SEARCH

SEARCH BY CITATION

It is an understandably daunting task to find oneself following both the Editor of HEPATOLOGY and the immediate Past President of the American Association for the Study of Liver Diseases (AASLD) for the newly established monthly Associate Editor Commentary, and merely because of alphabetic happenstance at that, but the challenge is accepted, humbly. To have an opportunity to discuss the continuing role for careful, quality liver tissue evaluation in patient management and scientific studies is to be relished, particularly in our time of increasingly sophisticated tools for clinical testing and basic science investigations. It is rather apparent that the entire field of hepatology is in the midst of an exciting time of growth and opportunity; from my personal perspective, rather than feeling threatened that pathologists may be losing ground to noninvasive testing, I would posit that careful tissue evaluation can and should continue to serve multiple functions in the multifaceted collective progress of our field, as discussed.

First, the noninvasive serologically based testing requires careful analysis of adequately sized liver biopsies by knowledgeable hepatopathologists for validation. Next, and perhaps more importantly, as the noninvasive tests are being developed, the resulting algorithms for clinical care often retain the use of a carefully timed liver biopsy in selected patients, just as the tests that we use today do. Even with the advanced imaging modalities available today, liver biopsy is recommended by both the Practice Guidelines of the AASLD1 and others2 for 1- to 2-cm or >2-cm lesions in cirrhotic livers with atypical vascular patterns1 or hypovascular arterial phase patterns.2 Pathology-based studies have resulted in refined concepts of preneoplastic lesions and their diagnostic criteria; these have been recently reviewed by two authoritative groups.3, 4 In medical liver disease, examples for selected liver biopsies include primary biliary cirrhosis and acute hepatitis. Current clinical testing for both is sensitive and specific; however, in the situations in which the test results or clinical course is atypical, liver biopsy evaluation is likely to be undertaken.

The noninvasive serologic and imaging tests for fibrosis have a defined and growing role in screening for evidence (or lack thereof) of extremes: absence or advanced fibrosis. None of the tests to date have been shown to correlate with disease activity or the intermediate (and potentially more reversible) stages of fibrosis. However, in a recent long-term follow-up study of the natural history of nonalcoholic fatty liver disease (NAFLD), it was shown that (1) in comparison with the control population, long-term mortality was increased in the subset of individuals with steatohepatitis (not “just” fatty liver) in the initial biopsy and (2) there was no risk of progression of fibrosis in subjects when the initial biopsy had less than periportal (that is, stage 1c or 2) fibrosis.5 In practice and in clinical trials, there may also be a role for these assays as a means of repeated follow-up testing during intervention. While it is recognized, it is worth recalling that these tests were not intended to replace detailed histologic evaluation of necroinflammatory activity of disease or the organ's architectural integrity; the latter includes not only the presence of newly formed fibrous connective tissue elements but also the complex vascular and parenchymal remodeling that results therefrom.

Finally, the possibility of concurrent processes, such as steatosis or steatohepatitis, the evaluation of subtle alterations of parenchymal or duct morphology, the presence of ductular reaction, the cellular localization of iron deposits, and the presence of dysplasia or unsuspected malignancy are not at all assessable by any of the current noninvasive tests. The fact that the lead-off speaker for each of the five sections of the 2007 AASLD Post Graduate Course was a pathologist demonstrates the understanding and appreciation of the integration of our specialties in clinical care and translational research by the organizers of the course.

Pathologists remain uniquely situated in both the clinical and experimental settings to fulfill an important role in quality control and validation of the increasingly sophisticated molecular assays available for tissue analysis. Even in our own studies utilizing tissue microarray, pathologists are striving to evaluate larger numbers and smaller fragments of tissue for markers of interest. The results are meaningless, however, if the clinical context is not understood and the area(s) from which the tissue came have not been well characterized. Furthermore, while it should be self-evident that unless the appropriate tissue specimen is obtained and utilized, the results of any test or experiment are to be questioned, the fact remains that not all tissue samples are appropriately triaged so that optimal preservation and processing of targets of interest occur. It is true that much more work can now be done with formalin-fixed, paraffin-embedded tissues than previously thought possible with techniques of protein (antigen) and nucleic acid retrieval. However, demonstration of true microvesicular steatosis, high-quality ultrastructural evaluation, and most reliable RNA analysis, for example, continue to require rapid and proper processing of freshly devitalized tissue. Communication with the prospective pathologist in advance of tissue procurement is thus a key feature in well-designed studies.

A recent review of the continuing value of histologic evaluation in tumor pathology6 noted that the use of commonly utilized histochemical stains in pathology practice naturally evolved as an integral component of tissue evaluation. In liver pathology, the routine “special stains” were developed as methods for detection of otherwise nonvisualizable intracellular and structural elements. As “old-fashioned” as it may be, the simple evaluation of an iron stain, such as Perls' stain, a periodic acid-Schiff stain with diastase, a reticulin stain, or a connective tissue stain in liver biopsy evaluation can serve as screening for significant inclusions or architectural alterations for nonspecialized pathologists and as a refined evaluation for focused evaluations.7 For instance, the periodic acid-Schiff–diastase stain not only highlights globules in periportal hepatocytes that are suggestive of alpha-1-antitrypsin, but also highlights basement membrane material. The stain aids in the analysis of bile ducts and their lesions, in deposition of basement membrane in perisinusoidal spaces and in the ceroid pigment within phagocytic Kupffer cells. A simple histochemical stain for copper aids in confirmation of chronic cholestatic processes.

