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Elevated ascitic fluid fibronectin concentration
219
JournalofHepatology, 1986; 3:219-222 Elsevier
HEP 000211
Elevated Ascitic Fluid Fibronectin Concentration A Non-Specific Finding Bruce A. Runyon Departmentof Internal Medicine, Universityof New Mexico, Albuquerque, NM 87131 (U.S.A.)
(Received 17 January, 1986) (Accepted 24 February, 1986)
Summary Ascitic fluid fibronectin concentration was measured in 111 specimens by laser nephelometry. Sterile, portal hypertension-related fluid fibronectin concentration (24 + 14/~g/ml) was significantly lower than the concentration in infected, portal hypertension-related ascites (49 + 44/zg/ml, P < 0.001), peritoneal carcinomatosis (123 + 45/zg/ml, P < 0.001), massive liver metastases-related ascites (55 + 21/~g/ml, P < 0.001), as well as in ascites of other types (94 + 42/~g/ml, P < 0.001). The percentage of samples with fibronectin concentration, > 75/zg/ml, was 0% for sterile, portal hypertension-relatedascites, 28% for infected, portal hypertension-related ascites, 89% for peritoneal carcinomatosis, 20% for massive liver metastases-related ascites, and 72% for ascites of other types. Ascitic fluid fibronectin concentration correlated in a linear fashion with ascitic fluid total protein (r = 0.81, P < 0.001). Fibronectin concentration in ascites appears to be elevated under a variety of conditions and does not appear to be a specific marker for cancer.
Introduction Plasma fibronectin was proposed as a marker for cancer just a few years ago [1,2]. Subsequent studies have not confirmed the expected utility of this test in the diagnosis of cancer [3,4]. Ascitic fluid fibronectin has recently been proposed as a marker for malignant ascites [5,6]. This study was performed in an at-
tempt to confirm this observation in a different group of ascites patients.
Methods Patients hospitalized with ascites at the Long Beach Veterans Administration Medical Center, the
This work was supported in part by Grant 5-M01RR00997-08 from the National Institutes of Health. Address reprint requests to: B.A. Runyon, M.D., Gastroenterology Division, Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131, U.S.A., Tel. (505) 277-4755. 0168-8278/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
220
B.A. RUNYON et al.
University of New Mexico Hospital, and the Albuquerque Veterans Administration Medical Center were candidates for participation in this study. Patients signed informed consent prior to abdominal paracentesis. This study was approved by the Human Research Review Committee of all institutions. Ascitic fluid samples were classified as sterile, portal hypertension-related (PHT) ascites due to parenchymal liver disease if there was biopsy confirmation of cirrhosis or alcoholic hepatitis and/or clinical evidence of chronic liver disease and/or a serum-ascites albumin gradient of greater than 1.1 g/dl [7] and the sample was sterile with a polymorphonuclear neutrophil (PMN) count less than 250 cells/ram 3. The sample was classified as infected, portal hypertension-related type (SBP) if the above criteria for parenchymal liver disease were met and the sample was bacterial culture-positive with a PMN count >250 cells/mm 3 or the sample was culture-negative with a PMN count >500 cells/mm 3 [8]. The sample was classified as peritoneal carcinomatosis (CA) type if there was a positive ascitic fluid cytology for malignancy and a low albumin gradient (<1.1 g/dl). The sample was categorized as massive liver metastasis (MLM) type if the patient had a known primary tumor, scan evidence of massive tumor in the liver, negative ascitic fluid cytology and a high albumin gradient (>1.1 g/dl). Fluids were classified as miscellaneous (MISC) type if they did not meet the criteria for any of the above categories. Ascitic fluid was obtained using sterile technique. A portion was sent for routine analysis including measurement of albumin concentration by the bromcresol green method. The remainder was stored at -70 °C in small aliquots. The samples were thawed (to 37 °C) only once, immediately before measure-
ment of fibronectin concentration using a Hyland Laser Nephelometer (Hyland Laboratories, Costa Mesa, CA). A Cappel Laboratories Fibronectin Kit (Cappel Laboratories, Westchester, PA) was used.as a source of fibronectin antibody and antigen standards. Student's t-test and linear regression analysis (comparing ascitic fluid fibronectin concentrations to ascitic fluid total protein concentration) were used for statistical analysis. A P value of <0.05 was considered significant.
