Regression of Severe Fibrotic Liver Disease in 2 Children With Neonatal Hemochromatosis

Neonatal hemochromatosis (NH) has been defined clinically as severe neonatal liver disease in association with extrahepatic siderosis in a distribution similar to that seen in hereditary hemochromatosis (1,2). Considerable evidence exists to suggest that it is a gestational disease in which fetal liver injury is the dominant feature (3). NH is associated with severe fetal liver injury, and one of its most common presentations is late second and third trimester fetal loss, as evidenced by the gestational histories of women who have had a baby diagnosed with NH (4). Most affected live-born babies show evidence of fetal insult (eg, intrauterine growth restriction, oligohydramnios) and premature birth is common. The presenting findings are those of acute liver failure and usually multiple organ failure. Liver disease generally is apparent within hours of birth, though in rare cases the liver disease takes a subacute course and manifests days to weeks after birth. The typical histopathology of the liver shows injury that is severe and out of proportion to that seen in other forms of hemochromatosis (1,2,5). Cirrhosis is evident in nearly all of the cases. The liver is generally small and deeply bile stained. Its contours may be irregular as a result of hepatocellular loss, collapse of stroma, and varying degrees of regeneration. Fibrosis is pronounced, particularly in the lobule and around the central vein. Regenerative nodules are usually present. In some instances, almost no well-preserved hepatocytes remain. The residual and/or regenerating hepatocytes may exhibit either giant cell or pseudoacinar transformation with canalicular bile plugs. Residual hepatocytes often show siderosis, whereas Kupffer cells are spared. The siderosis is coarsely granular, which is in contrast to the hazy iron staining of normal newborn liver. Considerable acute and chronic inflammation are usually evident. The pattern of injury is strongly reminiscent of subacute or acute hepatic failure in postnatal life. The prognosis in severe NH is generally poor. The average life expectancy of a severely affected baby is days to a few weeks. Current medical treatment is less than satisfactory (6,7). A cocktail containing both antioxidants and an iron chelator has been used to treat NH. Success rates with medical treatment have been reported to be 10% to 20%, and it is not clear what factors determine a successful outcome. Recent reports suggest that indeed this treatment does not alter the course of NH, in particular in babies with severe disease (8,9). However, it is clear that babies with clinical liver failure due to NH can recover fully with medical support. The most widely accepted definition of cirrhosis was established by the World Health Organization as a diffuse process characterized by fibrosis and the conversion of the normal liver architecture into structurally abnormal nodules (10). As a diffuse process, it disrupts the parenchymal architecture of the entire liver. It has long been maintained that once cirrhosis was established, it was irreversible. However, it has become clear that liver fibrosis is a dynamic and therefore potentially reversible process. Full “reversal” of cirrhosis, with complete restoration of normal architecture following established cirrhosis, is a concept that has been met with skepticism (11), whereas “regression” of fibrosis or cirrhosis, meaning reduction in a quantitative fibrosis score, is considered to be an important outcome measure in medical trials (12). Such regression has been documented in several liver diseases with treatment. We report on the regression of severe fibrotic liver disease in 2 siblings with NH. These babies were born to a mother whose previous baby had neonatal liver disease ascribed to NH and who received protocol-based gestational therapy with intravenous immunoglobulin (IVIg) to prevent its lethal recurrence (4). These 2 babies had clinical liver disease, albeit not as severe as the previous baby, and both made a full clinical recovery. The protocol provided the opportunity to obtain liver histopathology from these babies as neonates, which showed multifocal parenchymal collapse with advanced widespread fibrosis and focal nodule formation, and in follow-up some years later, which showed regression of severe fibrosis. The significance of these cases is that they question the commonly held belief that advanced fibrosis is irreversible. CASE 1 The patient was a boy born at 33 weeks of gestation by cesarean section for the indication of oligohydramnios. The mother was gravida 3 para 2; her first pregnancy ended as a spontaneous abortion, and the product of the second pregnancy had NH and died at 3 months of age after liver transplant. In this pregnancy, she had received gestational therapy with IVIg. Although the patient was clinically healthy, he had evidence of liver disease as demonstrated by peak values in the first week of life: serum ferritin of 1086 ng/mL (22–322), α-fetoprotein of 558,600 ng/mL (<80,000), international normalized ratio of 2.4, alanine aminotransferase of 52 IU/L (2–30), total bilirubin 15.8 mg/dL (0.2–1.0), and direct bilirubin 6.3 mg/dL (<0.3). A diagnosis of NH was made on the basis of maternal history with the patient's clinical and laboratory information, and he was treated with a combination of antioxidants and iron chelation from day 2. He was transferred to the authors' institution at 12 days of age. Chelation was subsequently discontinued at 5 weeks of age. A liver biopsy, which is described in the “Results” section, was performed at 31 days of age to evaluate the severity of liver injury and guide further care. He was discharged home on day 36 and at 6-year follow-up has continued to do well with a normal physical examination, no evidence of liver disease, and normal development. A follow-up liver biopsy was performed at 3½ years of age to