Methods

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Patients

We focus on 5 of 19 patients with biopsy-proven precirrhotic primary biliary cirrhosis. Eighteen of the 19 patients were positive for antimitochondrial antibody, all had had primary biliary cirrhosis for at least 1 year, and all received low-dose methotrexate therapy. Nine of these patients have been described in an earlier report [8]. Three of the 5 patients we describe here had not responded to colchicine or had responded only partially. The 5 patients had received maintenance therapy with methotrexate for at least 6 years; the other 14 patients in the group had received methotrexate for 1 to 6 years.

Intervention

Methotrexate was given orally at a dosage of 15 mg/wk, divided into three 5-mg doses taken 12 hours apart over one 24-hour period.

Histologic Studies

Liver biopsy slides were coded and read independently by two senior pathologists who had no clinical information other than the fact that the patients had primary biliary cirrhosis. Biopsy specimens were paired by patient, but the pathologists were not aware of the timing of the biopsies (that is, initial compared with final). The length of the biopsy specimens and the number of portal triads on each slide were recorded. The specimens were graded according to histologic stage [9]; modified Knodell score, in which bile duct changes were graded and added to the total score [8]; and a score of global change (unchanged, 0; probably improved, +1; and unequivocally improved, +2) [4]. After each pathologist had read the slides independently, they met to resolve any differences and record the final histologic scores.

Case Reports

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Patient 1

A 50-year-old woman was referred because of primary biliary cirrhosis that had developed 2 years earlier. The patient had been receiving cholestyramine, one packet three times per day, for 1 year. When primary biliary cirrhosis was first diagnosed, the alkaline phosphatase level was 750 IU (12.5 µkat/L) and the patient was positive for antimitochondrial antibody (titer > 1:640). When the patient was seen at the New England Medical Center in Boston, results of physical examination were normal. At this point, the alkaline phosphatase level was 1245 IU (20.75 µkat/L), the serum bilirubin level was 1.9 mg/dL (32.5 µmol/L), and the alanine aminotransferase level was 235 U/L (3.9 µkat/L). A liver biopsy specimen showed stage II to III primary biliary cirrhosis with many florid bile duct lesions (Figure 1, panel A). The patient was prescribed colchicine, 0.6 mg twice daily; cholestyramine therapy was continued.

View larger version (127K): [in this window] [in a new window]   Figure 1. Liver biopsy specimens before and after methotrexate therapy for primary biliary cirrhosis. A. Patient 1 before colchicine therapy, 0.6 mg twice daily. Several portal triads are greatly enlarged, scarred, and infiltrated with mononuclear inflammatory cells. Bridging of adjacent portal triads is seen. Degenerating bile ducts are at the center of the inflammatory infiltrate. Specimen is typical of stage II to III disease. (Masson trichrome; original magnification, x94.) B. Patient 1 after 11 years of treatment: 5 years with colchicine alone; 3 years with colchicine, 0.6 mg twice daily, and methotrexate, 15 mg/wk; and 3 years with colchicine, methotrexate, and ursodeoxycholic acid. Normal parenchyma, minimally scarred portal triads, and no inflammation or any feature of disease are shown. (Masson trichrome; original magnification, x60.) C. Patient 2 before treatment with methotrexate, 15 mg/wk. Portal triads are greatly expanded and infiltrated with mononuclear inflammatory cells. Florid bile duct lesion is visible (arrow). The bridging of enlarged portal triads is a finding consistent with stage III disease. (Hematoxylin and eosin; original magnification, x60.) D. Patient 2 after 4 years of treatment with methotrexate, 15 mg/wk. Normal parenchyma; inflammation confined to the portal triads (some of which are moderately enlarged). Normal bile ducts; no bridging of adjacent portal triads. (Masson trichrome; original magnification, x60.) E. Patient 3 five years after diagnosis of disease and 1 year after treatment with colchicine, 0.6 mg twice daily. Portal triads are greatly enlarged, somewhat edematous, and infiltrated with mononuclear inflammatory cells. Bridging of adjacent triads and early nodule formation are evident. Specimen shows stage III to IV disease. (Masson trichrome; original magnification, x94.) F. Patient 3 after 6 years of methotrexate therapy. Specimen is normal except for portal triads that are minimally enlarged and infiltrated with mononuclear cells. No features of disease are seen. (Masson trichrome; original magnification, x94.).

