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Epithelial Cell Adhesion Molecule-positive Circulating Tumor Cells as Predictive Biomarker in Patients With Prostate Cancer
Medical Oncology Epithelial Cell Adhesion Molecule-positive Circulating Tumor Cells as Predictive Biomarker in Patients With Prostate Cancer Robert J. Amato, Vladislava Melnikova, Yujian Zhang, Wen Liu, Somyata Saxena, Parth K. Shah, Brett T. Jensen, Karen E. Torres, and Darren W. Davis OBJECTIVE
MATERIALS AND METHODS
RESULTS
CONCLUSION
To assess the use of circulating tumor cells (CTCs) as a longitudinal endpoint factor for clinical monitoring of patients with prostate cancer and to evaluate the association among the baseline CTC number, various clinical characteristics, and survival. The CTCs were enumerated using the CellSearch Food and Drug Administrationecleared CTC kit in 202 patients with prostate cancer. Variables, including metastatic site, prostate-specific antigen level, Gleason score, testosterone level, and use of androgen treatment, were tested for association with the CTC number. The probability of patient survival over time was estimated using the Kaplan-Meier method. The baseline CTC numbers were strongly associated with survival (P <.0001), with overall survival significantly poorer in patients with 5 CTCs. Significantly greater CTC numbers were observed in patients with bone metastasis (mean 41.12 CTCs) than in those with lymph node metastasis (mean 2.53 CTC, P ¼ .026). Analysis of the association between the CTC count and prostate-specific antigen level revealed a weak positive correlation (correlation coefficient r ¼ 0.2695, P ¼ .0007). The CTC number also correlated with the Gleason score (P ¼ .0138) and lower testosterone level (P <.0001). Patients without androgen depletion had significantly lower CTC numbers (mean 2.70) than those with androgen depletion (mean 26.39, P <.0001). The baseline CTC counts were predictive of patient survival and correlated significantly with the clinical characteristics of patients with prostate cancer. Our study results have confirmed previous findings that support the use of CTC enumeration as a prognostic biomarker for patients with prostate cancer. UROLOGY 81: 1303e1307, 2013. 2013 Elsevier Inc.
P
rostate cancer is the most common nondermatologic malignancy in men and the second leading cause of male cancer-related deaths in the United States.1,2 The prostate-specific antigen (PSA) blood level is widely used as a diagnostic, predictive, and prognostic marker of prostate cancer.3,4 The PSA level, however, might not accurately reflect the disease status,5 because increases in the PSA level are only slightly associated with survival. Hence, more reliable indicators of prostate cancer disease status are needed. The shedding of tumor cells into the circulation results in metastatic cancer spread.6,7 The presence of circulating tumor cells (CTCs) in the peripheral circulation during the early stages of cancer development has increasingly been Financial Disclosure: The authors declare that they have no relevant financial interests. From the Division of Oncology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX; the UT Memorial Hermann Cancer Center, Houston, TX; and ApoCell, Incorporated, Houston, TX Reprint requests: Robert J. Amato, D.O., University of Texas Memorial Hermann Cancer Center, 6410 Fannin Street, Suite 830, Houston, TX 77030. E-mail: robert. [email protected] Submitted: March 12, 2012, accepted (with revisions): October 24, 2012
ª 2013 Elsevier Inc. All Rights Reserved
considered a hallmark of cancer progression. The CTC level has been correlated with the prognosis in patients with prostate cancer.8-10 In addition to providing prognostic information for prostate cancer, molecular analysis of CTCs has the potential to predict the sensitivity, or resistance, to drug treatment.11-14 In recent years, several CTC isolation techniques have been reported, but only one method, CellSearch (Veridex, Raritan, NJ), is analytically valid and has been cleared by the Food and Drug Administration.15 Most CTC detection methods such as MACs, CellSearch (Veridex), and the CTC-chip depend on immunomagnetic separation, in which positive selection of epithelial cell adhesion molecule-expressing CTCs allows them to be distinguished from normal blood cells.16-19 The enriched cells are further classified as CTCs on the basis of morphologic limits and criteria for cytokeratin staining, nucleus displaying, and exclusion of white blood cells (CD45 staining). Studies of patients with progressive metastatic breast, colorectal, or prostate cancer have shown that CTCs are indicators of prognosis before and after therapy using discrete disease cutoff points 0090-4295/12/$36.00 http://dx.doi.org/10.1016/j.urology.2012.10.041
1303
(5 CTCs/7.5 mL blood vs 4 CTCs) to define favorable and unfavorable groups.8,10,20 We evaluated the CTC numbers in patients who had received previous treatment of prostate cancer by radical prostatectomy, radiotherapy, hormonal therapy, or chemotherapy. The relationship between CTC number and patient survival was explored, as were patterns of metastatic spread and other measures of disease burden, including the PSA level.
MATERIAL AND METHODS Patients and Study Design Blood samples from 202 patients with prostate cancer were analyzed using the Food and Drug Administrationecleared CellSearch (Veridex) prognostic CTC testing method. The data recorded included treatment of the primary tumor at diagnosis and all subsequent systemic therapies. A physical examination and laboratory studies, including complete blood count and chemistry panel (PSA, alkaline phosphatase, lactate dehydrogenase, and testosterone measurement), were performed at the time of blood sampling. The distribution of the soft tissue disease was determined using computed tomography and/or magnetic resonance imaging and evaluated for the presence of lymph nodes and organ involvement. Radionuclide bone scans were evaluated for the presence or absence of metastatic bone disease. For patients with metastases, the disease extent was estimated using the bone scan index to assess the proportion of bony skeleton involvement.