determine the extent of residual liver disease that could not be otherwise detected. CASE 2 The patient was born at 37 weeks of gestation by an elective caesarean section, almost 2 years following the birth of her brother (case 1). Again, her mother had received gestational therapy with IVIg. She was transferred to the authors' institution on day 2. She was clinically asymptomatic, but showed evidence of liver involvement as demonstrated by peak values in the first week of life: serum ferritin 8107 ng/mL (22–322), α-fetoprotein 230,280 ng/mL (<80,000), international normalized ratio 2.2, alanine aminotransferase 28 IU/L (2–30), total bilirubin 5.5 mg/dL (0.2–1.0), and direct bilirubin 3.1 mg/dL (<0.3). A liver biopsy, described in the Results section, was performed at 1 week of age to evaluate the severity of liver injury. On the basis of this biopsy showing severe liver injury, in combination with the clinical and laboratory information, medical therapy with chelation/antioxidants was instituted. She was transferred to her local hospital to continue treatment and was discharged home at 40 days of age. At 4-year follow-up, she has continued to do well, with a normal physical examination, no evidence of liver disease, and normal development. A follow-up liver biopsy was performed at 2 years of age. METHODS FOR ANALYSIS OF HISTOPATHOLOGY Needle biopsy samples were fixed in 10% buffered formalin, and 5-μm-thick sections were obtained from paraffin-embedded tissue. Hematoxylin-eosin stains were used for a general evaluation of histological features, and Prussian blue stain for siderosis. For evaluation of fibrosis, samples were stained with Masson trichrome stain. Fibrosis was scored as previously described by Batts et al (13). Morphometric analysis was performed as follows: Digital images were captured from each sample at ×200 magnification including all lobular zones to avoid selection bias. The images were opened in Photoshop 7.0 (Adobe Systems, San Jose, CA), and collagen fibers were selected by their blue color on the trichrome-stained sections, using the “magic wand” function of the software. The extent of the selected areas was controlled using the “tolerance” option of that function, to ensure inclusion of spectral variance within the same range of color. The nonselected (ie, the noncollagenous) areas were then deleted, and the image was converted to black and white, so that the end result was an image in which the areas of interest (ie, collagenous tissue) were black on a totally white background, a requirement of our morphometry software. Measurement of the pixels occupied by black as a percentage of the total area was performed using the Photoshop plug-in Image Processing Tool Kit (Reindeer Graphics, Asheville, NC). The result was then recorded as the percentage of collagenous tissue in each image. The mean value per tissue sample was calculated from the results of all of the images derived from the sample. RESULTS The initial (31 days) liver biopsy from case 1 (Figure 1A) showed marked alteration of architecture consistent with advanced portal and pericellular fibrosis with focal formation of regenerative nodules. Fibrosis was extensive and pan-lobular, and nodules of hepatocytes surrounded by fibrosis were evident. The follow-up (3.5 years) biopsy (Figure 1B) showed normal hepatic morphology with normal lobular architecture, no lobular fibrosis, and normal portal areas. The fibrosis score (13) for the initial biopsy was 4 and for the follow-up biopsy was 0. The mean percentage of surface area represented by fibrous tissue in the initial biopsy was 11% and in the follow-up biopsy was 2.7%. Prussian blue stain was negative on the follow-up biopsy.FIG. 1: A, Biopsy from the neonatal period shows advanced portal fibrosis with focal formation of regenerative nodules (trichrome stain, original magnification ×10). B, Follow-up liver biopsy at 3.5 years of age shows a portal tract (darker area) with no significant expansion. There is no significant parenchymal fibrosis (trichrome stain, original magnification ×10).The initial (7 days) biopsy from case 2 (Figure 2A) showed advanced portal fibrosis with septa formation, multifocal parenchymal collapse, parenchymal fibrosis, and regenerative nodules. The follow-up (2 years) biopsy (Figure 2B) showed normal hepatic morphology with normal lobular architecture, no lobular fibrosis, and normal portal areas. The fibrosis score for the initial biopsy was 4 and for the follow-up biopsy was 0. The mean percentage of surface area represented by fibrous tissue in the initial biopsy was 30.8% and in the follow-up biopsy was 2.2%. Prussian blue stain was negative on the follow-up biopsy.FIG. 2: A, Biopsy from the neonatal period shows advanced portal fibrosis with focal formation of regenerative nodules (trichrome stain, original magnification ×10). B, Follow-up liver biopsy at 2 years of age shows mild expansion of a portal tract due to fibrosis, along with a small portal-to-portal fibrous bridge.DISCUSSION These 2 patients had NH with widespread fibrosis and focal nodule formation, despite their mother having received gestational treatment with IVIg, which is intended to prevent recurrent NH or lessen its severity. They also had liver dysfunction meeting accepted criteria for diagnosis of neonatal liver failure (14). They were, however, clinically stable and recovered with support and chelation/antioxidant therapy. Liver biopsies obtained from both neonates demonstrated advanced portal and pericellular fibrosis with regenerative nodules, whereas repeat biopsies after full clinical recovery showed normal hepatic architecture with no residual disease. These pairs of biopsies fairly demonstrate complete regression of severe fibrotic liver disease in NH. Survival of babies with NH, whether spontaneous or as a result of medical therapy, is somewhat unusual and yet is requisite to examining the extent or completeness of recovery. Indeed, there are few reports that