Increases in Quantitative D-Dimer Levels Correlate With Progressive ...

Coagulation and Transfusion Medicine / CTC and Quantitative D-Dimer Levels in Prostate Cancer

Increases in Quantitative D-Dimer Levels Correlate With Progressive Disease Better Than Circulating Tumor Cell Counts in Patients With Refractory Prostate Cancer Joseph D. Khoury, MD,1 Dorothy M. Adcock, MD,2 Fanny Chan, MD,1 James T. Symanowski, PhD,3 Stefan Tiefenbacher, PhD,2 Oscar Goodman, MD, PhD,4 Lazara Paz, MS,1 Yupo Ma, MD,5 David C. Ward, PhD,6 Nicholas J. Vogelzang, MD,7 and Louis M. Fink, MD1 Key Words: Prostate cancer; D-dimer; Circulating tumor cells; Prostate-specific antigen DOI: 10.1309/AJCPH92SXYLIKKTS

Abstract Changes in quantitative D-dimer levels, circulating tumor cell (CTC) counts, and prostate-specific antigen (PSA) levels were measured in 28 patients with refractory castration-resistant prostate cancer to assess their concordance during the course of therapy and their relationship with risk of progressive disease. A significant correlation was identified between changes in PSA and both CTC counts and D-dimer levels (r = 0.67 and 0.58, respectively; P < .001). In addition, there was a significant correlation between changes in CTC count and D-dimer level (r = 0.62; P < .001). A significantly stronger concordance between these biomarkers was noted for increasing values (sensitivity, 72%-77.8%) compared with decreasing values (specificity, 43.8%-71.4%). Notably, increases in PSA and D-dimer levels, not CTC counts, were associated with increased risks for progressive disease (P < .024). Increases in quantitative D-dimer levels correlate with progressive disease better than CTC counts in patients with refractory prostate cancer.

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Am J Clin Pathol 2010;134:964-969 DOI: 10.1309/AJCPH92SXYLIKKTS

Prostate cancer (PCa) is the most common epithelial malignancy in men and the second leading cause of cancerrelated deaths in the United States.1 A subset of PCa is characterized by de novo or progressive resistance to hormonal therapy, commonly referred to as castration-resistant PCa. Although measurements based on quantification of serum prostate-specific antigen (PSA) levels remain widely used to assess disease progression in patients with PCa,2,3 their role appears limited in castration-resistant cases.4 The usefulness of radiographic tools to assess clinical end points in PCa is also generally limited.5,6 Accordingly, there remains a clinical need for biomarkers that can complement or surpass PSA in assessing disease status, especially in patients with PCa who fail first-line therapy and are castration-resistant. Circulating tumor cell (CTC) enumeration has recently emerged as a useful prognostic tool in patients with prostate, breast, or colon cancer.7-9 Notably, several studies have demonstrated that CTC enumeration is an accurate and independent predictor of overall survival in castration-resistant PCa.9-14 Patients with unfavorable pretherapy or posttherapy CTC counts (>5 CTCs/7.5 mL of whole blood) were found to have a significantly shorter median overall survival in comparison with patients with favorable counts (≤5 CTCs/7.5 mL of whole blood).11 In addition, relative to PSA monitoring, determination of CTC counts may allow earlier clinical decisions regarding response to therapeutic interventions in patients with PCa.9,10,12,13 Hypercoagulability with venous thromboembolism is frequent in a variety of cancers.15 As by-products of fibrin degradation, plasma D-dimer levels are generally elevated in association with hypercoagulable conditions. Because hypercoagulability associated with cancer increases in severity © American Society for Clinical Pathology

Coagulation and Transfusion Medicine / Original Article

with disease progression, quantitative assessment of plasma D-dimer levels has been suggested as a measure of disease status in patients with malignant neoplasms.15-18 Indeed, D-dimer levels have been found to rise proportionally to the extent and severity of disease in patients with PCa.19,20 However, D-dimer levels may be elevated in a variety of other conditions such as trauma, surgery, and inflammatory processes, thereby limiting assay specificity. The use of quantitative D-dimer determinations in conjunction with PSA and CTC count (hypothetically to enhance specificity) has not been evaluated in patients with refractory PCa. In this study, we assessed the relationship between quantitative D-dimer levels, CTC counts, and PSA levels in patients with refractory, castration-resistant PCa and asked whether changes in these parameters are concordant when measured in longitudinal studies during the course of therapy. In addition, we asked whether quantitative D-dimer levels and CTC enumeration, alone or in combination, enhance the usefulness of the PSA level in assessing disease status in this category of patients with PCa.

