Bone Marrow Micrometastases and Markers of Angiogenesis in Esophageal Cancer

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Bone Marrow Micrometastases and Markers of Angiogenesis in Esophageal Cancer Gary M. Spence, MD, Alastair N. J. Graham, MD, Kathleen Mulholland, MRC(Path), Perry Maxwell, PhD, W. Glenn McCluggage, MD, James M. Sloan, MD, and James A. McGuigan, FRCS Departments of Thoracic Surgery and Pathology, Royal Victoria Hospital, Belfast, Northern Ireland

Background. Tumor angiogenesis is critical for metastasis development. The detection of bone marrow micrometastases may indicate a metastatic phenotype. We aim to establish if the detection of bone marrow micrometastases associates with elevated markers of angiogenesis and adverse histopathologic features of esophageal cancer. Methods. Bone marrow aspirates from 49 patients with esophageal cancer were assessed and assigned to be positive or negative for micrometastases. Routine histologic assessment of the primary tumor was also undertaken. Circulating and tumor levels of the angiogenic cytokine vascular endothelial growth factor were determined in plasma and tumor homogenate. Intratumor microvessel density was evaluated by counting antiCD34 positive neovessels. Results. Twenty-two patients were positive for bone marrow micrometastases (44.9%). The detection of micrometastases was associated with advanced T stage (T3/4 vs

T1/2; p ⴝ 0.023), circumferential margin involvement (p ⴝ 0.002) and lymphovascular invasion (p ⴝ 0.024). Plasma vascular endothelial growth factor was significantly more elevated in micrometastatic-positive patients than in those without micrometastases (p ⴝ 0.018). No difference was noted in tumor vascular endothelial growth factor expression. For adenocarcinomas alone, intratumor microvessel density was significantly higher in micrometastatic positive cases (p ⴝ 0.03). This was not the case for squamous cell carcinomas. Conclusions. The detection of bone marrow micrometastases is associated with esophageal tumors of advanced T stage and specifically for adenocarcinomas with tumor vascularity. Plasma vascular endothelial growth factor is elevated in micrometastatic positive cases and might be derived from sources other than the primary tumor. (Ann Thorac Surg 2004;78:1944 –50) © 2004 by The Society of Thoracic Surgeons

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repeatable surrogate marker of angiogenic activity and tumor progression [4]. The systemic dissemination of tumor cells is an essential prerequisite for metastasis formation. Subclinical metastatic disease, undetectable by conventional staging and imaging techniques, may be present at the time of diagnosis [9, 10]. The detection of cancer cells in bone marrow samples suggests the existence of otherwise undetectable systemic micrometastases [11] and might indicate patients at risk of developing clinically important metastatic disease [12, 13]. Cancers arising in the esophagus and at the esophagogastric junction are increasing in prominence in the United States and Europe. This is due to the rising incidence of the adenocarcinoma cell type [14, 15]. Surgery remains the mainstay of treatment, but the prognosis for patients with esophageal cancer treated by surgery alone is poor. The 5-year survival rate remains disappointingly low, averaging 20% in most series [16]. In addition to conventional chemotherapy and radiation therapy, novel therapies such as those that target the tumor vasculature are being developed [17]. Disease-free and overall survival has been shown to be significantly shorter in esophageal cancer patients who have bone marrow micrometastases (BMMs) [18, 19].

umor angiogenesis, the process by which tumors acquire a vascular network, is a critical component of the metastatic cascade and is considered to be one of the hallmarks of cancer [1]. Highly vascular tumors have a greater potential to produce metastases than less angiogenic tumors [2]. Numerous animal and clinical human studies correlate microvascular density [3] and angiogenic growth factor expression [4] with the development of metastasis and poor patient outcome. Presently the most widely accepted marker of tumor angiogenesis is quantification of the number of microvessels per unit area on a representative histopathologic section [5]. This measure is known as the intratumor microvessel density (IMD). Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that has a pivotal role in regulating tumor angiogenesis [6]. Quantitative tissue expression of VEGF is related to IMD and prognosis in esophageal [7] and gastric cancers [8]. VEGF is also detectable in the circulation of patients with cancer and may be a reliable, Accepted for publication March 31, 2004. Address reprint requests to Dr Spence, Department of Thoracic Surgery, Royal Victoria Hospital, Belfast BT12 6BA, Northern Ireland; e-mail: [email protected].

