Detection of Circulating Tumor Cells in Patients With Adrenocortical Carcinoma: A Monocentric Preliminary Study
Pamela Pinzani,* Cristian Scatena,* Francesca Salvianti, Elisa Corsini, Letizia Canu, Giada Poli, Milena Paglierani, Valentina Piccini, Mario Pazzagli, Gabriella Nesi, Massimo Mannelli, and Michaela Luconi
Clinical Biochemistry (P.P., F.S., M.Paz.) and Endocrinology (E.C., L.C., G.P., V.P., M.M., M.L.) Units, Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence 50139, Italy; Division of Pathological Anatomy (C.S., M.Pag., G.N.), Department of Surgery and Translational Medicine, University of Florence, Florence 50139, Italy; and Istituto Toscano Tumori (M.M.), Florence 50139, Italy
Context: Adrenocortical carcinoma (ACC) is a rare malignancy, the prognosis of which is mainly dependent on stage at diagnosis. The identification of disease-associated markers for early diag- nosis and drug monitoring is mandatory. Circulating tumor cells (CTCs) are released into the blood- stream from primary tumor/metastasis. CTC detection in blood samples may have enormous po- tential for assisting in the diagnosis of malignancy, estimating prognosis, and monitoring the disease.
Objective: The aim of the study was to investigate the presence of CTCs in blood samples of patients with ACC or benign adrenocortical adenoma (ACA).
Setting: We conducted the study at a university hospital.
Intervention: CTC analysis was performed in blood samples from 14 ACC patients and 10 ACA patients. CTCs were isolated on the basis of cell size by filtration through ScreenCell devices, followed by iden- tification according to validated morphometric criteria and immunocytochemistry.
Main Outcome Measure: We measured the difference in CTC detection between ACC and ACA.
Results: CTCs were detected in all ACC samples, but not in ACA samples. Immunocytochemistry confirmed the adrenocortical origin. When ACC patients were stratified according to the median value of tumor diameter and metastatic condition, a statistically significant difference was found in the number of CTCs detected after surgery. A significant correlation between the number of CTCs in postsurgical samples and clinical parameters was found for tumor diameter alone.
Conclusions: Our findings provide the first evidence for adrenocortical tumors that CTCs may represent a useful marker to support differential diagnosis between ACC and ACA. The correlation with some clinical parameters suggests a possible relevance of CTC analysis for prognosis and noninvasive mon- itoring of disease progression and drug response. (J Clin Endocrinol Metab 98: 3731-3738, 2013)
A drenocortical carcinoma (ACC) is a rare and very aggressive endocrine tumor with a poor prognosis, mainly dependent on tumor stage at diagnosis. Early di-
agnosis followed by surgical tumor removal, possibly as- sociated to adjuvant mitotane therapy (1), has been proven as the best option for ACC treatment. The mean
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A.
Copyright @ 2013 by The Endocrine Society
Received February 14, 2013. Accepted June 26, 2013.
First Published Online July 8, 2013
* P.P. and C.S. contributed equally to the work. Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; CTC, cir- culating tumor cell; EpCAM, epithelial cell adhesion molecule; SF-1, steroidogenic factor 1; TBS, Tris-buffered saline.
5-year survival rate ranges between 16 and 38%, although it drops to less than 10% in metastatic disease (2, 3). Con- sidering that an early diagnosis is pivotal to the prognosis, the identification of sensitive, specific, and noninvasive biomarkers is mandatory to significantly improve the clin- ical management along with the survival rate and quality of life of ACC patients. The best biomarkers should not only be able to discriminate between benign and malig- nant adrenocortical masses, but also to provide prognostic penetrance, enabling noninvasive follow-up once the tu- mor has been surgically removed. Detection of circulating tumor cells (CTCs) in peripheral blood is a reliable tool for prognosis and follow-up in several solid cancers (4, 5), including rare tumors of neuroendocrine origin (6). CTCs are neoplastic cells originating from either primary tumor or metastases, and they circulate freely in the peripheral blood of cancer patients (4, 7). Tumor-induced angiogen- esis and invasion processes allow localized tumors with high invasive potential to release CTCs into peripheral circulation before any detectable metastasis is established. CTC detection may therefore have enormous potential in diagnosing malignancy, estimating prognosis, and moni- toring disease recurrence and response to anticancer ther- apy (8).
No attempt has so far been made to detect and char- acterize CTCs in blood samples of patients affected by ACC or adrenocortical adenoma (ACA).
