Histopathologic Diagnosis of Chronic Graft-versus-Host Disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: II. Pathology Working Group Report

Liver 

The diagnosis of liver GVHD is based on the global assessment of immune-mediated destructive damage to small bile ducts and ductules, together with cholestatic and inflammatory changes, after consideration of confounding causes of liver disease from infection or drug injury. Characteristic bile duct changes may be absent or may affect only a minority of portal spaces if the liver biopsy is performed soon after the onset of liver dysfunction related to GVHD [13]. The duration of active liver GVHD, the anti-inflammatory effects of immunosuppression, and the anticholestatic influence of ursodeoxycholic acid affect these alterations.

There is no clear dichotomy between acute and chronic GVHD in the liver. Long-term persistence of GVHD increases the amount of portal fibrosis [13]. Patients with acute hepatitis after donor lymphocyte infusions or tapering of immunosuppressive medications have more necroinflammatory activity and portal inflammation than typically seen in patients who are receiving immunosuppression [8, 14]. Liver biopsy samples vary greatly in the number of portal spaces that can be evaluated. The greater the number, the more accurate the overall assessment. The minimum diagnostic threshold relies on a global assessment of characteristic withered interlobular bile ducts with nuclear and cytoplasmic alterations, with or without lymphocytic ductitis (Figure 1). Although these changes may not be present in every portal space, they should be representative of the overall picture. Unlike some other epithelia, apoptotic bile duct changes are infrequent.

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Figure 1. Hepatic GVHD. A, Late onset of acute GVHD, day 123. The expanded portal space contains a mixed infiltrate of lymphocytes and scattered eosinophils. The interlobular bile duct shows destructive changes of GVHD with infiltration by lymphocytes, segmental loss of nuclei, cytoplasmic vacuolization, and nuclear dyspolarity. Ductular proliferation at the margins of the portal space also shows some features of GVHD (original magnification, ×250). B, Refractory chronic GVHD, day 556. Interlobular bile ducts have a characteristic withered appearance with dyspolarity, dropout of nuclei, nuclear enlargement, anisonucleosis, infiltrating lymphocytes, and eosinophilia of the cytoplasm. The fibrotic portal space contains scattered lymphoid cells, and periportal hepatocytes show changes of chronic cholestasis with cytoplasmic ballooning (original magnification, ×250). C, Refractory untreated chronic GVHD, day 350. Portal spaces have marked ductopenia with a loss of bile ducts, a lymphoplasmacytic infiltrate, and fibrosis with focal bridging (not shown; original magnification, ×250).

Lymphocytic infiltration, nuclear and cytoplasmic alterations, and proliferative ductular reactions along the margins of portal spaces, previously described as bile ductule proliferation (see Web link; liver worksheet), are similar to those in affected interlobular bile ducts. The ductular reaction seems to represent both a reparative effort and a secondary target of GVHD. Data are insufficient to determine whether quantitative immunohistologic assessment of bile duct loss by staining for cytokeratins 7 or 19 or replicative senescence by P21 staining provides additional information above and beyond the usual histologic evaluation [15].

Refractoriness or a delay in starting treatment is associated with greater loss of bile ducts and a longer time to recovery [8]. Refractory GVHD in the liver usually produces a picture of chronic cholestasis with ductopenia and, rarely, a picture of bile ductular proliferation (reaction) with or without bridging fibrosis [6, 8, 16]. In young pediatric patients with chronic liver disorders, the developing hepatobiliary tract is especially vulnerable to injury and prone to fibrosis [17].

Although rare cases of cirrhosis have been attributed to chronic GVHD [6, 16, 18], most were reported before the identification of HCV infection, which occurred in up to 32% of patients who received HCT before the advent of blood-product screening for HCV [19]. Longitudinal studies indicate that nearly all cases of cirrhosis that develop after HCT are caused by chronic HCV [20]. The long-term cumulative incidence of cirrhosis from HCV acquired after HCT is 24% at 20 years [21]. The usual manifestation of HCV is a mild self-limited increase of serum aminotransferases during tapering of immunosuppression [19]. If no other signs of GVHD are present, a liver biopsy may be necessary. Although HCV causes inflammation and reactive bile duct changes [6, 13, 22], the withered degenerative bile duct changes of GVHD are qualitatively different from those caused by HCV (Figure 1A and B).