© 2004 by The Society of Thoracic Surgeons Published by Elsevier Inc

0003-4975/04/$30.00 doi:10.1016/j.athoracsur.2004.03.099

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Clinical Staging Protocol Abbreviations and Acronyms BMM ⫽ bone marrow micrometastasis BMM⫹ ⫽ bone marrow micrometastasis positive BMM– ⫽ bone marrow micrometastasis negative CT ⫽ computed tomography ELISA ⫽ enzyme linked immunosorbent assay G ⫽ tumor histological grade IMD ⫽ intratumor microvessel density IQR ⫽ interquartile range LVI ⫽ lymphovascular invasion PLR ⫽ peritumoral lymphocytic reaction P-VEGF ⫽ plasma vascular endothelial growth factor SEM ⫽ standard error of the mean T-VEGF ⫽ tumor vascular endothelial growth factor TNM ⫽ tumor node metastasis VEGF ⫽ vascular endothelial growth factor

Although still being debated, patient staging that is based on the presence or absence of bone marrow micrometastases (BMM⫹/BMM–) may be a valuable early marker of poor outcome, against which novel tumor-derived molecular prognosticators may be assessed. In two published clinical studies that investigated the relationship between angiogenesis and the detection of disseminated tumor cells in bone marrow— one in patients with breast cancer [20] and the other in gastric cancer [21]—an association was discovered between elevated IMD and the presence of bone marrow micrometastases. The significance of angiogenesis in micrometastasis development remains incompletely assessed for patients who have cancer of the esophagus. We therefore examined the bone marrow of such patients for the presence of cancer cells to see if a relationship existed between indicators of a tumor’s angiogenic status and the presence of BMMs. The angiogenic markers measured included IMD, tumor VEGF levels (T-VEGF), and plasma VEGF (P-VEGF) levels.

Patients and Methods Patients Between February 1999 and August 2000, 49 patients (8 women) scheduled to undergo surgical resection of carcinomas situated in the thoracic esophagus or at the esophagogastric junction were recruited to the study prospectively. The mean age at the time of operation was 67 years (range 39 to 83 years). Informed consent was obtained from all patients and ethical approval for the study was obtained from the local research ethics committee. Patients were excluded if they had a past history of malignant disease or of an active angiogenic-related disease process. These included inflammatory arthritis, psoriasis, active ulceration, and critical cardiovascular disease. Patients were also excluded if neoadjuvant chemotherapy had been administered before surgery.

All patients underwent clinical staging by physical examination, upper gastrointestinal endoscopy, chest roentgenogram, and contrast-enhanced computed tomography (CT) of the thorax and upper abdomen. Bronchoscopy, isotope bone scanning, and laparoscopy were used selectively, depending on history, clinical examination, and the results of CT.

Operative Intervention Resection with curative intent was achieved in 42 patients (85.7%). Twenty-three underwent a total thoracic esophagectomy through a left thoracolaparotomy that incorporated a cervical anastomosis. A further 7 patients underwent an esophagogastrectomy through a left thoracolaparotomy only. Two patients had a three-stage esophagectomy, 8 had a two-stage esophagectomy and 2 had a transhiatal esophagectomy. No tissue was obtained from the 7 patients who underwent exploratory surgery only.

Pathologic Staging Protocol The pathologic stage of disease was classified according to the Union Internationale Contre le Cancer tumor node metastasis (TNM) classification and current stage groupings [22].