Isolation of CTCs from the other circulating elements can be achieved with various methods (5), either immu- nological or physical. Immunological techniques are based on the separation of CTCs through their expression of epithelial cell-specific markers (epithelial adhesion mol- ecules, such as epithelial cell adhesion molecule [EpCAM]) or tumor-specific markers (5, 9). Physical methods are based on cell separation according to size or migration along a density gradient. Among them, blood filtration allows CTC isolation on the basis of their larger size over other blood cells. The latter method has the advantage of isolating intact CTCs, but needs further morphological analysis to identify CTCs, whereas immunocytochemistry is recommended for cell origin characterization. This tech- nique shows high sensitivity, detecting even 1 single tumor cell from 1 mL of blood in a background of 106-107 nor- mal blood cells (10).
In this study, we evaluated CTC presence in blood sam- ples of 14 patients with ACC and 10 patients with ACA using a cytomorphological technique based on filtration, specifically the ScreenCell device system (ScreenCell), fol- lowed by immunocytochemical characterization with the same markers employed in tumor tissue for ACC diagno- sis. Moreover, we tried to correlate the number of CTCs
detected in postsurgical blood samples with some clinical parameters of ACC.
Patients and Methods
Patients
All patients gave their written informed consent to the study, which was approved by the Local Ethical Committee. The study includes 24 patients evaluated at our university hospital for ad- renocortical tumors (14 ACC and 10 ACA).
Blood sample collection
In each patient, 6 mL of blood were collected in EDTA tubes. Sampling was performed before surgery (n = 3 ACC and 10 ACA patients) or at different time points during postsurgical fol- low-up (n = 14 ACC and 2 ACA). All blood samples were pro- cessed within 3 hours after collection and then evaluated for CTC presence.
CTC analysis
CTC analysis was performed through 3 sequential steps con- sisting of isolation from blood by filtration on ScreenCell Cyto filtration devices, followed by CTC identification through vali- dated morphometric criteria (10, 11), and finally identification of cell origin by immunocytochemistry using antibodies against adrenocortical markers.
1. Isolation
Blood was filtered by the ScreenCell Cyto filtration devices according to the procedure previously described (12). Briefly, before filtration and in order to lyse red blood cells, 3-mL blood samples were diluted in 4 mL of a specific dilution buffer for fixed cells (ScreenCell FC dilution buffer; ScreenCell). After filtration, an additional 1 mL of PBS was filtered to remove red blood cell debris. Filtration was usually completed within approximately 50 seconds. The filter was then disassembled from the filtration module and allowed to air dry. For each patient’s blood sample, filtration was performed in duplicate.
2. Identification
Cytological studies, including staining and immunocyto- chemistry, were conducted directly on the filter. The track- etched filters were stained with hematoxylin solution S (Merck KGaA), applied to the membrane for 1 minute, and Shandon eosin Y aqueous (Thermo Electron Corporation, Thermo Fisher Scientific Inc) for 45 seconds. For microscopic observation, the ScreenCell Cyto filter was placed on a standard microscopy glass slide, and a 7-mm circular cover slip (Menzel-Glaser) was laid on the filter with the appropriate mounting medium.
CTCs were identified according to the following morpholog- ical criteria: cell size ≥ 16 um, nucleocytoplasmic ratio ≥ 50%, irregular nuclear shape, hyperchromatic nucleus, and basophilic cytoplasm. Under these criteria, red cells and platelets were not entrapped in the filters, and leukocytes could be excluded (10, 11).
3. Cytological characterization
For immunostaining, the ScreenCell Cyto filters were hy- drated with Tris-buffered saline (TBS; pH 7.4). The excess TBS was removed with absorbent paper, and the filters were put on the paraffin film in a humid chamber. Each spot was incubated for 5 minutes at room temperature with 70 uL of permeabilizing buffer. All antibodies required heat-induced epitope retrieval, so the Metafilter spots were treated in a bath containing the Target Retrieval Solution (S2367; Dako) (pH 9.0) at 99℃ for 20 minutes.