Assessment of the response to therapy requires integration of the clinical and pathologic data, especially with liver biopsies, where improvement in liver tests and histology may take months. The extent to which improvement in clinical features correlates with repair and regeneration of bile ducts is not known. In an anecdotal case with complete ductopenia, liver tests returned to normal after 1 year [8].

Gastrointestinal Tract 

Upper esophageal webs are a diagnostic clinical feature of chronic GVHD [9]. In contrast, no histologic changes in the gastrointestinal tract are specific for chronic GVHD. Features that reflect the duration of the disease, fibrosis within the lamina propria, crypt loss, lymphocytic-plasmacytic inflammation, colonic Paneth cell metaplasia, or submucosal or serosal fibrosis are sequelae of refractory acute or late acute GVHD [2, 3, 7, 23] (Figure 2).

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Figure 2. Gastrointestinal GVHD. A, Persistent GVHD in the colon, day 87. The colonic biopsy specimen has numerous contiguous apoptotic changes (arrows; original magnification, ×250). B, Late acute GVHD in the stomach, day 133. The biopsy sample shows pronounced lymphocytic and prominent eosinophilic infiltration with destruction of gastric antral glands and formation of crypt abscesses (original magnification, ×250).

Consensus as to whether clinical, endoscopic, or histologic data define the gold standard for the diagnosis of gastrointestinal GVHD has not been reached. Large discrepancies can occur between gross endoscopic and histologic findings [24]. Endoscopy can visualize the full extent of the changes, whereas biopsy is subject to the vagaries of sampling and histologic preparation. When a gut biopsy sample fails to show features of GVHD, the probability of GVHD remains high in patients at risk who have typical signs and symptoms and gross endoscopic appearance (mucosal edema) or ultrasound evidence of mucosal edema, provided that all studies for infection are negative. After an extended discussion of this issue between clinicians and pathologists, it was recommended that patients with only clinical evidence of gut GVHD could be eligible for clinical trials, but that the analysis should be stratified according to presence or absence of histopathologic findings of GVHD.

The histologic threshold for a minimal diagnosis of GVHD in endoscopic biopsy samples was subject to some differences of opinion regarding any apoptosis versus apoptosis in each piece. The histologic diagnostic threshold ranged from rare isolated apoptotic enterocytes to extensive lymphocytic infiltration with glandular or crypt destruction and crypt abscesses filled with apoptotic debris (apoptotic crypt abscess; Figure 2A). Eosinophils contribute to the injury, with variable scant to heavy infiltration and destruction of glands with crypt abscess formation (Figure 2B) [25]. Focally enhanced gastritis, characterized by small collections of lymphocytes, histiocytes with or without neutrophils surrounding small groups of foveolae or gastric glands, is found in GVHD and inflammatory bowel disease [26]. There was some disagreement about whether focally enhanced gastritis should be regarded as “consistent with GVHD” in the setting of HCT.

In the absence of confounding features (drug reaction or infections), several variations of minimum criteria thresholds requiring apoptotic enterocytes (excluding those on the surface) were suggested for a diagnosis of “consistent with GVHD”: at least 1 apoptotic body per biopsy piece (University of Michigan), the total number of apoptotic bodies at least to equal the number of pieces (Fred Hutchinson Cancer Research Center), or scattered apoptotic bodies in more than 1 crypt (M.D. Anderson). None of these variations specifies the number of serial sections that should be cut and examined. Although this issue has not been formally studied, clearly false-negative conclusions may result from examination of too few serial sections. Because GVHD may have a patchy distribution with variable evidence of apoptosis, at least 8, and up to 20, serial sections should be analyzed to avoid missing infrequent apoptotic changes or cytomegalovirus (CMV) viral inclusions. The use of apoptotic stains (eg, caspase 3) is neither recommended nor proven to be more useful than good-quality H&E serial sections.

