Versus-Host Disease in Man - Europe PMC

The Skin Biopsy in the Diagnosis of Acute GraftVersus-Host Disease in Man George E. Sale, MD, Kenneth G. Lerner, MD, Edward A. Barker, MD, Howard M. Shulman, MD, and E. Donnall Thomas, MD

Criteria for diagnosis of acute graft-versus-host disease (GVHD) using skin biopsies were derived from a) general experience with more than 300 human marrow grafts and b) the results of "blind" studies of skin biopsy specimens of patients grafted with either allogeneic or syngeneic marrow. Large doses of cytotoxic drugs and irradiation given before grafting can produce transient skin changes interfering significantly with the diagnosis of GVHD. Artifacts can also cause difficulty. Epidermal cytologic atypia, dyskeratosis, and satellitosis were present both in allografted patients with acute clinical GVHD of multiple systems and in patients given autologous or syngeneic marrow. Due to the marked overlap in histopathologic findings between these two types of skin injury, frequent serial skin biopsies must be combined with all other available clinical and biopsy data to provide reliable diagnosis of acute GVHD in man. (Am J Pathol 89:621-636, 1977)

THE TRANSPLANTATION OF BONE MIARROW following supralethal doses of radiation or chemotherapy is a successful means of treating some patients wvith aplastic anemia or acute leukemia.' Among the life-threatening complications of this procedure in genetically nonidentical donor-recipient pairs is graft-versus-host disease (GVHD), in which it is presumed that immunologically competent donor cells mount an immunologic reaction against the host. Target organs for this reaction are primarily I-mphoid cells, skin, liver, and gut.2-7 Nluch of the literature pertaining to GV'HD of the skin is descriptive. " Pathogenetic mechanisms have been deduced from observations of necrosis of individual epidermal cells, usually at the basal layer of epidermis. The migration of mononuclear cells into epidermis, with occasional satellitosis of mononuclear cells around individual dying epidermal cells, has been cited as the basic lesion of graft-versus-host reaction (GVHR) in skin.'"' Woodruff coined the term "aggressor lymphocyte" to describe this satellitosis phenomenon, which he demonstrated in light microscopic studies of primate skin.10 Electron microscopic studies confirmed a satelFrom the Departments of Pathology and Medicine. Disvision of Oncology. University of W ashington School of Medicine. The Fred Hutchinson Cancer Research Center. and The Harbor\-ies- Medical Center. Seattle. WN-ashington Supported by Grant CA-18029 from the National Cancer Institute; Dr. Lerner and Dr Sale are supported in part b%- Fellowships from the American Cancer Society. Dr Thomas is a recipient of Research Career Aw-ard AI-0i2425 from the National Institute of Allergy and Infectious Diseases

Accepted for publication July 27. 1977.

Address reprint requests to Dr George E Sale. Division of Oncology. Fred Hutchinson Cancer Research Center. 1124 Columbia Street. Seattle. WVA 98104 621

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litosis phenomenon around dying epithelial cells of tongue and lips in GVHD." The accurate diagnosis of GVHD is important because the GVHR and the immunosuppressive agents used in its treatment render the host immunologically incompetent and vulnerable to opportunistic infections. The clinical grading system is based on an overall evaluation of clinical, laboratory, and histologic parameters.1', Clinical data contributing to the diagnosis of GVHD include skin rash involving trunk, palms, soles, and ears; abdominal cramping and diarrhea; and abnormal liver function tests, particularly alkaline phosphatase and bilirubin elevations. Skin, liver, and gut (usually rectum and occasionally small bowel) biopsies are useful procedures. In their histologic grading system, Lerner et al.12 attempt to identify changes in the GVHD process as follows: Grade I, vacuolar degeneration of epidermal basal cells and acanthocytes; Grade II, vacuolar change with spongiosis and dyskeratosis or "eosinophilic bodies"; Grade III, epidermolysis and bulla formation plus Grade II changes; and Grade IV, total denudation of epithelium. The safest and hence most frequently employed biopsy procedure is the skin biopsy. This paper addresses the histologic criteria used in skin biopsies for the evaluation of GVHD. We have found that cytotoxic drugs and radiation as well as artifactual changes produce histologic changes that are difficult to distinguish from those of GVHD. Changes of Grade II or above are necessary but not sufficient in establishing the diagnosis of GVHD.