After being washed quickly in a bath containing distilled wa- ter, each filter was incubated overnight with 70 µL monoclonal mouse antihuman MART-1/Melan A (clone A103; Ventana Medical Systems), monoclonal mouse antihuman synaptophysin (clone MRQ-40; Ventana) and polyclonal anti-steroidogenic factor 1 (anti-SF-1; catalog no. 07-618; Upstate, Millipore) an- tibodies ready to use. The filters were then washed once with TBS for 1 minute and immersed in a bath containing distilled water. Staining was achieved by treating each spot with 70 LL En Vision Detection System Peroxidase/DAB, Rabbit/Mouse (K5007; Dako) for 40 minutes at room temperature, followed by the chromogen 3.3’ diaminobenzidine (Dako) for 10 minutes at room temperature. Each filter was then placed on paraffin film, and the nuclei were slightly counterstained with Mayer’s hema- toxylin for 6 minutes. Finally, the filters were rinsed with running water and dried for 10 minutes at room temperature.
Histological analysis and immunohistochemistry of the primary tumor
Histological diagnosis was performed by the reference pa- thologist on tumor tissue removed at surgery (n = 14 ACC and
3 ACA). In 7 patients affected by nonhypersecreting adrenal incidentaloma, the diagnosis of ACA was established by com- puted tomography/magnetic resonance imaging tumor charac- teristics and unchanged imaging characteristics at least 1 year after diagnosis.
Tumor specimens were evaluated according to the Weiss Sys- tem, which combines 9 morphological parameters: 3 related to tumor structure (description of cytoplasm, diffuse architecture, and necrosis), 3 related to cytology (atypia, atypical mitotic fig- ures, and mitotic count), and 3 related to invasion (veins, sinu- soids, and tumor capsule). The presence of 3 or more criteria highly correlates with malignant behavior (13).
Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissues using antibodies directed against adrenocortical markers such as MART-1, inhibin-a, and synap- tophysin to define the adrenocortical origin of the tumor. Ki67 index was evaluated as a proliferation marker to assess ACC prognosis (14, 15). Immunohistochemistry analysis with mouse antihuman Ki67 monoclonal MIB1 antibody (Dako) was per- formed with the Ventana Benchmark XT system (Ventana Med- ical Systems). Nuclei were hematoxylin-counterstained. Ki67- positive nuclei were counted on 1000 tumor cells, and Ki67 was expressed as the percentage of proliferating cells. Negative con- trols were achieved by omitting the primary antibody.
Tumor stage was assessed according to the revised TNM clas- sification of ACC proposed by the European Network for the Study of Adrenal Tumors (16).
Statistical analysis
All data were expressed as mean ± SD and median [inter- quartile range]. Statistical analysis was performed by SPSS ver-
| Mean + SD, Median [Interquartile Range] | No. of Patients | % | |
|---|---|---|---|
| Age at surgery, y | 44 ± 18, 47 [25-59] | 14 | 100 |
| Sex | |||
| Male | 5 | 36 | |
| Female | 9 | 64 | |
| Adrenal | |||
| Left | 5 | 36 | |
| Right | 9 | 64 | |
| Secretion | 9 | 64 | |
| Cortisol | 6 | 67 | |
| Androgens | 6 | 67 | |
| DHEAS | 1 | 11 | |
| Progestins | 1 | 11 | |
| Tumor diameter, cm | 10.1 ± 5.7, 8.8 [5.7-14.6] | 14 | 100 |
| Ki67, % | 27.4 ± 20.7, 20.0 [12.5-40.0] | 14 | 100 |
| WEISS | 6.6 ± 1.6, 7 [6-8] | 12 | 86 |
| Stage | |||
| 1 | 2 | 14.3 | |
| 2 | 5 | 35.7 | |
| 3 | 3 | 21.4 | |
| 4 | 4 | 28.6 | |
| Metastases | Lung, liver, bone, pancreas | 4 | 28.6 |
| Surgery | 14 | 100 | |
| MTT therapy | 13 | 93 | |
| Other chemotherapies (EDP) | 5 | 35.7 | |
| Radiotherapy | 0 | 0 | |
| Follow-up from surgery, mo | 32.6 ± 20.7, 22.5 [16.2-54.0] | 14 | 100 |
| Survival | 11 | 79 |
Abbreviations: EDP, etoposide-doxorubicin-cisplatin combined chemotherapy; MTT, mitotane; DHEAS, dehydroepiandrosterone sulfate. Mean ± SD and median [interquartile range] values for the indicated parameters are reported, along with the number of patients and their percentage.
sion 18.0 for Windows (Statistical Package for the Social Sci- ences). P values of less than .05 were considered statistically significant. Univariate correlation was carried out using Pear- son’s test. Groups of data were compared using the nonpara- metric Mann-Whitney U test or Student’s t test for independent values, when appropriate.
