| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Oncology, Markers, Clinical Correlates |
1 Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Switzerland; 2 Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark; and 3 Institute of Medical Oncology, Inselspital, Berne, Switzerland
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Experimental Design: CCK gene expression was evaluated with in situ hybridization or reverse transcription-PCR in tumor tissue. CCK precursors and bioactive CCK were measured with specific RIAs in tumor tissue, in cell culture medium, and in plasma of Ewing sarcoma patients before and after chemotherapy as well as after tumor recurrence.
Results: CCK mRNA was identified in 12 Ewing sarcoma biopsies sampled in two series and in four Ewing sarcoma cell lines but not in unrelated neoplasia. Immunoreactive proCCK was identified in the culturing medium of all Ewing sarcoma cell lines but not in the media from unrelated tumor cell lines. Moreover, in plasma from Ewing sarcoma patients, precursors and mature forms of CCK, in particular proCCK, were detected; several fold elevation of the total proCCK product was found in plasma from patients before treatment and after tumor recurrence, whereas successful chemotherapy reduced proCCK to basal concentrations. Plasma concentrations of proCCK paralleled the respective tumor size.
Conclusions: This is the first study that consistently documents an altered CCK metabolism in human cancer; Ewing sarcomas synthesize and secrete proCCK that can be identified in plasma as circulating tumor marker.
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Recently, however, mRNA for CCK has been detected in Ewing sarcomas (5 , 6) , a tumor of mesenchymal origin. Ewing sarcoma is yet an unclear entity, difficult to classify (7 , 8) , and for which new parameters are needed to better understand its biology and improve its diagnosis and therapy. One such parameter is the t(11;22) translocation (9 , 10) with the resulting fusion oncoprotein EWS-FLI 1 (11) in 90% of the Ewing sarcomas (11) and the t(21;22) translocation with the EWS-ERG fusion protein in 5–10% of the sarcomas (12) . Another example is the recent finding of gastrin and CCK mRNA in tumor tissues, which may be of prime interest for the understanding of the biology of this sarcoma; however, it is even more relevant, from a clinical point of view, to examine whether CCK and gastrin peptides from the Ewing tumors are released into the circulation in diagnostic amounts.
The present report is an analysis of the CCK gene expression in Ewing sarcoma cell lines and in several patients with Ewing sarcomas, in comparison with tumors unrelated to Ewing sarcoma. First, cell lines expressing the EWS-FLI 1 and EWS-ERG fusion proteins were examined with respect to CCK peptide synthesis and release. Furthermore, CCK mRNA as well as peptide precursors, processing intermediates and mature peptides (2) were identified in the tumor tissue of Ewing sarcoma patients. As final proof, precursor and mature forms of CCK were measured in the circulation of three patients before and during therapy and in one of the patients after tumor recurrence.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Patients
In five Ewing sarcoma patients, part of the tumor biopsy collected for diagnostic purposes before treatment or part of the surgically resected tumor after chemotherapy was frozen in dry ice and stored at –80°C. These samples were used for mRNA detection by in situ hybridization and/or for measuring the tissue peptide contents by RIA. In two of these patients, as well as a third one with no tumor tissue collected, blood samples were taken before, during, and/or at the end of therapy to quantify the plasma peptide content.
One of the patients (1ZEW) was a 22-year-old man admitted with increasingly severe knee and hip joint pain. The radiological examinations at admission showed a process in the hip and femur bone with infiltration in the soft tissues. Magnetic resonance imaging (MRI) examination identified a large circular tumor around the bone of approximately 60 x 60 x 40 mm (144,000 mm3). The other staging examinations showed no metastases. The tumor biopsy showed, by histomorphological and immunohistochemical criteria, the presence of a Ewing sarcoma (13) . The diagnosis was cytogenetically confirmed by detection of a t(11;22) translocation. An induction chemotherapy started on day 5 after diagnosis with vincristin (2 mg for 1 day), ifosfamid (5.2 g/day for 3 days), doxorubicin (35 mg/day for 3 days), and etoposid (250 mg/day for 3 days). After two cycles, the radiological evaluation with MRI on day 38 showed a decrease of the tumor mass to approximately 24,000 mm3 (40 x 30 x 20 mm) in the right femur, corresponding to a partial remission. After two additional cycles, the tumor had shrunk to 25 x 15 x 10 mm (3750 mm3) on day 89. A surgical tumor resection was considered. But at that time, the MRI evaluation showed two small new lesions in the contralateral pelvic bones, one in the spina iliaca anterior inferior (15 x 10 mm) and one in the os ileum (5 x 5 mm). Therefore, instead of surgical resection of the primary tumor, chemotherapy was continued. However, after three more cycles, the MRI on day 157 showed an increase of the soft tissue mass in the femur area of 60 x 40 x 25 mm (60,000 mm3), whereas the two small pelvic lesions did not increase beyond the first detection. The patient received on the same day a first high-dose chemotherapy with alkeran (60 mg/day for 4 days) and etopophos (750 mg/day for 4 days), and a stem cell infusion. The MRI evaluation on day 196 showed a further tumor progression in all sites: femur, 60 x 40 x 30 mm (72000 mm3); spina iliaca anterior inferior, 20 x 20 x 20 mm (8000 mm3); and os ileum, 30 x 25 x 20 mm (15000 mm3). The patient was then symptomatic with bone pain in the right hip and femur. A palliative radiotherapy started on day 196. The following weeks, the patient’s pain increased, and the conventional X-ray examination showed a progressive disease in all sites. The patient died a few weeks later, 287 days after diagnosis. Blood samples for CCK measurements were taken before therapy, 3 months after tumor volume-reducing chemotherapy on day 88 and after tumor recurrence on day 217.
