Circulating Tumour Cell Popularity In Cancer Diagnosis Biology Essay

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Circulating Tumor Cells are gaining popularity in diagnosis and prognosis of various cancers. Detection, monitoring and molecular analysis of such cells will provide the powerful, efficient and non invasive approach to detect the disease, its prognosis and response to various therapeutic modalities. Endothelial cells initially arise from organ-confined lesion but eventually spread to distant places responsible for the lethality of the cancer by causing metastases. Hence detection of CTC's also assists in targeting the metastatic cells resulting in modification of therapeutic approach of the physicians to such patients. The process of metastatic cells to leave the primary lesion to enter blood stream and getting settled to a distant place is not well documented. A mouse model of tumor spread has implicated the mechanism of mesenchymal transition in which epithelial cells adopt migratory cell fate. Although mouse model differs considerably from human model [1].

Prostate cancer is the most common nondermatologic malignancy in men and the second leading cause of death in United states. In 2008 186.320 new cases of prostate cancer were diagnosed while 28.660 deaths due to prostate cancer were reported [2]. Helabi et al and others have extensively analyzed a variety of prognostic factors in prostate cancer patients. These includes pain interference scores, performance status, age, prostate-specific antigen (PSA), lactate dehydrogenase (LDH), and Gleason score [3]. Recently Circulating tumor cells have been proposed a new diagnostic, prognostic and predictive factor in Castration resistant prostate cancer (CRPC). Moreno et al first determine the significance of CTCs in prostate and other cancer by the use of PCR to detect the PSA -positive cells in circulation of patients with prostate cancer [4]. Prostate circulating tumor cells (PCTC) have been often found in metastatic prostate cancer. These cells have been thought to contribute to metastatic progression although PCTCs are rare. These days CTCs are used clinically as a prognostic indicator of prostate cancer patient survival [6,7,8].

Although challenging, detection of CTCs in peripheral blood in cancer patients holds great promise and various techniques have been devised in detection and molecular characterization of CTCs. Clinical application of CTCs analyses has also been discussed in various studies in the context of predicting prognosis and selecting the most efficient therapy in dealing such patients. Klaus P and Catherine A-P in their article reviewed the various techniques in detection and molecular characteristics of CTCs. Sensitive and specific analytical methods are usually combined with enrichment procedures in the detection of CTCs. They include density gradient configuration, immunogenetic procedures with antibodies against epithelial tumor-associated antigen or the common leukocyte antigen (CD45) and filtration as well. Over the past six years Cell Search technique has gained popularity. In addition a microfluidic platform called CTC chip has also been applied to detection and analysis of CTCs [9]. The other important techniques for the detection of CTCs as has been mentioned by Klaus P and Catherien A-P is given in table 1 [9].

Table 1. Current Techniques for CTC detection.

Assay System

Source/Volume

Tumour Origin

Enrichment

Detection

Comments

CellSearchTM system[10]

Blood/

7.5 mL

Breast, colon,

prostate

EpCAM-Ab

coupled ferrofluid

Positive marker:

CK Negative marker:

CD45 Nucleus: DAPI

Semi-automated system with

FDA approval for metastatic

breast colon and prostate cancer

EPISPOT assay[14]

Blood,

BM/ 10 mL

Breast, colon,

prostate,

thyroid gland

Depletion of

CD45+ cells

Secretion of proteins:

CK19, MUC1, Cath-D

(breast); CK19 (colon);

PSA (prostate);

TG (thyroid)

Detection of viable epithelial

secreting cells; unbiased

enrichment independent of

CTC/DTC phenotype

CTC-chip microchip[11,12,13]

Blood/

0.9 mL

NSCLC, pancreatic,

breast,

prostate, colon

EpCAM-Ab

coupled

microposts

Positive marker:

CK Negative marker:

CD45 Nucleus: DAPI

High detection rate

(approximately 100%)

even in M0 patients warrants

further investigations on

assay specificity

Vita-AssayTM or

Collagen Adhesion

Matrix (CAM)

Technology[15]

Blood/

3 mL

Breast, prostate,

ovaries

Ingestion of

fluorescent

CAM fragments

(CAM+)

Positive marker: EpCAM,

ESA, pan-CK 4, 5, 6, 8,

10, 13 and 18 (CK)

Negative marker: CD45

Detection of CTCs with the

invasive phenotype in blood

MagSweeper[16]