Results
Patient characteristics
The enrolled cohort of 24 adrenal tumor patients con- sisted of 14 patients with ACC and 10 with ACA whose main characteristics are detailed in Tables 1 and 2, respectively.
Of the 14 ACC patients, 5 (36%) were male, and 9 (64%) presented a secreting ACC. Mean + SD age at diagnosis was 44 ± 18 years. Stages at diagnosis were as follows: stage 1, 2 patients (14.3%); stage 2, 5 patients (35.7%); stage 3, 3 patients (21.4%); and stage 4, 4 patients (28.6%). All pa- tients underwent adrenalectomy. After surgery, 13 (93%) were administered adjuvant mitotane therapy. Among these, five also received etoposide-doxorubicin-cisplatin combined chemotherapy. None underwent radiotherapy. Survival rate was 79% with a mean ± SD follow-up of 32.6 ± 20.7 months from surgery.
Of the 10 ACA patients, 5 (50%) were males and 3 (30%) had a cortisol-secreting tumor. Mean + SD age at diagnosis was 59 + 14 years. Adrenalectomy was per- formed in the 3 patients with cortisol-secreting tumors. The mean ± SD duration of follow-up was 29.3 ± 16.4 months after diagnosis.
Detection of circulating ACC cells
CTCs were isolated and detected in all patients affected by ACC after hematoxylin/eosin staining of filters (Figure
1, A-F). Tumor cells were observed mostly as isolated units (Figure 1). On the other hand, CTCs were not found in the blood of ACA patients.
CTCs were detected in all patients tested before sur- gery (mean CTCs/3 mL = 14.5 ± 14.6; n = 3 patients and 3 samples) and in all patients tested in the postsur- gical period (mean CTCs/3 mL = 3.9 ± 7.1; median CTCs/3 mL = 1.9 [interquartile range, 0.8-4.5]; n = 14 patients and 21 samples). The presurgery blood samples were collected at hospital recovery (12-24 h before sur- gery). The postsurgery blood samples were collected 17 ± 15 months (mean ± SD) after surgery. In 2 patients affected by stage 2 ACC and positive for CTCs shortly after surgery, CTCs were not detected in blood samples drawn 12 and 24 months after surgery, respectively.
No CTCs were detected in presurgical blood samples from any of the ACA patients analyzed (n = 10). The presurgery blood samples were collected at hospital re- covery (12-24 h before surgery) in patients who were go- ing to be operated (n = 3 patients; Table 2) or in nonop- erated patients during a control visit. In 2 ACA patients, we also obtained blood samples 2 months after surgery. These blood samples remained CTC negative.
Immunocytochemical analysis of the filters performed using antibodies against MART-1 (Figure 1G), synapto- physin (Figure 1H), and SF-1 (Figure 1I) demonstrated a marked positivity of CTCs, confirming their adrenocor- tical nature (n = 14 patients).
Surgery affects the number of CTCs
In 3 of the 14 patients analyzed, we obtained presur- gical as well as postsurgical blood samples at different follow-up times (0, 2, 6, and 12 mo). When presurgical and postsurgical samples from the same patient were com-
| Mean + SD, Median [Interquartile Range] | No. of Patients | % | |
|---|---|---|---|
| Age at diagnosis, y | 59 + 14, 65 [52-68] | 10 | 100 |
| Sex | |||
| Male | 5 | 50 | |
| Female | 5 | 50 | |
| Adrenal | |||
| Bilateral | 1 | 10 | |
| Left | 2 | 20 | |
| Right | 7 | 70 | |
| Secretion | 3 | 30 | |
| Cortisol | 3 | 100 | |
| Tumor diameter, cmª | 2.9 ± 0.9, 3.0 [2.3-3.5] | 10 | 100 |
| Follow-up surgery/diagnosis, mo | 29.3 ± 16.4, 27.5 [14.0-42.7] | 10 | 100 |
| Surgery | 3 | 30 | |
| Survival | 10 | 100 |
Mean + SD and median [interquartile range] values for the indicated parameters are reported, along with the number of patients and their percentage.
a Evaluated by computed tomography/magnetic resonance imaging scan.
A
B
C
%
·
D
E
8
p
0
G
H
pared, a statistically significant decrease in the number of CTCs was noted (Figure 2A, Student’s t test for unpaired samples; P = . 02). In 2 of the 3 patients, the CTC number considerably decreased after surgery and remained stable, whereas in the third patient, surgery did not seem to affect CTCs (Figure 2B). No significant correlation between the CTC number and the length of follow-up was evident. The characteristics of these patients are described in Supplemen-
tal Table 1 (published on The Endo- crine Society’s Journals Online web site at http://jcem.endojournals.org).
