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Previous Volume 328:1592-1598 June 3, 1993 Number 22 Next

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ABSTRACT

Background Hepatic veno-occlusive disease and idiopathic interstitial pneumonitis are major causes of morbidity and mortality after bone marrow transplantation. Fibrosis is a characteristic of both conditions, and transforming growth factor (TGF) has been implicated in the pathogenesis of fibrosis.

Methods Using acid-ethanol extraction to remove TGF from human plasma and a mink-lung epithelial-cell growth-inhibition assay to measure TGF activity, we quantified plasma TGF in 10 normal subjects and 41 patients before and after they underwent high-dose chemotherapy and autologous bone marrow transplantation for advanced breast cancer.

Results There was no difference in pretransplantation TGF levels between the controls and the patients who did not have hepatic veno-occlusive disease or idiopathic interstitial pneumonitis after transplantation. In contrast, pretransplantation TGF levels were significantly higher in patients in whom hepatic veno-occlusive disease or idiopathic interstitial pneumonitis developed than in the controls or the patients without these conditions. The predictive value for the development of either condition was 90 percent or more when pretransplantation plasma TGF levels were more than 2 SD above the mean established in the controls.

Conclusions The plasma TGF concentration measured after induction chemotherapy but before high-dose chemotherapy and autologous bone marrow transplantation strongly correlates with the risk of hepatic veno-occlusive disease and idiopathic interstitial pneumonitis after these treatments.

Similarly, pulmonary complications of bone marrow-transplantation are a major source of morbidity, occurring in 40 to 60 percent of patients⁸. Noninfectious pulmonary complications (idiopathic interstitial pneumonitis) occur in 10 to 25 percent of bone marrow-transplant recipients⁸. This syndrome is characterized by dyspnea, fever, and hypoxemia, with or without diffuse interstitial infiltrates on chest radiography. It occurs 40 to 75 days after transplantation; the mortality rates are high. Both the chemotherapy and the radiotherapy used in the conditioning regimens have been implicated in the development of liver and lung damage after bone marrow transplantation^1,8,9.

Fibrosis is a prominent feature in both the lungs and the liver in patients with these complications^10,11. Recently, efforts have been directed at elucidating the molecular mechanisms of these fibrotic reactions. Transforming growth factor (TGF) stimulates fibroblasts to migrate to the site of injury, proliferate, and produce collagen; it also inhibits collagen degradation¹². Thus, it plays an important part in normal wound healing^13,14 as well as in abnormal fibrogenesis. TGF has been implicated in the causation of chronic pulmonary fibrosis in rats and mice exposed to bleomycin or cyclophosphamide^{15,16,17,18,19,20} and in the development of hepatic fibrosis in rats exposed to radiation²¹ or carbon tetrachloride^22,23. TGF may also have a role in fibrotic liver and lung diseases in humans,^24,25,26,27 such as chronic hepatitis,²⁸ idiopathic pulmonary fibrosis,^29,30 and systemic sclerosis^31,32,33. Inhibition of TGF activity can prevent the development of chronic hepatitis,²⁸ acute mesangial proliferative glomerulonephritis,³⁴ and the cirrhotic effects of carbon tetrachloride,³⁵ providing further evidence for the role of TGF in these fibrotic conditions.

Because the level of expression of the gene for TGF1 is elevated in both animals and humans with fibrotic liver or lung diseases,^28,30 we postulated that an increase in the release and activation of TGF1 in fibrotic tissue would also result in an increase in the circulating level of this growth factor. It may be possible to use the plasma concentration of TGF proteins measured before the administration of high-dose chemotherapy to identify patients most prone to the development of lung or liver injury after bone marrow transplantation.

Methods

Patients

At the time of this analysis, 102 women with adenocarcinoma of the breast had been treated according to research protocols for bone marrow transplantation at Duke University Medical Center. All patients had either stage IV disease (metastases) or advanced stage II or III disease (more than 10 positive lymph nodes found after axillary dissection) and underwent four cycles of induction chemotherapy followed by high-dose chemotherapy and autologous bone marrow transplantation (Figure 1). The details of this treatment regimen have been previously reported³⁶. In brief, the induction regimen consisted of cyclophosphamide, doxorubicin (Adriamycin), and fluorouracil (for stage II or III disease) or doxorubicin, fluorouracil, and methotrexate (for stage IV disease). The high-dose chemotherapy consisted of carmustine, cyclophosphamide, and cisplatin. Radiation therapy was directed at the sites of known metastases (stage IV disease) or to the ipsilateral chest wall, internal mammary nodes, and supraclavicular lymph nodes (stage II or III disease) after autologous bone marrow transplantation.