The other patient (2REW) was a 13-year-old boy admitted with pain and swelling of the left distal forearm. MRI performed at admission showed a soft tissue tumor with cortical bone reaction, of a size of 35 x 30 x 30 mm (31,500 mm3). Other staging examinations were negative. The tumor biopsy fulfilled the histomorphological and immunohistochemical diagnosis criteria of a Ewing sarcoma. The diagnosis was cytogenetically confirmed by detection of a t(11;22) translocation. The patient was treated with induction-chemotherapy from day 17 to day 86 after diagnosis, with vincristin, doxorubicin, cyclophosphamid, and ifosfamid. An MRI examination, performed on day 100, detected a significant decrease of the tumor size, which had shrunk to 25 x 15 x 15 mm (5,625 mm3). A wide tumor resection with a fibula transplantation followed on day 122. Afterward, the patient received a consolidation-chemotherapy until day 139. Six months later, under a maintenance chemotherapy with doxorubicin, ifosphamid, vincristin, cyclophosphamid, and etopophos, neither clinical nor MRI examination revealed any tumor presence. Blood samples for CCK measurements were taken before therapy on day 9 after diagnosis and after 3.5 months therapy on day 121. Further blood samples were taken during clinical remission at various intervals.
The third patient (3HEW) was a 27-year-old man admitted in a bad performance status with dyspnoe and pain in the right elbow. The staging examinations showed a tumor in the right ulna of 40 x 30 x 25 mm (30,000 mm3) and a huge tumor mass and fluid filling the whole left chest (size not measurable). The tumor biopsy of the ulnar mass was diagnosed histopathologically as a Ewing sarcoma. A chemotherapy with Oncovin, Bleomyin, Etopothis, and Holoxan was started. The patient left the hospital 21 days later folowing the end of the second chemotherapy cycle. Forty-two days after the diagnosis, the radiological evaluation showed a massive reduction in the tumor mass in the right ulna (20 x 10 x 15 mm, 3000 mm3). The chest computed tomography scan showed a partial tumor response with a residual mass in the left paracardial and pleural chest of 35 x 40 x 40 mm (56,000 mm3). No other tumor was visible. The re-staging examinations showed a tumor reduction of 90% after 5 months and seven cycles of chemotherapy. At that stage, a tumor debulking was done in the left thorax. Histological examination of the tissue did not show residual tumor. In the right ulna, a radiotherapy of 66 Gy was carried on. Two months later, the patient got an intensified chemotherapy with Melphalan and Busulfan, as well as a consolidation therapy with peripheral stem cell support. The restating examinations by MRI and computed tomography showed a complete remission 9 months after initial diagnosis. Blood samples for CCK measurements were taken at diagnosis before therapy, and 8 months after chemotherapy during the phase of complete remission.
In addition, 13 young healthy volunteers were enrolled for the assessment of the normal preprandial plasma concentrations of amidated and sulfated CCK as well as of proCCK. Moreover, six patients suffering from mesenchymal tumors other than Ewing sarcomas (one osteosarcoma, two neuroblastomas, one nephroblastoma, one rhabdomyosarcoma, and one synovial sarcoma) were enrolled for the measurement of proCCK plasma concentrations at the time of diagnosis.