Isolation and Enumeration of CTCs The CellSearch system (Veridex) was used for the isolation and enumeration of the CTCs. Blood (7.5 mL) was collected from patients in 10-mL CellSave tubes and processed within 24 hours using the CellSearch System (Veridex) with the Food and Drug Administrationecleared CTC Kit, which isolates epithelial cell adhesion molecule-positive cells using a ferromagnetic immunoabsorption assay. Analyses of the CTCs were performed using the CellTracks Analyzer II, and the final CTC images were scored using a Veridex-certified analyst to unambiguously identify the CTCs. CTC enumeration was reported as the number of epithelial cell adhesion molecule-enriched, cytokeratin-positive, CD45-negative, and 4’,6-diamidino-2-phenylindoleepositive cells/7.5 mL of blood.
Statistical Analysis Statistical analysis was performed to evaluate the endpoints. Overall survival (OS) was measured from the date of the first initiation of therapy to the date of death or the last follow-up examination. Statistical analysis was performed using the MedCalc, version 11.4.4.0, software program (MedCalc Software, Mariakerke, Belgium). Survival distributions were estimated using the Kaplan-Meier method. The patients lost to follow-up were counted as censored values (indicated by tick marks on the graphs). Cox proportional hazards regression analysis was performed to test the association between the number of CTCs and the PSA level. For categorical variables, such as the Gleason score, metastatic site, and androgen depletion treatment, statistical significance was tested with analysis of variance (Kruskal-Wallis test) using GraphPad Instat, version 3.10 (GraphPad Instat, La Jolla, CA), and MedCalc, version 11.4.4.0 (MedCalc Software). 1304
Table 1. Patient characteristics (n ¼ 202) Characteristic Value Age at diagnosis (y) Mean Range Race White Black Hispanic Asian NA Primary therapy Radical prostatectomy Radiotherapy No primary therapy Other surgery Cryosurgery Unknown Previous systemic therapy First-line chemotherapy 2 Chemotherapy regimens No therapy Gleason score 5-6 7 8 9 10 NA Metastatic sites Bone only Lymph node only Bone plus lymph node No metastasis Unknown Other/mixed Baseline PSA (ng/mL) Median Range Alkaline phosphatase (U/L) Median Range LDH (U/L) Median Range Testosterone (ng/dL) Median Range CTC range 0 <5 5-100 >100
71 48-94 156 17 14 3 12
(77) (8) (7) (2) (6)
108 40 38 6 3 7
(53) (20) (19) (3) (1) (4)
86 (43) 60 (30) 56 (28) 14 51 27 49 6 55
(7) (25) (13) (25) (3) (27)
40 15 24 87 5 31
(20) (7) (12) (43) (3) (15)
1 0.01-1961 73 30-1214 179 92-610 16 0.02-886.1 156 18 21 7
(77) (9) (10) (4)
CTC, circulating tumor cell; LDH, lactate dehydrogenase; NA, not available; PSA, prostate-specific antigen. Data presented as n (%), unless otherwise noted.
RESULTS Patient Characteristics The present retrospective study evaluated 202 patients. The patient characteristics are detailed in Table 1. Of these patients, 164 (81%) had received previous local treatment of prostate cancer, including prostatectomy in 108, radiotherapy in 40, and other therapy in 16. Of the 202 patients, 86 (43%) received first-line chemotherapy, UROLOGY 81 (6), 2013
Kaplan-Meier analysis 100 Group CTC: 0
Survival Probability (%)
80
CTC: < 5 CTC: 5 - 100 CTC: >100
60
40
20
0 40
50
60
70
80
90 100
Time (months) CTC: 0
CTC: < 5
CTC: 5 - 100
CTC: >100
156
18
21
7
0
18
21
7
Not yet reached (range, 48.42-94.69)
78.54 (range, 58.7788.45)
73.33 (range, 49.1085.77)
70.71 (range, 5280.48)
8
4
9
6
Sample Size Detectable CTC Median (months) Deaths P Value
< .0001
Figure 1. Kaplan-Meier analysis showing lower overall survival in patients with 5 circulating tumor cells (CTCs)/7.5 mL blood. Survival rate calculated from baseline blood sample.