Results One hundred eleven samples from 72 patients were analyzed (Table 1, Fig. 1). In 4 specimens (all P H T type) from 3 patients, flbronectin could not be measured because of opacity of the specimen. Four patients whose ascites was samples when it was sterile, subsequently were sampled after the fluid became infected. The miscellaneous category included 3 patients with cardiac ascites, 2 patients with pancreatic ascites, 2 patients with non-nephrotic renal failure, 1 • patient with tricuspid insufficiency as well as chronic active hepatitis, and 3 samples from 2 patients with ascites of unclear etiology. Uninfected portal hypertension-related (PHT) ascitic fluid samples had significantly (P < 0.001) lower fibronectin concentration than any of the other categories, either individually or in aggregate. In individual patients who had serial samples obtained before infection of the fluid and/or during infection and/or after initiation of treatment of the infection, the fibronectin concentration in general rose
TABLE 1 ASCITIC FLUID FIBRONECTIN CONCENTRATIONS Ascites type Number of specimens Number of patients Fibronectin (.ug/ml) % of specimens with fibronectin >75/tg/ml
PHT 53 37 24 + 14 0
SBP 29 15 49 + 44 28
CA 9 7 123 + 45 89
MLM 5 4 55 + 21 20
MISC 11 10 94 + 42 72
ASCITIC FLUID FIBRONECTIN
221
zoc IZ 0 noB IT
El J u I-
PHT
SBP
CA
MLM
MI SC
Fig. 1. Fibronectin concentration in ascitic fluid of various types. PHT = sterile portal hypertension-related, SBP = infected portalhypertension-related, CA = peritoneal carcinomatosis, MLM = massiveliver metastasis, MISC = miscellaneous. during active infection and declined after treatment began. However, these perturbations were not statistically significant. In contrast there was a significant (P < 0.001) difference overall between the P H T and SBP categories as mentioned above. All P H T samples contained less than 75 #g/ml (the reported 'cut-off' separating benign and malignant samples). Not all of the malignancy-related samples had high fibronectin levels. In fact, only 9/14 (64%) of the total group of malignant (CA + MLM) samples had a level greater than 75 #/ml. There was no 'cut-off' that completely separated benign from malignant type samples. MLM samples had a significantly lower fibronectin concentration (P < 0.02) than CA samples. Linear regression analysis of fibronectin versus total protein concentration revealed a correlation coefficient of 0.81 (P < 0.001).
Discussion
The concentration of fibronectin in ascitic fluid appears to be helpful in separating P H T type ascites from other types of ascites. However, since infected ascites, pancreatic ascites, cardiac ascites, and malignant ascites can contain high levels of fibronectin, this test does not appear to be specific for any one diagnosis. In addition, fibronectin correlates directly
with the total protein concentration of the ascitic fluid. It is unclear why these data differ from the data that are in the literature [5,6]. In the other two studies the ascitic fluid fibronectin concentration was found to separate benign from malignant specimens with nearly perfect accuracy. In one of these studies [6] only cirrhotic and malignant ascites were discussed. No other control groups, e.g. cardiac, pancreatic ascites, etc. were mentioned. If such fluids would have been tested, perhaps they would have been found to have elevated fibronectin concentrations as in the current series. Neither of the other two series clearly separated their cancer patients into peritoneal carcinomatosis or massive liver metastasis groups. In the current series, this separation appeared to be justified and real because of the significant difference in fibronectin concentration between the two groups. Since ascitic fluid in patients with MLM characteristically contains little protein [9], and since in some MLM patients the scan may reveal only splenomegaly without focal defects (unpublished observations), perhaps some patients with MLM in the other two series were misclassified as having cirrhotic ascites. The Scholmerich and Deverbizier series report 62.5% and 66% sensitivity of cytology in the diagnosis of malignant ascites, respectively [5,6]. In the current series cytology was 64% sensitive overall; all of
222 the peritoneal carcinomatosis patients had positive cytologies (by study inclusion criteria) and none of the massive liver metastasis patients had positive cytologies (by study inclusion criteria). The consistency of the 'sensitivity' of cytology in detecting malignancy-related ascites may simply reflect the consistency of the make-up of the various series. Two-thirds of the patients have peritoneal carcinomatosis. The vast majority of these have positive cytologies. One-third of the patients have massive liver metastases and negative cytologies. If fibronectin is released into ascites by tumor cells lining the peritoneum and not released from tumor cells that are in the liver, one would expect that only patients with peritoneal carcinomatosis would have elevated ascitic fluid fibronectin concentrations. In view of the many problems in measurement of fibronectin, as outlined by Zerlauth et al. [3], it is not surprising that differences exist between series. Apparently plasma fibronectin concentration is affected
B.A. RUNYON et al. by age, nutrition, drugs and inflammation. Perhaps all of these difficult-to-control variables in addition to unrecognized differences in specimen handling, storage, or testing may explain the differences in ascitic fluid results between series. Scholmerich et al. state in their paper that 'a specific monovalent antibody' against fibronectin was used in their analysis [5]. The meaning of the word monovalent is not clear in this context. In fact the catalog number of the Boehringer antibody that was used in their study is the catalog number of a polyclonal antifibronectin antibody - - similar to the antibody used in the current study. On the basis of the data in the current series, the diagnosis of malignant ascites cannot be made by measurement of the fibronectin concentration alone. 'Cancer screening tests' of serum, e.g. carcino-embryonic antigen, alpha-fetoprotein and fibronectin have been fraught with hazard. The diagnosis of cancer should be based on histology not serology.