The volunteers, the patients, or their parents gave their informed consent for these studies. The investigations were performed after approval by the local institutional review boards.
From an additional 12 patients with various types of sarcoma (Table 1)
, RNA was purified from tumor biopsies and was stored at –80°C. The samples were examined for the fusion proteins. Seven samples had an EWS-FLI 1 fusion. In one patient (ES5), a sample of a tumor was resected 9 months after successful chemotherapy, at a time when no residual neoplastic cells were present. In three cases, the PAX3-FKHR fusion associated with rhabdomyosarcoma were found, and in the last case, an EWS-WT1 fusion was found, confirming the diagnosis of desmoplastic round cell tumor. All samples were examined for CCK mRNA using reverse transcription-PCR (RT-PCR; Table 1
).
|
CCK mRNA Detection in Tumor Tissue
Consecutive cryostat sections of Ewing sarcoma tissues were used for CCK mRNA detection by in situ hybridization. The protocol followed was essentially that described in detail previously (5)
. Oligonucleotide probes complementary to the nucleotides coding for amino acids 1–11 or 80–95 of the human CCK gene were synthesized and purified on a 20% polyacrylamide, 8 mol/L urea sequencing gel (Microsynth, Balgach, Switzerland). They were labeled at the end 3' by using [32P or 33P]-
-dATP (3,000 or 2,000 Ci/mmol, respectively; NEN Life Science, Boston, MA) and terminal deoxynucleotidyltransferase (Boehringer, Mannheim, Germany) to specific activities of 0.9–2.0 x 104 Ci/mmol.
The absorbance was measured in the autoradiograms over the tumor area with a computer-assisted image-processing system, as described previously (5) . A tumor was considered positive when the absorbance measured in a normally hybridized section was at least twice that measured in a parallel section in which hybridization was blocked with 20-fold excess of the corresponding probe. Tumor sections were hybridized with an oligonucleotide complementary to nucleotide 45–92 of the human ß-actin mRNA to confirm and normalize the presence of mRNA in the tumors analyzed.
Tissue and Plasma Concentrations of proCCK and Its Products
Extraction of Tissue and Plasma.
Frozen tumor tissue was extracted in boiling redistilled water (10 ml/g tissue) and subsequently in 0.5 mol/L acetic acid (10 ml/g tissue), as described previously (14)
. The plasma samples were extracted in ethanol (96%, v/v), as described elsewhere (15
, 16)
and submitted to RIA after reconstitution of the dried sample to the initial volume (400 µL) with 20 mmol/L sodium veronal buffer (pH 8.4). When tryptic cleavage was to follow as part of the processing-independent analysis for measurement of the "total prohormone product," the dried sample was reconstituted to half the initial volume (i.e., 200 µL) with 0.1 mmol/L of a sodium phosphate buffer (pH 7.5; ref. 15
).
Tryptic Cleavage for Processing-Independent Analysis.
Samples of 200 µL tissue or plasma extracts were incubated with equal volumes of trypsin [0.2 mg/ml N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin, 0.1 mol/L sodium phosphate buffer (pH 7.5); Worthington Diagnostic Systems Inc., Lakewood, NJ] for 30 min. The cleavage was terminated by boiling. The samples were then centrifuged, and the supernatants were assayed as described in the following (14
, 15)
.
Radioimmunoassay.
The total proCCK product was quantitated in trypsinized samples by RIA using antibody no. 89009, fragment 62–71 of human proCCK as standard and the same monoiodinated fragment as tracer (14)
. Mature, bioactive CCK peptides (i.e., proCCK products that are both carboxyamidated and O-sulfated such as CCK-58, -33, -22 and -8; Fig. 1
) were quantitated in non-trypsinized samples by RIA using antibody no. 92128, O-sulfated CCK-8 as standard, and O-sulfated monoiodinated CCK-8 as tracer (16)
. For both assays the intra- and intervariation of plasma measurement were <15% (15
, 16)
.
|
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. CCK mRNA was detected in all tumors that were shown to express the EWS-FLI1 fusion protein. One desmoplastic small round cell tumor, with the EWS-WT1 fusion protein, also had CCK mRNA. Conversely, neither the successfully treated Ewing sarcoma nor the three rhabdomyosarcomas expressed CCK mRNA; they also lacked the EWS fusion protein (Table 1)
.
