versus-host disease in man

The Skin Biopsy in the Diagnosis of Acute Graft-Versus-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 biopsieswere derived from a) general experience with more than 300 human marrow grafts andb) the results of "blind" studies of skin biopsy specimens of patients grafted with eitherallogeneic or syngeneic marrow. Large doses of cytotoxic drugs and irradiation givenbefore grafting can produce transient skin changes interfering significantly with thediagnosis of GVHD. Artifacts can also cause difficulty. Epidermal cytologic atypia,dyskeratosis, and satellitosis were present both in allografted patients with acuteclinical GVHD of multiple systems and in patients given autologous or syngeneicmarrow. Due to the marked overlap in histopathologic findings between these twotypes of skin injury, frequent serial skin biopsies must be combined with all otheravailable 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 supralethaldoses of radiation or chemotherapy is a successful means of treating somepatients wvith aplastic anemia or acute leukemia.' Among the life-threat-ening complications of this procedure in genetically nonidenticaldonor-recipient pairs is graft-versus-host disease (GVHD), in which itis presumed that immunologically competent donor cells mount animmunologic reaction against the host. Target organs for this reactionare 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 necro-sis of individual epidermal cells, usually at the basal layer of epidermis.The migration of mononuclear cells into epidermis, with occasional satel-litosis of mononuclear cells around individual dying epidermal cells, hasbeen cited as the basic lesion of graft-versus-host reaction (GVHR) inskin.'"' Woodruff coined the term "aggressor lymphocyte" to describethis satellitosis phenomenon, which he demonstrated in light microscopicstudies of primate skin.10 Electron microscopic studies confirmed a satel-

From the Departments of Pathology and Medicine. Disvision of Oncology. University of W ashing-ton School of Medicine. The Fred Hutchinson Cancer Research Center. and The Harbor\-ies- MedicalCenter. Seattle. WN-ashington

Supported by Grant CA-18029 from the National Cancer Institute; Dr. Lerner and Dr Sale aresupported in part b%- Fellowships from the American Cancer Society. Dr Thomas is a recipient ofResearch 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 98104621

622 SALE ET AL. American Journalof Pathology

litosis phenomenon around dying epithelial cells of tongue and lips inGVHD."The accurate diagnosis of GVHD is important because the GVHR and

the immunosuppressive agents used in its treatment render the hostimmunologically 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 thediagnosis of GVHD include skin rash involving trunk, palms, soles, andears; abdominal cramping and diarrhea; and abnormal liver functiontests, particularly alkaline phosphatase and bilirubin elevations. Skin,liver, and gut (usually rectum and occasionally small bowel) biopsies areuseful procedures. In their histologic grading system, Lerner et al.12attempt to identify changes in the GVHD process as follows: GradeI, vacuolar degeneration of epidermal basal cells and acanthocytes; GradeII, vacuolar change with spongiosis and dyskeratosis or "eosinophilicbodies"; Grade III, epidermolysis and bulla formation plus Grade IIchanges; and Grade IV, total denudation of epithelium. The safest andhence most frequently employed biopsy procedure is the skin biopsy. Thispaper addresses the histologic criteria used in skin biopsies for the evalua-tion of GVHD. We have found that cytotoxic drugs and radiation as wellas artifactual changes produce histologic changes that are difficult todistinguish from those of GVHD. Changes of Grade II or above arenecessary but not sufficient in establishing the diagnosis of GVHD.

Materals and MethodsDefinitions

We define Day 0 as the last day of administration of cytotoxic agents. This is the sameday as grafting in all the marrow transplant patients. When the term "cytotoxic" refers to agroup of patients, it denotes those incapable of acute GVHD, i.e., those who receivedsyngeneic marrow grafts.

Preliminary StudiesSkin 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) tenbiopsy 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 drugsfollowed by 1000 rad TBI.13

Artifactual ChangesArtifactual changes in skin biopsy specimens due to problems in handling were also

examined. Multiple 3-mm punch biopsy specimens were obtained from normal skin atautopsy and submitted in triplicate for a) rapid fixation in neutral buffered formalin atroom temperature; b) rapid fixation in cold (4 C) neutral buffered formalin; c) incubation

Vol. 89, No. 3 ACUTE GVH DISEASE 623December 1977

Table 1-Cytotoxic Group in Initial Blind Study

No. of biopsies No. of patients Preconditioning regimen

5 5 CY,TBI1 1 (a) CY, TBI, HYD1 1 CY, DAUN, TBI2 1(a) CY, HYD, DAUN, TBI1 1 (a) BCNU, CY, TBI2 1 (a) ADR, BCNU, CY, HN2, TBI2 1 Ara-C, CY, DAUN, TBI1 1 Ara-C, CY, DAUN, HYD, TBI1 1 ADR, Ara-C, CY, TBI

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 xylocaineintradermal wheal with a tuberculin syringe and needle to duplicate the effects of localanesthesia. All specimens were studied by light microscopy.