View larger version (44K): [in this window] [in a new window]   Figure 1. Treatment Protocol and Plasma Sampling in 102 Women with Breast Cancer. AFM denotes a regimen of doxorubicin (Adriamycin), fluorouracil, and methotrexate; CAF, a regimen of cyclophosphamide, doxorubicin, and fluorouracil; and high-dose chemotherapy, a regimen of cyclophosphamide, carmustine, and cisplatin. Transplantation of autologous bone marrow was carried out after the high-dose chemotherapy.

 
Of the 102 patients treated, 12 subsequently had hepatic veno-occlusive disease, 19 had pulmonary fibrosis, and the remaining 71 had neither. Both conditions were defined clinically. Hepatic veno-occlusive disease was indicated by the development of weight gain, hepatomegaly, ascites, and hyperbilirubinemia one to three weeks after transplantation. Pulmonary fibrosis was indicated by dyspnea, fever, and hypoxemia with or without diffuse interstitial infiltrates on chest radiography, beginning 40 to 75 days after transplantation. Other causes of these two syndromes had to be excluded in order to accept these diagnoses. Biopsy was not required. All patients with hepatic veno-occlusive disease or pulmonary fibrosis were included in this analysis. A sample of 10 patients who had neither condition (a sample matching the number of controls, described below) was randomly selected from among all patients enrolled under these protocols whose plasma samples were stored in the archives of the cryopreservation laboratory. Specimens were coded, and TGF levels measured, without the investigators' prior knowledge of whether or not the patient had hepatic veno-occlusive disease or pulmonary fibrosis. After the plasma TGF levels in the samples were measured, the code was broken and the data were grouped for analysis according to the patients' status for toxic complications (see below).

Plasma samples were obtained twice. The first sample was obtained after induction chemotherapy but before the administration of high-dose chemotherapy and autologous bone marrow transplantation. The second sample was obtained after the high-dose chemotherapy and transplantation (Figure 1), between 12 and 37 days after the operation, depending on the availability of adequate samples. The findings in the transplant recipients were compared with those in controls -- 10 normal blood donors whose plasma was obtained from the American Red Cross (Charlotte, N.C.).

Extraction of TGF from Plasma

Total TGF (both active and inactive forms) was isolated from the plasma through acid-ethanol extraction^37,38. Because this extraction procedure activates TGF, we could not determine the amount of active and inactive TGF present in the samples. To extract TGF, 4 ml of an acid-ethanol solution (375 ml of 95 percent ethanol, 7.5 ml of 12 N hydrochloric acid, 33 mg of phenylmethylsulfanylfluoride, and 1.9 mg of pepstatin) was added to a 1-ml plasma sample previously diluted by a factor of 2 with distilled water. The samples were incubated overnight at 4 °C, then centrifuged at 20,000 x g for 30 minutes at 4 °C. The supernatant was removed and stored at 4 °C, and the remainder of the sample was reextracted and centrifuged. The two supernatants were then combined, and the pH was adjusted to 5.2 to 5.3 with ammonium hydroxide. One milliliter of 2 M ammonium hydroxide was added to 85 ml of supernatant and diluted by a factor of 3 with cold (4 °C) 100 percent ethanol. This solution was incubated at -20 °C for at least two days and then centrifuged. The pellet was resuspended in 4 ml of 1 M acetic acid, dialyzed overnight in 1 percent acetic acid, divided into aliquots, lyophilized, and stored at -20 °C.

Assay for TGF

Plasma levels of TGF were quantified with the use of an assay measuring the inhibition of the growth of mink-lung epithelial cells³⁹. Because this assay is not capable of discriminating among the three isoforms of TGF, throughout this paper we simply use the term "TGF." In brief, after the MV 1 Lu mink-lung epithelial cells (CCL-64) were subjected to trypsinization and suspended in the assay medium, they were plated at a concentration of 10⁵ cells per milliliter. After incubation at 37 °C for 1 hour, TGF test samples and standards of known TGF1 concentration were added to the wells and incubated at 37 °C for 22 hours. The extent of DNA synthesis was then determined by incubating the cells with ³H-labeled thymidine at 37 °C for an additional four hours. The cells were finally fixed for one hour at room temperature in 1.0 ml of methanol-acetic acid solution (3:1 vol/vol) and washed twice in 80 percent methanol. They were then solubilized in 0.3 N sodium hydroxide, and the radiolabeled DNA was extracted by precipitation with trichloroacetic acid. The amount of radioactivity in the cells exposed to the test samples and TGF1 standards was determined with a liquid-scintillation counter. This assay was able to detect amounts of TGF ranging from 0.05 to 0.5 ng per milliliter (0.2 to 2 x 10^-8 mmol per liter), with 50 percent inhibition occurring at a concentration of 0.1 ng per milliliter (0.4 x 10^-8 mmol per liter). The samples were serially diluted until the quantities of TGF present were in the linear portion of the sigmoid-shaped curve for the TGF standard. Actual TGF levels were then calculated by multiplying the measured TGF concentration by the dilution factor. Test samples were always assayed with samples containing known quantities of TGF to ensure the reliability of the bioassay.