Materials and Methods Patient Group This study was approved by the institutional review board overseeing human research at Nevada Cancer Institute, Las Vegas. The study group consisted of 28 patients with castrationresistant PCa seen between 2007 and 2009. All patients in this cohort were resistant to standard first-line or second-line hormonal therapy. Blood samples were collected from each of the patients at each regular visit to assess disease status through monitoring PSA and CTC levels. A total of 69 samples from the 28 patients were analyzed. In addition, for the purpose of this study, the interval overall clinical status of the disease between visits was assessed during the study period and classified as progressive disease (PD) or nonprogressive disease (non-PD) based on review of clinical progress notes, imaging findings indicative of enlarging or new tumor sites, and, to a lesser extent, laboratory studies (eg, lactate dehydrogenase level). Serum PSA Level Determination Serum total PSA levels were determined using an ADVIA Centaur automated platform (Siemens, Deerfield, IL) using a 2-site sandwich immunoassay and direct chemiluminescence. Assessment was performed following the manufacturer’s recommendations. D-Dimer Quantification Quantification of plasma D-dimer levels was carried out using the Asserachrom test kit (Diagnostica Stago, Parsippany, NJ), an enzyme-linked immunoassay. Samples in which the

D-dimer level exceeded the linearity range of the assay (9.6 μg/mL) were diluted, and the final D-dimer level was calculated accordingly. CTC Enumeration CTCs were detected in whole blood by immunomagnetic isolation and immunofluorescent staining using the CellSearch system (Veridex, San Diego, CA). Briefly, 10 mL of blood is drawn into a tube containing cell preservatives and maintained at room temperature, and an aliquot of 7.7 mL is processed and analyzed within 24 hours. The sample is incubated with epithelial cell adhesion molecule (EpCAM) antibody–covered ferroparticles at room temperature and processed on the CellTracks Autoprep (Veridex). Circulating epithelial cells expressing EpCAM are isolated by a magnetic field without centrifugation. After the supernatant containing unbound cells is removed, the enriched samples are processed for fluorescence. Nucleic acid is stained with 4,6-diamidino2-phenylindole (DAPI), and epithelial cells are stained with anti–cytokeratin-diamidino-2-phenylindole. Leukocytes are excluded with an allophycocyanin-conjugated anti-CD45 antibody. Stained cells are analyzed on a fluorescence microscope using the Cell Track Analyzer II (Veridex). CTCs are defined as cytokeratin+, DAPI+ cells lacking CD45 staining. The counts are performed by a technician, trained by Veridex, and interpreted by a pathologist (L.M.F.). Statistical Analysis Exploratory statistical analyses were conducted to assess the association between interval changes in biomarkers and the corresponding clinical assessments. For each patient and interval, the changes in log10 transformation of biomarker values were calculated for PSA level, CTC count, and D-dimer level. Analysis of variance was conducted to compare the biomarker changes in the PD group with changes in the nonPD group. Logistic regression was conducted to assess the association between the biomarker changes and the probability of PD. Biomarker changes were further dichotomized as increasing or decreasing. These changes were also used in the logistic regression analysis. Sensitivity and specificity were calculated to assess the concordance between the directional changes in biomarkers (increasing or decreasing) and the interval clinical status groups (PD or non-PD). Pearson correlation coefficients were calculated for the interval changes: PSA level vs CTC count, PSA level vs D-dimer level, and CTC count vs D-dimer level. Concordance among biomarker changes was also calculated.

Results Patients had a median age of 63.4 years (range, 53-78 years). For the entire group, PSA values ranged from 0.1 to

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9,051.4 ng/mL, quantitative D-dimer levels ranged from 0.125 to 15.608 μg/mL, and CTC counts ranged from 0 to 4,732 cells/7.5 mL of whole blood. The median interval between blood collections for biomarker analyses ranged between 105 and 110 days. A significant correlation was identified between changes in PSA levels and changes in CTC counts and D-dimer levels (r = 0.67 and 0.58, respectively; P < .001) ❚Figure 1A❚. In addition, there was a significant correlation between changes in CTC counts and D-dimer levels (r = 0.62; P < .001) ❚Figure 1B❚. The identified correlation between changes in PSA and D-dimer levels was in keeping with previously published observations.20 Concordance in the directional changes (increasing vs decreasing) among the 3 biomarkers ❚Table 1❚ and ❚Table 2❚ reflected the Pearson correlations. It is interesting to note that there was stronger concordance between these biomarkers for increasing values (sensitivity, 72.0%-77.8%) than for decreasing values (specificity, 43.8%-71.4%). Changes in D-dimer levels, CTC counts, and PSA levels in the intervals between blood collections were analyzed as a function of the assessed disease status (PD vs non-PD). The data are summarized in ❚Table 3❚. In general, all 3 biomarkers trended upward as disease progressed (PD), whereas they remained overall relatively unchanged in the non-PD group. Differences between the PD and the non-PD groups showed stronger trends for PSA and D-dimer changes (P < .013) than for changes in CTC counts (P = .150). In the 14 intervals associated with PD, 13 had increasing PSA levels, 10 had increasing CTC counts, and 13 had increasing quantitative D-dimer measurements, with 92.9% sensitivity for each of D-dimer and PSA levels and 71.4% for CTC counts ❚Table 4❚. On the other