Correlation of CTC values with clinicopathological prognostic parameters
8 To ascertain any association be- tween the CTC number in postsur- gical blood samples and the main F clinicopathological characteristics of ACC patients, we performed uni- variate regression analysis between CTC number/3 mL and available pa- rameters-namely, patient age, tu- mor diameter, Ki67, stage, and Weiss score, using the first sample available at follow-up (mean ± SD = 15 ± 11 mo of follow-up). A statis- tically significant linear correlation was found only with the tumor di- ameter (R2 = 0.362; R = 0.602; P = .023; n = 14), but not with the other parameters analyzed such as Ki67 (R2 = 0.147; R = 0.384; P = . 196; n = 13), age, stage, and Weiss score (data not shown).
When patients were stratified into 2 classes according to the tumor diameter median value in the ACC cohort and to metastatic condition (stage 4), a statistically significant difference was found in the number of postsurgical CTCs. CTC mean number ± SD per 3 mL was 8.3 ± 11.2 vs 1.8 ± 2.0 (P = . 006) for tumor diameter ≥ 8.8 cm and < 8.8 cm, respectively, and was 11.7 ± 14.5 vs 2.1 ± 2.1 (P =. 031)
A
B
35
35
·- PZ n1
30
30
o-PZ n2
mean CTC number/ 3ml
PZ n3
25
CTC number/ 3 ml
25
20
20
15
15
10
10
5
5
0
0
PRE-SURGERY
POST-SURGERY
0
2
4
6
8
10
12
14
Time (months)
for stage 4 and stages < 4, respectively (Table 3). Finally, there was no statistically significant difference in the mean number of CTCs in postsurgical samples between alive and deceased patients (data not shown).
Discussion
In this study, we demonstrated the ability of the ScreenCell method to detect CTCs of adrenocortical origin dependent on cell size in blood samples from ACC patients after sur- gical removal of the tumor, with no positivity in ACA samples. Our analysis revealed that CTC positivity was found in all presurgical blood samples, as well as in all postsurgical blood samples in metastatic patients. More- over, the false-positive outcome among the benign adre- nocortical tumors was zero, thereby suggesting the high specificity and sensitivity of the method. Interestingly, CTCs were not found in ACA patients after surgery (2-mo follow-up), thus excluding that intraoperative manipula- tion of the adrenal mass may cause tumor cell dissemina- tion, as has been suggested for other solid tumors (11, 17, 18). However, in the absence of long-term follow-up, these studies failed to demonstrate any cause-and-effect relationship between surgical indirect cell dissemination and the development of metastasis. Further longitudinal studies on larger cohorts of ACC patients operated in var- ious surgical centers are needed to evaluate the clinical impact of different surgical procedures (open vs video- assisted) in shedding adrenocortical cancer cells into the circulation.
Our findings indicated that CTC retrieval from periph- eral blood by minimally invasive procedures could be a valid and sensitive marker to support the differential di- agnosis between malignant and benign adrenocortical tu- mors. The importance of this diagnostic biomarker is even more relevant in adrenocortical tumors because the prog- nosis is strictly dependent on early diagnosis. Indeed, up to now, ACC diagnosis was only possible after surgical re- moval of the mass and histological confirmation.
The ScreenCell method allows separation of CTCs from blood based on cell size and morphological criteria,
with subsequent specific characterization to identify the adrenocortical origin. We chose this method of separation to avoid CTC selection on the basis of the expression of specific markers, thus allowing the capture of all CTCs present in blood samples, irrespective of surface markers. In fact, other separation methodologies based on cell sur- face expression of epithelial markers, such as EpCAM (4, 5, 19), might underestimate CTCs derived from adrenal carcinomas, which have been demonstrated to be negative for EpCAM (20).
Immunocytochemistry performed on enriched CTCs confirmed the ACC origin because they were positive for markers routinely used for characterization of primary adrenocortical tumors (MART-1 and synaptophysin), and in particular displayed nuclear positivity for SF-1, which is strongly expressed in ACC (21) and H295R (22), with a positive correlation with tumor aggressiveness.
Metastatic cells from various tumors have often been demonstrated to express phenotypic and genotypic char- acteristics at variance with the primary tumor (9). Thus, continuous monitoring and characterization of such dif- ferences on isolated CTCs from blood samples during pa- tient follow-up may be relevant for modulating personal- ized anticancer therapies specific for metastatic rather than for the primary tumor cells (19, 23).