To determine whether the inhibitory effect of the test samples was due specifically to TGF, a neutralizing antibody that recognized TGF (R&D Systems, Minneapolis) was added to all the test samples one hour before they were added to the mink-lung cells. Because the antibody was not specific for an individual isoform of TGF, we could not determine the relative contributions of the three isoforms to the total plasma concentration. In all test samples the TGF antibody was able to neutralize completely the inhibition of 50 percent of the cell growth (data not shown).

Statistical Analysis

Plasma TGF was measured in the controls and patients both before and after bone marrow transplantation. Analysis of variance and Scheffe's method of multiple comparisons were used to compare mean values determined before and after transplantation in patients according to whether they subsequently had hepatic veno-occlusive disease, pulmonary fibrosis, or neither condition. Sensitivity, specificity, and predictive values (positive and negative) were calculated on the basis of a cutoff value for plasma TGF of 10 ng per milliliter (4 x 10^-7 mmol per liter), which was 2 SD above the mean determined in the controls (6 ng per milliliter [2.4 x 10^-7 mmol per liter]).

The clinical variables determined in each patient are shown in Table 1. These data were analyzed in the same way as the TGF measurements⁴⁰. No clinical information was available for the controls because they were anonymous blood donors. Laboratory values were measured on or as close as possible to the dates on which plasma samples were obtained for measurement of TGF (Figure 1), to determine whether there were any differences between the patients in whom hepatic veno-occlusive disease or pulmonary fibrosis developed and the patients without these complications.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics Evaluated in 102 Women with Breast Cancer.

 
Results

The characteristics of the 41 patients who underwent autologous bone marrow transplantation for advanced breast cancer are shown in Table 2. The patients in whom pulmonary fibrosis or hepatic veno-occlusive disease later developed and the patients without these complications were similar in all respects except that the group with hepatic veno-occlusive disease included patients with distant metastases who had received chemotherapy or radiotherapy before they were enrolled in the transplantation program. The mortality rates for pulmonary fibrosis and hepatic veno-occlusive disease were 26 percent and 17 percent, respectively (Table 2).

View this table: [in this window] [in a new window]   Table 2. Characteristics of Patients Undergoing Autologous Bone Marrow Transplantation for Breast Cancer.

 
The TGF concentrations in each patient and control are shown in Figure 2; the solid line at 10 ng per milliliter represents the TGF level 2 SD above the mean value of 6.1 ng per milliliter (2.4 x 10^-7 mmol per liter) determined in the controls (10 healthy blood donors). The mean TGF concentrations in each study group are shown in Figure 3. When we compared the TGF levels measured in the patients before transplantation with the levels in the controls, we found no significant difference (P>0.1) between the controls and the patients who did not have hepatic veno-occlusive disease or pulmonary fibrosis after transplantation. In contrast, the pretransplantation TGF levels in patients who later had hepatic veno-occlusive disease or pulmonary fibrosis were significantly higher (P = 0.003) than those in the controls and the patients without fibrotic changes in their lungs or liver.

View larger version (27K): [in this window] [in a new window]   Figure 2. Individual TGF Plasma Concentrations in the Four Study Groups. Healthy blood donors served as controls. One group of patients did not have pulmonary fibrosis or hepatic veno-occlusive disease after high-dose chemotherapy and autologous bone marrow transplantation (No fibrosis); the two other groups had either hepatic veno-occlusive disease (Liver fibrosis) or pulmonary fibrosis (Lung fibrosis). TGF was measured before (circles) and after (squares) high-dose chemotherapy and transplantation (Figure 1 shows the timing of the regimens). The solid horizontal line indicates the value (10 ng per milliliter, or 2 SD above the mean value determined in the controls) that was used as a cutoff point to determine the ability of pretransplantation TGF measurement to predict the development of hepatic or pulmonary toxicity after transplantation. To convert values for TGF to millimoles per liter, multiply by 4 x 10-8.