CCK mRNA was also found in the four Ewing sarcoma cell lines but not in the two rhabdomyosarcoma cell lines that were tested (Table 2)
. The content of CCK peptides and their precursors were measured in the cell lines as well as the culturing medium harvested after one day, where the cells were still in growth phase. The cells secreted up to 2,800 pmol/L proCCK to the media, whereas no proCCK was found in the culturing medium from the SJCRH30 and RD cells (Table 2)
.
|
. It was found in abundance in four of five tumors; the fifth case with lower levels originated from a sample obtained after chemotherapy (19EW; Table 3
), i.e., a sample with large areas of fibrotic tissue and only a few tumor cells. Figure 2
shows an example of a Ewing sarcoma (patient 1ZEW) with abundant CCK mRNA. In all cases with sufficient material, the tumor concentrations of CCK peptides were measured in parallel by specific RIAs. The processing scheme shown in Fig. 1
illustrates the measured proCCK products, and Table 3
shows the peptide concentrations in the tumors. In general, the precursor forms of CCK dominate in these samples, an observation often made in neoplasms (2)
in which prohormone processing is attenuated. There was a good correlation between mRNA and prohormone contents in the tumor samples. The tumor concentration of proCCK in the case of a tumor sample obtained after chemotherapy was the lowest of all (19EW; Table 3
), in close agreement with the CCK mRNA values.
|
|
; Fig. 3
). The peptide concentrations in plasma were also assessed in the same three patients after several months of a therapy, which had reduced the tumor size by 97%, 82%, and 100%, respectively. In one of the patients (1ZEW), a tumor recurrence was observed in the following period despite extensive therapy. A further plasma sample was taken in this patient during that period of tumor growth for CCK measurements. Table 4
summarizes the results. The most salient findings are several fold-elevated concentrations of the total proCCK product in plasma before chemotherapy in all patients. These concentrations are reduced in parallel to the tumor volume to almost basal values during chemotherapy. However, in the patient with tumor recurrence, the proCCK concentration increased again during tumor progression to values up to 20 times larger than basal values (Table 4)
. The correlation between plasma proCCK concentration and tumor size in the course of the disease in this patient is illustrated in Fig. 3
. In the tumor patients, the ratio of precursor versus mature forms shifts toward immature precursor forms. For comparison, it should be noticed that the concentrations of the mature amidated and O-sulfated CCK peptides do not change during treatment or after tumor recurrence (Table 4)
. The basal concentrations of proCCK in plasma were measured in a normal population of 13 healthy young adults in the morning before a meal (Table 4)
. Moreover, the proCCK concentrations were measured before therapy in six patients with tumors not related to Ewing sarcomas and were found to be almost twice as high as the values in normal volunteers (Table 4)
. Plasma CCK concentrations in patient 2REW during clinical remission were highly variable (Table 4)
and included values higher than those of control individuals.
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although bioactive, carboxyamidated and O-sulfated CCK peptides are apparently not produced in diagnostically significant quantities in Ewing sarcomas; proCCK and its non-amidated processing intermediates are synthesized and released from these neoplastic cells; and proCCK is released into the culture medium from Ewing sarcoma cell lines in large amounts. In patients, proCCK is also released into the circulation in amounts sufficient to elevate the plasma concentrations. When the plasma concentrations in Ewing sarcoma patients are compared with the concentrations in young healthy adults (Table 4)
, it is evident that it is primarily the "proCCK product" that is elevated in the sarcoma patients, whereas the concentrations of amidated, bioactive peptide remain normal. Precursors such as proCCK have no established physiological effects in comparison with their carboxyamidated and tyrosyl-sulfated end-products, which have been shown to stimulate cellular growth, including tumor growth (4)
. One should therefore not expect the increased amount of proCCK and its processing intermediates in circulation to affect CCK target tissues.
In patients in whom both tumor and plasma were analyzed, the tumors having reached a considerable size at the time of investigation represent the likely origin of the increase of plasma proCCK concentrations. Moreover, two arguments strongly suggest that the elevated proCCK concentrations in plasma of the patients originate from the Ewing sarcoma tissue: (a) successful chemotherapy progressively reduced and almost normalized the proCCK concentrations in circulation in parallel with tumor size reduction, and (b) during recurrence of the tumor in one of the patients, a 19-fold increase over basal levels of proCCK in plasma occurred, again in parallel with the increase in tumor size. This indicates that proCCK in plasma may be considered as a useful marker for Ewing sarcoma (i.e., a marker that reflects the size of the tumor burden).