Blood/

9 mL

Breast

EpCAM-Ab

coupled ferrofluid

Microscope visualisation

Isolation of CepCs with

a high degree of purity

ISET [17,18]

Blood/

5 mL

Breast

Size

Positive marker:

CK Nucleus:

Mayer's haematoxylin

Sensitivity threshold of

one carcinoma cell per

millilitre of blood; HER2

amplification determined

by real-time PCR on DNA

extracted from CK-positive

immunostained cells (CTCs)

collected by laser

microdissection from

selected ISET-positive filters

Laser scanning

cytometry

Maintrac1[19]

Blood,

BM/ 10 mL

Breast, lung

RBC lysis

Positive marker:

EpCAM, Negative

marker: CD45

High incidence of positive

events up to three logs higher

CTC counts than those

obtained with other

techniques warrants further

investigations on assay

specificity

Ikoniscope1

imaging system[20,21]

Blood/

1 mL

Prostate,

colon, ovaries

Ficoll-Isopaque or

filtration with

track-etched

membranes

Positive markers:

EpCAM, CK7/8 PSA

(prostate only) FISH:

chromosomes 7 and

8 Nucleus: DAPI

Two epithelial specific Abs

and FISH to detect chromosomal

abnormalities in CTCs

Ariol1 system[20,21]

Blood/

7.5 mL

Breast

RBC lysis, then

CK-Ab+EpCAM-Ab

coupled microbeads

Positive markers: CK8,

18 and 19 Negative

marker: CD45 Nucleus:

DAPI

Detection of EpCAM+

and EpCAM- CTCs

Abbreviations: EpCAM, epithelial cell adhesion molecule; Ab, antibody; CK, cytokeratin; DAPI, 40,6-diamidino-2-phenylindole; EPISPOT, EPithelial ImmunoSPOT; BM, bone marrow; MUC1, mucine 1; Cath-D, cathepsin D; PSA, prostate-specific antigen; TG, thyroglobulin; CTCs, circulating tumour cells; NSCLC, non-small-cell lung cancer; CepCs,circulating epithelial cells; ISET, isolation by size of epithelial tumour cells; RBC, red blood cell; FISH, fluorescent in situ hybridisation. ( Table courtesy to Klaus P and Catherien A-P)

The most successful technique for CTCs detection is to make use of the fact that the epithelial cells that are shed from primary lesion express the cell adhesion protein EpCAM that is absent from normal blood cell. Capturing of CTCs involves the use of antibodies against EpCAM and treating them against the blood sample of the patient. These antibodies are usually tagged with magnetic particles and they are separated by creating the magnetic field. In the final process the CTCs are distinguished from contaminated white blood cells by staining the isolated cells with antibodies against other epithelial markers like cytokeratins and CLA (CD45). This method has low yield (1CTC/ml) and purity of 0.1% [5]. CTC-chip identifies the cytokerating positive circulating tumor cells in almost all patients in different metastatic cancer patients. This method creates a median yield of 50 cells/ml and purity of 1-80% [11]. Considering the rarity of CTCs in the circulation of metastatic patients, such high yield and purity of the CTC-chip is of great benefit in early detection of micrometastases and modeling the therapeutic approach and assessing the prognosis of such patients. Removing the white blood cells from the blood sample and leaving behind the CTCs (The antibody based negative selection) has also been employed as the potential technique to isolate CTCs. A it was thought that the isolation of CTCs will not be affected by the unidentified epithelial markers on the CTCs. But due to very rare prevalence of CTCs in blood sample and inability to isolate white blood cells from the sample even with the most rigorous techniques this approach only cause very low purity of CTCs isolation[22]. Other methods without antibody usage has been devised. Filters of different pore size has also been used to isolate the CTCs. This is based on the hypothesis that CTCs are often larger in sized compared to normal leukocytes. But the tumour cells in various malignancies are of variable size and often as large as the normal cells, this technique does not offer great promise [17, 18, 23]. The other promising techniques involve the laser scanning and using nucleated RBCs to isolate CTCs [19,20,21]. Techniques like microfluidic CTCs capturing allow us to isolate the viable CTCs, that offer great promise and insight about tumor cell signaling and their drug susceptibility [1]. Various isolation techniques are responsible for the clinical and biological insight into CTCs. The pilot studies of microfluidic-based CTC detection appeared to be particularly useful to isolate the higher number of CTCs that can be monitored at various stages of the tumor progression. In initial study response to chemotherapy in various malignancies was associated with considerable decline in CTCs [1, 11]. This study leads to the conclusion that CTCs measurement with cancer therapy can guide us to determine the successful therapies more effectively. Molecular analysis of the CTCs provides the non invasive approach to the determine the genotype of the CTCs over the course of the therapy that can to determine the genetic abnormalities in response to the therapy that can ultimately leads to the drug resistance or subtherapeutic response. In some patients with localized prostate cancer CTCs were detected long before the evidence of metastases. Presence or absence of CTCs have not been correlated with different markers of tumor grading, suggesting that CTCs enumeration and quantification early may be an independent measure of vascular invasiveness. Hence in patients with borderline PSA measuring CTCs can provide us with valuable insight[1].