References
1 Todd HD, Coffee MS, Waalkes TP, Abeloff MD, et al. Serum levels of fibronectin and a fibronectin-like DNA-binding protein in patients with various disease. J Nat Can Inst 1980; 65: 901-904. 2 Zardi L, Cecconi C, Barbieri O, Carnemolla B, et al. Concentration of fibronectin in plasma of tumor-bearing mice and synthesis by Ehrlich ascites tumor cell. Cancer Res 1979; 39: 3774-3777. 3 Zerlauth G, Wolf G. Plasma fibronectin as a marker for cancer and other diseases. Amer J Med 1984; 77: 685-689. 4 Choate J, Mosher DF. Fibronectin concentration in plasma of patients with breast cancer, colon cancer and acute leukemia. Cancer 1983; 51: 1142-1147. 5 Scholmerich J, Volk BA, Kottgen E, Ehlers S, et al. Fibro-
6 • 7 8 9
nectin concentration in ascites differentiates between malignant and non-malignant ascites. Gastroenterology 1984; 87: 1160-1164. Deverbizier G, Beachant M, Chapron A, Touchard G, et al. Fibronectin, a marker for malignant ascites. Lancet 1985; ii: 1104. Pare P, Talbot J, Hoefs JC. Serum-ascites albumin concentration gradient: physiologic approach to the differential diagnosis of ascites. Gastroenterology 1983; 85: 240-244. Runyon BA, Hoefs JC. Culture-negative neutrocytic ascites; a variant of spontaneous bacterial peritonitis. HepatoIogy 1984; 4: 1209-1211. Runyon BA, Hoers JC. Ascitic fluid caused by massive liver metastases: Its prevalence and characterization. Hepatology 1985; 5:1000 (Abstract).
JournalofHepatology, 1986; 3:219-222 Elsevier
HEP 000211
Elevated Ascitic Fluid Fibronectin Concentration A Non-Specific Finding Bruce A. Runyon Departmentof Internal Medicine, Universityof New Mexico, Albuquerque, NM 87131 (U.S.A.)
(Received 17 January, 1986) (Accepted 24 February, 1986)
Summary Ascitic fluid fibronectin concentration was measured in 111 specimens by laser nephelometry. Sterile, portal hypertension-related fluid fibronectin concentration (24 + 14/~g/ml) was significantly lower than the concentration in infected, portal hypertension-related ascites (49 + 44/zg/ml, P < 0.001), peritoneal carcinomatosis (123 + 45/zg/ml, P < 0.001), massive liver metastases-related ascites (55 + 21/~g/ml, P < 0.001), as well as in ascites of other types (94 + 42/~g/ml, P < 0.001). The percentage of samples with fibronectin concentration, > 75/zg/ml, was 0% for sterile, portal hypertension-relatedascites, 28% for infected, portal hypertension-related ascites, 89% for peritoneal carcinomatosis, 20% for massive liver metastases-related ascites, and 72% for ascites of other types. Ascitic fluid fibronectin concentration correlated in a linear fashion with ascitic fluid total protein (r = 0.81, P < 0.001). Fibronectin concentration in ascites appears to be elevated under a variety of conditions and does not appear to be a specific marker for cancer.
Introduction Plasma fibronectin was proposed as a marker for cancer just a few years ago [1,2]. Subsequent studies have not confirmed the expected utility of this test in the diagnosis of cancer [3,4]. Ascitic fluid fibronectin has recently been proposed as a marker for malignant ascites [5,6]. This study was performed in an at-
tempt to confirm this observation in a different group of ascites patients.
Methods Patients hospitalized with ascites at the Long Beach Veterans Administration Medical Center, the
This work was supported in part by Grant 5-M01RR00997-08 from the National Institutes of Health. Address reprint requests to: B.A. Runyon, M.D., Gastroenterology Division, Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131, U.S.A., Tel. (505) 277-4755. 0168-8278/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
220
B.A. RUNYON et al.