60 (30%) received 2 chemotherapy regimens, and 56 (27%) had received no previous systemic therapy. Metastatic disease was absent in 87 patients (43%), 40 (20%) had bone-only metastases, 15 (7%) had lymph node-only metastases, and 24 (12%) had both bone and lymph node metastases. The baseline PSA, alkaline phosphatase, lactate dehydrogenase, testosterone, and CTC levels are listed by range. CTC Counts The data from all 202 patients were successfully analyzed using the CellSearch CTC enumeration kit (Veridex). The CTC counts for these patients are listed in Table 1. The distribution of CTC counts is listed in Table 1 and shown in Figure 1. Of the 202 patients, 156 (77%) had undetectable CTC counts. Of 46 patients with detectable CTC counts, 18 (9%) had <5 CTCs, 21 (10%) had a CTC count of 5-100, and 7 (3%) had a CTC count >100. CTC Count as OS Predictor The Kaplan-Meier curves for OS stratified by the CTC range are depicted in Figure 1. Four groups of patients were compared: group 1, 0 CTCs/7.5 mL blood; group 2, <5 CTCs/7.5 mL blood; group 3, 5-100 CTCs/7.5 mL blood; and group 4, >100 CTCs/7.5 mL blood. The median OS UROLOGY 81 (6), 2013
for group 1 was not reached. The median OS for groups 2, 3, and 4 was 78.54, 73.33, and 70.71 months, respectively. Correlations in Disease Distribution and Disease Extent Greater CTC numbers were detected in patients with bone-only metastases (mean 41.12 CTCs, median 0 CTCs) or with bone and lymph node metastases (mean 57.04 CTCs, median 0 CTCs) compared with those with lymph node-only metastases (mean 2.53 CTCs, median 0 CTCs; Fig. 2). Androgen-depleted patients had a greater mean CTC count (mean 26.4, median 0, range 0-1134) compared with the nonandrogen-depleted patients (mean 2.7, median 0, range 0-28; Fig. 3A). The mean testosterone level for the androgen-depleted patients was 9.9 ng/dL (median 3.9, range 0.02-46) and for the nonandrogen-depleted patients was 392.1 ng/dL (median 406.4, range 50-886.1; Fig. 3B). Of the patients with metastases and androgen depletion, 3 had bone metastases, 3 had bone and lymph node metastases, and 4 had lymph node-only metastases. To test the correlation between the CTC counts and Gleason score, 147 patients were divided into 3 groups according to Gleason score cutoff of 5; 14 patients had a Gleason score of 6 (no patients with detectable CTCs), 1305
Metastatic groups
A
1000
Mean CTC Counts 50 40
CTC Count
800
20
CTC
600
30
10
400 Androgen Depletion
200
Androgen Depletion No. of Patients
0
Bone Only
Both Lymph Node Only
Bone Only
Lymph Node Only
Both
Total no.
40
15
24
Detectable CTC
13
4
11
41.12
2.53
57.04
SD
0
0
0
160.25
6.68
156.40
.3176
Figure 2. Graph and tabular data showing metastatic groups (bone only, lymph node only, and both), with entire circulating tumor cell (CTC) range (0->1000) included for analysis. A total of 79 patients were analyzed stratified by their metastatic category. Differences were not statistically significant (P ¼ .3176). (Color version available onlnie.)
51 patients had a Gleason score of 7 (8 patients with detectable CTCs), and 82 patients had a Gleason score of 8 (25 patients with detectable CTCs). A significant difference in CTC counts was found among patients with Gleason score <6 (mean 0 CTCs, median 0 CTCs), Gleason score 7 (mean 1.43 CTCs, median 0 CTCs; standard deviation 7.59), and Gleason score >8 (mean 38.42 CTCs, median 0 CTCs, standard deviation 171.12; P <.0138; Supplemental Fig. 1). An analysis of the association between CTC counts and PSA level in 155 patients revealed a weak positive correlation between the CTC number and PSA level (r ¼ 0.2695; P <.0007; 95% confidence interval for r 0.1168-0.4097; Supplemental Fig. 2).
COMMENT The results of the present research have provided evidence that supports the use of CTC enumeration as a prognostic biomarker for patients with prostate cancer. The power of the baseline CTC count to discriminate between low and high survival was significant. The CTC numbers correlated strongly with progression-free survival, and OS was significantly poorer in patients with 5 CTCs, indicating that CTCs reflect the intrinsic biology of tumor progression. Greater CTC levels were detected in patients with bone metastasis compared with patients with lymph node metastasis. The association between the baseline CTC 1306
41
No. Patients with detectable CTC
12 (15.8%)
11 (26.8%)
CTC = 0
64 (84.2%)
30 (73.2%)
CTC < 5
4 (5.3%)
5 (12.2%)
CTC = 5-100
6 (7.9%)
6 (14.6%)
CTC > 100
2 (2.6%)
0 (0.0%)
26.4 (±17.2)
2.7 (±1.0)
0 (0-1134)
0 (0-28)
Mean (±SEM)
B
Testosterone estosterone Levels
1000
Testosterone (ng/dL)
Median
Non-Androgen Depletion
76
Median (min-max)
Mean
P Value
Non-Androgen Depletion
800 600 400 200
Androgen Depletion
Non-Androgen Depletion
Androgen Depletion No. of Patients Mean (±SEM) Median (min-max)
Non-Androgen Depletion
76
41
9.9 (±1.4)
392.1 (±29.3)
3.9 (0.02-46)
406.4 (50-886.1)
* indicates p<.0001
Figure 3. (A) Difference in circulating tumor cell (CTC) numbers between nonandrogen-depleted patients and androgen-depleted patients. Entire CTC range (0->1000) included for analysis. (B) A total of 117 patients had complete testosterone data and their data were analyzed, showing a statistically significant difference between groups (P ¼ .0001).