During the next 5 years, the alkaline phosphatase level gradually decreased to 400 IU (6.67 µkat/L), the serum bilirubin level decreased to 1 mg/dL (17.1 µmol/L), and the alanine aminotransferase level decreased to 80 U/L (1.33 µkat/L). The patient remained positive for antimitochondrial antibody. A repeated liver biopsy specimen showed decreased inflammation in portal triads but was still consistent with stage II disease. The patient began receiving oral methotrexate, 15 mg/wk.

After the patient had received methotrexate for 1 year, cholestyramine therapy was discontinued; pruritus did not recur. After 1 year, during which the patient received the combination of methotrexate and colchicine, blood test results had improved: The alkaline phosphatase level was 200 IU (3.33 µkat/L), the bilirubin level was 0.6 mg/dL (10.3 µmol/L), and the alanine aminotransferase level was 50 U/L (0.83 µkat/L). A repeated liver biopsy specimen showed only minimal portal fibrosis and inflammation. Because the alkaline phosphatase level was still elevated, the patient began receiving ursodiol, 300 mg twice daily. The alkaline phosphatase level decreased to normal during the next 12 months. After 3 years of treatment with all three medications, liver function test results were normal: The alkaline phosphatase level was 102 IU (1.7 µkat/L), the bilirubin level was 0.5 mg/dL (8.5 µkat/L), and the alanine aminotransferase level was 19 U/L (0.32 µkat/L). A liver biopsy specimen obtained after 11 years of treatment (6 years of methotrexate therapy) was normal except for scattered foci of nonspecific inflammation in portal triads (Figure 1, panel B). The patient remains positive for antimitochondrial antibody after 14 years of treatment.

Patient 2

A 34-year-old woman was referred because of a 1-year history of primary biliary cirrhosis. She reported pruritus and fatigue and was receiving hydroxyzine for pruritus. Results of physical examination showed striking bilateral xanthelasmas, diffuse excoriations, and an enlarged liver that was palpated 6 cm below the right costal margin. The serum alkaline phosphatase level was 962 IU (16 µkat/L), the serum bilirubin level was 1.8 mg/dL (30.8 µmol/L), and the alanine aminotransferase level was 251 U/L (4.2 µkat/L). Results of tests for antimitochondrial antibody were positive (titer, 1:1024). A liver biopsy specimen showed stage III primary biliary cirrhosis with scattered florid bile duct lesions (Figure 1, panel C). The patient was prescribed oral methotrexate, 15 mg/wk.

After 2 months of treatment, blood test results began to gradually but steadily improve. After 6 months of methotrexate therapy, the serum alkaline phosphatase level was 490 IU (8.2 µkat/L), the serum bilirubin level was 0.7 mg/dL (12.0 µmol/L), and the alanine aminotransferase level was 145 U/L (2.42 µkat/L). After 8 months of therapy, pruritus and fatigue had disappeared and the patient could discontinue hydroxyzine therapy. Percutaneous liver biopsies done at years 1, 2, and 4 showed that inflammation and fibrosis had gradually diminished. After 4 years of methotrexate therapy, liver histologic findings were considerably improved. Scattered foci of inflammation were seen in portal triads, inflammation slightly extended into the periportal parenchyma, no portal-to-portal bridging inflammation or fibrosis was seen, and no florid bile duct lesions were present (Figure 1, panel D). The biopsy specimen was consistent with stage I primary biliary cirrhosis. Results of physical examination were normal. The xanthelasmas had disappeared, and the liver was no longer enlarged. After 6 years of methotrexate therapy, all liver function test results were normal: The serum alkaline phosphatase level was 94 IU (1.57 µkat/L), the serum bilirubin level was 0.5 mg/dL (8.5 µmol/L), and the alanine aminotransferase level was 17 U/L (0.28 µkat/L). Results of tests for antimitochondrial antibody remained positive. The patient moved to another state and was lost to follow-up for 1.5 years. She discontinued methotrexate therapy and remained asymptomatic. After therapy was discontinued, however, the serum alkaline phosphatase level increased to 398 IU (6.63 µkat/L) and the alanine aminotransferase level increased to 75 U/L (1.25 µkat/L). The bilirubin level remained normal (0.3 mg/dL [5.1 µmol/L]). The patient has resumed methotrexate therapy.