 

View larger version (27K): [in this window] [in a new window]   Figure 3. Mean (±SEM) TGF Concentrations in the Controls and Patients. The legend to Figure 2 describes the four study groups. TGF was measured before (solid bars) and after (hatched bars) bone marrow transplantation (Fig. 1 shows the times of plasma sampling). The numbers above the bars are the numbers of subjects in each group in whom TGF was measured. The patients with fibrosis had significantly higher plasma TGF levels before transplantation (P = 0.003) than either the controls or the patients without fibrosis. To convert values for TGF to millimoles per liter, multiply by 4 x 10-8.

 
After autologous bone marrow transplantation, there was a significant decrease (P<0.001) in the TGF levels of patients with subsequent hepatic veno-occlusive disease or pulmonary fibrosis (Figure 2 and Figure 3). This decrease paralleled a marked decrease in the platelet count after the high-dose chemotherapy (Table 3). In contrast, the plasma TGF levels remained unchanged (P>0.1) in the patients who did not have hepatic veno-occlusive disease or pulmonary fibrosis, even though their platelet counts decreased to the same extent as the counts of the patients who did have these complications. Although there was a significant difference in pretransplantation TGF levels between the groups with hepatic veno-occlusive disease and pulmonary fibrosis and the group without these developments, as noted above, there was no significant (P>0.1) difference in the post-transplantation levels among these three groups.

View this table: [in this window] [in a new window]   Table 3. Mean (±SEM) TGF Levels and Platelet Counts before and after Autologous Bone Marrow Transplantation.

 
To test the usefulness of the TGF level as an indicator of an increased risk of hepatic veno-occlusive disease or pulmonary fibrosis after high-dose chemotherapy and autologous bone marrow transplantation, the sensitivity, specificity, and the positive and negative predictive values of this marker were calculated. To make these calculations, the upper limit of normal TGF levels in plasma was set at 10 ng per milliliter, which was 2 SD above the mean value in normal subjects (Figure 2). The resulting values (Table 4) showed that the TGF level measured in plasma after induction chemotherapy but before high-dose chemotherapy and transplantation was a very good indicator of which patients would subsequently have pulmonary fibrosis or hepatic veno-occlusive disease (or both) after chemotherapy and transplantation. If the plasma concentration of TGF was greater than 10 ng per milliliter, it was possible to predict with more than 90 percent accuracy that either hepatic veno-occlusive disease or pulmonary fibrosis would develop (i.e., the positive predictive value was >90 percent).

View this table: [in this window] [in a new window]   Table 4. Pretransplantation Measurement of Plasma TGF as a Predictor of Liver and Lung Fibrosis after High-Dose Chemotherapy and Autologous Bone Marrow Transplantation.

 
We also attempted to determine whether the development of pulmonary fibrosis or hepatic veno-occlusive disease was associated with any of the variables listed in Table 1. We could find no significant difference in the mean values for these clinical factors between the patients with these complications after transplantation and those without them (P>0.1 in all cases). Furthermore, there was no correlation between any of the factors and the pretransplantation TGF levels in the three groups of patients (data not shown).

To explore the possibility that TGF might be produced by the tumor, the relation between tumor burden, as measured by the maximal tumor dimension and the number of lymph nodes involved by cancer, and the plasma TGF concentration before transplantation was determined (Table 5). There were no significant differences in pretransplantation TGF levels when the patients were compared according to the number of involved lymph nodes or the greatest measurable tumor dimension (before induction chemotherapy).

View this table: [in this window] [in a new window]   Table 5. TGF() Concentration in Relation to Tumor Burden in Patients with Liver or Lung Fibrosis.

 
We also considered the possibility that patients with stage IV cancer who had previously received chemotherapy (before their enrollment in the bone marrow transplantation study) might be at increased risk for toxic complications, as compared with patients who had not previously received chemotherapy. This comparison could be made only in the group with hepatic veno-occlusive disease, since no patient in the other groups had been previously treated with chemotherapy. We found no difference (P>0.1) in the pretransplantation TGF levels between the patients who had received previous chemotherapy and those who had not, which suggested that previous chemotherapy did not necessarily increase the risk of hepatic veno-occlusive disease in this group.