Retrieval of Tissue Samples PRIMARY TUMOR. Tissue blocks were randomly obtained from each tumor specimen. Half of each block was placed in 10% unbuffered formal saline for 24 hours before being embedded in paraffin wax (Surgipath Medical Industries Inc, Richmond, IL). The other half of each block was placed in polypropylene tubes (Nalge Nunc Laboratory Supplies & Instruments, Hereford, UK), snap-frozen and stored in liquid nitrogen. Because of small tumor size, the material available from two patients was inadequate to facilitate bisection of tumor blocks. Consequently the complete block was processed solely for paraffin wax embedding. The presence of tumor tissue in each block was confirmed by histologic assessment of a representative tissue section. BONE MARROW. Bone marrow aspiration was performed with a single-use bone marrow aspiration needle (Allegiance Healthcare Corporation, McGaw, IL) under general anesthesia at the time of surgery. A single bone marrow aspirate was obtained from the left posterior iliac crest before tumor manipulation. Ten milliliters of bone marrow was aspirated into a syringe containing 2500 IU heparin and added to 10 mL phosphate buffered saline for preparation of cytospins. PLASMA. Ten mL of peripheral venous blood was obtained from each patient the day before surgery and immediately added to 0.25 mL sodium ethylenediamine tetraacetic acid inhibit clotting. Without delay, the blood sample was transported on ice directly to the laboratory where it underwent centrifugation at 13,600g for 3 min-

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utes at 4°C to separate the plasma and cell fractions. The plasma was removed and stored at ⫺80°C until analysis. The time between venesection and plasma storage was consistent and prompt for all patients.

Detection of Bone Marrow Micrometastases PREPARATION OF BONE MARROW CYTOSPINS. Cytospins were prepared by density gradient centrifugation through Ficoll-Hypaque (Pharmacia, Freiburg Germany) [23]. Resuspended bone marrow samples were adjusted to a final concentration of 1 ⫻ 106 nucleated cells per 150 ␮L before being spun onto aminopropyltriethoxysilane coated slides and stored in 95% alcohol at room temperature until immunocytochemical assessment. CYTOSPIN ASSESSMENT BY IMMUNOCYTOCHEMISTRY. Cytokeratins are proteins that comprise the epithelial cell cytoskeleton [24]. Anticytokeratin antibodies can be used in the detection of micrometastatic disease in breast, prostatic, and gastrointestinal malignancy [24]. Cytospins were stained with anticytokeratin antibodies, CAM 5.2 at 2.5 ␮g/mL (BDIS, San Jose, CA) and AE1/AE3 at 0.214 ␮g/mL (Dako, Carpenteria, CA) using alkaline phosphatase-antialkaline phosphatase methodology (Dako). Two cytospins (2 ⫻ 106 cells) were screened for each antibody. Cytokeratin-positive cells were identified by light microscopy and confirmed to have morphologic characteristics of malignant cells by an experienced cytopathologist (WGMcC).

Intratumor Microvessel Density Sections from each primary tumor block were immunostained with anti-CD34 clone number QBEND-10 (Serotec, Oxford, UK), an endothelial cell marker, at 0.2 ␮g/mL using a standard streptavidin-biotin complex immunoperoxidase method for paraffin wax-embedded sections (Dako). The tumor microvessel count was assessed using “hotspot” methodology after the technique described by Graham and colleagues [23]. IMD (expressed as microvessels per mm2) was then derived by dividing the microvessel count by the fixed field size (0.41 mm2).

T-VEGF VEGF protein expression in esophageal tumor blocks was measured using the technique described by Crew and colleagues [25] optimized for use in our laboratory. Briefly, snap-frozen nonfixed tumor blocks were homogenized. After ultracentrifugation, the resulting cell cytosolic fraction was quantitatively assayed for T-VEGF using a commercially available enzyme linked immunosorbent assay (ELISA) kit specific for Quantikine human VEGF (R & D Systems, Minneapolis, MN). T-VEGF levels were standardized for total protein concentration to allow for the effect of varying sized tumor blocks. Total protein was quantified using a technique based on the Bradford colorimetric method for total protein quantification [26]. A commercial protein assay reagent was employed (Coomassie Plus Protein

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Assay Reagent, Pierce, IL). Results were expressed as pg VEGF/mg total protein.

P-VEGF P-VEGF levels were measured as described in a previous report [27] by using the same commercial ELISA kit that was used to measure T-VEGF. P-VEGF concentrations are expressed as pg/mL.