Patient 3

A 42-year-old woman was referred because of primary biliary cirrhosis that had developed 4 years earlier. She had had the Raynaud phenomenon for 12 years. A maternal aunt had died of primary biliary cirrhosis. The patient's serum alkaline phosphatase level was 263 IU (4.38 µkat/L), the serum bilirubin level was 0.6 mg/dL (10.3 µmol/L), and the alanine aminotransferase level was 57 U/L (0.95 µkat/L). Testing by enzyme-linked immunosorbent assay showed strong positivity for antimitochondrial antibody: 0.35 OD (normal < 0.10 ODs) [10]. A percutaneous liver biopsy specimen revealed stage III primary biliary cirrhosis. The patient was prescribed colchicine, 0.6 mg twice daily. She remained asymptomatic, but blood test results did not improve. Repeated liver biopsy specimens obtained after 1 year showed worsening portal and periportal inflammation and the same degree of portal fibrosis (Figure 1, panel E). The patient began receiving oral methotrexate, 15 mg/wk, and remained asymptomatic.

Two months after methotrexate therapy began, blood test results gradually improved. After 6 years of methotrexate and colchicine therapy, the serum alkaline phosphatase level had decreased to 96 IU (1.6 µkat/L), the serum bilirubin level remained 0.6 mg/dL (10.3 µmol/L), and the alanine aminotransferase level was 34 U/L (0.57 µkat/L). Serial liver biopsy specimens, obtained at years 1, 2, 4, and 6 of methotrexate therapy, showed a steady decrease in inflammation and fibrosis. After 6 years of methotrexate and colchicine therapy, liver histologic findings are normal except for scattered foci of portal inflammation (Figure 1, panel F). Bile ducts are normal in appearance and number. The patient remains positive for antimitochondrial antibody.

Results

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All five patients who responded biochemically and histologically had well-documented primary biliary cirrhosis that had been present for an average of 3.2 years (range, 1 to 7 years) before methotrexate therapy was started (Table 1). In each patient, symptoms have now remitted, results of biochemical tests of liver function have become normal, and liver histologic findings have improved (Table 2).

All baseline liver biopsy specimens had shown intense portal inflammation and florid bile duct lesions. Granulomas were seen in three baseline specimens. Subsequent liver biopsy specimens consistently showed decreased inflammation, the disappearance of florid bile duct lesions and granulomas, and a decrease in fibrosis. Table 3 quantifies the histologic improvement, as determined by two senior pathologists who read the specimens separately and under code. The independent interpretations of these pathologists were nearly identical. The mean length of the biopsy specimens was 26.6 mm (range, 15 to 46 mm). A mean of 9.5 portal triads (range, 6 to 14) was seen per specimen. The large size of the specimens and adequate number of portal triads make sampling artifact unlikely. Piecemeal necrosis, portal inflammation, lobular inflammation, portal fibrosis, and bile duct damage were unequivocally reduced. Each final specimen contained an almost normal number of bile ducts. The mean Knodell score decreased from 15.1 to 5.8, and the mean histologic stage decreased from 2.5 to 1. The pathologists also assessed the specimens globally [4]. Four specimens had dramatically improved and were close to normal, and one had probably improved. Serial liver biopsy specimens ( 5) were obtained for two of the five patients; all specimens showed continuing improvement in histologic findings during methotrexate therapy.

The other 14 patients in this cohort had decreases in serum alkaline phosphatase levels. Thirteen patients had sustained decreases in alanine aminotransferase levels, and the 6 patients whose baseline serum bilirubin levels were elevated had initial decreases in serum bilirubin levels (Table 4). Seven of these 14 patients have had sustained improvements in biochemical test results for 3 to 6 years. In 5 of these 7 patients, liver histologic findings have stabilized or improved (as shown by decreased inflammation); in the other 2, the histologic stage has worsened (from stage III to IV). The condition of 2 of the 7 patients deteriorated after 3 years of methotrexate therapy. In these patients, serum bilirubin levels increased and hemorrhage from esophageal varices occurred. The patients were referred for liver transplantation. Four of the remaining 5 patients have discontinued methotrexate therapy: 2 because of personal reasons (intention to become pregnant and desire to use only herbal medicines, respectively), 1 because of methotrexate toxicity (pancytopenia), and 1 because of complications of diabetes mellitus and the need for extensive vascular surgery. One patient has been lost to follow-up.

Discussion

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On the basis of the criteria by which liver disease is usually judged, all five patients who received methotrexate for at least 6 years are in remission. We found no previous reports of patients with primary biliary cirrhosis whose disease has gone into remission, either spontaneously or in response to treatment [5]. In one study [4], colchicine seemed to decrease the rate of death related to liver failure and to improve results of biochemical tests of liver function, but it had no beneficial effect on symptoms or histologic findings. Of the five patients in our study who have gone into remission while receiving methotrexate, patient 1 partially responded to colchicine and patients 3 and 4 did not respond.