discuss the long-term outcome of babies surviving NH without transplantation in more than cursory detail, and only 2 that describe the hepatic histopathology after clinical recovery. In a recent large series, only 2 of 19 babies survived with medical therapy, 1 with and 1 without chelation/antioxidant treatment (9). These babies underwent liver biopsy at 7 and 8 months of age, which showed fibrotic liver with postnecrotic bridging septa in 1 and micronodular cirrhosis in the other. In both, there was evidence of remodeling and no evidence of ongoing injury, inflammation, and fibrosis. Neither baby had a previous biopsy to assess the degree of initial injury and fibrosis, nor a longer term follow-up biopsy to determine completeness of recovery. In another brief report, the sibling of a child who died in liver failure due to NH spontaneously recovered with medical support (15). The initial biopsy was reported as showing giant cell hepatitis, which is consistent with being mildly affected with NH, and follow-up biopsy at 16 months of age showed fibrosis, with no further detail provided. Our well-documented cases add considerably to the sparse knowledge regarding the potential for histological recovery in NH. Liver biopsy histology is the standard for scoring fibrosis and diagnosing cirrhosis (10,16). The first biopsies from these babies showed clear advanced and widespread fibrosis with focal regenerative nodule formation, and the follow-up biopsies showed a clear lack of fibrosis, indeed normal histology. The potential for sampling error always exists when using a biopsy to estimate the pathological condition of the entire liver (17). It would seem that the potential for erroneous grading and staging of the initial biopsies is small. They showed severe necrosis, inflammation, and fibrosis throughout the biopsy sample. Given that most information about the liver pathology in NH comes from autopsy materials and hepatic explants, it is known that the liver is always injured and scarred throughout. There is no record of limited or focal injury in this disease. Thus, it is safe to assume that the entire liver looked like what was seen in the biopsy. However, the follow-up biopsies could represent an area of focal recovery, whereas the rest of the liver could be cirrhotic. Against this possibility is the reality that these patients had fully recovered clinically and biochemically, and had normal physical examinations. Furthermore, both patients showed full regression of advanced fibrosis. Sampling error is not usually unidirectional. Another issue in interpreting liver biopsies for changes in fibrosis related to treatment protocols is interobserver variation, which can be substantial (17). In trials of hepatitis C treatment, rates of disagreement as high as 33% to 55% are observed when considering specimens showing a 1-grade change in fibrosis score (17). Disagreement falls to near zero when the score changes by 3 points. It is unlikely that the findings in these 2 children would be subject to error for this reason, although they were not interpreted in any blinded fashion as would be expected in a randomized treatment trial. Regression of fibrosis/cirrhosis as evidenced by a reduction in the fibrosis score by 2 or more grades has been documented in several liver diseases. Notably, patients with autoimmune hepatitis may have a dramatic reduction in the fibrosis score with immune therapy (18). Also, patients with thalassemia and advanced liver disease from iron overload may have significant regression of fibrosis after successful bone marrow transplant and removal of iron (19). These 2 conditions share some similarities with NH. Autoimmune hepatitis is an aggressive disease with extensive inflammation and active fibrogenesis. Treating the immune process eliminates the inflammation and stimulus for ongoing fibrosis. There is substantial evidence that NH is an alloimmune disease, mediated by maternal IgG antibody. If the liver damage is not so severe, permitting the baby to survive, the source of immune-mediated liver injury is eliminated by separation from the placenta (2,4). Of course, eliminating the need for transfusion and removing excess iron deletes the toxin responsible for liver injury in secondary iron overload. It is not clear what role iron plays in the liver injury associated with NH, but it is possible that the removal of excess iron by chelation may increase the potential for recovery. Most important in these 3 conditions, significant regression of fibrosis appears to be possible if the source of injury is eliminated. NH represents a special case that may be more likely to show regression of fibrosis/cirrhosis than other liver diseases. First, the fibrosis, although extensive, cannot be long-standing and may therefore be in a more plastic state than in chronic end-stage liver disease. It is thought that the earliest that NH significantly injures the liver is at 18 to 24 weeks of gestation (2,3,20). Thus, at birth at 34 to 40 weeks, the longest possible duration of injury is only about 20 weeks. Furthermore, there is no knowledge regarding when sufficient populations of hepatic stellate cells develop during fetal life to produce fibrosis, or when the immune components of the fetal liver develop to the degree that stellate cell activation is efficient. All of the scarring evident at birth may be only a few weeks in the making. Second, the neonatal liver is generally thought to be innately more plastic and capable of recovery from insult than the liver of older individuals. For example, the massive liver resections performed for treatment of hepatoblastoma are well tolerated by young children but would not be tolerated by adults at all. Conclusions In conclusion, our 2 well-documented cases show the potential for histological recovery in neonatal hemochromatosis; however, more careful follow-up of other treated babies is indicated to better understand this process in neonatal hemochromatosis.