number and PSA level was weak. The difference in CTC levels in patients with bone metastasis relative to those with metastasis in the lymph nodes and the modest association between the CTC number and PSA level suggests that the PSA level alone does not provide predictive power and that future treatment decision should be determined by a combination of multiple biomarkers, such as lactate dehydrogenase, albumin, and PSA, sedimentation rate, and CTC count, which should be monitored concurrently. In contrast to the PSA levels, the baseline Gleason scores correlated with the CTC counts, revealing a significant difference in CTC counts among patient groups with a Gleason score UROLOGY 81 (6), 2013
of <6, 7, and >8. These results are in agreement with previous findings by Okegawa et al,21 who reported on a larger proportion of patients with 2, 5, 10, and 50 CTCs who also had a Gleason score of 8-10. Goodman et al23 demonstrated a trend toward increased median CTC counts in patients with a greater Gleason score, although this trend was not statistically significant. Significantly greater CTCs were observed in patients with androgen depletion compared with those without androgen depletion, indicating an increase in CTC burden with disease progression. Our findings are consistent with previous observations that an elevated CTC count results from metastatic spread, such that osseous sites are seeded hematogenously and soft tissue disease spreads predominantly by way of lymphatic routes.22 Although previous work has shown a significant association between the CTC and PSA levels, we found that the correlation between the CTC and PSA levels was modest. Our findings are in agreement with recent findings from Goodman et al23 and Scher et al,24 who showed elevated PSA levels are only slightly associated with survival. Collectively, these data reinforce the idea that the PSA level alone is not of sufficient prognostic value and must be considered in conjunction with additional biomarkers.
CONCLUSION Future advancements in patient care will depend on analysis of multiple markers from primary, metastatic, and circulating tumor cells. Patient-tailored therapy requires the ability to identify the target of interest in a sample that reflects the patient’s disease progression at the time treatment is being considered. Measuring CTC levels, the “liquid biopsy,” has the potential to be an informative biomarker in monitoring metastatic disease progression. In addition to providing pretreatment prognostic information, CTCs are a rich source of disease-specific biomarkers and can be interrogated at the deoxyribonucleic acid, ribonucleic acid, and protein levels. Acknowledgment. To Kenna Anderes, Ph.D., Vice President Scientific Affairs at ApoCell, and Mika Stepankiw for providing editorial assistance, with thanks. References 1. Jemal A, Siegel R, Xu J, et al. Cancer statistics, 2010. CA Cancer J Clin. 2010;60:277-300. 2. Scher HI, Sawyers CL. Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis. J Clin Oncol. 2005;23:8253-8261. 3. Lilja H, Ulmert D, Vickers AJ. Prostate-specific antigen and prostate cancer: prediction, detection and monitoring. Nature Rev Cancer. 2008;8:268-278. 4. Schroder FH, Hugosson J, Roobol MJ, et al. Screening and prostatecancer mortality in a randomized European study. N Engl J Med. 2009;360:1320-1328. 5. Scher HI, Morris MJ, Kelly WK, et al. Prostate cancer clinical trial end points: “RECIST”ing a step backwards. Clin Cancer Res. 2005; 11:5223-5232.
UROLOGY 81 (6), 2013
6. Eccles SA, Welch DR. Metastasis: recent discoveries and novel treatment strategies. Lancet. 2007;369:1742-1757. 7. Nguyen DX, Bos PD, Massague J. Metastasis: from dissemination to organ-specific colonization. Nature Rev Cancer. 2009;9:274-284. 8. de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castrationresistant prostate cancer. Clin Cancer Res. 2008;14:6302-6309. 9. Olmos D, Arkenau HT, Ang JE, et al. Circulating tumour cell (CTC) counts as intermediate end points in castration-resistant prostate cancer (CRPC): a single-centre experience. Ann Oncol. 2009;20:27-33. 10. Scher HI, Jia X, de Bono JS, et al. Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. Lancet Oncol. 2009;10:233-239. 11. Attard G, Swennenhuis JF, Olmos D, et al. Characterization of ERG, AR and PTEN gene status in circulating tumor cells from patients with castration-resistant prostate cancer. Cancer Res. 2009; 69:2912-2918. 12. King JC, Xu J, Wongvipat J, et al. Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis. Nature Genet. 2009;41:524-526. 13. Leversha MA, Han J, Asgari Z, et al. Fluorescence in situ hybridization analysis of circulating tumor cells in metastatic prostate cancer. Clin Cancer Res. 2009;15:2091-2097. 14. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science. 2005;310:644-648. 15. 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. 16. Griwatz C, Brandt B, Assmann G, et al. An immunological enrichment method for epithelial cells from peripheral blood. J Immunol Methods. 1995;183:251-265. 17. Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007;450:1235-1239. 18. Riethdorf S, Fritsche H, Muller V, et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer Res. 2007;13:920-928. 19. Sequist LV, Nagrath S, Toner M, et al. The CTC-chip: an exciting new tool to detect circulating tumor cells in lung cancer patients. J Thorac Oncol. 2009;4:281-283. 20. Cohen SJ, Punt CJ, Iannotti N, et al. Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol. 2008;26:3213-3221. 21. Okegawa T, Nutahara K, Higashihara E. Immunomagnetic quantification of circulating tumor cells as a prognostic factor of androgen deprivation responsiveness in patients with hormone naive metastatic prostate cancer. J Urol. 2008;180: 1342-1347. 22. Danila DC, Heller G, Gignac GA, et al. Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer. Clin Cancer Res. 2007;13:7053-7058. 23. Goodman OB Jr, Symanowski JT, Loudyi A, et al. Circulating tumor cells as a predictive biomarker in patients with hormonesensitive prostate cancer. Clin Genitourin Cancer. 2011;9:31-38. 24. Scher HI, Warren M, Heller G. The association between measures of progression and survival in castrate-metastatic prostate cancer. Clin Cancer Res. 2007;13:1488-1492.