Materals and Methods Definitions

We define Day 0 as the last day of administration of cytotoxic agents. This is the same day as grafting in all the marrow transplant patients. When the term "cytotoxic" refers to a group of patients, it denotes those incapable of acute GVHD, i.e., those who received syngeneic marrow grafts. Preliminary Studies Skin biopsy specimens were obtained from three groups of allografted patients: a) six biopsy specimens from 3 patients treated with 1000 rad total body irradiation (TBI), b) ten biopsy specimens from 7 patients treated with cyclophosphamide (CY) (50 mg/kg X 4), and c) 43 biopsy specimens from 27 patients treated with one or more cytotoxic drugs followed by 1000 rad TBI.13 Artifactual Changes

Artifactual changes in skin biopsy specimens due to problems in handling were also examined. Multiple 3-mm punch biopsy specimens were obtained from normal skin at autopsy and submitted in triplicate for a) rapid fixation in neutral buffered formalin at room temperature; b) rapid fixation in cold (4 C) neutral buffered formalin; c) incubation

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Table 1-Cytotoxic Group in Initial Blind Study No. of biopsies

No. of patients

Preconditioning regimen

5 1 1 2

5 1 (a) 1 1(a) 1 (a) 1 (a) 1 1 1

CY,TBI CY, TBI, HYD CY, DAUN, TBI CY, HYD, DAUN, TBI BCNU, CY, TBI ADR, BCNU, CY, HN2, TBI Ara-C, CY, DAUN, TBI Ara-C, CY, DAUN, HYD, TBI ADR, Ara-C, CY, TBI

1 2 2 1 1

16

13

Included are 9 recipients of identical twin marrow and 4 recipients of autologous marrow. CY = cyclophosphamide; TBI = total body irradiation; (a) = autologous marrow recipient; HYD = hydroxyurea; DAUN = daunomycin; BCNU = 1,3-bis(2-chloroethyl)-1-nitrosourea; ADR = adriamycin; HN2 = nitrogen mustard; Ara-C = cytosine arabinoside.

in distilled water for 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours; d) crushing; e) incubation in isotonic saline for 4 hours at 60 C; and f) production of a large xylocaine intradermal wheal with a tuberculin syringe and needle to duplicate the effects of local anesthesia. All specimens were studied by light microscopy. Init Bld Sub Fortv-nine skin biopsy specimens taken from marrow transplant patients were coded and studied "blindly" by three pathologists. These patients were selected only by the availability of biopsy material in our files at the time the study began. The cytotoxic group consisted of 10 specimens from identical tuin marrow recipients and six specimens from autologous marrow recipients (Table 1). This group is theoretically incapable of developing GVHD. The biopsy specimens were obtaitied between Days 0 and 26 after grafting. The clinical GVHD group consisted of 33 specimens from allografted patients who received marrow matched for HLA antigens and compatible by mixed leukocvte culture techniques (Table 2). All patients in this group had a clinical diagnosis of GVHD. Biopsies were performed on this group between Days 1 and 96 after grafting. Repeat biopsy specimens from a few patients were available. Each of three pathologists made a diagnosis and graded 13 histologic criteria (Table 3) on a scale of 0 to 8, using a previously agreed upon grading svstem. Atl sts A follow-up analvsis of eosinophilic bodies was performed blindly by one pathologist to

determine the frequency of satellitosis around both nucleated and anucleate eosinophilic bodies in both groups in the initial study. Woodruff stated that the anucleate type mav be specific for GVHD, based on work in primates.'1 When an additional group of biopsy specimens from patients incapable of GVHD (recipients of conventional cancer chemotherapy as well as svngeneic marrow) was collected, another histologic study was done to determine which of the specimens showed histologic changes compatible with Grade II GVHD. (We had bv that time decided that Grade I changes were of little use.) The previous studies suggested that most of the confusing histologic changes should be gone by 2 weeks. Therefore, the specimens were