Low yield of isolated CTCs (median _< 1 CTC/ml) by most of the methods has limited in the use of CTC enumeration for prognostic purpose rather than measuring therapeutic response by quantitating CTCs in patients. Thus so far, monitoring CTCs to guide therapeutic response has not been established. And in metastatic patients high CTC count is associated with poor prognosis . CTC-Chip is a microfluidic device that has high yield and percentage purity. The CTC-Chip has a surface area of 970 mm2 that contain chemically active 78,000 micropost coated with EpCAM. The flow kinetics of the device allows the minimal sheer force and maximum contact between cells and microposts. This allows the isolation of EpCAM expressing CTC from leukocytes and red blood cells [13]. Nagrath S. et al in their initial cohort detected CTCs by EpCAM mediated capturing (86 cells/ml) in patients with different metastatic cancers. They followed this step with staining with an antibody to cytokeratin [11]. Scott S.L. et al[13] has focused on the detection of CTCs by chip method in patients with localized and metastatic prostate cancer because of the unique tumor marker ( PSA) and potential application of CTCs detection to the management of prostate cancer. In about half of the metastatic prostate cancer, fusion of androgen responsive TMPRSS2 promoter with ERG(an ETS family transcription Factor) occurs in chromosomal translocation. Primary prostate cancer may harbor multiple cancerous foci with distinct translocations. So the metastatic causing cells may not be identified at the time of diagnosis [24,25,26]. They made use of the available markers for prostate cancer to establish the imaging strategy for the precise, automated quantification and molecular characterization of CTCs. Instead of staining with cytokeratin, the CTCs were stained for PSA expression after being captured by microposts coated with antibody to EpCAM, that offers a signal-to-noise ratio that is ideal for optimizing image analyses. The threshold CTC number chosen to optimally distinguish between patients with prostate cancer and cancer-free males (14 CTCs/ml) may underestimate CTCs in some cancer patients with a few true PSA-positive

cells, but it ensures a low likelihood of false positives. The number of CTCs identified by PSA staining were correlating with serum PSA concentrations during longitudinal follow-up of such patients that were being treated with ADT. Hence both of these markers(CTC and PSA) reflect tumor cell response to ongoing therapy. Yet in their study CTC numbers and serum PSA concentrations among different patients were minimally correlated. In addition to the overall tumor burden measured traditionally by serum PSA, CTC enumeration can reflect tumor cell invasion and tumor vascularity. Scott S.L.[13] were able to estimate the half life of CTCs in blood circulation after surgical resection of primary tumor. They observed a very rapid decline in CTCs following prostatectomy in 6 out of 8 patients indicating that if all sources of the tumor have been removed the CTCs in the circulation would be eliminated within 24 hrs. In contrast, 2 out of 8 patients failed to show the rapid decline in CTCs in circulation and 6 out of 19 patients had a transient presence of CTCs upto 3 months depicting that the source of such cells would be extraprostatic sites that continue to shed CTCs into the circulation after the resection of primary tumor. According to them this hypothesis is consistent with the fact that the patients with apparently localized prostate cancer may have cancer cells in bone marrow, as detected visually or by R-T- PCR [27,28,29]. IT is hypothesized that site of micrometastases has been established early and if confirmed in future this hypothesis would also support the application of CTC monitoring as a marker of localized invasive disease before the establishment of the viable metastatic lesions. A fraction of prostate CTCs represent proliferative cells that raise the possibility that the CTCs may represent the proliferative state of localized and metastatic cancer [13].