In metastatic patients, CTCs isolated in postsurgical blood samples are likely to derive from metastases or tu- mor recurrence. Conversely, the origin of CTCs still de- tectable in 90% of disease-free patients even after ex- tended follow-up is unclear. In breast cancer patients, tumor cell detection has been described in both blood (CTCs) and bone marrow samples even at longer fol- low-up (median, 40 mo) from primary operation (24), suggesting a long-lasting reminiscence of the bulk of cells spilled out from the primary tumor before its removal. Due to this persistent presence of CTCs in the bloodstream, it would probably be more important to evaluate over time the change in the number of CTCs, rather than the abso- lute number. In the 3 patients studied at different time points, the number of CTCs after surgery either remained stable, as in the case of stage 1 and 2 patients, or signifi-
| Tumor | Stage < 4 | Stage 4 | P | Diameter < 8.8 cm | Diameter ≥ 8.8 cm | P |
|---|---|---|---|---|---|---|
| Postsurgical CTCs, n/3 mL | ||||||
| Mean (SD) | 2.1 (2.1) | 11.7 (14.5) | .031 | 1.8 (2.0) | 8.3 (11.2) | .006 |
| Median [interquartile range] | 1.1 [0.7-3.0] | 5.8 [2.4-27.0] | 1.0 [0.5-2.3] | 3.0 [2.2-9.0] | ||
| Patients, n (%) | 10 (71) | 4 (29) | 7 (50) | 7 (50) |
Patients (n = 14) were stratified in 2 classes for stage and diameter using stage-4 or diameter median value as cutoff. Mean (SD) and median [interquartile range] values for postsurgical CTC are reported, along with the number of patients and their percentage. The range of follow-up was 2-36 months from surgery. Statistical difference between mean values in the 2 classes was evaluated using the nonparametric Mann-Whitney U test for independent values.
cantly decreased compared to presurgical samples, as in the case of the stage 4 patient. Although based on a limited number of patients, this may confirm the absence of sur- gical dissemination as well as the fact that mass removal may reduce the number of CTCs entering the blood- stream. In some patients, CTCs became undetectable dur- ing follow-up, although a significant correlation between the number of CTCs and follow-up duration could not be found.
When correlating CTC detection in postsurgical blood samples with clinical parameters of the tumor, a signifi- cant correlation was found with tumor diameter, but not with Ki67. Indeed, cell metastatic potential may be inde- pendent from the proliferative characteristics of the tu- mor, of which Ki67 can be considered a valid marker. Conversely, tumor diameter has been demonstrated as one of the best predictors of malignancy (25, 26) and an in- dependent parameter of survival. Indeed, large tumors with diameter > 12 cm have been associated with lower survival after complete resection (27). The tumor diameter consequently represents a good independent parameter to be correlated with the number of CTCs detaching from the primary mass. A significant correlation between tumor diameter and the number of CTCs has been observed in liver (28) and gastric tumors (29).
The other interesting finding is the statistically signifi- cant difference found in the mean number of CTCs in metastatic vs nonmetastatic patients. The prognostic value of CTCs has already been recognized in non-small- cell lung cancer because metastatic and nonmetastatic pa- tients significantly differed in the CTC mean number (30). A cutoff higher than 5 CTCs per 7.5 mL was the strongest predictor of overall survival on multivariate analysis in non-small-cell lung (28), breast (4), and prostate (31, 32) cancer and metastatic melanoma (33). A recent meta-anal- ysis, conducted on articles published between January 1990 and January 2012, has pointed out the clinical prog- nostic power of CTCs for overall, disease-free, and pro- gression-free survival in early and metastatic stages of breast cancer, irrespective of the CTC detection method and time point of blood withdrawal (34). However, some warnings on the prognostic potential of this new bio- marker have to be considered, due to the heterogeneity of the studies performed, characterized by intra- and inter- study variability, at least in melanoma meta-analysis (35). The clinical meaning of CTCs found in stage 1 and 2 pa- tients is at present unclear. Further studies with larger cohorts of patients at different stages of ACC are required to define a potentially prognostic threshold for ACC.