Statistical Analysis Statistical analysis was undertaken using SPSS version 9 for Windows (SPSS Inc, Chicago, IL). Graphs were constructed using GraphPad Prism version 3.0 for Windows (GraphPad Software, San Diego, CA). Data are expressed as mean (SEM) or median {IQR}, where appropriate. Categorical variables were compared with the ␹2 test unless, where stated, by the Fisher exact test. Continuous variables that followed a parametric distribution were compared using the independent samples t test. Nonparametric data were compared using the MannWhitney U test. Correlation analysis was undertaken using Spearman rank correlation. Statistical significance was accepted if p was less than 0.05.

Results Detection of BMM After the morphologic assessment of cytospins immunostained with CAM 5.2 and AE1/AE3, micrometastases was detected in the bone marrow (BMM⫹) of 22 patients (44.9%); and 55.1% were classified as being bone marrow micrometastasis negative (BMM–). Cytokeratin-positive cells were detected in the bone marrow of 19 patients (45.2%) of the 42 patients who underwent an attempted curative surgical resection. There was no difference in the detection of BMMs between those patients who were operable and the 7 who were not (p ⫽ 0.999, Fisher exact test).

BMM and Pathological Disease Stage There was no difference in the size of the primary tumor between those patients who were BMM⫹ and those who were BMM–; the median tumor area was 9 {7–18} cm2 vs 9 {2.7–21} cm2, respectively ( z ⫽ ⫺0.835, p ⫽ 0.404, Mann Whitney U test). The incidence of disseminated tumor cells in bone marrow and their relationship to primary tumor staging, histopathologic features, and margin involvement of the resected tumor specimen are shown in Tables 1, 2, and 3, respectively.

IMD and BMM Taking all esophageal tumor specimens, no significant difference was noted in IMD between those patients that were BMM– and those that were BMM⫹ (333 (29) vs 406 (48) microvessels/mm2; t ⫽ ⫺1.341, p ⫽ 0.187; independent samples t test with 40 df). A study of lung cancer showed that adenocarcinomas have higher IMD than squamous cell cancers [23]. We have demonstrated similar findings in esophageal cancer

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Table 1. Disseminated Tumor Cells in Bone Marrow—Relationship to Tumor Staging in Resectable Esophageal Carcinoma (n ⫽ 42)a BMM⫹ (%) T1 T2 T3 T4 N0 N1 M0 M1 Stage Stage Stage Stage

I II III IV

0 (0%) 2 (22%) 17 (58.6%) 0 5 (35.7%) 14 (50%) 19 (46.3%) 0 (0%) 1 (16.7%) 4 (40%) 13 (54.2%) 1 (50%)

Total cases 4 9 29 0 14 28 41 1 6 10 24 2

Table 3. Disseminated Tumor Cells in Bone Marrow ⫺ Relationship to Surgical Limits for Patients With Resectable Esophageal Carcinoma (n ⫽ 42)

p value

BMM⫹ (%)

c

0.023 (T1/T2 vs. T3/T4) ... ... 0.584 ... 0.548b ... 0.267 (Stage I/II vs. III/IV) ...

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Circumferential margin⫹a Circumferential Margin⫺ Resection Margin⫹ Resection Margin⫺ a b

17 (65.4%) 2 (12.5%) 0 (0%) 19 (47.5%)

Total cases

p value

26 16 2 40

0.002c 0.492b

Tumor present at/or within 1 mm of the inked circumferential margin. c Fisher exact test. Significant at p ⬍ 0.05.

of 49) who had micrometastases detectable in their bone marrow (Fig 2).

T-VEGF and P-VEGF

a

Accurate postsurgical stage data available for resectable carcinomas b c only. Fisher exact test. Significant at p ⬍ 0.05.

comparing adenocarcinoma and squamous cell (413 (29) vs 193 (14) microvessels/mm2, respectively; t ⫽ 3.844, p ⬍ 0.001, independent samples t test with 40 df). Consequently, we decided to analyze the influence of IMD on bone marrow status for each cell type in turn. High tumor vascularity is associated with detection of BMMs for adenocarcinomas (Fig 1). This is not seen for squamous cell carcinomas (Fig 1).

T-VEGF and BMM Forty esophageal tumors were assessed for quantitative T-VEGF protein expression and 19 (47.5%) were BMM⫹. No significant difference in T-VEGF was found between BMM– and BMM⫹ patients (213 {128 –511} vs 278 {154 – 395} pg VEGF/mg total protein, respectively; z ⫽ ⫺0.149, p ⫽ 0.882, Mann Whitney U test).