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Table 2-Patients With Clinicopathologic Syndrome of GVHD Diagnosis

Aplastic anemia

Total Acute leukemia

Total

No.of No.of biopsies patients

Preconditloning regimen*

3 5 1 4 1 14

2 4 1 1 1 9

TBI CY CY, TBI PRO,ATG,TBI PRO, ATG, CY

11 4 1 1 1 1 19

7 1 1 1 1 1

CY, TBI Ara-C, CY, DAUN, TBI CY, DAUN, TBI CY, DAUN, PRO, TBI CY, HYD, TBI BCNU, CY, TBI

12

* TBI = total body irradiation; CY = cyclophosphamide; PRO = procarbazine; ATG = antithymocyte globulin; Ara-C = cytosine arabinoside; DAUN = daunomycin; HYD = hydroxyurea; BCNU = 1,3-bis(2-chloroethyl)-1-nitrosourea.

divided into those taken before and those taken after 14 days after the last dose of cytotoxic agent

or

TBI.

When multiple specimens were available from syngeneically grafted patients, these specimens were analyzed sequentially (Table 4) to study the disappearance rate of these changes. In another study, biopsy specimens from two allografted groups were compared: specimens from patients with a definite clinical GVHD syndrome were compared with specimens from patients who had no convincing evidence of GVHD but who underwent biopsies for transient skin rashes (Table 5). The study was intended to determine, within rough limits, at what time after grafting the differential diagnosis might become relatively easy, assuming that the non-GVHD group would reflect the approximate time after grafting when the effect of cytotoxic agents could be expected to disappear. Thirty-three biopsy specimens from patients judged to have GVHD of at least two systems (drawn from Table 3-Histologic Criteria Used in Blind Study of Marrow Transplant Patients A) Epidermis 1) Basal cell vacuolization 2) Spongiosis 3) Mononuclear cell infiltration 4) Pyknosis 5) Eosinophilic bodies (individual necrotic cells) 6) Cell dropout 7) Fungal infection 8) Increased nuclear:cytoplasmic ratio 9) Nuclear membrane irregularity 10) Presence of binucleate or multinucleate epidermal cells B) Dermis 1) Edema 2) Mononuclear cell infiltration 3) Pigment deposition


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Table 4-Serial Skin Histology in Patients Receiving Syngeneic Marrow Unique patient number

Pregraft regimen

ADR, HN2, BCNU, CY, TBI DAUN, CY, Ara-C, TBI HYD, CY, DAUN, TBI BCNU, CY, TBI CY, TBI

44 293 364 389 559 560 563 594 662

DMM X 2

CY, TBI CY,TBI CY, TBI

Day postgraft on which skin specimen was obtained 8, 15,27* 0, 3 10,26,30,58* 17, 144* 10, 21 6,14,72* 18,28 10,28 14,21

ADR = adriamycin; HN2 = nitrogen mustard; BCNU = 1,3-bis(2-chloroethyl)-1-nitrosourea; CY = cyclophosphamide; TBI = total body irradiation; DAUN = daunomycin; Ara-C = cytosine arabinoside; HYD = hydroxyurea; DMM = dimethyl myleran. * Necropsy specimen.

the initial blind stud!-) w-ere compared with 20 biopsy specimens from patients who never manifested clinical esvidence of GV'HD. The specimens analvzed were from tw o postgraft periods. 15 to 28 days postgraft and 29 days or longer postgraft.

Results Prelminary

Observations

The skin biopsy specimens from patients receiving 1000 rad TBI alone sho-ed verv mild and transient epithelial atypia lasting 4 to 7 davs. CY alone produced mild atypia up to 2 weeks after grafting. Mlultiple cytotoxic drug regimens given to allografted leukemia patients were associated with marked epithelial atvpia and some individual cell necrosis lasting from 13 to 40 days after grafting. These histologic features rendered the differential diagnosis very difficult. Table 5-Biopsies From Allografted Patients Who Never Developed GVHD Syndrome

Preconditioning regimens

CY

CY, TBI BCNU, CY, TBI PRO, ATG, CY, TBI PRO, ATG, CY Total

Number

9 7 1 1 1 19

Included are 30 biopsies from 11 patients with aplastic anemia and 8 with acute leukemia. CY = cyclophosphamide; TBI = total body irradiation; BCNU = 1,3-bis(2-chloroethyl)-1nitrosourea; PRO = procarbazine; ATG = antithymocyte globulin.