In another study Scott S.L. et al demonstrated the application of a microfluidic vortex generator, the HB-Chip that uses the herringbone grooves within the transparent wall of the device to creat micorvortices that direct the target cells toward capture on the antibody-coated walls, thus allowing higher blood volume throughput and increased CTC capture efficiency and purity compared to CTC-Chip method. HB-Chip also successfully isolated CTCs from 14 of 15 (93%) with metastatic prostate cancer (median 63 cells/ml) and allows RNA-based detection of TMPRSS2-ERG fusion transcript, and shows a further improvement over previous CTC-Chip. HB-Chip has made it possible to do the morphological analysis in detail. The combined use of immunofluorescene and light microscopy-based stain alos provide identity of the so-called "double positive" cells that are positive for both epithelial and hematopoietic markers. It is thought that these ''double positive" cells have the morphology of mononuclear leukocytes that made them excluded from CTC enumeration that are thought to be "single positive" cells. Isolation of CTCs by using HB-Chip also resulted in an isolation of CTC clusters ( upto 14 CTCs) from blood sample of patients with metastatic prostate cancer. Such clusters have not been reported using immunomagnetic bead platform, may be due to inability to withstand the multiple steps associated with batch purification and they were not evident when CTC-Chip method was used, as the the tightly packed spacing of microposts may have prevented their passage. Such clusters of CTCs may provide the insight into the process of metastases in human cancer. Circulating CTC clusters could constitute microemboli breaking off from primary site or they can result from intravascular proliferation of the tumor cells (Proliferating CTCs). CTC clusters have potential to lodge in distal capillary beds and initiating the metastatic lesion [30].

Okegawa T. et al in their study tried to determine if CTCs can predict the PSA failure in metastatic prostate cancer before anti-androgen therapy. They used immunogenetic quantification for CTC enumeration (Cell Search Method). They also compared CTC count with other prognostic factors in such patients. CTCs enumeration by cell search method have shown prognostic significance. They utilized PC3 (PSA nonexpression cells), LNCaP(PSA expression cells) and DU145 (PSA nonexpression cells) They Detected approximately 69% PC3, 95% LNCaP and 89% DU145. As cells search system detected PSA nonexpression prostate cancer cells, this is believed to be superior to the serum PSA value. Patients with CTCs < 5/7.5 ml blood had a median androgen deprivation responsiveness time of longer than 32 months while it was 17 months in patients with CTCs > 5 or more. In their study the Gleason Score, PSA nadir (cutoff value of 0.2ng/ml) retained their prognostic significance that was determined by multivariate analysis. This study suggested that CTCs in patients with hormone naïve metastatic prostate cancer is relatively associated with short androgen responsiveness. So CTCs may be independent predictor of androgen deprivation responsiveness in such patients [31].

Murray N.P et al determined the frequency of primary circulating prostate cells with prostate cancer ct the time of diagnosis, relation with pathological staging, association with micrometastases and subclassification with CD82. They used immunocytochemistry to detect PSA positive and CD82 positive clone by developing antibodies to the expressed markers. They suggested that at the time of diagnosis it is possible to predict the patient that would be micrometastases negative with sensitivity of 87 and specificity of 74. They also identified the expression of CD82 on prostate cancer cells that can determine that the patients with CD82 expression of cells are less likely to have bone marrow micrometastases . It was previously thought that CD82 expression is responsible for the inhibition of the metastases development whereas they determined that it only inhibits the bone marrow implantation. Thus CD82 expression on circulating cells can guide the therapeutic approach and timing in such patients. The prognostic factors for extra- capsular extension are serum PSA, gleason score and clinical staging. The CPC (circulating prostate cells)detection and subclassification to CD82 positive and CD82 negative is independent of other prognostic factors and can predict the presence or absence of micrometastases. Such findings can be utilized to determine the patients that would be benefitted with radical prostatectomy. Further the patients with CPCs negative for CD82 should be evaluated micrometastases presence and adjuvant therapy [32].