The minimal diagnostic criteria are also subject to false-positive interpretation, because apoptosis in the gut epithelium is not limited to GVHD. Cryptosporidia and heavy CMV infection of the gut mucosa are well-known causes of apoptosis [3, 27]. Immunohistologic examination for CMV may be needed in selected cases to distinguish CMV from GVHD. Extensive apoptosis in the absence of identifiable CMV antigens by immunohistology is most likely to represent GVHD. Focal colonic ulcerations with marked apoptosis and acute and chronic inflammation coupled with normal mucosal biopsy specimens from sites distant from the lesions should raise the possibility of colitis due to mycophenolate mofetil exposure [28]. Apoptosis has also been reported with the use of proton pump inhibitors used to treat gastric disorders [29]. Small numbers of apoptotic changes in esophageal mucosal biopsy specimens can arise from a variety of chronic inflammatory conditions. Unless there is a combination of lichenoid interface changes with apoptosis along the basilar portion of the mucosa, the diagnosis of esophageal GVHD should be made with some qualification (see “Standardized reporting of GVHD in the ‘Final Diagnosis’”).

With the exception of diffuse ulceration or ulcerated stenotic segments verified by endoscopic or radiographic evaluations [30], it is unclear whether there is any additional prognostic value in the Lerner et al. [31] histologic grading scheme for acute GVHD above and beyond clinical parameters. Some centers use the histologic grading scheme routinely, whereas others do not. Any proposed grading or staging scheme for gut GVHD should be a clinicopathologic composite score that reflects the overall extent of symptoms and damage seen by endoscopy and imaging studies, together with the degree of histologic mucosal destruction or fibrosis.

Skin 

The minimal histologic criteria for active GVHD require apoptosis within the basilar or lower spinosum layers of the epidermis (Figure 3A) [2, 5, 31]. The archetypical features of both acute and chronic GVHD are superficial interface dermatitis either (1) with a lichenoid pattern with lymphocytic inflammation with or without lymphocyte satellitosis or (2) with predominately vacuolar change in the basilar layer [4, 5]. As a note of caution, because no single histologic feature is pathognomonic of GVHD, the major overall inflammatory reaction pattern should be factored into the final interpretation [32]. Thus exuberant superficial spongiotic dermatitis with marked spongiosis (intraepidermal edema) and lymphocytic infiltration into the epidermis with only a rare apoptotic keratinocyte is much more likely to represent an allergic reaction than GVHD. The inconstant finding of lymphocyte satellitosis (lymphocytes surrounding an apoptotic keratinocyte in the epidermis or appendages) provides evidence that the dermatitis is caused by GVHD.

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Figure 3. Progression of histologic changes from acute to chronic cutaneous GVHD. A, Screening skin biopsy, day 85. A focal apoptotic body formation is present at the tips of rete ridges (arrow) with focal surrounding lymphocytic satellitosis (original magnification, ×400). B, Lichen planus–like chronic GVHD, day 426. The thickened epidermis displays orthokeratosis, hypergranulosis, and acanthosis. The striking lichenoid reaction along the damaged basal layer includes a prominent lymphocytic inflammation and infiltration, apoptotic changes, loss of rete ridges, and prominence of the superficial vascularity (original magnification, ×100). C, Progression of GVHD from panel A into a sclerotic stage, day 382. A zone of dense, relatively avascular homogenized collagen has replaced the papillary and upper reticular dermis (original magnification, ×63). D, High-power view shows a hyperkeratotic epidermis with flattening of the rete ridges, vacuolar changes, and lymphocytic infiltration along the basal layer, with disruption of the epidermal melanin unit and with coarse clumps of melanin in the epidermis and incontinent melanin pigment in the sclerotic papillary dermis (original magnification, ×160).