The main limitation of our study is the small number of patients enrolled and scanty presurgical data. Such limi- tations are mainly due to the rarity of ACC and to the fact
that most patients had already undergone surgery when enrolled in the study. Another limiting point is the vari- ability of the CTC number found in different blood sam- ples collected during follow-up. Indeed, the number of CTCs may also be affected by the discontinuous shedding of CTCs from primary and metastatic lesions, as already described for tumors at other sites (35). Multiple sampling is therefore required to limit such variability and improve the reliability of CTC detection. Finally, we here report the results obtained by blood filtering after cell fixation, which prevented us from evaluating CTC viability. Cell viability is crucial to better analyze cell biological charac- teristics, metastatic potential, and sensitivity to chemotherapy.
In conclusion, our findings provide the first evidence that CTCs may be a valid and useful presurgical marker to support differential diagnosis between benign and malig- nant adrenocortical tumors. These cells seem to correlate with some clinical parameters of ACC, such as stage and tumor diameter, suggesting that this so-called “liquid bi- opsy” might be a useful mini-invasive tool for prognosis and for monitoring progression and response to treat- ments. Moreover, in the near future, evaluation of the molecular expression profile of CTCs may help to develop tailored antimetastatic therapies in ACC. Further studies, performed on larger cohorts of patients and on blood sam- ples taken before surgery and at different follow-up inter- vals, are required to definitively validate the prognostic value of this novel biomarker in ACC.
Acknowledgments
We thank Dr Andrea Valeri (Azienda Ospedaliero Universitaria Careggi) and Prof Giuliano Perigli (University of Florence) for performing adrenal surgery. We are indebted to Dr Enzo Lalli (University of Nice, France) for providing anti-SF-1 antibody.
Address all correspondence and requests for reprints to: Mas- simo Mannelli, MD, Department of Experimental and Clinical Biomedical Sciences, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italia. E-mail: massimo.mannelli@unifi.it.
The research leading to these results received funding from the Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 259735 of the European Network for the Study of Adrenal Tumors (ENS@T)-Cancer and from the Fondo per gli Investimenti della Ricerca di Base fund of the Italian Min- istry of University, Research, and Instruction (prot no. RBAP1153LS).
L.C., E.C., M.L., M.M., G.N., and G.P. are members of the ENS@T.
Disclosure Summary: The authors have nothing to disclose.
References
1. Terzolo M, Angeli A, Fassnacht M, et al. Adjuvant mitotane treat- ment for adrenocortical carcinoma. N Engl J Med. 2007;356:2372- 2380.
2. Fassnacht M, Allolio B. Clinical management of adrenocortical car- cinoma. Best Pract Res Clin Endocrinol Metab. 2009;23:273-289.
3. Icard P, Goudet P, Charpenay C, et al. Adrenocortical carcinomas: surgical trends and results of a 253-patient series from the French Association of Endocrine Surgeons study group. World J Surg. 2001; 25:891-897.
4. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004;351:781-791.
5. Sun YF, Yang XR, Zhou J, Qiu SJ, Fan J, Xu Y. Circulating tumor cells: advances in detection methods, biological issues, and clinical relevance. J Cancer Res Clin Oncol. 2011;137:1151-1173.
6. Khan MS, Tsigani T, Rashid M, et al. Circulating tumor cells and EpCAM expression in neuroendocrine tumors. Clin Cancer Res. 2011;17:337-345.
7. Allard WJ, Matera J, Miller MC, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004;10: 6897-6904.
8. Riethdorf S, Wikman H, Pantel K. Review: biological relevance of disseminated tumor cells in cancer patients. Int J Cancer. 2008;123: 1991-2006.
9. Pantel K, Alix-Panabières C. Circulating tumour cells in cancer pa- tients: challenges and perspectives. Trends Mol Med. 2010;16:398- 406.
10. Vona G, Sabile A, Louha M, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and mo- lecular characterization of circulating tumor cells. Am J Pathol. 2000;156:57-63.
11. De Giorgi V, Pinzani P, Salvianti F, et al. Application of a filtration- and isolation-by-size technique for the detection of circulating tu- mor cells in cutaneous melanoma. J Invest Dermatol. 2010;130: 2440-2447.
12. Desitter I, Guerrouahen BS, Benali-Furet N, et al. A new device for rapid isolation by size and characterization of rare circulating tumor cells. Anticancer Res. 2011;31:427-441.
13. Lau SK, Weiss LM. The Weiss system for evaluating adrenocortical neoplasms: 25 years later. Hum Pathol. 2009;40:757-768.