Discussion

Hepatic veno-occlusive disease and pulmonary fibrosis are major causes of morbidity and mortality after bone marrow transplantation for cancer. Many clinical factors define patient populations at increased risk for the development of these complications,^{1,2,41,42,43,44,45,46} but none of these clinical factors have been useful in assessing the risk in an individual patient. Our study indicates that the plasma TGF concentration, if measured after induction chemotherapy, strongly correlates with the development of pulmonary fibrosis or hepatic veno-occlusive disease after high-dose chemotherapy and autologous bone marrow transplantation.

Patients most prone to pulmonary fibrosis or hepatic veno-occlusive disease after high-dose chemotherapy and autologous bone marrow transplantation have elevated TGF levels before transplantation. A positive test for TGF has a positive predictive value of more than 90 percent for the development of either hepatic veno-occlusive disease or pulmonary fibrosis in a given patient. It may be possible to use TGF plasma levels to individualize therapy and thus reduce the risk of both complications.

We chose the assay system we used in this study because of its ability to detect very low levels of TGF. Although enzyme-linked immunosorbent assays for the quantification of TGFb⁴⁷ are less sensitive than our biologic assay, our results demonstrate that enzyme-linked immunosorbent assays should have sufficient sensitivity to permit rapid screening for patients most prone to fibrotic changes (i.e., patients with plasma levels of TGF1 above 10 mg per milliliter [4 x 10^-7 mmol per liter]).

The cause of elevated plasma levels of TGF in patients who ultimately have hepatic or pulmonary fibrosis is not known. Platelets are the principal source of TGF in humans, but an artifactual disruption of platelets seems unlikely. For TGF levels to become falsely elevated in the patients we studied, blood samples would have had to have been obtained shortly after platelet destruction occurred, since the half-life of TGF in the blood is only a few minutes. Also, the putative destruction of platelets by drugs or venipuncture would have had to have occurred only in the patients who ultimately had fibrosis. Finally, all patients treated with high-dose chemotherapy and autologous bone marrow transplantation had a decrease in TGF concurrent with chemotherapy-induced thrombocytopenia.

The elevation of plasma levels of TGF in patients with hepatic veno-occlusive disease or pulmonary fibrosis also does not appear to be related to their tumor burden. Some factor other than the tumor is apparently responsible for the elevated TGF levels in patients with these complications.

Increased synthesis or activation of TGF or decreased degradation of this growth factor (or some combination of these processes) is a possible response to induction chemotherapy in patients who subsequently have hepatic veno-occlusive disease or pulmonary fibrosis. Hoyt and Lazo²⁰ have shown that strain-specific variations in TGF messenger RNA in the lungs of mice correlate with differences in susceptibility to cyclophosphamide-induced pulmonary fibrosis. Genetic differences may also occur in human responses to chemotherapeutic agents.

TGF is normally secreted from cells as a glycosylated latent complex that contains phosphorylated mannose residues⁴⁸. It must be dissociated from this complex to become biologically active. The latent complex of TGF1 binds to the receptor that accepts both glycoproteins containing mannose-6-phosphate and insulin-like growth factor II,⁴⁹ and this binding has been shown to facilitate the activation of the TGF1 molecule by proteolytic enzymes¹⁹. It is possible that this activation process is augmented in patients in whom hepatic veno-occlusive disease or pulmonary fibrosis develops, and consequently more mature TGF would be present in the plasma. We have observed an increased concentration of TGF in hepatocytes with increased numbers of mannose-6-phosphate-insulin-like growth factor II receptors when the liver is undergoing regeneration⁵⁰ or has been exposed to the liver-tumor promoter phenobarbital⁵¹. Whether a concomitant increase in the level of TGF1 and the number of mannose-6-phosphate-insulin-like growth factor II receptors also occurs in the liver and lungs of humans after exposure to chemotherapeutic agents, radiation, or other insults resulting in fibrosis is unknown.

Supported by grants (CA-40172 and CA-25951) from the National Cancer Institute.

We are indebted to Karen Hillary for technical assistance, to Denise Crawford for assistance in data acquisition, to Richard Dodge for statistical advice, and to Roxanne Scroggs for assistance in the preparation of the manuscript.

Source Information

From the Department of Radiation Oncology (M.S.A., H.R., R.L.J.) and the Department of Medicine, Division of Hematology-Oncology (W.P. Peters, W.P. Petros), Duke University Medical School, Durham, N.C.

Address reprint requests to Dr. Jirtle at Box 3433, Duke University Medical Center, Durham, NC 27710.

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