A

❚Table 1❚ Comparison of CTC Counts and D-Dimer Levels With PSA Measurements CTC Count

D-Dimer

PSA

Increase

Decrease

Increase

Decrease

Increase Decrease Sensitivity (%) Specificity (%)

21 4 77.8 71.4

6 10

21 6 77.8 57.1

6 8

CTC, circulating tumor cell; PSA, prostate-specific antigen.

❚Table 2❚ Comparison of CTC Counts and D-Dimer Levels* D-Dimer CTC Count

Increase

Decrease

Increase Decrease

18 9

7 7

CTC, circulating tumor cell. * Sensitivity, 72.0 (%); specificity, 43.8 (%).

hand, the concordance between the biomarker changes in the intervals associated with non-PD was markedly weaker, with 48.1% specificity for each of D-dimer and PSA levels and 44.4% for CTC counts (Table 4). Correlation between disease status and changes in biomarker levels was further assessed by using logistic regression. Results are summarized in ❚Table 5❚. Two sets of models

B 3.00

1.20

2.00

0.80 0.40

1.00

0.00 0.00 –0.40 –1.00

–0.80

–2.00

–1.20 –1.80

–3.00 –1.80

–1.20

–0.60

0.00

0.60

Change in Log PSA

1.20

1.80

–3.00

–2.00

–1.00

0.00

1.00

2.00

3.00

Change in Log CTC

❚Figure 1❚ Correlation between prostate-specific antigen (PSA) changes and changes in D-dimer (closed circles) and circulating tumor cell (CTC) count (open circles; A) and between CTC count changes and changes in D-dimer (B). Numbers on the y-axis represent changes in log10 transformed values of CTC or D-dimer. A, D-dimer, r = 0.58. CTC count, r = 0.67. B, D-dimer, r = 0.62. 966 966




❚Table 3❚ Analysis of Variance of the Changes in PSA Level, CTC Count, and D-Dimer Level on Interval Disease Status Clinical Outcome Non-PD Mean Median Maximum Minimum No. of cases PD Mean Median Maximum Minimum No. of cases P

PSA Level

CTC Count

D-Dimer Level

–0.056 0.055 1.079 –1.783 27

–0.041 0.301 2.750 –2.812 27

0.016 0.011 1.052 –1.380 27

0.506 0.480 1.681 –0.049 14 .004

0.536 0.500 2.189 –0.749 14 .150

0.352 0.240 1.052 –0.013 14 .013

CTC, circulating tumor cell; PD, progressive disease; PSA, prostate-specific antigen.

were analyzed: (1) using directional changes in the biomarkers (increase vs decrease) and (2) using the change in the log transformation of the biomarkers. For both parameterizations, increases in PSA and D-dimer levels were associated with increased risks for PD (P < .024), and all odds ratios were greater than 11. CTC counts were not predictive of an increased risk for PD.

Discussion D-dimers are degradation products of cross-linked fibrin. The presence of D-dimers in plasma is an indirect marker of coagulation activation followed by reactive thrombolysis.15 Elevated plasma D-dimer levels can be seen in patients with cancer because procoagulant factors in various types of cancer lead to constitutive activation of the coagulation cascade with resultant thrombin generation followed by fibrin formation.19 Fibrin may also conversely form a protective shield on malignant tumor cells, making them resistant to endogenous defense mechanisms and promoting angiogenesis, tumor invasion, and metastasis.12,15,19-26 The clinical correlate of these phenomena is that quantitative determinations of D-dimer levels