No correlation was found between tumor expression of VEGF and circulating plasma levels (␳ ⫽ ⫺0.117, p ⫽ 0.473, Spearman rank test).

Comment The finding of disseminated tumor cells in the bone marrow samples of 44.9% of patients with invasive esophageal cancer (45.2% of those that were resectable) compares closely with prevalence rates obtained from aspirated iliac crest marrow and immunocytochemical methodology that have been reported previously in the literature. Thorban and colleagues discovered bone marrow micrometastases in 41.1% of patients with squamous cell cancers of the esophagus [18] and a meta-analysis of assorted types of carcinoma found a median prevalence of approximately 35% [28]. The BMM detection rate was found to be increased in

P-VEGF and BMM Compared with BMM–patients, P-VEGF was significantly elevated in the 22 esophageal cancer patients (out Table 2. Disseminated Tumor Cells in Bone Marrow—Relationship to Histopathological Features of the Esophageal Tumor BMM⫹(%) Total cases Squamous cell Adenocarcinoma Middle/Lower esophagus OGJ/Cardia G1 G2 G3/G4 LVI⫹ LVI⫺ PLR⫹ PLR⫺

8 (66.7%) 14 (37.8%) 9 (56.3%) 13 (39.4%) 2 (100%) 9 (47.4%) 8 (38.1%) 19 (52.8%) 0 (0%) 15 (42.9%) 4 (57.1%)

12 37 16 33 2 19 21 36 6 35 7

p value 0.158 0.42 0.535a (G1/G2 vs G3/G4) 0.024abc 0.682ab

a b Considering resectable carcinomas only (n ⫽ 42). Fisher exact c test. Significant at p ⬍ 0.05. G ⫽ tumor histological grade; LVI ⫽ lymphovascular invasion; PLR ⫽ peritumoral lymphocytic reaction.

Fig 1. Intratumor microvessel density (IMD) versus status of bone marrow micrometastases (BMM) (subgroup analysis by cell-type). Squamous cell carcinomas are depicted in black, adenocarcinomas in white. The bar represents the mean value, error bar represents mean plus standard error of the mean. *IMD was significantly higher in patients who were BMM⫹ than in patients who were BMM⫺ for adenocarcinomas (487 (48) versus 359 (32), t ⫽ ⫺2.278, p ⫽ 0.030, independent samples t test with 31 df). No difference was noted between BMM⫹ and BMM⫺ for squamous cell carcinomas (180 (17) versus 211 (22), t ⫽ 1.127, p ⫽ 0.297, independent samples t test with 7 df).

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Fig 2. Scatter plot of plasma vascular endothelial growth factor (PVEGF) versus status of bone marrow micrometastases (BMM). *BMM⫺ versus BMM⫹ (10 {0 –22} versus 20 {13– 47} pg/mL, respectively, z ⫽ ⫺2.370, p ⫽ 0.018, Mann Whitney U test).

patients who had tumors of advanced T stage (Table 1) and in those who had circumferential margin involvement (Table 3). In contrast, BMM⫹ patients did not have tumors of larger mucosal surface area than BMM– patients, nor was there a relationship between lymph node positivity and detection of BMMs (Table 1). Similar findings have been described previously in breast cancer [29]. Braun and colleagues found that the detection of BMMs was associated with advanced T stage but not lymph node involvement [29]. They postulated that alternative pathways exist for tumor cell dissemination that might result in distinct patterns of metastasis [29]. As such, it may be speculated that two distinct routes to metastasis also exist for esophageal cancer. Tumor cells may disseminate initially to the lymphatics or directly by way of the microcirculation. The collective importance of both pathways as a means to tumor cell dissemination is confirmed by the finding that BMMs are more common in patients with esophageal tumors that have lymphatic or vascular invasion noted on conventional histologic analysis. Indeed, no case in which lymphovascular invasion was absent had micrometastases detected in the bone marrow (Table 2). With regard to the association between tumor angiogenic markers and detection of BMMs, the study findings are more equivocal. Although a correlation between IMD and BMMs could be demonstrated for esophageal adenocarcinomas (Fig 1), IMD disparity between cell types, insufficient numbers in the squamous cell subgroup, and biologic variation are all potential reasons why BMMs were not associated with IMD when all patients were collectively assessed. Another possibility is that metastasis formation is a complex multistage process and angiogenesis may not be the rate-limiting step to tumor cell dissemination in squamous cell cancers. Some other component of the metastatic process, such as loss of cell– cell adhesion or degradation of the extracellular matrix, may hold that position. The positive correlation between high tumor vascular-