University of New Mexico Hospital, and the Albuquerque Veterans Administration Medical Center were candidates for participation in this study. Patients signed informed consent prior to abdominal paracentesis. This study was approved by the Human Research Review Committee of all institutions. Ascitic fluid samples were classified as sterile, portal hypertension-related (PHT) ascites due to parenchymal liver disease if there was biopsy confirmation of cirrhosis or alcoholic hepatitis and/or clinical evidence of chronic liver disease and/or a serum-ascites albumin gradient of greater than 1.1 g/dl [7] and the sample was sterile with a polymorphonuclear neutrophil (PMN) count less than 250 cells/ram 3. The sample was classified as infected, portal hypertension-related type (SBP) if the above criteria for parenchymal liver disease were met and the sample was bacterial culture-positive with a PMN count >250 cells/mm 3 or the sample was culture-negative with a PMN count >500 cells/mm 3 [8]. The sample was classified as peritoneal carcinomatosis (CA) type if there was a positive ascitic fluid cytology for malignancy and a low albumin gradient (<1.1 g/dl). The sample was categorized as massive liver metastasis (MLM) type if the patient had a known primary tumor, scan evidence of massive tumor in the liver, negative ascitic fluid cytology and a high albumin gradient (>1.1 g/dl). Fluids were classified as miscellaneous (MISC) type if they did not meet the criteria for any of the above categories. Ascitic fluid was obtained using sterile technique. A portion was sent for routine analysis including measurement of albumin concentration by the bromcresol green method. The remainder was stored at -70 °C in small aliquots. The samples were thawed (to 37 °C) only once, immediately before measure-
ment of fibronectin concentration using a Hyland Laser Nephelometer (Hyland Laboratories, Costa Mesa, CA). A Cappel Laboratories Fibronectin Kit (Cappel Laboratories, Westchester, PA) was used.as a source of fibronectin antibody and antigen standards. Student's t-test and linear regression analysis (comparing ascitic fluid fibronectin concentrations to ascitic fluid total protein concentration) were used for statistical analysis. A P value of <0.05 was considered significant.
Results One hundred eleven samples from 72 patients were analyzed (Table 1, Fig. 1). In 4 specimens (all P H T type) from 3 patients, flbronectin could not be measured because of opacity of the specimen. Four patients whose ascites was samples when it was sterile, subsequently were sampled after the fluid became infected. The miscellaneous category included 3 patients with cardiac ascites, 2 patients with pancreatic ascites, 2 patients with non-nephrotic renal failure, 1 • patient with tricuspid insufficiency as well as chronic active hepatitis, and 3 samples from 2 patients with ascites of unclear etiology. Uninfected portal hypertension-related (PHT) ascitic fluid samples had significantly (P < 0.001) lower fibronectin concentration than any of the other categories, either individually or in aggregate. In individual patients who had serial samples obtained before infection of the fluid and/or during infection and/or after initiation of treatment of the infection, the fibronectin concentration in general rose
TABLE 1 ASCITIC FLUID FIBRONECTIN CONCENTRATIONS Ascites type Number of specimens Number of patients Fibronectin (.ug/ml) % of specimens with fibronectin >75/tg/ml
PHT 53 37 24 + 14 0
SBP 29 15 49 + 44 28
CA 9 7 123 + 45 89
MLM 5 4 55 + 21 20
MISC 11 10 94 + 42 72
ASCITIC FLUID FIBRONECTIN
221
zoc IZ 0 noB IT
El J u I-
PHT
SBP
CA
MLM
MI SC
Fig. 1. Fibronectin concentration in ascitic fluid of various types. PHT = sterile portal hypertension-related, SBP = infected portalhypertension-related, CA = peritoneal carcinomatosis, MLM = massiveliver metastasis, MISC = miscellaneous. during active infection and declined after treatment began. However, these perturbations were not statistically significant. In contrast there was a significant (P < 0.001) difference overall between the P H T and SBP categories as mentioned above. All P H T samples contained less than 75 #g/ml (the reported 'cut-off' separating benign and malignant samples). Not all of the malignancy-related samples had high fibronectin levels. In fact, only 9/14 (64%) of the total group of malignant (CA + MLM) samples had a level greater than 75 #/ml. There was no 'cut-off' that completely separated benign from malignant type samples. MLM samples had a significantly lower fibronectin concentration (P < 0.02) than CA samples. Linear regression analysis of fibronectin versus total protein concentration revealed a correlation coefficient of 0.81 (P < 0.001).
