The added value of circulating tumor cells examination in ovarian cancer staging

Introduction

Ovarian cancer (OC) is the second most common malignancy of the female reproductive system in Western civilized countries [1]. Delayed diagnosis of (OC) is usually a cause of its high mortality. OC counts for one of the most aggressive gynecological malignancies. Only 25% of OC are diagnosed while the malignancy is still confined to the ovary, and the cure rate in these patients can reach 90%. If the disease spread already by the time of diagnosis, the probability of cure is really low [2]. For that reason, noninvasive biomarkers may be used to help with diagnostic and treatment decisions to ensure optimal patient management in OC.

Circulating tumor cells (CTCs) have demonstrated predictive and prognostic value in patients with metastatic breast, prostate, and colorectal cancers [3-5]. Results from prospective trials in each of these cancer modalities showed that patients with elevated CTCs at any time during the course of the treatment had a significantly shorter progression free survival (PFS) and overall survival (OS) than patients without CTCs in their peripheral blood [6-9]. Several studies have been conducted so far to assess CTCs in a similar context in OC. The results are summarized in Romero-Laorden review and suggest that CTCs and DTCs are associated with poor clinical outcomes in OC [10]. To implement CTCs examination as a prognostic tool in the clinical setting further multi-center clinical studies need to be conducted and at least two CTC-isolation methods need to be compared side by side to confirm their CTCs capture efficiency. Our study examined CTCs incidence and their association with clinicopathological characteristics of OC in patients with newly diagnosed or previously non-diagnosed OC at the time of surgical intervention. The objective of this study was to isolate and cultivate CTCs in OC patients by a recently introduced size-based separation method. The presence of CTCs was further evaluated in relation to CA125 values, the presence of ascites, age and other factors.

The focus was on the relation between CTCs and CA125. CA125 is the gold standard tumor marker in OC. Serum level of CA125 is used to monitor response to chemotherapy, relapse, and disease progression in OC patients. Given this, it seems reasonable to examine if CA125 may be used as a prognostic indicator in correlation to CTCs. CA125 level less than 35 U/mL is now considered normal [11,12]. When stratified by disease stage, elevated levels were found in more than 90% of patients with advanced stage ovarian cancer but in only 50% of patients with stage I disease [15-17]. Most reports indicate that a rise in CA125 level precedes clinical detection by about 3 months [18]. Despite the limitations in the interpretation of a solitary CA125 value, this biomarker is widely used to prospectively evaluate therapeutic efficacy and monitor disease status among ovarian cancer patients [19,20].

CA125 antigen is a serum marker which has been sufficiently well validated to be of use in routine clinical care [14]. We expect that based on further clinical studies, CTCs may become as important as CA125 in OC diagnosis and treatment? We hypothesized that CTCs and CA125 are independent factors in OC patients, adding new information values to OC staging. If this hypothesis is confirmed, CTCs may become a standard in OC management algorithm.

Materials and methods

Patients: To date 118 patients with diagnosed OC have been enrolled in the study in accordance with the Declaration of Helsinki. All patients were candidates for surgery or surgical diagnostics. Based on informed consent, clinical data were collected from all participating patients. The patient stage characteristics are shown in Table 1 and Figure 1. For each patient, approximately 8 mL of venous blood was drawn from the antecubital veins and placed into S-Monovette tubes (Sarstedt AG & Co., Numbrecht, Germany) containing 1.6 mg EDTA/mL blood as an anticoagulant. The samples were processed at room temperature using an isolation procedure completed within 24 hours after the blood draw.

CTCs enrichment and culture: A new size-based separation method for viable CTC enrichment from peripheral blood has recently been introduced (MetaCell^®, MetaCell s.r.o., Ostrava, Czech Republic) [20,21]. The size-based enrichment process is based on the filtration of peripheral blood through a porous polycarbonate membrane (with pores of 8 μm diameter). The minimum and maximum volume of the filtered peripheral blood may be adjusted up to 50 mL of fluid. The standard 8 mL peripheral blood sample from patients suffering from OC was transferred into the filtration tube. Gradual transfer of the blood in several steps is preferred to prevent blood clotting on the membrane filter. The peripheral blood flow is supported by capillary action of the absorbent touching the membrane filter. The filtered CTCs were observed immediately after filtration on the membrane. The control of CTCs presence immediately after isolation eliminates false negative results of the examination. The membrane filter is kept in a plastic ring that is transferred into the 6-well cultivation plate, 4 mL RPMI media is added to the filter top and CTCs are cultured on the membrane in vitro under standard cell-culture conditions (37°C, 5% atmospheric CO₂) and observed by inverted microscope (Figure 3). The CTCs were grown in FBS-enriched RPMI medium (10%) for a minimum of 3-14 days on the membrane. The cultured cells were analyzed by means of vital fluorescent microscopy using unspecific nuclear (NucBlue^TM) and cytoplasmatic (Celltracker^TM) stain and by histochemistry (May-Grunwald staining).