Many of the current molecular techniques no longer require any link with morphologic confirmation or evaluation, including reverse-transcription polymerase chain reaction, comparative genomic hybridization, genetic expression profiling microarrays, and the various -omic assays. As has been the case for well-trained pathologists in the past, the current challenge continues to be as stated: “correlat[ation] of morphologic phenotypes with the genotype signatures upon which the … phenotype is based.”6 Only with dedicated and concerted efforts of collaboration with our clinical and scientific colleagues will this challenge be met.

Nonalcoholic Fatty Liver Disease (NAFLD)

  1. Top of page
  2. Nonalcoholic Fatty Liver Disease (NAFLD)
  3. Animal Models: Is It Time for Consideration of Uniform Terminology?
  4. References

There is no question that liver biopsy cannot and should not be used as a clinical “screening tool” in any disease, including NAFLD. However, as yet, there are no reliable surrogate serum markers or imaging studies that can reliably replace the multifaceted information obtainable from an adequate biopsy specimen: diagnosis of the process(es) of fatty liver, steatohepatitis, or steatohepatitis with fibrosis; determination of severity of necroinflammatory lesions (grade) or architectural alterations as well as the presence and location of fibrosis (stage); and consideration of the presence of comorbid processes. A recent review in HEPATOLOGY8 highlighted the existing and developing biomarkers of activity and fibrosis related to NAFLD as well as the scientific rationale for each. The proposed “noninvasive” assays are best utilized in patients in whom the diagnosis of NAFLD (alone) has been firmly clinically established; as noted, they are best at detecting advanced stages of necroinflammatory activity or architectural remodeling (that is, bridging fibrosis and/or cirrhosis). However, additional clinical conditions, such as Gilbert's syndrome, cholestasis, elevated haptoglobin, bilirubin, or apolipoprotein A1, may be limiting factors for the use of some of the popular assays.

Even a dedicated pathologist realizes that there are difficulties related to liver biopsy evaluation in NAFLD; many are addressable. Perhaps one of the more interesting is the variability of disease manifestations, particularly in a disease process for which there is no absolute diagnostic test. Parenchymal variability includes reported differences in histologic manifestations between adult NAFLD and many cases of pediatric/adolescent NAFLD. Details of gender, ethnicity, age, and metabolic status notwithstanding, the differences in the pattern(s) of injury, amounts and deposition of steatosis, presence of ballooning, presence of lobular and portal inflammation, and patterns of fibrosis have been well documented in the literature.9 Thus, a “unifying” concept of pathophysiology must acknowledge this “pediatric pattern.” The second manifestation of (potential) parenchymal variability in NAFLD is that documented within adult livers either within the same lobe (right)10 or between the right and left.11, 12 Details of these studies are worth recalling. One study was performed with percutaneous 16-gauge biopsy in obese patients (median body mass index, 31.6 kg/m2),10 whereas the other two were intraoperative tru-cut biopsies, 16-gauge12 and 14-gauge,11 in morbidly obese individuals undergoing bariatric surgery (median body mass index, 50 and 46.2 kg/m2, respectively). A fourth study compared lesions of NAFLD with lesions of chronic hepatitis C from retrospectively reviewed series of percutaneous liver biopsies performed for clinical care.13 The primary outcomes of these studies were as follows: (1) there is parenchymal heterogeneity in fibrosis in NAFLD, but it is less of a factor in scoring with longer (>1.6 cm13 and 22 mm11) and larger gauge biopsy samples11; (2) there is more intraparenchymal variability in lobular inflammation than in steatosis10–12; and (3) the parenchymal variability is greater for fibrosis than for the amount of steatosis but less for lobular inflammation.10, 12 One study found fibrosis to be the feature with the highest concordance between lobes (98%).11 Hepatocyte ballooning concordance was variable; there was concurrence that ballooning was not as predictably present in the samples as steatosis. A subtle but clearly significant difference in the studies was the biopsy needle: a 14-gauge needle with a mean length of 22 mm in one11 versus 16-gauge needles with mean lengths of 1412 and 20 mm10 in the two others reported. The resultant surface area, therefore, of the first11 was calculated to be 46.2 mm2 versus 23.1 and 33 mm2 for the latter two, respectively. Similarly improved results in fibrosis sampling have been shown in correlation with liver biopsy size (length and width) in chronic viral hepatitis.14, 15

These studies raise a series of as yet unanswered questions about NAFLD. First, should we expect the disease process to be the “same” throughout the liver parenchyma? We accept the fact that there is parenchymal (regional and zonal) variability of lipid, glucose, and insulin metabolism and enzymatic expression, for example, so why should we expect an injury pattern that is likely a pathologic manifestation of these processes to be uniform throughout the parenchyma? Second, is NAFLD truly “an” entity shared between all obese and lean individuals, or are there differences that we must carefully characterize and then learn to accept among (1) adults who are truly morbidly obese and those who are “only” overweight or obese, (2) children and adolescents compared with adults, (3) various ethnic groups, and (4) other subjects with NAFLD-like histologic manifestations from processes seemingly not related to insulin resistance?