14. Morimoto R, Satoh F, Murakami O, et al. Immunohistochemistry of a proliferation marker Ki67/MIB1 in adrenocortical carcinomas: Ki67/MIB1 labeling index is a predictor for recurrence of adreno- cortical carcinomas. Endocr J. 2008;55:49-55.
15. Fassnacht M, Allolio B. What is the best approach to an apparently nonmetastatic adrenocortical carcinoma? Clin Endocrinol (Oxf). 2010;73:561-565.
16. Fassnacht M, Johanssen S, Quinkler M, et al. Limited prognostic value of the 2004 International Union Against Cancer staging clas- sification for adrenocortical carcinoma: proposal for a Revised TNM Classification. Cancer. 2009;115:243-250.
17. Weitz J, Kienle P, Lacroix J, et al. Dissemination of tumor cells in patients undergoing surgery for colorectal cancer. Clin Cancer Res. 1998;4:343-348.
18. Weitz J, Herfarth C. Surgical strategies and minimal residual disease detection. Semin Surg Oncol. 2001;20:329-333.
19. Alix-Panabières C, Pantel K. Circulating tumor cells: liquid biopsy of cancer. Clin Chem. 2013;59:110-118.
20. Went PT, Lugli A, Meier S, et al. Frequent EpCAM protein expres- sion in human carcinomas. Hum Pathol. 2004;35:122-128.
21. Duregon E, Volante M, Giorcelli J, Terzolo M, Lalli E, Papotti M. Diagnostic and prognostic role of steroidogenic factor 1 in adreno- cortical carcinoma: a validation study focusing on clinical and pathologic correlates. Hum Pathol. 2013;44:822-828.
22. Doghman M, Karpova T, Rodrigues GA, et al. Increased steroido- genic factor-1 dosage triggers adrenocortical cell proliferation and cancer. Mol Endocrinol. 2007;21:2968-2987.
23. Alix-Panabières C, Schwarzenbach H, Pantel K. Circulating tumor cells and circulating tumor DNA. Annu Rev Med. 2012;63:199- 215.
24. Wiedswang G, Borgen E, Schirmer C, et al. Comparison of the clin- ical significance of occult tumor cells in blood and bone marrow in breast cancer. Int J Cancer. 2006;118:2013-2019.
25. Allolio B, Hahner S, Weismann D, Fassnacht M. Management of adrenocortical carcinoma. Clin Endocrinol (Oxf). 2004;60:273- 287.
26. Mantero F, Terzolo M, Arnaldi G, et al. A survey on adrenal inci- dentaloma in Italy. Study Group on Adrenal Tumors of the Italian Society of Endocrinology. J Clin Endocrinol Metab. 2000;85:637- 644.
27. Stojadinovic A, Ghossein RA, Hoos A, et al. Adrenocortical carci- noma: clinical, morphologic, and molecular characterization. J Clin Oncol. 2002;20:941-950.
28. Xu W, Cao L, Chen L, et al. Isolation of circulating tumor cells in patients with hepatocellular carcinoma using a novel cell separation strategy. Clin Cancer Res. 2011;17:3783-3793.
29. Wu CH, Lin SR, Hsieh JS, et al. Molecular detection of disseminated tumor cells in the peripheral blood of patients with gastric cancer: evaluation of their prognostic significance. Dis Markers. 2006;22: 103-109.
30. Krebs MG, Sloane R, Priest L, et al. Evaluation and prognostic significance of circulating tumor cells in patients with non-small-cell lung cancer. J Clin Oncol. 2011;29:1556-1563.
31. de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castration-re- sistant prostate cancer. Clin Cancer Res. 2008;14:6302-6309.
32. Miller MC, Doyle GV, Terstappen LW. Significance of circulating tumor cells detected by the CellSearch system in patients with met- astatic breast colorectal and prostate cancer. J Oncol. 2010;2010: 61742.
33. Hoshimoto S, Faries MB, Morton DL, et al. Assessment of prog- nostic circulating tumor cells in a phase III trial of adjuvant immu- notherapy after complete resection of stage IV melanoma. Ann Surg. 2012;255:357-362.
34. Zhang L, Riethdorf S, Wu G, et al. Meta-analysis of the prognostic value of circulating tumor cells in breast cancer. Clin Cancer Res. 2012;18:5701-5710.
35. Mocellin S, Hoon D, Ambrosi A, Nitti D, Rossi CR. The prognostic value of circulating tumor cells in patients with melanoma: a sys- tematic review and meta-analysis. Clin Cancer Res. 2006;12:4605- 4613.