Ali A. et al determined the incidence of CTCs in patients with low volume prostate cancer (less than 0.5 cc) the CTCs were isolated using cell search system. Anti CD45 antihuman immunoglobulins enriched system and fluorescence microscopy was utilized. They found out that the patients with CTCs had detectable PSA levels with and without transient PSA spikes. This study also provided the evidence that circulating epithelial cells might be used for follow-up of patients with low volume prostate cancer. However this did not predict PSA failure in their patients. Further they also predicted that combining CTC detection with PSA testing may allow anticipating the biochemical recurrence in low volume and anatomically confined tumors [33]. Goodman B.O. et al used cell search method of CTC enumeration to determine to determine their significance in prediction and prognosis of metastatic prostatic cancer. They did CTC enumeration in 100 patients with castration-resistant prostate cancer and correlated them with typical tumor markers such as PSA, LDH and radiological and clinicopathological characteristics. They followed their patients for 26 months to interpret their results. They found out that CTC count gives survival prediction (p<0.002) but it also interrelated significantly with the other biomarkers of tumor progression and patient survival. They identified the new cut-off value of 4CTC/7.5 ml to correlate with the survival outcome and prediction of metastases development whereas previously it was thought to be 5 CTCs/7.5ml. Although the optimal cut-off value was never determined previously. Patients younger that 60 years or with high gleason score are more likely to have elevated CTC count indicative of the aggressive disease. They also determined that the correlation of disease extent with CTC count of 4 or greater as well as lack of surrogacy of radiographic response for survival outcomes raises the possibility that CTC monitoring could reduce the need for routine radiographic monitoring after the establishment of metastatic disease radiographically. Also CTC count of 4 or more in otherwise asymptomatic patient can replace the radiographic imaging as the presence of metastatic disease has been assured [34].

Johann S. De Bono et al did CTCs enumeration by using Cell Search System and suggested that CTCs are the independent and most accurate predictors of over survival (OS) in patients with Castration Resistant Prostate Cancer with metastases. This prospective study particularly showed that CTCs predict overall survival after initial therapy in CRPC patients with metastases. This study also suggested that if CTC counts prove to be a surrogate of outcome, this could potentially assist in guiding early discontinuation of the ineffective therapy. Discontinuation of the ineffective treatment can reduce the potential drug toxicity, can be cost effective and can allow patient to receive effective treatment. Authors have suggested using CTC count as an exploratory end point in several clinical trials of novel therapeutic agents in CRPC treatment. They themselves used this end point in clinical trials involving abiraterone acetate, an inhibitor of the enzyme CYP17 and androgen synthesis and of a human monoclonal antibody to the insulin-like growth factor-1 receptor. They envisioned that the CTC count will become a vital component to evaluate anti-tumor activity of novel agents in phase II trial to make the selection of agents for phase III more optimal. Further they also suggested that comparison of CTCs before and after therapy constitute a predictor of outcome [35, 36,37]. Olmos D. et al also evaluated the association of CTCs with before and after treatment with overall survival in patients with CRPC. They also made use of the cell search system for CTC enumeration. A CTC count of > 5 was an independent prognostic factor at all time points evaluated. Patients with baseline > 5 had shorter overall survival (OS) than patients with < 5 CTC count. They also suggested that characterizing CTC changes over the course of therapy can be useful. For instance a fall in CTC count by 30% following therapy is associated with improved prognosis compared to those with stable or increased CTC count. Early availability of CTC could decrease the use of ineffective treatments. In combination with other parameters (PSA and LDH) in a composite model, changes in CTC count more effectively be used as an intermediate end point in determining effective therapy at appropriate time [38].

Coding Mutations in the Androgen receptors (AR) have been identified in many patients with advanced prostate cancer and thought to be responsible for Castration -resistant prostate cancer (CRPC). Circulating Tumor Cells provide an excellent source to determine such mutations in patients with CRPC. Jiang Y. et al in their study utilized Cell search system for CTC enumeration in patients with CRPC and with the help of Cell search profile Kit plus Qiagen's AllPrep DNA/RNA Micro Kit for the measurement CTC count and isolation of nucleic acids. They amplified AR gene by using PCR and mutation status was analyzed by HPLC technology and direct sequencing. Out of 35 CRPC patients 20 patients showed AR mutation, 19 missense mutations, 2 silent mutations, 5 deletions and 1 insertion. Many AR mutations were identified in surgical biopsies or at autopsy and they were associated with resistance to androgen-directed therapies. They concluded that AR mutations can be identified in CTC-enriched peripheral blood samples from CRPC patients thus providing us the perspective of understanding CTCs and mechanism of tumor progression and metastasis in CRPC. They suggested that this approach may offer clinical and practical advantages over the research approaches that rely on sequential imaging or biopsies of the patients with metastases cancer [39].