Alternatively the enriched CTCs fraction can be transferred from the membrane and cultured directly on any plastic surface or a microscopic slide. Microscopic slide culture is preferred if immunohistochemistry/immunofluorescence analysis is planned. If an intermediate CTCs-analysis is awaited, the CTCs-fraction is transferred in PBS (1.5 mL) to a cytospin slide. The slide is then dried for 24 hours and analyzed immunohistochemically.

Results

We report successful CTCs isolation in 77 out of 118 patients with OC (65.2%), capturing cells with proliferation potential in different histology OC subtypes. The cells enriched by size-based filtration remained in good fitness, unaffected by any antibodies or lysing solutions, which enabled theirs in vitro culture. The CTCs were cultured in vitro in short-term cultures (3-5 days) for further downstream applications.

CTCs and clinicopathological criteria

The frequency of CTC positivity is summarized for different patient subgroups in Tables 1 and 2. As next CTC presence is put into correlation with other clinicopathological criteria in Table 3. Interestingly, under all the more serious disease conditions like higher FIGO stages, higher Grade, CTCs can be detected in more than 2/3 of the patients (more than 60% of tested patients are CTC-positive).

Table 2.

Clinicopathological characteristics and CTCs presence

		%	CTCs positive	%	CTCs negative	%	P-Value
CTC (N)	118
CTC positive	73	61.8
CTC negative	45	38.1
CA125 (N)	85
CA125 (1)	58	68.2	36	62.1	22	37.9
CA125 (0)	27	31.8	14	51.9	13	48.1	0.3729
Ascites (N)	112
Ascites (1)	53	47.3	39	73.6	14	26.4
Ascites (0)	59	52.7	31	52.5	28	47.5	0.0216
Peritoneal carcinomatosis (N)	110
Peritoneal carcinomatosis (1)	61	55.5	43	70.5	18	29.5
Peritoneal carcinomatosis (0)	49	44.5	25	51	24	49	0.0367
Lymph node tested (N)	80
Lymph node involvement (1)	23	28.7	16	69.6	7	30.4
Lymph node involvement (0)	57	71.3	33	57.9	24	42.1	0.3322
Residual disease (N)	100
Residual disease (1)	56	56	42	75	14	25
Residual disease (0)	44	44	24	54.5	20	45.5	0.0321
Ascites (1) (N)	53
Peritoneal carcinomatosis (1)	48	90.6	34	70.8	14	29.2
Peritoneal carcinomatosis (0)	5	9.4	3	60	2	40	0.6156

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Table 3.

Correlation analysis of CTCs and other clinicopathological characteristics. Pearson correlation coefficients in the table are shown if reaching the level of significance (p< 0.05)

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As shown in Table 2, CTCs were found in 30% of OC patients diagnosed with lymph node involvement. No significant correlation between CTC and LN-involvement has been reported in our OC group. 73.6% of the patients with ascites were CTC-positive. Up to 70% of OC patients with peritoneal carcinomatosis were CTC-positive. Patients with residual disease after surgery were also CTC-positive in 75%. Ascites was observed in 70% (48 patients) out of group of patient with peritoneal carcinomatosis data. As next, significance has been shown for correlation of CTCs and ascites, CTCs and peritoneal carcinomatosis, CTCs and residual disease.

In the context of CTC and CA125 testing the patients were divided into two subgroups based on the relative CA125 values. Only relative numbers for CA125 were taken into the analysis (elevated CA125=1, normal CA125=0). The results report that CTCs and CA125 behave like two independent biomarkers in the presented study. The independence has been confirmed by Chi-square test and correlation analysis (Pearson correlation coefficient) (Table 3). Pearson correlation coefficients for CTCs and other clinicopathological characteristics are shown only if reaching the level of significance (P<0.05). Another result of interest shown in the Table 3 is the correlation of peritoneal carcinomatosis and residual disease with elevated CA125 values.

Additionally, cluster analysis of the tested clinicopathological parameters has been provided to demonstrate how different the group of OC patient undergoing surgery may be. Clearly there are 4 main clusters of the OC patients (see Figure 4A). There is a relatively big subgroup exhibiting no prognostically negative clinicopathological characteristic except of CTC-presence (3^rd cluster from the left). Similarly, there is a cluster defined by peritoneal disease only (1^st cluster from the left).

Figure 4.

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If as next variable CA125 (new CA125=relative CA125 value) is added to the cluster analysis, the subgroup of patients can be divided clearly into 2 distinct subclusters (Figure 4B). The first one is represented by elevated CA125 values, the second one is represented only by CTC presence. These facts may be used for better clinicopathological characterization of the OC patients, dividing them into the subgroups with hematogenous spread only and peritoneal spread only.