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Artifact Studies

Only two of the artifact manipulations mimicked changes of GVHD. Osmotic damage induced by incubating a skin biopsy specimen in distilled water produced basal cell cytoplasmic vacuolization and rupture but required 30 minutes of incubation to become evident and 2 hours to become marked. Such osmotic damage mimicked Grade I GVHD, although, as expected, inflammatory cells were totally absent. Production of a large xylocaine intradermal wheal (8 to 10 mm in diameter) produced artifactual edema of the dermal-epidermal junction and some intercellular edema in the lower epidermis. Rare basal cells appeared swollen. Initial Blind Study Observer Consistence

In the initial blind study all three observers agreed exactly on the diagnosis of GVHD vs cytotoxic effects in only 31% of cases; two of three agreed in another 51%. Three different diagnoses were given in 14% of cases. Observer 2 had a greater tendency to diagnose GVHD than the other two observers but also made the highest number of false-positive diagnoses in the cytotoxic group. Observer 1 made no false-positive diagnoses but made the lowest number of positive diagnoses in the GVHD group. Thus, although the three observers had different thresholds for the diagnosis of GVHD and cytotoxicity effects, these differences conferred no significant increase in diagnostic acumen to any of the observers. The study provided an internal control for difference in threshold, which demonstrated rather emphatically the overlap of findings for the two injury types. Diagnoses

The diagnoses given by the three pathologists are listed in Tables 6 and 7. A diagnosis of "GVHD Grade I" is considered diagnostic in Table 6. "Grade I GVHD" diagnoses are ignored in Table 7; only diagnoses of Grade II or higher are included. As can be seen, we were unable to separate the two groups effectively regardless of the "grade threshold." The diagnostic categories of "GVHD," "cytotoxic effects," and "GVHD and cytotoxic effects" have approximately equal distributions in each group. Histologic Criteria

We expected nuclear/cytoplasmic ratio increase, binucleation or multinucleation, and irregularity of nuclear membranes to correlate with cyto-

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Table 6-Tabulation of Diagnoses in Initial Blind Study: Low Threshold (Considering Grade I GVHD Changes as Diagnostic)

Cytotoxic group

GVHD group

No. of

No. of

Diagnosis GVHD Cytotoxicity effects Both or either Neither Total

diagnoses

%

diagnoses

%

41

42 16

9 13 14 13 49

18 27 28

16 19 22 98

19 23

27

toxic drug administration, as has been suggested by Koss." We consisused these three criteria for diagnosing cvtotoxic agent effects in the

tentlv

blind study. However, e could find little difference between the cvtotoxic group and the GVHD group with respect to these same three criteria.

The five criteria wve thought would distinguish the GVHD group were basal cell vacuolization, epidermal inflammatory infiltrates, eosinophilic bodies, pyknosis, and cell dropout. However, the cvtotoxic group did not differ from the GVHD group with respect to these criteria either. We expressed these tw-o groups of criteria separately as an "'atvpia score" (or chemotherapy score) and a "GVHD score." Atvpia score was the average grade of the three atypia criteria. GVHD score was the average grade of the five GVHD criteria. When these scores for each biopsy specimen were plotted against each other on a scatter diagram, thex tended to scatter almost at random with a correlation coefficient of 0.49. The marked similaritv in skin histology of allografted and autografted patients at comparable postgrafting intervals (26 and 30 days) is illustrated in Figures 1 and 2. Table 7-Tabulation of Diagnoses in Initial Blind Study: High Threshold (Diagnosis Not Made of GVHD With Less Than Grade II Level Changes)

Cytotoxic group

GVHD group

Diagnosis GVHD

Cytotoxicity effects Both or either Neither Total

No.of diagnoses 29 24 11 34 98

% 30 24 11

35

No.of diagnoses

%

7 18 9 15 49

14 37 18 31

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Table 8-Number of Biopsy Specimens Having Histologic Changes Consistent With Grade II GVHD