Polymerase Chain reaction (PCR) amplification of the mRNA in circulating tumor cells is gaining favor in CTC enumeration and molecular characterization. PCR is more sensitive than conventional technique for CTC detection. It allows the identification of one tumor cell diluted to 1 ml of blood. Many studies suggested that PCR is more specific in detection of CTC and micrometastases in many solid tumors like melanoma, prostate and thyroid. Many studies suggested the PCR as the predictor of outcome in prostate cancer. PCR is an PCR is an in vitro method that enzymatically amplifies specific DNA sequences by using oligonucleotide primers. Tumor specific abnormalities are present in DNA or RNA of the tumor cells. PCR amplifies such abnormalities for detection, quantification and molecular characterization of Circulating Tumor Cells. Amplification of the mRNA is many occult malignancies involve the use of Reverse Tnanscriptase- PCR (RT-PCR). This technique is mainly applied for the detection of CTCs and micrometastases. This technique makes use of the fact that many malignant cells expresses the tissue specific markers e.g. PSA has been useful marker in detection and monitoring of Prostate cancer. PCR technique is better than Protein-based assay. RNA is very unstable in extracellular environment and its detection should indicate the presence of tumor cells in examined tissue. Protein-based assay are not capable of single molecular detection of mRNA as well as by PCR technique tissue specific mRNA can indicate the presence of tumor cells even if the protein-based assay showed negative result. For example PSA mRNA transcripts have been detected in patients with poorly differentiated carcinoma cells that do not express PSA proteins. Various markers have been used by PCR detection of CTCs and micrometastases in prostate cancer [40,41,42].

Table 2: Prostate Specific markers For PCR

Tumor Markers

Correlation with disease Pathology

Correlation with Clinical Stage

Correlation with Disease Progression.

PSA[43.44]

Prostate Specific ,Androgen Dependant mostly, Increased in well differentiated tumor cells

Increasing tumor grading

Extraprostate extension, tumor invasion, Surgical failure, biochemical recurrence.

PSMA [45,46]

Highly Prostate Specific, downregulated by androgens, increased in poorly differentiated tumor cells.

Pathological Stage

Extra capsular extension.

PSCA [47,48]

Mainly Prostate specific, overly expressed in advance poorly differentiated, androgen independent and metastatic cancer

Increasing tumor stage and gleason score.

Extra prostatic extension, increase recurrence risk, low disease-progression-free survival.

CK19 [49]

Specific for all tumor cells with epithelial origin

Presence of distant metastases

Poor clinical outcome, distant metastases

PTHrP [50]

Increased in poorly differentiated tumor cells

Increasing tumor grade

Bone metastases, tumor invasiveness and metastatic potential

Abbreviations: PSA: Prostate Specific antigen, PSMA: Prostate specific membrane antigen, PCSA: Prostate stem cell antigen, PTHrP: Parathyroid hormone-related peptide. Table courtesy to Panteleakou et al [42].

The CTCs detected by RT-PCR can help in detecting the patients with localized prostate cancer and can help to monitor the disease activity in patients with metastatic disease. Various studies has been done using RT-PCR technique to detect localized and metastatic prostate cancer and its molecular characterization.

Table 3: Detection of circulating tumor cells and bone marrow micrometastases in prostate carcinome.

Study

Markers

Sample

Localized PC*

Metastatic PC**

Katz et al. [43]

PSA mRNA

Blood

25/65(38%)

14/18 (78%)

Israeli et al. [52]

PSA mRNA

PSMA nRNA

Blood

Blood

0/18(0%)

13/18(72%)

6/24(25%)

16/24(67%)

Seiden et al. [54]

PSA mRNA

Blood

3/41(7%)

11/35(31%)

Ghossein et al. [51]

PSA mRNA

Blood

4/25(16%)

26/76(34%)

Sokoloff et al. [55]

PSA mRNA

PSMA mRNA

Blood

Blood

43/69(62%)

12/69(17%)

29/33(88%)

13/33(39%)

Corey et al. [53]

PSAmRNA

Blood

Bone marrow

12/63(19%)

45/63(71%)

6/13(46%)

10/13(77%)

Wood et al. [56]

PSAmRNA

Bone marrow

39/86(45%)

--

Gao et al. [57]

PSA mRNA

Blood

25/84(30%)

3/8(37.5%)

Ellis et al. [58]

PSA mRNA

Blood

13/75(17%)

--

Ennis et al. [59]

PSA mRNA

Blood

55/201(27%)

--

*Localized PC includes stage A, B(Clinically organ confined disease only)

**Metastatic PC includes patients with stage D1-D3 disease (D1, pelvic lymph node metastases; D2, distant metastases without prior hormonal therapy; D3, D2 disease refractory to hormonal therapy) in all the listed studies except in the study by Israeli et al. In this study, 3 patients with D0 disease (elevated serum tumor markers only) were also included as metastatic PC.(Table Courtesy to Ghossein RA et al. [40].