The histologic manifestations of chronic cutaneous GVHD evolve over time, are modified by treatment, and to some extent overlap with those of acute GVHD. The histologic counterparts to the proposed diagnostic clinical definitions of cutaneous chronic GVHD include several different histologic patterns (Figure 3B). The lichen-planus type eruptions (initially classified as early generalized extensive chronic GVHD [1, 4]) refer to a specific constellation with epidermal thickening by acanthosis (hyperplasia) with orthokeratosis (stratum corneum) and parakeratosis, hypergranulosis, a bandlike infiltrate along the dermal-epidermal junction, extensive apoptosis and vacuolization of basilar keratinocytes, saw-toothed (short blunted) rete ridges, and inflammation around the dermal adnexa. This constellation, especially when accompanied by lymphoplasmacytic inflammation around the eccrine coils, is highly specific for chronic GVHD, but at the expense of reduced sensitivity. Because the skin lesions of chronic GVHD are not synchronous, the presence or absence of chronic GVHD features in a biopsy sample can be influenced by sampling or partial thickness. In practice, members of the dermatopathology subcommittee regarded the combination of epidermal compact orthokeratosis, hypergranulosis, and acanthosis as features that favor or are consistent with chronic lichenoid GVHD (Figure 3). As a note of caution, a lesser degree of this combination of features can occur occasionally in skin biopsy specimens from patients with severe clinical acute GVHD.

In the initial descriptions of sclerotic or late chronic GVHD, the fibrosis that followed the lichenoid stage had a top-down progression from the papillary through the reticular dermis (Figure 3C and D) [1, 5]. Some patients develop diffuse dermal sclerosis without an apparent inflammatory lichenoid phase. The suggested minimal criterion for the diagnosis of cutaneous sclerotic chronic GVHD is homogenization (sclerosis) of most of the papillary dermis or reticular dermis. In the morpheic variant of chronic cutaneous GVHD, sclerosis may be largely confined to the reticular dermis and underlying fascia with little or no epidermal involvement. Another variant, fasciitis, may show fibrous thickening only in the fascia, with adjacent inflammation but without any epidermal or dermal involvement [33] (Figure 4).

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Figure 4. Morpheic GVHD lesion, day 607. A, Low power shows a thickened dermis with sclerotic widening of the lower reticular dermis and fascia (original magnification, ×20). B, The epidermis shows activity of GVHD with scattered apoptotic bodies. Note that the papillary dermis is not sclerotic, in contrast to the deep dermis and fascia in panel D (original magnification, ×400). C, Syringitis: eccrine coils are infiltrated by lymphocytes with a loss of adjacent fat tissue replaced by fibrous tissue (original magnification, ×200). D, Interface of the reticular dermis and the fascia shows fasciitis with fibrous thickening of the septa, lymphocytic panniculitis, and formation of lymphoid follicles (original magnification, ×63).

The cutaneous pathology group addressed the changes in chronic GVHD histologic characteristics as related to immunosuppressive treatment. After treatment, skin biopsy samples display a combination of residual damage, with loss of rete ridges and dermal appendages, some increase in papillary or dermal sclerosis, and reduced to absent lichenoid inflammation. The hallmark of an incomplete response indicating ongoing GVHD activity is residual apoptotic changes in the epidermis or appendages. After treatment, the histologic significance of minor residual perivascular lymphocytic inflammation or persistent epidermal vacuolar degeneration requires additional study, as does the assessment of activity in patients who have received psoralen and UVA irradiation or who have established deep dermal sclerosis or morpheic chronic GVHD. The diagnosis and staging committee recommended that keratinocyte apoptosis without other chronic GVHD features in day 80 to 100 screening skin biopsy samples does not indicate chronic GVHD and does not necessarily predict that a flare may follow cessation of immunosuppressive therapy.