Day after last dose of cytotoxic agent

0-14 Recipients of marrow from identical twins Recipients of conventional chemotherapy for various malignancies Recipients of own stored marrow (autografts)

15 or later

4/13

2/9 (positive biopsies Days 18 and 19)

1/13

1/4 (positive biopsy Day 15)

1/2

6/6 (range, Days 15-31)

Individual criteria thought to be useful in the diagnosis of GVHD were separately analyzed. Eosinophilic bodies were most frequent in the autografted group, which is a group receiving a high drug dosage. Eosinophilic bodies were also present in 3 of 5 identical twins treated only with CY (120 mg/kg) and 1000 rad TBI. The allografts were not distinguished from the syngeneic grafts. Epidermal inflammatory infiltrates of mononuclear cells were most frequent and extensive in the autografts, least in twins, and intermediate in the GVHD group. They were by no means a constant or marked feature in the GVHD group and, therefore, were of no distinguishing value. Minimal to moderate dermal perivascular mononuclear cell infiltrates were present in virtually all of the biopsy specimens and, therefore, conferred no discriminatory power. Additional Studies

Satellitosis Study. Satellitosis of mononuclear cells around degenerating epidermal cells was found in 14 of 33 (42%) of the GVHD patients' specimens and 8 of 16 (50%) of the cytotoxic patients' specimens. If this designation is limited to satellitosis of mononuclear cells around an anucleate red or eosinophilic mass, then only 4 of 33 (12%) of the GVHD group and 4 of 16 (25 % ) of the cytotoxic group demonstrated this feature. Additional Epidermal Toxicity Studies. Results of the expanded study of drug and irradiation skin toxicity are summarized in Table 8. Seventeen biopsy specimens were from patients receiving chemotherapy for disorders such as leukemia and testicular carcinoma. Only two of these specimens showed epidermal changes mimicking Grade II GVHD; changes in one of these were noted on Day 15. Specimens from 6 recipients of marrow from an identical twin showed Grade II changes: 2 of these were as late as Days 18 and 19. Patients receiving their own stored marrow


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showed the most extensive and long-lasting changes. Grade II changes in seven of eight specimens from this group were noted as late as Dav 31. The chemotherapy regimens in this group were generallv more extensive and were usuallv preceded by long-term maintenance chemotherapy. The largest subgroup of biopsy specimens available from patients homogenous with regard to preconditioning regimen includes specimens from identical twins treated with CY (60 mg/kg X 2) plus 1000 rad TBI. In this group, four specimens exhibited changes consistent with Grade II GVHD (Davs 7, 11, 11, and 19) and 10 specimens lacked such changes (Days 8, 10, 10, 14, 14, 18, 21, 21, 27, and 28). Thus, the confusing changes ranged from Day 7 to Day 19, but only approximately one-half of the patients on whom biopsies were performed during this period had such changes. The 10 negative specimens were obtained between Day 8 and Day 28. Serial Biopsies After Syngeneic and Autologous Grafting. All identical twin and autograft cases with serial skin specimens were reviewed (Table 4). Twenty-two specimens from 6 identical twin marrow recipients and 3 autologous marrow recipients were available. All of the data were consistent with epidermal damage, peaking at 1 to 2 weeks and resolving by 3 to 4 weeks. All 3 autografted patients showed Grade II to III changes which began to recede after 3 weeks. All of these patients had at least occasional individual necrotic epidermal cells on one or more specimens. Five of them had changes severe enough to be confused with GVHD. GCVHD vs Non-GV'HD Allografted Patients. The group of patients which never showed clinical GVHD syndrome was very clearly negative for convincing skin biopsy changes beyond the 14th day. Only one biopsy specimen (of nine) from this group, taken before Day 29, showed Grade I changes, and none of the 24 biopsies available after Day 28 in this group showed epidermal changes of Grade I. In contrast, before Day 29, three of eight biopsv specimens from patients with GVHD syndrome had Grade II changes, three had Grade I changes, and two had neither. After Dav 29, 8 of 12 specimens from the GVHD group had Grade II changes. It appears that histologic Grade II changes in most allografted patients bevond Week 3 to Week 4 are verv likelv to be associated with the clinical syndrome of GVDH. Additional Cincal Observations

Heterogeneity in cvtotoxic regimens used in leukemia patients has produced many small groups of 1 or 2 patients whose skin manifestations have been informative. Patients receiving 1,3-bis(2-chloroethyl)-1-nitrosourea or hvdroxvurea in addition to CY and TBI have had skin changes lasting as long as 43 days, which we have had difficultv interpreting.