Discussion
The concentration of fibronectin in ascitic fluid appears to be helpful in separating P H T type ascites from other types of ascites. However, since infected ascites, pancreatic ascites, cardiac ascites, and malignant ascites can contain high levels of fibronectin, this test does not appear to be specific for any one diagnosis. In addition, fibronectin correlates directly
with the total protein concentration of the ascitic fluid. It is unclear why these data differ from the data that are in the literature [5,6]. In the other two studies the ascitic fluid fibronectin concentration was found to separate benign from malignant specimens with nearly perfect accuracy. In one of these studies [6] only cirrhotic and malignant ascites were discussed. No other control groups, e.g. cardiac, pancreatic ascites, etc. were mentioned. If such fluids would have been tested, perhaps they would have been found to have elevated fibronectin concentrations as in the current series. Neither of the other two series clearly separated their cancer patients into peritoneal carcinomatosis or massive liver metastasis groups. In the current series, this separation appeared to be justified and real because of the significant difference in fibronectin concentration between the two groups. Since ascitic fluid in patients with MLM characteristically contains little protein [9], and since in some MLM patients the scan may reveal only splenomegaly without focal defects (unpublished observations), perhaps some patients with MLM in the other two series were misclassified as having cirrhotic ascites. The Scholmerich and Deverbizier series report 62.5% and 66% sensitivity of cytology in the diagnosis of malignant ascites, respectively [5,6]. In the current series cytology was 64% sensitive overall; all of
222 the peritoneal carcinomatosis patients had positive cytologies (by study inclusion criteria) and none of the massive liver metastasis patients had positive cytologies (by study inclusion criteria). The consistency of the 'sensitivity' of cytology in detecting malignancy-related ascites may simply reflect the consistency of the make-up of the various series. Two-thirds of the patients have peritoneal carcinomatosis. The vast majority of these have positive cytologies. One-third of the patients have massive liver metastases and negative cytologies. If fibronectin is released into ascites by tumor cells lining the peritoneum and not released from tumor cells that are in the liver, one would expect that only patients with peritoneal carcinomatosis would have elevated ascitic fluid fibronectin concentrations. In view of the many problems in measurement of fibronectin, as outlined by Zerlauth et al. [3], it is not surprising that differences exist between series. Apparently plasma fibronectin concentration is affected
B.A. RUNYON et al. by age, nutrition, drugs and inflammation. Perhaps all of these difficult-to-control variables in addition to unrecognized differences in specimen handling, storage, or testing may explain the differences in ascitic fluid results between series. Scholmerich et al. state in their paper that 'a specific monovalent antibody' against fibronectin was used in their analysis [5]. The meaning of the word monovalent is not clear in this context. In fact the catalog number of the Boehringer antibody that was used in their study is the catalog number of a polyclonal antifibronectin antibody - - similar to the antibody used in the current study. On the basis of the data in the current series, the diagnosis of malignant ascites cannot be made by measurement of the fibronectin concentration alone. 'Cancer screening tests' of serum, e.g. carcino-embryonic antigen, alpha-fetoprotein and fibronectin have been fraught with hazard. The diagnosis of cancer should be based on histology not serology.
References
1 Todd HD, Coffee MS, Waalkes TP, Abeloff MD, et al. Serum levels of fibronectin and a fibronectin-like DNA-binding protein in patients with various disease. J Nat Can Inst 1980; 65: 901-904. 2 Zardi L, Cecconi C, Barbieri O, Carnemolla B, et al. Concentration of fibronectin in plasma of tumor-bearing mice and synthesis by Ehrlich ascites tumor cell. Cancer Res 1979; 39: 3774-3777. 3 Zerlauth G, Wolf G. Plasma fibronectin as a marker for cancer and other diseases. Amer J Med 1984; 77: 685-689. 4 Choate J, Mosher DF. Fibronectin concentration in plasma of patients with breast cancer, colon cancer and acute leukemia. Cancer 1983; 51: 1142-1147. 5 Scholmerich J, Volk BA, Kottgen E, Ehlers S, et al. Fibro-
6 • 7 8 9
nectin concentration in ascites differentiates between malignant and non-malignant ascites. Gastroenterology 1984; 87: 1160-1164. Deverbizier G, Beachant M, Chapron A, Touchard G, et al. Fibronectin, a marker for malignant ascites. Lancet 1985; ii: 1104. Pare P, Talbot J, Hoefs JC. Serum-ascites albumin concentration gradient: physiologic approach to the differential diagnosis of ascites. Gastroenterology 1983; 85: 240-244. Runyon BA, Hoefs JC. Culture-negative neutrocytic ascites; a variant of spontaneous bacterial peritonitis. HepatoIogy 1984; 4: 1209-1211. Runyon BA, Hoers JC. Ascitic fluid caused by massive liver metastases: Its prevalence and characterization. Hepatology 1985; 5:1000 (Abstract).