APPENDIX SUPPLEMENTARY DATA
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.urology. 2012.10.041. 1307
MATERIALS AND METHODS
RESULTS
CONCLUSION
To assess the use of circulating tumor cells (CTCs) as a longitudinal endpoint factor for clinical monitoring of patients with prostate cancer and to evaluate the association among the baseline CTC number, various clinical characteristics, and survival. The CTCs were enumerated using the CellSearch Food and Drug Administrationecleared CTC kit in 202 patients with prostate cancer. Variables, including metastatic site, prostate-specific antigen level, Gleason score, testosterone level, and use of androgen treatment, were tested for association with the CTC number. The probability of patient survival over time was estimated using the Kaplan-Meier method. The baseline CTC numbers were strongly associated with survival (P <.0001), with overall survival significantly poorer in patients with 5 CTCs. Significantly greater CTC numbers were observed in patients with bone metastasis (mean 41.12 CTCs) than in those with lymph node metastasis (mean 2.53 CTC, P ¼ .026). Analysis of the association between the CTC count and prostate-specific antigen level revealed a weak positive correlation (correlation coefficient r ¼ 0.2695, P ¼ .0007). The CTC number also correlated with the Gleason score (P ¼ .0138) and lower testosterone level (P <.0001). Patients without androgen depletion had significantly lower CTC numbers (mean 2.70) than those with androgen depletion (mean 26.39, P <.0001). The baseline CTC counts were predictive of patient survival and correlated significantly with the clinical characteristics of patients with prostate cancer. Our study results have confirmed previous findings that support the use of CTC enumeration as a prognostic biomarker for patients with prostate cancer. UROLOGY 81: 1303e1307, 2013. 2013 Elsevier Inc.
P
rostate cancer is the most common nondermatologic malignancy in men and the second leading cause of male cancer-related deaths in the United States.1,2 The prostate-specific antigen (PSA) blood level is widely used as a diagnostic, predictive, and prognostic marker of prostate cancer.3,4 The PSA level, however, might not accurately reflect the disease status,5 because increases in the PSA level are only slightly associated with survival. Hence, more reliable indicators of prostate cancer disease status are needed. The shedding of tumor cells into the circulation results in metastatic cancer spread.6,7 The presence of circulating tumor cells (CTCs) in the peripheral circulation during the early stages of cancer development has increasingly been Financial Disclosure: The authors declare that they have no relevant financial interests. From the Division of Oncology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX; the UT Memorial Hermann Cancer Center, Houston, TX; and ApoCell, Incorporated, Houston, TX Reprint requests: Robert J. Amato, D.O., University of Texas Memorial Hermann Cancer Center, 6410 Fannin Street, Suite 830, Houston, TX 77030. E-mail: robert. [email protected] Submitted: March 12, 2012, accepted (with revisions): October 24, 2012
ª 2013 Elsevier Inc. All Rights Reserved
considered a hallmark of cancer progression. The CTC level has been correlated with the prognosis in patients with prostate cancer.8-10 In addition to providing prognostic information for prostate cancer, molecular analysis of CTCs has the potential to predict the sensitivity, or resistance, to drug treatment.11-14 In recent years, several CTC isolation techniques have been reported, but only one method, CellSearch (Veridex, Raritan, NJ), is analytically valid and has been cleared by the Food and Drug Administration.15 Most CTC detection methods such as MACs, CellSearch (Veridex), and the CTC-chip depend on immunomagnetic separation, in which positive selection of epithelial cell adhesion molecule-expressing CTCs allows them to be distinguished from normal blood cells.16-19 The enriched cells are further classified as CTCs on the basis of morphologic limits and criteria for cytokeratin staining, nucleus displaying, and exclusion of white blood cells (CD45 staining). Studies of patients with progressive metastatic breast, colorectal, or prostate cancer have shown that CTCs are indicators of prognosis before and after therapy using discrete disease cutoff points 0090-4295/12/$36.00 http://dx.doi.org/10.1016/j.urology.2012.10.041
1303
(5 CTCs/7.5 mL blood vs 4 CTCs) to define favorable and unfavorable groups.8,10,20 We evaluated the CTC numbers in patients who had received previous treatment of prostate cancer by radical prostatectomy, radiotherapy, hormonal therapy, or chemotherapy. The relationship between CTC number and patient survival was explored, as were patterns of metastatic spread and other measures of disease burden, including the PSA level.
MATERIAL AND METHODS Patients and Study Design Blood samples from 202 patients with prostate cancer were analyzed using the Food and Drug Administrationecleared CellSearch (Veridex) prognostic CTC testing method. The data recorded included treatment of the primary tumor at diagnosis and all subsequent systemic therapies. A physical examination and laboratory studies, including complete blood count and chemistry panel (PSA, alkaline phosphatase, lactate dehydrogenase, and testosterone measurement), were performed at the time of blood sampling. The distribution of the soft tissue disease was determined using computed tomography and/or magnetic resonance imaging and evaluated for the presence of lymph nodes and organ involvement. Radionuclide bone scans were evaluated for the presence or absence of metastatic bone disease. For patients with metastases, the disease extent was estimated using the bone scan index to assess the proportion of bony skeleton involvement.