The cutaneous subgroup (E.F.) proposed a working definition of marked apoptotic activity in the skin as >5 epidermal apoptotic bodies per section from a 4-mm punch biopsy. Previous attempts to quantify the inflammatory changes of lichen planus–like chronic GVHD did not correlate with the response to therapy, whereas continued histologic signs of activity after treatment increased the likelihood of some irreversible damage to tissues. Clinicopathologic correlation from prospectively obtained data will be used to evaluate these guidelines for assessing activity (see “Standardized reporting of GVHD in the ‘Final Diagnosis’”).

Oropharynx, Vulva, and Eye 

On the basis of studies of oral labial biopsy samples taken 80 to 100 days after HCT, patients without any signs or biopsy evidence of GVHD may have chronic inflammation without apoptotic changes in the mucosa and minor salivary glands. These changes were attributed to chemotherapy or irradiation in the conditioning regimen [34]. Accordingly, lip biopsies to screen for chronic GVHD were rarely performed before day 80. Even so, some patients with aplasia who received only cyclophosphamide before HCT developed typical gross changes of oral chronic GVHD before day 80. A similar situation may also apply to reduced-intensity conditioning.

The minimal histologic criteria for oral chronic GVHD have remained unchanged: localized or generalized epithelial changes (lichenoid interface inflammation, exocytosis, and apoptosis) similar to those described in cutaneous GVHD or the presence of intralobular, periductal lymphocytes with or without plasma cells and exocytosis of lymphocytes (without neutrophils) into intralobular ducts and acini (Figure 5). A later variation proposed the minimal criteria as >3 mucosal apoptotic bodies and, for salivary changes, a >10% loss of acinar tissue or ductal epithelial cell necrosis [35]. Periductal fibrosis (not generalized interstitial fibrosis) is often present. Horn et al. [36] developed a histologic grading system for chronic GVHD of minor salivary glands based on the degree of lymphocytic infiltration and destruction of glandular acini. This GVHD grading schema and others like it most accurately reflect the stage of the disease.

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Figure 5. Oral GVHD. A, Oral mucosal biopsy sample, day 75. This view shows lymphocytic infiltrate along the basal layer of the mucosa (original magnification, ×160). B, High-powered view shows apoptotic changes along the tip of a rete ridge (arrow; original magnification, ×400). C, Minor salivary gland, day 364. Early lobular involvement shows focal periductal lymphocytic infiltrates with minimal loss of acinar tissue (original magnification, ×63). D, High-powered view of an intralobular duct with marked lymphocytic infiltration, cytoplasmic vacuolization, and focal destruction of the ductular epithelium (original magnification, ×160).

Persistent salivary dysfunction after treatment of chronic GVHD is related to continuing lymphocytic inflammation and absent recovery of minor salivary secretory units [37]. In children, oncocytic ductal metaplasia may be an additional feature favoring GVHD (T. Morton, University of Washington School of Dentistry, Seattle, WA, personal communication, 2005). Similar findings are commonly seen in oral biopsy specimens of adults older than 40 years and are considered evidence of previous ductal damage. Moderate to intense periductal and periacinar fibroblastic stroma is evidence of previous inflammation or chronic GVHD activity, whereas dense fibrous tissue with destruction of acinal tissue and duct ectasia may be only a marker for previous damage. The assessment of GVHD activity should focus on lobules that are not completely fibrotic. In these nodules, fibroplasia, acinar and periductal inflammation, and damage to ducts indicate GVHD activity. Finally, clinicians and pathologist should be aware that premalignant dysplasias and oral cancers, a leading cause of secondary malignancies after allogeneic transplantation, often present with a lichenoid appearance [38, 39].