Curiously, proCCK concentrations in plasma were found to be twice as high in tumor patients bearing various types of mesenchymal tumors unrelated to Ewing sarcomas. The reason for that is presently unknown; it is possibly related to slight metabolic disturbances linked to tumor burden. Also unclear is the variability of proCCK levels during clinical remission in one of the patients (2REW) undergoing long-term chemotherapy, with one of the proCCK values (20.4 pmol/L) being clearly off side. Because there are large variabilities in proCCK levels, it may be difficult to define a precise cutoff point between "normal" and "sarcoma" blood levels.
Because the antral hormone, gastrin, is homologous to CCK, and because the gastrin gene is expressed also in Ewing sarcomas (5 , 6) , we examined the occurrence of gastrin mRNA and peptides in tumor tissue as well as measured the concentrations of progastrin and mature gastrin peptides in plasma. Although gastrin mRNA and peptides invariably were found in the tumors, the levels were always considerably lower than those of CCK mRNA and peptides, and many fold lower than in antroduodenal G cells. Accordingly, also the plasma levels of progastrin and the amidated gastrins were not enhanced in the tumor patients (data not shown). Progastrin measurements (18) , therefore, cannot be used either to diagnose or to follow the development of Ewing sarcoma. However, the present study provides the first evidence that a group of human tumors possesses the ability to synthesize simultaneously the homologous hormones, CCK and gastrin, an observation never made before in human tumors despite extensive research (4) .
It is presently unclear why proCCK is being produced in tumors derived from the mesenchyma, because there is no evidence of any physiological involvement of the CCK system in this tissue. However, it is well known that mesenchymal tumors can produce certain hormones (i.e., insulin) ectopically. ProCCK production by Ewing sarcomas may be another example of that. The production of a precursor hormone like proCCK rather than the biologically active CCK is not unusual in tumors; for instance, it is known that progastrin and glycine-extended gastrins are preferentially expressed in undifferentiated gastrointestinal cancers (for review, see ref. 4 ). Although there is no evidence for proCCK to play a major role in the normal CCK physiology, it is unknown yet whether proCCK or its processing intermediates may nevertheless have an effect on tumor tissue, as has been reported recently for progastrin (19 , 20) in gastrointestinal cancers.
The rarity of Ewing sarcomas has made it difficult to recruit rapidly a large number of patients for a prospective investigation. Nevertheless, the following arguments suggest that the present data may be of general value for diagnosis and therapy of Ewing sarcomas: (a) all human Ewing sarcomas tested up to now (16 primary tumors; 8 cell lines) express the CCK gene abundantly (refs. 5 and 6 and the present study); (b) elevated proCCK expression was identified in all Ewing sarcoma tissues analyzed by us up to now, in particular in those tissues taken before the start of a specific oncological treatment; (c) elevated proCCK levels were found to be released in the medium from the four Ewing sarcoma cell lines; and (d) the three patients of this series from which plasma could be collected during treatment both displayed several fold-elevated plasma proCCK concentrations that varied in parallel with the tumor burden. The data presented in this report should therefore trigger a large-scale study. A confirmation that Ewing sarcoma patients in general have elevated plasma proCCK concentrations would mean that measurement of the total proCCK products in plasma may not only be used to diagnose Ewing sarcoma at early stages but also to monitor the efficiency of chemotherapy and/or the degree of surgical removal of the tumor. Plasma proCCK may also represent an early marker for recurrence of Ewing sarcomas. A great advantage of such a marker is the relatively easy and non-invasive method of measurement (14 , 15) .
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Jens F. Rehfeld, Department of Clinical Biochemistry, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Phone: 45-35-45-30-18; Fax: 45-35-45-46-40. E-mail: rehfeld{at}rh.dk
Received 7/ 9/03; revised 4/26/04; accepted 5/12/04.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. Carrillo, E. Garcia-Aragoncillo, D. Azorin, N. Agra, A. Sastre, I. Gonzalez-Mediero, P. Garcia-Miguel, A. Pestana, S. Gallego, D. Segura, et al. Cholecystokinin Down-Regulation by RNA Interference Impairs Ewing Tumor Growth Clin. Cancer Res., April 15, 2007; 13(8): 2429 - 2440. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Borglum, J. F. Rehfeld, L. B. Drivsholm, and L. Hilsted Processing-Independent Quantitation of Chromogranin A in Plasma from Patients with Neuroendocrine Tumors and Small-Cell Lung Carcinomas Clin. Chem., March 1, 2007; 53(3): 438 - 446. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
). Arrows represent the various chemotherapy cycles. The horizontal arrow represents the period of palliative radiotherapy. The horizontally dotted line represents the mean proCCK concentration in a fasting normal population.