Reverse transcriptase - polymerase chain reaction for prostate specific antigen has also been used for molecular prognosis of prostate cancer. Positive RT-PCR correlates with poorer failure free survival. Katz et al. first demonstrated the utility of the RT-PCR in making decision for radical prostatectomy. Patients with positive RT-PCR were considered to be the poor candidates for radical prostatectomy [43]. RT-PCR for PSA mRNA has also been successful in detecting occult malignancy in bone marrow and lymph nodes and this technique has been proved to be more sensitive than the conventional techniques like immunohistochemistry and standard histopathology. This is particularly useful in determining the lymph node metastases in localized disease [53, 56 ]. Panteleakou Z. et al suggested that RT-PCR detection of CTCs in prostate cancer can be used as an additional tool that can be used in cases of "high probability for systemic disease", such as in patients with gleason scores of 7-10, PSA 20 ng/ml or more or "suspicious uptakes" on bone scans. Further PCR technique using various prostate specific markers mRNA can help to select for systemic therapy, an adjuvant or neoadjuvant approach to apparently localize prostate cancer. Further it can help to determine other therapies for cure like radical prostatectomy and/or radiotherapy [42].

Table 4: Molecular Prognosis in Prostate Carcinoma using Reverse Tnanscriptase-Polymerase Chain Reaction for Prostate Specific Antigen.

Study

Patient Population

Sample

End Point

Univariate RR

P Value

Olsson et al. [61]

Localized PC

Blood

Failure Free Survival

3.6

0.0286

Wood et al. [56]

Localized PC

Bone marrow

Failure Free Survival

NA

0.004

Gao et al. [57]

Localized PC

Blood

Failure Free Survival

NA

0.598

Ghossein R.A. et al.[60]

Metastatic AIPC

Blood

Overall Survival

2.25

0.028

AIPC: androgen independent prostate carcinoma, NA, not available, PC prostate cancer, RR, relative risk, Failure was defined as serumprostatic specific antigen (PSA) >0.2 ng/ml on one occasion after radical prostatectomy(RP) in the study by Olsson et al.[61] and on two occasions in the study by Gao et al. [57]. Failure was defined as serum PSA >0.4 ng/ml or local recurrence on digital rectal exam after RP in the study by wood et al [56]. In these studies, samples for RT-PCR were taken from patients before RO. Although no RR was given in the study by Wood et al. RT-PCR positively did correlate with poorer failure-free survival. Only those studies using Kaplan-Meier survival analysis are included in given table. Table courtesy to Ghossien et al.

Mostret B. et al. suggested that establishing miRNA in circulation (cell free or on CTCs) can offer great advantage than determining it on primary tumor tissue. miRNA profiling on Circulating tumor cells can guide the therapeutic approach and can help to determine the molecular prognosis of patients with solid tumors. Identifying miRNA can help to stratify patients according to the prognosis. Not all miRNA can be measured in peripheral circulation. Since at least 100 different mRNA circulate in blood, measuring cell free miRNA is highly likely to produce false-negative results. Measuring cell free miRNA may not be reliable to represent metastatic or localized disease, Hence CTCs are preferred for miRNA detection and molecular analysis. Further miRNA has a benefit to correlate a miRNA signal to a CTC count that can aid in the interpretation of epithelial specificity [62]. TMPRSS2:ERG fusion gene in circulating tumor cells (CTC) of patients with prostate cancer has a potential in monitoring tumor metastases. Mao X. et al analyzed the frequency of TMPRSS2:ERG and TMPRSS2:ETV1 transcripts in 27 prostate cancer biopsies from prostatectomies and TMPRSS2:ERG transcripts in CTC isolated from 15 patients with advanced androgen independent disease using RT-PCR. FISH was analyzed to genomic truncation of ERG, which is the result of TMPRSS2:ETV1. By FISH they analyzed chromosomal rearrangements affecting the ERG gene in 6 of 10 CTC samples, including 1 case with associated TMPRSS2:ERG fusion at the primary site. However TMPRSS2:ERG transcripts were not detected in any of 15 CTC samples, including 10 cases analyzed by FISH. They concluded in this study that detection of genomic truncation of ERG gene by FISH analysis could be useful for monitoring the appearance of CTC and can detect the early signs of prostate cancer metastases [63].