Isolation and Enumeration of CTCs The CellSearch system (Veridex) was used for the isolation and enumeration of the CTCs. Blood (7.5 mL) was collected from patients in 10-mL CellSave tubes and processed within 24 hours using the CellSearch System (Veridex) with the Food and Drug Administrationecleared CTC Kit, which isolates epithelial cell adhesion molecule-positive cells using a ferromagnetic immunoabsorption assay. Analyses of the CTCs were performed using the CellTracks Analyzer II, and the final CTC images were scored using a Veridex-certified analyst to unambiguously identify the CTCs. CTC enumeration was reported as the number of epithelial cell adhesion molecule-enriched, cytokeratin-positive, CD45-negative, and 4’,6-diamidino-2-phenylindoleepositive cells/7.5 mL of blood.
Statistical Analysis Statistical analysis was performed to evaluate the endpoints. Overall survival (OS) was measured from the date of the first initiation of therapy to the date of death or the last follow-up examination. Statistical analysis was performed using the MedCalc, version 11.4.4.0, software program (MedCalc Software, Mariakerke, Belgium). Survival distributions were estimated using the Kaplan-Meier method. The patients lost to follow-up were counted as censored values (indicated by tick marks on the graphs). Cox proportional hazards regression analysis was performed to test the association between the number of CTCs and the PSA level. For categorical variables, such as the Gleason score, metastatic site, and androgen depletion treatment, statistical significance was tested with analysis of variance (Kruskal-Wallis test) using GraphPad Instat, version 3.10 (GraphPad Instat, La Jolla, CA), and MedCalc, version 11.4.4.0 (MedCalc Software). 1304
Table 1. Patient characteristics (n ¼ 202) Characteristic Value Age at diagnosis (y) Mean Range Race White Black Hispanic Asian NA Primary therapy Radical prostatectomy Radiotherapy No primary therapy Other surgery Cryosurgery Unknown Previous systemic therapy First-line chemotherapy 2 Chemotherapy regimens No therapy Gleason score 5-6 7 8 9 10 NA Metastatic sites Bone only Lymph node only Bone plus lymph node No metastasis Unknown Other/mixed Baseline PSA (ng/mL) Median Range Alkaline phosphatase (U/L) Median Range LDH (U/L) Median Range Testosterone (ng/dL) Median Range CTC range 0 <5 5-100 >100
71 48-94 156 17 14 3 12
(77) (8) (7) (2) (6)
108 40 38 6 3 7
(53) (20) (19) (3) (1) (4)
86 (43) 60 (30) 56 (28) 14 51 27 49 6 55
(7) (25) (13) (25) (3) (27)
40 15 24 87 5 31
(20) (7) (12) (43) (3) (15)
1 0.01-1961 73 30-1214 179 92-610 16 0.02-886.1 156 18 21 7
(77) (9) (10) (4)
CTC, circulating tumor cell; LDH, lactate dehydrogenase; NA, not available; PSA, prostate-specific antigen. Data presented as n (%), unless otherwise noted.
RESULTS Patient Characteristics The present retrospective study evaluated 202 patients. The patient characteristics are detailed in Table 1. Of these patients, 164 (81%) had received previous local treatment of prostate cancer, including prostatectomy in 108, radiotherapy in 40, and other therapy in 16. Of the 202 patients, 86 (43%) received first-line chemotherapy, UROLOGY 81 (6), 2013
Kaplan-Meier analysis 100 Group CTC: 0
Survival Probability (%)
80
CTC: < 5 CTC: 5 - 100 CTC: >100
60
40
20
0 40
50
60
70
80
90 100
Time (months) CTC: 0
CTC: < 5
CTC: 5 - 100
CTC: >100
156
18
21
7
0
18
21
7
Not yet reached (range, 48.42-94.69)
78.54 (range, 58.7788.45)
73.33 (range, 49.1085.77)
70.71 (range, 5280.48)
8
4
9
6
Sample Size Detectable CTC Median (months) Deaths P Value
< .0001
Figure 1. Kaplan-Meier analysis showing lower overall survival in patients with 5 circulating tumor cells (CTCs)/7.5 mL blood. Survival rate calculated from baseline blood sample.