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These are very similar to changes reported with bleomycin and long-term hydroxyurea.'5- ' Patients with chronic myelogenous leukemia who have been on long-term busulfan therapy seem to have a higher incidence of skin complications whether they receive allografts, autografts, or twin grafts, suggesting that long-term chemotherapy predisposes skin to such damage. Data obtained from examination of biopsy specimens from these patients must be interpreted cautiously with regard to GVHD. Discussion

Direct dose-related cytopathic effects of cytotoxic agents on epithelial surfaces include those on skin.'4 The hyperplastic epidermis of patients with psoriasis is sensitive to low-dose methotrexate or hydroxyurea, as manifested by individual cell necrosis indistinguishable from that seen in patients with GVHD.'6"8," Normal skin undergoes similar damage during therapy with bleomycin,' 520'2' adriamycin,22 radiation coupled with acti1 nomycin D or adriamycin, 23,24 and long-term hydroxyurea."7 Our biopsies on autografted patients produced evidence of marked acute cytotoxicity effects mimicking those attributed to GVHD in allografted patients. We expected to be able to distinguish the two by using criteria such as cytologic atypia (to identify cytotoxic effects) and eosinophilic bodies, satellitosis, and epidermal inflammatory infiltrates (to identify GVHD). Instead, the two proved histologically indistinguishable when subjected to double-blind analysis. The two complexes of histologic variables which we expected to be separable and specific (one for GVHD and the other for chemotherapy and irradiation damage) segregated together. This suggested a) that the two injurious stimuli produce nearly identical effects and b) that in the allografted patients with GVHD the effects of the two injuries are additive. The relative nonspecificity of dyskeratosis (indistinguishable from eosinophilic body formation) was not unexpected. Satellitosis was equally present in patients with GVHD damage and those with cytotoxic damage. Thus, one must factor time after the drug regimen and grafting into the diagnostic equation. Cytologic atypia of mild to moderate degree is not specific for cytotoxic therapy; it is probably a feature of regeneration after injury. The artifact study dictates special caution in interpretation of milder changes, which could be mimicked by local anesthesia or osmotic damage. The histologic threshold for diagnosis was therefore raised by our study. Since satellitosis can be observed in patients with syngeneic marrow grafts, it seems risky to assume that such a finding in the allografted patient necessarily represents immune cell-mediated cytotoxicity. It may represent the natural fate of injured epidermal cells regardless of the


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cause. These findings underscore the fact that the mechanism of epidermal cell injurv in cutaneous GVHD or GVHR is not understood.',' Further studies are needed to examine serum factors active against isolated epidermal cells in vitro 2I and immunoglobulins or other molecules deposited at the dermal-epidermal junction.28'" The practical implications of the present study for diagnostic strategy on the marrow transplantation ward are as follows: 1. Grade I changes should not be considered adequate except in lateonset GVHD (perhaps after Day 35 or 40), when effects of cytotoxic agents have usually disappeared, and if artifacts are minimized by proper specimen handling. 2. Eosinophilic bodies (dyskeratotic cells), with or without satellitosis, are a necessary, but not a sufficient, criterion for GVHD diagnosis, since cvtotoxic agents alone can produce them. However, in a patient receiving onlv CY (60 mg/kg X 2) plus 1000 rad TBI, eosinophilic bodies are unusual beyond 19 days after the graft. 3. Persistence in obtaining two or more serial biopsy specimens is necessarv in some cases for the diagnosis of GVHD. An equivocal biopsy should be repeated in 2 to 3 days. The probability that epidermal damage is due to cytotoxic agents should be waning as the probability that the damage is due to GVHD increases. 4. As a result of the nonspecificitv demonstrated by this study, a clinicopathologic correlation should be made bv the pathologist and the clinician, assessing rectal and liver biopsy data when available, as well as clinical data. The diagnosis of human GVHD is a complex clinicopathologic skill requiring considerable experience. There as yet appears to be no pathognomonic single clinical or histologic feature in the acute phase. Therefore, there are no shortcuts to careful serial gross or microscopic observations of skin or to consideration of all other available relevant data.