Several physical properties distinguish TCCs from normal blood cells. These include the larger size of most epithelial cells and difference on charge, density, migratory properties and cell specific properties. Difference in cell size has also been used in devising a way for CTC isolation and enumeration. Isolation of CTCs due to their increased size, compared with leukocytes has been applied using several different filtration processes [64]. Lin H.K. et al reported the development of novel parylene membrane filter-based portable microdevice for size based isolation with high recovery rate and direct on-chip characterization of captured CTC from human peripheral blood. The filter-based microdevice is both a capture and analysis platform, capable of multiplexed imaging and genetic analysis. Such microdevice has the potential to enable routine CTC analysis for effective management of cancer patients. Microfabricated parylene membrane filter device with a simple manual syringe injection system capture CTCs directly from peripheral blood with minimal processing and is capable of >90% recovery with high enrichment factor. They suggested that this system has a superior recovery rate compared to Cell Search System. And CTC recovery seems superior to affinity based microchip system. They proposed that this portable-filter based CTC enrichment microdevice could provide the cost-effective method to detect CTC with high recovery rate to assess metastases and monitoring the therapeutic response in such patients. Further, since almost all solid tumor cells are larger than normal cells of the blood, this microdevice should be used broadly than affinity based methods. This system can help to carry out repeat tumor biopsies in patients undergoing therapy with simple blood test. This device provides a single-station capture, enrichment and molecular analysis tool for CTC characterization that can help to identify the therapeutic targets directly on the captured CTC [65].

Fig. 1. Illustration of device assembly. A, schematic drawing of a functional microdevice consists of parylene membrane filter sandwiched between rectangular PDMS slabs and clamped in between acrylic jigs with inlet and outlet for syringes. B, bright field image of an optically transparent parylene filter with uniformly shaped and spaced 8-μm pores. C, scanning electron microscope image of single cultured tumor cell captured on the membrane.(Courtesy to Lin H.K. et al)

Fig 2.Histogram showing performance comparison of membrane microfilter versus CellSearch assay in clinical samples. Solid and striped bars, number of CTCs detected using the commercially available CellSearch assay and the microdevice, respectively. The numbers of CTC-positive samples were 27 versus 14 (microdevice versus CellSearch) of 28 patients for prostate cancer, 10 versus 4 of 12 patients for colorectal cancer, 11 versus 6 of 11 patients for breast cancer, and 3 versus 2 of 6 patients for bladder cancer. Fig. courtesy to Lin H.K. et al [65].

Mostly, the techniques used for CTC isolation cannot capture live CTCs and mostly apply to tumor of epithelial origin. Tong Xu et al developed a novel platform that measure telomerase activity from live CTCs captured on parylene-C slot microfilter. It is a low pressure system that is capable of cell capture form 1 ml blood in less than5 minutes. It provides 90 % capture efficiency, 90% cell viability and 200-fold sample enrichment. Telomerase activity is the well recognized universal cancer marker. It was detected by using quantitative PCR from as few as 25 cancer cells added to 7.5 ml blood sample and captured on microfilter. They used qPCR-TRAP that can amplify the telomerase activity signal as few as one cancer cell and CTC-telomerase combination can be very useful in early detection of occult malignancy. They suggested that this platform can be used in advance research and improved patient care [66].

Jason P. et al demonstrated that Geometrically-enhanced differential immunocapture(GEDI) and an antibody for prostate specific membrane antigen(PSMA) can be used for high-purity capture of circulating tumor cells from peripheral blood of patients with castrate-resistant prostate cancer.This can be used for high efficacy capture of PCTCs. Leveraging the fluid mechanical design to control cell transport combined with high antibody specificity to target cell has shown exceptionally high cell capture efficiency and capturing of highly pure cell population(40% improvement compared to other techniques). This technique can potentially help in capturing PCTCs to study molecular mechanism of clinical drug resistance, individual patient's CTC profiling to generate subject specific chemotherapies and real-time monitoring of their efficacy by using non-invasive blood-based assay [67].

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