The same criteria described previously for oral and esophageal mucosa are used for histologic assessment of chronic GVHD in vulvar [40], conjunctival, and lacrimal biopsy specimens. Histopathologic findings of ocular GVHD have been described in the conjunctiva and in the lacrimal gland [34, 41, 42, 43, 44]. The alterations in lacrimal gland acinar tissue resemble those in minor salivary glands with prominent infiltration of mononuclear cells around medium-size ducts, with loss of acinar lobules replaced by fibrosis. Whereas lacrimal gland biopsy is relatively invasive and may result in decreased functional capacity, conjunctival biopsy samples may be obtained without much risk to the patient. Histologic evaluation of the conjunctiva may aid in the diagnosis and management of ocular GVHD in symptomatic patients with conjunctival disease [41, 45, 46]. Although the biopsy is not performed routinely, it may be particularly helpful in cases in which ocular GVHD is in question in symptomatic patients who have normal or unchanged Schirmer tests with or without GVHD of other organs. The conjunctival specimen may also be tested by using special stains for viral involvement when indicated. Routine survey biopsy, however, is thought to serve little benefit for early detection of ocular GVHD [41, 44]. Conjunctival histologic features for GVHD include lymphocyte exocytosis, satellitosis, vacuolization of the basal epithelium, and epithelial cell necrosis, similar to changes that are observed in other organs [41, 42, 43, 44, 45]. Other features are relatively nonspecific, including epithelial attenuation and goblet cell depletion, which are not sufficient for the diagnosis of ocular GVHD [43]. Corneal and conjunctival pseudomembranous histologic findings are clinical manifestations generally associated with an acute pattern of ocular GVHD [45, 46, 47].

Lungs 

The pathologic finding of obliterative bronchiolitis is considered to be a diagnostic feature of pulmonary chronic GVHD and resembles chronic lung allograft rejection [48]. Lung biopsy shows unequivocal dense eosinophilic scarring of the bronchioles, resulting in some degree of luminal narrowing (Figure 6). The process begins with inflammation around the small arteries and veins and beneath the respiratory epithelial lining in small airways. Eventually, the submucosal layer is replaced by fibrous tissue, and the lumen is obliterated (also called constrictive bronchiolitis). Secondary changes include distal mucostasis or aggregates of foamy macrophages. Inflammation is common but variable and insufficient for diagnosis. The extent and severity of changes should be correlated with functional studies, particularly if only a single affected airway is present in the biopsy specimen. Other causes, such as infection and chronic aspiration, should be excluded [48]. Interstitial lung disease is not considered to be a direct manifestation of GVHD. In the presence of other distinctive organ features and a constellation of suggestive pulmonary function tests and chest computed tomographic scans, a lung biopsy is not necessary to diagnose pulmonary chronic GVHD [9].

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Figure 6. Pulmonary GVHD with obliterative bronchiolitis: lung biopsy specimen, day 194. A small airway shows constriction of the bronchiole lumen by a subepithelial expansion of fibrous tissue. A lymphocytic infiltrate surrounds the outer bronchiole smooth muscle layer (original magnification, ×250; photo courtesy of Dr. Robert Hackman, of Fred Hutchinson Cancer Research Center).

Idiopathic bronchiolitis obliterans–organizing pneumonia (BOOP) is associated with both acute and chronic GVHD. BOOP is a clinicopathologic syndrome defined by plugs of granulation tissue that fill the lumens of the distal airways in a patchy distribution, extending into the alveolar ducts and alveolar sacs, and associated with chronic interstitial inflammation [49]. BOOP should be distinguished from obliterative bronchitis because BOOP has a different clinicopathologic presentation and a more favorable outcome.

Other Sites 

Several other sites of chronic GVHD are less commonly involved or subjected to biopsy. Myositis is clearly a phenomenon associated with chronic GVHD. A comprehensive description with comparison to other myositis entities has not been made. The skeletal muscle biopsy changes range from mild perimysial lymphocytic infiltrates to extensive endomysial inflammation with necrosis and regeneration of fibers [1].

Biopsies may be useful in the evaluation of other rare manifestations that may be related to chronic GVHD. These syndromes include nephrotic syndrome and glomerulonephritis, inflammatory neuropathies, and synovitis. Chronic GVHD can cause obliterative coronary artery changes that resemble transplant atherosclerosis.