The presented data could support the hypothesis that the elevated CA125 levels are indicators of peritoneal OC spread in general. On the other site, CTC-presence is representing another type of metastatic spread-a hematogenous. As independence of CTC and CA125 has been shown (additionally on Figures 5, 6 and 7), we may conclude that the both CTCs and CA125 tests may have its own role in the OC disease management.

Figure 5.

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Figure 6.

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Figure 7.

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Discussion

The theory of metastatic process is based on the fact that single tumor cells disseminate from the primary tumor either locally, leading to local metastases or through lymphatic vessels to regional lymph nodes or hematogenously to distant organs. Accurate diagnosis of disease stage can greatly influence the treatment modus in OC as well. It was believed that OC metastasizes via a passive mechanism by which ovarian cancer cells are shed from the primary tumor and carried by physiological movement and peritoneal fluid to the peritoneum and omentum. The discovery of hematogenous metastasis of OC via CTCs initiated rethinking of possible clinical implications of these findings for OCmanagement [23].

Patients with cancers that disseminate predominantly through the blood, such as breast, lung and prostate cancers, have substantially higher numbers of detectable CTCs [24]. So, it was surprising that the majority of patients (65.2%) investigated within planned surgical treatment in this study had detectable CTCs in their blood given that OC does not predominantly metastasize through the bloodstream.

Currently, no consensus on clinical utility has been reached for CTCs in OC patients. As reported in a review by Romero, more prospective validation and uniform methodology for CTCs detection is needed [10]. The CTC-examination may offer clinicians a more reliable method to identify disease aggressiveness earlier. The multicenter, randomized, exploratory study reported findings of patients with relapsed/recurrent advanced OC. Patients with baseline ≥2 CTCs had significantly shorter time to disease progression and shorter overall survival time than patients without CTCs in their blood. This was independent of baseline CA125 value, platinum therapy status, tumor cell type and grade, prior taxane treatment, ECOG score, largest size and number of lesions, presence of ascites, race, and age. These data demonstrate that the prognostic value of CTCs is independent of established factors confirming their biological significance [25]. The independence of CTCs information on other clinicopathological characteristics (e.g. lymph node involvement and CA125 values) was shown by our study as well.

Prognostic significance has been confirmed for CTCs in several studies using different CTC-separation techniques [26-28]. Braun et al. observed a decreased overall survival as well as an increased rate of early, distant metastatic disease in patients with OC and DTCs detected in bone marrow biopsy [29].

Taken together CTCs may represent an opportunity to assess cancer spread directly and earlier than recent methods, which classify tumor growths in general. A functional methodology to harvest separated tumor cells from blood may provide researchers with a population of viable and proliferating cells to examine gene expression profiles or gene mutations in cancer.

Differences in isolation techniques and early disease stages make it difficult to report on recommendations resulting from presented OC-CTC studies. Nevertheless, the detection of CTCs is often hampered by the heterogeneity of the primary tumor and by the loss of epithelial antigens as occurs during epithelial to mesenchymal transition [30]. EpCAM (epithelial cell adhesion molecule) is definitely not a perfect marker for CTCs detection due to the high variation in its gene expression between tumor subtypes and its illegitimate transcription from leukocytes [31]. Furthermore cytokeratins, generic markers for epithelial CTCs, are also found on healthy epithelial cells released into circulation as well as on a subpopulation of granulocytes [32,33]. For this reason, testing of other tumor-associated genes has been introduced into the CTCs evaluation process through our study. We were able to identify expression of tumor-associated genes on captured CTCs and confirm cancer origin of the captured cells by gene expression analysis.

As a result, even if the captured enriched CTC-population is contaminated by white blood cells, these can be eliminated by short-term in vitro culture. If still some white blood cells survive the short term in vitro culture, they may be identified by cytomorphological and gene expression analysis. Similarly, higher expression of tumor-associated genes can be detected if more cancerous cells are present in the enriched cell fraction used for testing.

Interestingly, under all the more serious disease conditions like higher FIGO stages, higher Grade, CTCs were detected in more than 2/3 of the patients (more than 60% of tested patients were CTC-positive) in our study.

Our study results suggest that MetaCell^® techniques may be used to capture, identify and enumerate CTCs in patients with OC. This may provide useful additional and independent prognostic information independently on standard CA125 biomarker. As concluded from our data elevated CA125 could characterize peritoneal spread of the OC. Then CTCs could be a sign of hematogenous spread solely. From that context there is an extremely interesting group of patients who do not display CA125 elevation and are CTC positive.

The ability to isolate CTCs at an early disease stage and independently from epithelial antigens is an important improvement. The treatment of OC is increasingly likely to evolve into a more individualized approach, based on a better understanding of the molecular composition of each patient’s tumor. In this context, easy access to CTCs, both at the time of diagnosis and relapse, should be an increasingly valuable tool in future years.

Our methodological approach may provide an opportunity for more personalized medicine where treatment for OC may be guided by information from an individual’s CTCs molecular profile.

Disclosure of conflict of interest

None.