60 (30%) received 2 chemotherapy regimens, and 56 (27%) had received no previous systemic therapy. Metastatic disease was absent in 87 patients (43%), 40 (20%) had bone-only metastases, 15 (7%) had lymph node-only metastases, and 24 (12%) had both bone and lymph node metastases. The baseline PSA, alkaline phosphatase, lactate dehydrogenase, testosterone, and CTC levels are listed by range. CTC Counts The data from all 202 patients were successfully analyzed using the CellSearch CTC enumeration kit (Veridex). The CTC counts for these patients are listed in Table 1. The distribution of CTC counts is listed in Table 1 and shown in Figure 1. Of the 202 patients, 156 (77%) had undetectable CTC counts. Of 46 patients with detectable CTC counts, 18 (9%) had <5 CTCs, 21 (10%) had a CTC count of 5-100, and 7 (3%) had a CTC count >100. CTC Count as OS Predictor The Kaplan-Meier curves for OS stratified by the CTC range are depicted in Figure 1. Four groups of patients were compared: group 1, 0 CTCs/7.5 mL blood; group 2, <5 CTCs/7.5 mL blood; group 3, 5-100 CTCs/7.5 mL blood; and group 4, >100 CTCs/7.5 mL blood. The median OS UROLOGY 81 (6), 2013
for group 1 was not reached. The median OS for groups 2, 3, and 4 was 78.54, 73.33, and 70.71 months, respectively. Correlations in Disease Distribution and Disease Extent Greater CTC numbers were detected in patients with bone-only metastases (mean 41.12 CTCs, median 0 CTCs) or with bone and lymph node metastases (mean 57.04 CTCs, median 0 CTCs) compared with those with lymph node-only metastases (mean 2.53 CTCs, median 0 CTCs; Fig. 2). Androgen-depleted patients had a greater mean CTC count (mean 26.4, median 0, range 0-1134) compared with the nonandrogen-depleted patients (mean 2.7, median 0, range 0-28; Fig. 3A). The mean testosterone level for the androgen-depleted patients was 9.9 ng/dL (median 3.9, range 0.02-46) and for the nonandrogen-depleted patients was 392.1 ng/dL (median 406.4, range 50-886.1; Fig. 3B). Of the patients with metastases and androgen depletion, 3 had bone metastases, 3 had bone and lymph node metastases, and 4 had lymph node-only metastases. To test the correlation between the CTC counts and Gleason score, 147 patients were divided into 3 groups according to Gleason score cutoff of 5; 14 patients had a Gleason score of 6 (no patients with detectable CTCs), 1305
Metastatic groups
A
1000
Mean CTC Counts 50 40
CTC Count
800
20
CTC
600
30
10
400 Androgen Depletion
200
Androgen Depletion No. of Patients
0
Bone Only
Both Lymph Node Only
Bone Only
Lymph Node Only
Both
Total no.
40
15
24
Detectable CTC
13
4
11
41.12
2.53
57.04
SD
0
0
0
160.25
6.68
156.40
.3176
Figure 2. Graph and tabular data showing metastatic groups (bone only, lymph node only, and both), with entire circulating tumor cell (CTC) range (0->1000) included for analysis. A total of 79 patients were analyzed stratified by their metastatic category. Differences were not statistically significant (P ¼ .3176). (Color version available onlnie.)
51 patients had a Gleason score of 7 (8 patients with detectable CTCs), and 82 patients had a Gleason score of 8 (25 patients with detectable CTCs). A significant difference in CTC counts was found among patients with Gleason score <6 (mean 0 CTCs, median 0 CTCs), Gleason score 7 (mean 1.43 CTCs, median 0 CTCs; standard deviation 7.59), and Gleason score >8 (mean 38.42 CTCs, median 0 CTCs, standard deviation 171.12; P <.0138; Supplemental Fig. 1). An analysis of the association between CTC counts and PSA level in 155 patients revealed a weak positive correlation between the CTC number and PSA level (r ¼ 0.2695; P <.0007; 95% confidence interval for r 0.1168-0.4097; Supplemental Fig. 2).
COMMENT The results of the present research have provided evidence that supports the use of CTC enumeration as a prognostic biomarker for patients with prostate cancer. The power of the baseline CTC count to discriminate between low and high survival was significant. The CTC numbers correlated strongly with progression-free survival, and OS was significantly poorer in patients with 5 CTCs, indicating that CTCs reflect the intrinsic biology of tumor progression. Greater CTC levels were detected in patients with bone metastasis compared with patients with lymph node metastasis. The association between the baseline CTC 1306
41
No. Patients with detectable CTC
12 (15.8%)
11 (26.8%)
CTC = 0
64 (84.2%)
30 (73.2%)
CTC < 5
4 (5.3%)
5 (12.2%)
CTC = 5-100
6 (7.9%)
6 (14.6%)
CTC > 100
2 (2.6%)
0 (0.0%)
26.4 (±17.2)
2.7 (±1.0)
0 (0-1134)
0 (0-28)
Mean (±SEM)
B
Testosterone estosterone Levels
1000
Testosterone (ng/dL)
Median
Non-Androgen Depletion
76
Median (min-max)
Mean
P Value
Non-Androgen Depletion
800 600 400 200
Androgen Depletion
Non-Androgen Depletion
Androgen Depletion No. of Patients Mean (±SEM) Median (min-max)
Non-Androgen Depletion
76
41
9.9 (±1.4)
392.1 (±29.3)
3.9 (0.02-46)
406.4 (50-886.1)
* indicates p<.0001
Figure 3. (A) Difference in circulating tumor cell (CTC) numbers between nonandrogen-depleted patients and androgen-depleted patients. Entire CTC range (0->1000) included for analysis. (B) A total of 117 patients had complete testosterone data and their data were analyzed, showing a statistically significant difference between groups (P ¼ .0001).