have been associated with tumor progression in prostate, lung, breast, and colorectal carcinoma.16-18,27 However, in patients with cancer, D-dimer levels may be also elevated in response to other conditions, such as deep vein thrombosis, pulmonary embolism, severe infection, inflammation, sepsis, surgery, and disseminated intravascular coagulation. In this study, with 1 exception, patients did not appear to have conditions, other than PCa, to explain elevated D-dimer levels. The exception was 1 patient who was diagnosed during the study interval with deep vein thrombosis and pulmonary embolism. The serum PSA level is one of the main measures of disease progression in castration-resistant PCa, albeit its prognostic value is generally limited.3,28-31 Despite its production by tumor cells and secretion in the circulation in most cases of PCa, serum PSA levels may be nondetectable or very low in patients with high-grade prostatic adenocarcinoma or other histologic variants such as small cell neuroendocrine carcinoma.4,5 These limitations of PSA have necessitated a quest for additional biomarkers of disease burden and prognosis in patients with PCa. CTC enumeration in whole blood has emerged during the past several years as a novel biomarker for patients with PCa and with other epithelial malignancies such as breast and colorectal carcinoma and has been approved by the US Food and Drug Administration as a predictive biomarker.9-13,32 While useful as a general biomarker of tumor burden in patients with epithelial malignancies, CTC counts do not seem to consistently correlate with standard measures of response to therapy33 and, in some contexts, miss CTCs that lack EpCAM expression.34,35 In view of the

❚Table 5❚ Logistic Regression for Risk of Disease Progression Increase/Decrease

Prostate-specific antigen Circulating tumor cell count D-dimer

Δ Changes of Log

P

Odds Ratio

P

Odds Ratio

.024 .327 .024

12.07 2.00 12.07

.014 .153 .024

11.46 1.55 15.31

❚Table 4❚ Concordance Between PSA Level, CTC Count, and D-Dimer Level With Interval Disease Status PSA Level

CTC Count

D-Dimer Level

Disease Status

Increase

Decrease

Increase

Decrease

Increase

Decrease

Progressive Nonprogressive Sensitivity (%) Specificity (%)

13 14 92.9 48.1

1 13

10 15 71.4 44.4

4 12

13 14 92.9 48.1

1 13

CTC, circulating tumor cell; PSA, prostate-specific antigen.


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limitations of PSA levels and CTC counts, our data suggest that quantitative D-dimer assessment offers an added value in monitoring disease progression in patients with castrationresistant PCa when coupled with these 2 biomarkers. A noteworthy finding in this study is the higher sensitivity and predictive ability of quantitative D-dimer assessment in comparison with CTC counts, especially when trending upward. This observation suggests that quantification of plasma D-dimers offers a better reflection of increasing tumor burden than enumeration of CTCs in patients with refractory castration-resistant PCa. It might be that the reason why D-dimer quantification seems to be better than CTC enumeration in predicting disease progression is related to the biologic factors that impact variations in these 2 biomarkers: release of tissue factor–bearing microparticles and shedding of tumor cells into the circulation, respectively. Microparticles are cell-derived submicron vesicles that express protein surface components from the parent cell from which they are derived. Tissue factor is a receptor protein capable of potently initiating blood coagulation in vivo.36 Tumor-derived tissue factor– bearing microparticles are associated with venous thromboembolism in patients with cancer and seem to be central to the pathogenesis of cancer-associated thrombosis.37 The release of microparticles into the circulation is hypothetically prone to relatively few microanatomic and immunologic impediments. On the other hand, for CTCs to enter the bloodstream, they would have to acquire the ability to separate from the tumor mass, penetrate blood vessels, and evade the immune system in the circulation before a persistent steady state is achieved at any given point during the disease course.38-40 We postulate that because it involves more stochastic steps, CTC detection and enumeration might not reflect tumor burden in patients with PD as linearly or as nimbly as D-dimer quantification. It is acknowledged that the results noted in this retrospective study represented a limited sample from nonuniformly treated patients and need to be further evaluated in the context of a controlled prospective clinical trial. One aim of such a prospective analysis would be to also determine whether data identified in our study suggesting a limited added advantage for CTC count over PSA and D-dimer levels may be extrapolated to a larger patient cohort. Confirming these data might have economic implications, if indeed D-dimer quantification is found to be equivalent or superior to CTC enumeration, in view of the higher cost and technical prerequisites of the latter. The use of PSA in conjunction with quantitative D-dimer seems to be a useful tool to estimate disease progression in patients with castration-resistant PCa. This biomarker combination seems more effective than PSA levels and CTC counts. Confirmation of whether D-dimer quantification may supplant CTC enumeration on the Veridex CellSearch system remains to be determined in a larger prospective study.

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From the Departments of 1Pathology, 3Biostatistics, and Nevada Cancer Institute, Las Vegas; 2Esoterix Coagulation, Englewood, CO; 5Department of Pathology, State University of New York at Stony Brook; 6Cancer Research Center of Hawaii, University of Hawaii, Honolulu; and 7Comprehensive Cancer Centers, Las Vegas.

4Hematology/Oncology

Supported in part by Department of Energy grant DE-FG0208ER64608 (Drs Fink, Ma, and Ward). Address reprint requests to Dr Khoury: Dept of Pathology, Nevada Cancer Institute, One Breakthrough Way, Las Vegas, NV 89135.

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