Finally, one of the most exciting breakthroughs in liver disease has been the multifaceted understanding of the nonstatic nature of fibrosis and parenchymal remodeling in chronic liver disease. The fact that these are potentially reversible processes, while a large driving force for development of therapeutic interventions, is often not accepted or recognized at the microscopic level. As highlighted by Wanless et al.16 in a study of regression of fibrosis in chronic hepatitis B, perhaps some of the parenchymal variability noted represents manifestations of this dynamic. Until we have finer tools for such evaluations, we can document only the lesions that we observe, but we must do so with care.

Animal Models: Is It Time for Consideration of Uniform Terminology?

  1. Top of page
  2. Nonalcoholic Fatty Liver Disease (NAFLD)
  3. Animal Models: Is It Time for Consideration of Uniform Terminology?
  4. References

Interestingly, little or no comment has been made regarding parenchymal variability in numerous animal (rodent) models utilized to study NAFLD. By their nature, these studies are quite different from the human situation: at the termination of experiments, animals are sacrificed, and thus sections from whole livers are available for evaluation. This situation has the potential to yield even more information regarding parenchymal variability, yet most reports give only brief written descriptions (or figure legends) of the noted lesions. For a pathologist, it is particularly challenging to garner information from the studies that supply only high-power images of a given field. Furthermore, when indications of hepatic steatosis can be understood only by use of special stains such as Oil Red O, there seems to be a problem with the fundamental “definition” of the process. This is a sensitive stain for detection of lipids. However, this stain is not employed in human NAFLD for a variety of reasons, including the lack of necessity, the technical inconvenience (as the stain must be done on frozen tissue sections, whether or not they have been formalin-fixed), and the risk of ruining the tissue for further appropriate microscopic analysis. The clinical setting for use of Oil Red O is to exclude a process of true microvesicular steatosis, a setting that does not mimic the chronic conditions of obesity and metabolic syndrome. Furthermore, the lesions by light microscopy are typically enlarged hepatocytes with either a vacuolated or foamy cytoplasmic appearance, often in the setting of necroinflammation (that is, lobular hepatitis). Thus, one does wonder why it is that Oil Red O has become almost a “norm” in rodent studies of NAFLD when light microscopy assessment and biochemical assays could serve equally well and could be potentially more informative.

Currently, one could say there is somewhat of a state of confusion as to which rodent model is truly that of human NAFLD and what criteria are to be considered for this diagnosis in animals. Is “ballooning” a predictable and reproducible lesion in animals? Do Mallory-Denk bodies occur as readily in rodents (without the use of other noxious agents) as they do in humans? Are they detectable without antibody confirmation? Does fibrosis occur from the steatohepatitic process as readily as in humans without additive toxic manipulations (which, of course, dampens one's enthusiasm for the model)? Is the pattern of fibrosis that of human disease? If so, is the pattern akin to the early stages of adult zone 3 pattern, the later stages of portal fibrosis, or the pediatric pattern? Is there a ductular reaction? Have glycogenated nuclei ever been observed in rodent models of NAFLD? A proposed alternative approach is to accept the fact that the variability of histologic findings among the various models is not necessarily an indication of (in)validity of any given model for the human disease but rather should be viewed as the histologic manifestation of the manipulations of that particular model. However, because of the importance of the disease, the need of an animal model to understand the pathophysiology, and the very real need for funding, many claims to “the model” have been made that with careful evaluation truly do not fit. Nonetheless, careful histopathologic evaluation and documentation of all manipulations in any model are needed if progress in the field is to occur and be carried into human studies.

An example with which I am familiar is the newly described ALIOS (American life-style induced obesity syndrome) model,17 which is based on the consumption of equivalent amounts of trans-fats and high-fructose corn syrup drinks of the American fast-food diet. One of the striking findings of this model is the parenchymal variability of the amount of steatosis between the subcapsular areas and the deeper, perihilar tissues as well as the zonality of types of steatosis.

In conclusion, I will acknowledge that among many hepatologists and pathologists, I realize I am “preaching to the choir.” As the future generations of clinical and academic hepatologists and pathologists are trained and focus on both patient care and translational research, let us all collectively be sure that they too are as grounded in the fundamental concepts of liver tissue analysis as their predecessors and mentors. Let us also realize that histologic observations are not static but will continue to evolve in concert with growth in newly discovered and honed techniques for tissue and molecular analysis. True scientific progress in our field continues to rely on concise, reproducible, scientifically meaningful, and integrated histologic documentation.

References

  1. Top of page
  2. Nonalcoholic Fatty Liver Disease (NAFLD)
  3. Animal Models: Is It Time for Consideration of Uniform Terminology?
  4. References