In several recent studies, ursodiol improved results of biochemical tests of liver function and slowed the rate of referral for liver transplantation compared with patients receiving placebo. However, ursodiol had either minor or no beneficial effect on liver histologic findings [3, 11-13]. In 12 of 65 patients in one recent study [14], blood test results returned to normal after 2 years of ursodiol therapy. Four of these patients had improved liver histologic findings. Ursodiol may also be effective in a subset of patients with primary biliary cirrhosis, but the 2-year follow-up of this study [14] is too short to determine this effectiveness with certainty.

The results seen with cyclosporine have been similarly disappointing [15]. Neither symptoms nor liver histologic results improved with this drug. The mortality rate and rate of referral for liver transplantation were slowed by cyclosporine only after adjustment for the fact that the patients randomly assigned to receive cyclosporine were sicker at baseline [15].

Because no previous reports have addressed spontaneous or treatment-related sustained remission in patients with primary biliary cirrhosis, many physicians will question whether the five patients described in our report truly have primary biliary cirrhosis. In support of the diagnosis, all biochemical test results showed a chronic cholestatic pattern, disease had been present for at least 1 year before treatment began ( 4 years in one patient), all five patients are positive for antimitochondrial antibody, and all patients had characteristic findings of primary biliary cirrhosis on liver biopsy [1, 9]. No patient was receiving medications at the onset of disease, and none had suggestion of any other type of liver disease. Thus, each patient meets all of the diagnostic criteria for primary biliary cirrhosis.

The five patients who responded dramatically to methotrexate may represent a subgroup of patients with this disease because not all patients with primary biliary cirrhosis seem to respond to this drug [8]. These five patients were selected from a group of 19 patients with primary biliary cirrhosis who were participating in an open-label trial of methotrexate. Of the other 14 patients, 5 have had sustained improvement in biochemical test results and stabilization of histologic findings after 3 to 6 years of treatment, and 2 have had histologic worsening despite biochemical improvement. The condition of 2 patients has deteriorated; these patients have been referred for liver transplantation. Four patients have discontinued methotrexate therapy because of reasons stated in the Results section, and 1 has been lost to follow-up.

The fact that only a subset of patients responds fully to methotrexate raises another question. Is primary biliary cirrhosis a syndrome rather than a disease? That is, are there different types or causes of primary biliary cirrhosis? Heretofore, primary biliary cirrhosis has been considered to be one disease. However, the fact that only some of these patients respond fully to methotrexate whereas others do not is reminiscent of the situation seen with chronic hepatitis more than 25 years ago, before hepatitis B and C were discovered. At that time, chronic hepatitis was thought to be one well-defined disease. Some physicians reported that patients with chronic hepatitis had a dramatic response to treatment with glucocorticoids, whereas others reported no response at all [16]. This controversy was not resolved until hepatitis B and C were discovered. It was then realized that patients who responded to glucocorticoids had autoimmune chronic active hepatitis, a disease quite different from chronic hepatitis B and C. However, until hepatitis B and C were defined, patients with autoimmune chronic hepatitis could not be distinguished from those with other types of chronic hepatitis except by their response to treatment and by the fact that some of the responders had greater elevations of aminotransferase or immunoglobulin levels than did nonresponders [16].

The above results suggest that some patients with primary biliary cirrhosis are treatable and are not doomed to progression of disease and eventual liver transplantation. The 7 to 14 years of observation of these patients (who received methotrexate for 6 to 7 years of this period) suggests that methotrexate does alter the natural history of the disease. This optimistic view is supported by the fact that symptoms have disappeared; biochemical test results have returned to normal; and, most importantly, histologic findings have improved and almost returned to normal. No patient had any signs of portal hypertension. Nevertheless, methotrexate must still be used with caution in patients with primary biliary cirrhosis because it is not well tolerated by all patients. The drug can be toxic and causes interstitial pneumonitis in 14% of patients with primary biliary cirrhosis [17]. In addition, results of other studies are at variance with ours; these investigators have questioned whether methotrexate adds efficacy to that provided by ursodiol [18]. Other researchers, however, have reported success with methotrexate [19, 20]. Our results suggest that the continued judicious use of methotrexate in ongoing prospective trials will demonstrate that in some patients, methotrexate can favorably alter the natural history of this hitherto untreatable disease.