Referwecs 1. 2.

3.

4. 5.

Thomas ED, Storb R, Clift RA, Fefer A, Johnson FL, Neiman PE, Lerner KG, Glucksberg H, Buckner CD: Bone-marrow Transplantation. N Engl J Med 292:832-843, 895-902, 1975 Mathe G, Amiel JL, Schwarzenberg L: Bone Marrow Transplantation and Leucocyte Transfusions. (American Lecture Living Chemistry Series.) Springfield, Ill., Charles C. Thomas, Publisher, 1971 Vries MJ de, Vos 0: Delayed mortality of radiation chimeras: A pathological and hematological studv. J Natl Cancer Inst 23:1403-1439, 1959 Vries MJ de, Crouch BG, Putten LM van, Bekkum DW van: Pathologic changes in irradiated monkeys treated with bone marrow. J Natl Cancer Inst 27:67-97, 1961 Glucksberg H, Storb R, Fefer A, Buckner CD, Neiman PE, Clift RA, Lemer KG,

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Thomas ED: Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation 18:295-304, 1974 Krueger GRF, Berard CW, DeLellis RA, Graw RG Jr, Yankee RA, Leventhal BG, Rogentine GN, Herzig GP, Halterman RH, Henderson ES: Graft-versus-host disease. Morphologic variation and differential diagnosis in 8 cases of HL-A matched bone marrow transplantation. Am J Pathol 63:179-202, 1971 Krueger GRF, Graw RG Jr, Rogentine GN, Darrow CC II, Neefe JR, Luetzeler J: Pathology of modified graft-versus-host disease in bone marrow allografted monkeys treated with antilymphocyte serum. Blut 30:19-30, 1975 Slavin RE, Santos GW: The graft versus host reaction in man after bone marrow

transplantation: Pathology, pathogenesis, clinical features, and implication. Clin Immunol Immunopathol 1:472-498, 1973 9. Slavin RE, Woodruff JM: The pathology of bone marrow transplantation. Pathology Annual, Vol 9. Edited by SD Sommers. New York, Appleton-Century-Crofts, 1974, pp 291-344 10. 11. 12.

13.

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Woodruff JM, Eltringham JR, Casey HW: Early secondary disease in the Rhesus monkey. I. A comparative histopathologic study. Lab Invest 20:499-511, 1969 Woodruff JM, Butcher WI, Hellerstein Li: Early secondary disease in the rhesus monkey. II. Electron microscopy of changes in mucous membranes and external epithelia as demonstrated in the tongue and lip. Lab Invest 27:85-98, 1972 Lerner KG, Kao GF, Storb R, Buckner CD, Clift RA, Thomas ED: Histopathology of graft-vs-host reaction (GvHR) in human recipients of marrow from HL-Amatched sibling donors. Transplant Proc 6:367-371, 1974 Thomas ED, Buckner CD, Banaji M, Clift RA, Fefer A, Flournoy N, Goodell BW, Hickman RO, Lerner KG, Neiman PE, Sale GE, Sanders JE, Singer J, Stevens M, Storb R, Weiden PL: One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 49:511-533, 1977 Koss LG: A light and electron microscopic study of the effects of a single dose of cyclophosphamide on various organs in the rat. I. The urinary bladder. Lab Invest 16:44-65, 1967

Blum RH, Carter SK, Agre K: A clinical review of bleomycin.-A new antineoplastic agent. Cancer 31:903-914, 1973 16. Smith C, Gelfant S: Effects of methotrexate and hydroxyurea on psoriatic epidermis. Preferential cytotoxic effects on psoriatic epidermis. Arch Dermatol 15.

110:70-72, 1974 17. 18. 19.

20. 21. 22.