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ity and the presence of BMMs (as seen for esophageal adenocarcinomas) is postulated to be because of a larger endothelial surface area with which potentially metastatic cells can interact. Supporting this theory is the finding of a positive association between tumor microvessel density and the number of circulating tumor cells detected intraoperatively in patients undergoing breast cancer surgery [30]. No difference in T-VEGF between BMM⫹ and BMM– cases was found. Although perhaps unexpected, it should not be surprising when one considers that alternative angiogenic factors may be involved in mediating tumor angiogenesis for patients with esophageal carcinoma. It is possible that VEGF is not the only, or even the pre-eminent, angiogenic growth factor responsible for neovascularization of primary esophageal tumors. Other cytokines such as thymidine phosphorylase [31] and transforming growth factor-␣ [32] may be more critical. In contrast, VEGF measured in the plasma of BMM⫹ esophageal cancer patients was significantly higher than that measured in the plasma of those who were BMM– (Fig 2). It is however, counter-intuitive that P-VEGF can influence detection of BMMs as a function of primary tumor angiogenesis when T-VEGF levels clearly do not. Thus, it may be proposed that the association between P-VEGF and bone marrow positivity might simply relate to a common correlation with advancing tumor T stage. We have not however, found this to be the case. Although it has been demonstrated that patients with advanced primary tumors are more likely to be BMM⫹ (Table 1), advanced T stage tumors do not have higher levels of P-VEGF (T1/T2 vs T3/T4, 13 {0 –23} vs 18 {0 –27} pg/mL, respectively; z ⫽ ⫺0.262, p ⫽ 0.793, Mann Whitney U test). Another explanation might be that in addition to the primary tumor, VEGF in the circulation may be derived, not only from nontumor-derived circulating elements such as platelets [27] but also from alternative sources such as systemically dispersed micrometastatic tumor deposits. This might also explain why no correlation was found between tumor and circulating VEGF levels. Micrometastases remain in a dormant state, during which proliferation and apoptosis are in balance [33]. A balance of proangiogenic and antiangiogenic factors may maintain the dormancy of micrometastases [34]. VEGF may be one such angiogenic stimulator secreted by micrometastatic tumor cells [34]. Thus disseminated micrometastatic cells may have a differing angiogenic cytokine profile to the primary tumor and in combination with platelets, which are known to be potent source of circulating VEGF [27], may contribute to P-VEGF levels. This study provides evidence that for esophageal adenocarcinomas, systemic tumor cell dissemination is positively associated with primary tumor neovascularity. Furthermore, we speculate that high circulating VEGF levels in the plasma of esophageal cancer patients may be linked to sources other than the primary tumor. The detection of micrometastases in the bone marrow may in itself be a useful adjunct to presurgical and postsurgical staging. Although not discriminatory with

regard to resectability in this study, the detection of BMMs at diagnosis may influence the surgeon to consider preoperative neoadjuvant therapy before surgical intervention. Proponents postulate that the detection of BMMs at the time of surgery indicates the presence of a metastatic phenotype. Others argue that such detection merely reflects transient shedding of cells from the primary tumor and does not indicate increased metastatic potential [35]. For patients in this study, long-term follow-up will be required to clarify if the detection of bone marrow micrometastases at the time of surgery has independent prognostic significance. The authors thank Mr Kieran G. McManus, Mr Eamon Mackle, and Mr R. John Moorehead for assistance with patient recruitment; Dr Marilyn A. Armstrong for assistance with VEGF assays; and Dr Gordon Cran for statistical advice. This work was supported by grants from the Royal Victoria Hospital and Ulster Hospital Research Funds, the Mason Medical Foundation, and the Ulster Cancer Foundation.

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20. 21. 22. 23.

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