutations in cancers of the biliary tract

Background

The pathogenesis of biliary cancers is ill-defined. This study investigates changes in gene expression and copy number in biliary cancers and correlates these changes with anatomical site of origin, histopathology and outcome.

Methods

We performed gene expression and CGH analysis on 34 biliary tract cancer specimens. Results were confirmed by RT-PCR. Clinical-pathologic correlation was made using functional over-representation analysis of the top 100 mutations associated with each variable.

Results

There were 545 genes with altered expression in extrahepatic cholangiocarcinoma, 2,354 in intrahepatic cholangiocarcinoma, and 1,281 in gallbladder cancer. Unsupervised hierarchical clustering analysis indicated there was no difference in the global gene expression patterns between each biliary cancer subgroup. CGH analysis revealed that short segments of chromosomes 1p, 3p, 6q, 8p, 9p, and 14q were commonly deleted across all cancer subtypes. Commonly amplified regions included segments of 1q, 3q, 5p, 7p, 7q, 8q, and 20q. Over-representation analysis revealed an association between altered expression of functional gene groupings and pathologic features.

Conclusion

This study defined regions of the genome associated with changes in DNA copy number and gene expression in specific subtypes of biliary cancers. The findings have implications for identification of therapeutic targets, screening, and prognostication.     

Circulating tumour cells are associated with increased risk of venous thromboembolism in metastatic breast cancer patients

Background:

Cancer is a risk factor for venous thromboembolism (VTE). Circulating tumour cells (CTCs) are an independent predictor of survival in metastatic breast cancer (MBC) patients. The aim of this study was to test the hypothesis that CTCs are associated with the risk of VTE in MBC patients.

Methods:

This retrospective study included 290 MBC patients treated in the MD Anderson Cancer Center from January 2004 to December 2007. Circulating tumour cells were detected and enumerated using the CellSearch system before starting new lines of therapy.

Results:

At a median follow-up of 12.5 months, 25 patients experienced VTE and 53 patients died without experiencing thrombosis. Cumulative incidence of thrombosis at 12 months was 8.5% (95% confidence interval (CI)=5.5%, 12.4%). Patients with CTCs ⩾1 and ⩾5 had a higher incidence of VTE compared with patients with 0 and <5 CTCs (12-month estimate, 11.7 and 11.6% vs 3 and 6.6%; P=0.006 and P=0.076, respectively). In the multivariate model, patients with CTCs⩾1 had a hazard ratio of VTE of 5.29 (95% CI=1.58, 17.7, P=0.007) compared with patients with no CTCs.

Conclusion:

These results suggest that CTCs in MBC patients are associated with increased risk of VTE. These patients should be followed up more closely for the risk of VTE.     

Circulating tumour cells detected by a novel adenovirus-mediated system may be a potent therapeutic marker in gynaecological cancers

Background:

Recently developed detection system for circulating tumour cells (CTCs) using a telomerase-specific replicative adenovirus generated nonspecific green fluorescent protein (GFP) signals because of the co-presence of white blood cells (WBCs) nonspecifically infected by viruses. Here, we established a unique detection system for CTCs that completely excludes nonspecific signals.

Methods:

Blood obtained from the patients was subjected to haemolytic processes to eliminate red blood cells. The cell pellets were then infected with OBP-401, fixed, incubated with fluorescence-labelled anti-CD45 antibody to mark white blood WBCs, and examined on slides under a microscope.

Results:

Preparatory experiments with cancer cells artificially added to healthy donor samples confirmed that CD45 labelling could distinguish GFP-positive cancer cells from WBCs. In 53 patients with gynaecological cancers, CTCs were detected in 21 patients (39.6%) when CD45-positive cells were excluded as WBCs among GFP-positive cells. No CTCs were detected in samples from healthy volunteers. There was no significant correlation between CTC counts and known clinicopathological factors. The CTCs rapidly vanished after surgery or chemotherapy in most patients whose treatments were effective. In contrast, the persistence of CTCs even after treatments was tightly associated with poor response to the treatments (P<0.005).

Conclusion:

The presence of CTCs in our system may potentially be a novel therapeutic marker in gynaecological cancers.     

International study on inter-reader variability for circulating tumor cells in breast cancer

Introduction

Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement.

Methods

CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test.

Results

For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90).

Conclusions

The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.