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Kennedy BJ, Smith LR, Goltz RW: Skin changes secondary to hydroxyurea therapy. Arch Dermatol 111:183-187, 1975 Weinstein GD, Velasco J: Selective action of methotrexate on psoriatic epidermal cells. J Invest Dermatol 59:121-127, 1972 Singer RM, Gelfant S: Continuous inhibition of DNA synthesis in mouse ear epidermis using hydroxyurea. Exp Cell Res 73:270-271, 1972 Baker JR, Fleischman RW, Thompson GR, Schaeppi U, Ilievski V, Cooney DA, Davis RD: Pathological effects of bleomycin on the skin of dogs and monkeys. Toxicol Appl Pharmacol 25:190-200, 1973 Cohen IS, Mosher MB, O'Keefe EJ, Klaus SN, DeConti RC: Cutaneous toxicity of bleomycin therapy. Arch Dermatol 107:553-555, 1973 Rudolph R, Stein RS, Pattillo RA: Skin ulcers due to adriamycin. Cancer 38:1087-1094, 1976 Greco FA, Brereton HD, Kent H, Zimbler H, Merrill J, Johnson RE: Adriamycin and enhanced radiation reaction in normal esophagus and skin. Ann Intern Med 85:294-298, 1976


24. 25. 26. 2 7. 28. 29.

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D'Angio GJ. Farber S, Maddock CL: Potentiation of x-ray effects by actinomycin D. Radiology 73:175-1 77, 1959 Grebe SC, Streilein JW: Graft-versus-host reactions: A review. Adv Immunol 22:119-221. 1976 Spielvogel RL, Ullman S, Goltz R': Skin changes in graft-vs-host disease. South Med J 69:1277-1281, 1976 Steinmuller D, WTunderlich JR: The use of freshly explanted mouse epidermal cells for the in vitro induction and detection of cell-mediated cvtotoxicity. Cell Immunol 24:146-163, 1976 Ullman S, Spielvogel RL, Kersey JH. Goltz RWV: Immunoglobulins and complement in skin in graft-versus-host disease. Ann Intern Med 85:205. 197-6 Merritt CB, Mann DL. Rogentine GN Jr: Cytotoxic antibody for epithelial cells in human graft versus host disease. Nature 232:63839. 1971

Akuowednents The authors are indebted to Nancy Flournov and Gars Schoch for help with data analysis. Janis Korbol for expert secretarial assistance, and the physicians. nurses. technicians and staff of the Fred Hutchinson Cancer Research Center for general assistance. especially Charles Mlahan and Robert Raff.

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[ Illustrations follow]

a

-

~~~~~~~~~~~~~~~~~~~~~~

Figure 1-Skin specimen from allografted patient with clinical GVHD syndrome 30 days after allograft for acute T-cell lymphoblastic leukemia preconditioned with 1 ,3-bis(2-chloroethyl)-1nitrosourea (400 mg/M2), CY (60 mg/kg x 2), and 1000 rad TBI. Note relatively sparse

infiltration of lymphocytes in upper dermis and lower epidermis, extensive basal cell vacuolization, and degeneration with some individual cell necrosis. Small nuclei adjacent to dying epidermal cells indicate satellitosis (arrow). There is also some epidermal hypertrophy, widening of the granular layer, and hyperkeratosis, all nonspecific epidermal responses. (Hematoxylin and eosin, x 250) Fgw 2-Skin specimen from patient with chronic myelogenous leukemia in blast crisis, who received his own stored bone marrow (autograft); biopsy was performed 26 days after grafting. Preconditioning regimen was hydroxyurea (195 mg/kg), Cy (120 mg/kg), adriamycin (1.8 mg/kg), cytosine arabinoside (17.5 mg/kg), and 1000 rad TBI. Note sparse infiltration of lymphocytes in upper dermis and lower epidermis, basal cell vacuolization, and necrosis forming eosinophilic bodies, with lymphocyte nuclei in basal layer occasionally adjacent to necrotic epidermal cells (arrow). Note strong similarities between the allografted patient's and autografted patient's skin histopathology. (Hematoxylin and eosin, x 200)

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[End of Article]