number and PSA level was weak. The difference in CTC levels in patients with bone metastasis relative to those with metastasis in the lymph nodes and the modest association between the CTC number and PSA level suggests that the PSA level alone does not provide predictive power and that future treatment decision should be determined by a combination of multiple biomarkers, such as lactate dehydrogenase, albumin, and PSA, sedimentation rate, and CTC count, which should be monitored concurrently. In contrast to the PSA levels, the baseline Gleason scores correlated with the CTC counts, revealing a significant difference in CTC counts among patient groups with a Gleason score UROLOGY 81 (6), 2013
of <6, 7, and >8. These results are in agreement with previous findings by Okegawa et al,21 who reported on a larger proportion of patients with 2, 5, 10, and 50 CTCs who also had a Gleason score of 8-10. Goodman et al23 demonstrated a trend toward increased median CTC counts in patients with a greater Gleason score, although this trend was not statistically significant. Significantly greater CTCs were observed in patients with androgen depletion compared with those without androgen depletion, indicating an increase in CTC burden with disease progression. Our findings are consistent with previous observations that an elevated CTC count results from metastatic spread, such that osseous sites are seeded hematogenously and soft tissue disease spreads predominantly by way of lymphatic routes.22 Although previous work has shown a significant association between the CTC and PSA levels, we found that the correlation between the CTC and PSA levels was modest. Our findings are in agreement with recent findings from Goodman et al23 and Scher et al,24 who showed elevated PSA levels are only slightly associated with survival. Collectively, these data reinforce the idea that the PSA level alone is not of sufficient prognostic value and must be considered in conjunction with additional biomarkers.
CONCLUSION Future advancements in patient care will depend on analysis of multiple markers from primary, metastatic, and circulating tumor cells. Patient-tailored therapy requires the ability to identify the target of interest in a sample that reflects the patient’s disease progression at the time treatment is being considered. Measuring CTC levels, the “liquid biopsy,” has the potential to be an informative biomarker in monitoring metastatic disease progression. In addition to providing pretreatment prognostic information, CTCs are a rich source of disease-specific biomarkers and can be interrogated at the deoxyribonucleic acid, ribonucleic acid, and protein levels. Acknowledgment. To Kenna Anderes, Ph.D., Vice President Scientific Affairs at ApoCell, and Mika Stepankiw for providing editorial assistance, with thanks. References 1. Jemal A, Siegel R, Xu J, et al. Cancer statistics, 2010. CA Cancer J Clin. 2010;60:277-300. 2. Scher HI, Sawyers CL. Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis. J Clin Oncol. 2005;23:8253-8261. 3. Lilja H, Ulmert D, Vickers AJ. Prostate-specific antigen and prostate cancer: prediction, detection and monitoring. Nature Rev Cancer. 2008;8:268-278. 4. Schroder FH, Hugosson J, Roobol MJ, et al. Screening and prostatecancer mortality in a randomized European study. N Engl J Med. 2009;360:1320-1328. 5. Scher HI, Morris MJ, Kelly WK, et al. Prostate cancer clinical trial end points: “RECIST”ing a step backwards. Clin Cancer Res. 2005; 11:5223-5232.
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6. Eccles SA, Welch DR. Metastasis: recent discoveries and novel treatment strategies. Lancet. 2007;369:1742-1757. 7. Nguyen DX, Bos PD, Massague J. Metastasis: from dissemination to organ-specific colonization. Nature Rev Cancer. 2009;9:274-284. 8. de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castrationresistant prostate cancer. Clin Cancer Res. 2008;14:6302-6309. 9. Olmos D, Arkenau HT, Ang JE, et al. Circulating tumour cell (CTC) counts as intermediate end points in castration-resistant prostate cancer (CRPC): a single-centre experience. Ann Oncol. 2009;20:27-33. 10. Scher HI, Jia X, de Bono JS, et al. Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. Lancet Oncol. 2009;10:233-239. 11. Attard G, Swennenhuis JF, Olmos D, et al. Characterization of ERG, AR and PTEN gene status in circulating tumor cells from patients with castration-resistant prostate cancer. Cancer Res. 2009; 69:2912-2918. 12. King JC, Xu J, Wongvipat J, et al. Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis. Nature Genet. 2009;41:524-526. 13. Leversha MA, Han J, Asgari Z, et al. Fluorescence in situ hybridization analysis of circulating tumor cells in metastatic prostate cancer. Clin Cancer Res. 2009;15:2091-2097. 14. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science. 2005;310:644-648. 15. 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. 16. Griwatz C, Brandt B, Assmann G, et al. An immunological enrichment method for epithelial cells from peripheral blood. J Immunol Methods. 1995;183:251-265. 17. Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007;450:1235-1239. 18. Riethdorf S, Fritsche H, Muller V, et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer Res. 2007;13:920-928. 19. Sequist LV, Nagrath S, Toner M, et al. The CTC-chip: an exciting new tool to detect circulating tumor cells in lung cancer patients. J Thorac Oncol. 2009;4:281-283. 20. Cohen SJ, Punt CJ, Iannotti N, et al. Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol. 2008;26:3213-3221. 21. Okegawa T, Nutahara K, Higashihara E. Immunomagnetic quantification of circulating tumor cells as a prognostic factor of androgen deprivation responsiveness in patients with hormone naive metastatic prostate cancer. J Urol. 2008;180: 1342-1347. 22. Danila DC, Heller G, Gignac GA, et al. Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer. Clin Cancer Res. 2007;13:7053-7058. 23. Goodman OB Jr, Symanowski JT, Loudyi A, et al. Circulating tumor cells as a predictive biomarker in patients with hormonesensitive prostate cancer. Clin Genitourin Cancer. 2011;9:31-38. 24. Scher HI, Warren M, Heller G. The association between measures of progression and survival in castrate-metastatic prostate cancer. Clin Cancer Res. 2007;13:1488-1492.
APPENDIX SUPPLEMENTARY DATA
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.urology. 2012.10.041. 1307