Init Bld SubFortv-nine skin biopsy specimens taken from marrow transplant patients were coded

and studied "blindly" by three pathologists. These patients were selected only by theavailability of biopsy material in our files at the time the study began. The cytotoxic groupconsisted of 10 specimens from identical tuin marrow recipients and six specimens fromautologous marrow recipients (Table 1). This group is theoretically incapable of devel-oping 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 culturetechniques (Table 2). All patients in this group had a clinical diagnosis of GVHD. Biopsieswere performed on this group between Days 1 and 96 after grafting. Repeat biopsyspecimens 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 stsA follow-up analvsis of eosinophilic bodies was performed blindly by one pathologist to

determine the frequency of satellitosis around both nucleated and anucleate eosinophilicbodies in both groups in the initial study. Woodruff stated that the anucleate type mav bespecific for GVHD, based on work in primates.'1When an additional group of biopsy specimens from patients incapable of GVHD

(recipients of conventional cancer chemotherapy as well as svngeneic marrow) was col-lected, another histologic study was done to determine which of the specimens showedhistologic changes compatible with Grade II GVHD. (We had bv that time decided thatGrade I changes were of little use.) The previous studies suggested that most of theconfusing histologic changes should be gone by 2 weeks. Therefore, the specimens were


Table 2-Patients With Clinicopathologic Syndrome of GVHD

No.of No.ofDiagnosis biopsies patients Preconditloning regimen*

Aplastic anemia 3 2 TBI5 4 CY1 1 CY, TBI4 1 PRO,ATG,TBI1 1 PRO, ATG, CY

Total 14 9

Acute leukemia 11 7 CY, TBI4 1 Ara-C, CY, DAUN, TBI1 1 CY, DAUN, TBI1 1 CY, DAUN, PRO, TBI1 1 CY, HYD, TBI1 1 BCNU, CY, TBI

Total 19 12

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

divided into those taken before and those taken after 14 days after the last dose of cytotoxicagent or TBI.When multiple specimens were available from syngeneically grafted patients, these

specimens were analyzed sequentially (Table 4) to study the disappearance rate of thesechanges.

In another study, biopsy specimens from two allografted groups were compared: speci-mens from patients with a definite clinical GVHD syndrome were compared with speci-mens from patients who had no convincing evidence of GVHD but who underwentbiopsies for transient skin rashes (Table 5). The study was intended to determine, withinrough limits, at what time after grafting the differential diagnosis might become relativelyeasy, assuming that the non-GVHD group would reflect the approximate time aftergrafting when the effect of cytotoxic agents could be expected to disappear. Thirty-threebiopsy 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) Epidermis1) Basal cell vacuolization2) Spongiosis3) Mononuclear cell infiltration4) Pyknosis5) Eosinophilic bodies (individual necrotic cells)6) Cell dropout7) Fungal infection8) Increased nuclear:cytoplasmic ratio9) Nuclear membrane irregularity

10) Presence of binucleate or multinucleate epidermal cellsB) Dermis

1) Edema2) Mononuclear cell infiltration3) Pigment deposition

Vol. 89, No. 3December 1977

ACUTE GVH DISEASE

Table 4-Serial Skin Histology in Patients Receiving Syngeneic Marrow

Unique Day postgraft onpatient which skin specimennumber Pregraft regimen was obtained

44 ADR, HN2, BCNU, CY, TBI 8, 15,27*293 DAUN, CY, Ara-C, TBI 0, 3364 HYD, CY, DAUN, TBI 10,26,30,58*389 BCNU, CY, TBI 17, 144*559 CY, TBI 10, 21560 DMM X 2 6,14,72*563 CY, TBI 18,28594 CY,TBI 10,28662 CY, TBI 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 = cytosinearabinoside; 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 postgraftperiods. 15 to 28 days postgraft and 29 days or longer postgraft.

Results

Prelminary Observations

The skin biopsy specimens from patients receiving 1000 rad TBI alonesho-ed verv mild and transient epithelial atypia lasting 4 to 7 davs. CYalone produced mild atypia up to 2 weeks after grafting. Mlultiple cyto-toxic drug regimens given to allografted leukemia patients were associ-

ated with marked epithelial atvpia and some individual cell necrosislasting from 13 to 40 days after grafting. These histologic features ren-

dered the differential diagnosis very difficult.

Table 5-Biopsies From Allografted Patients Who Never Developed GVHD Syndrome

Preconditioning regimens Number

CY 9CY, TBI 7BCNU, CY, TBI 1PRO, ATG, CY, TBI 1PRO, ATG, CY 1

Total 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)-1-nitrosourea; PRO = procarbazine; ATG = antithymocyte globulin.

625


Artifact Studies

Only two of the artifact manipulations mimicked changes of GVHD.Osmotic damage induced by incubating a skin biopsy specimen in dis-tilled water produced basal cell cytoplasmic vacuolization and rupturebut required 30 minutes of incubation to become evident and 2 hours tobecome marked. Such osmotic damage mimicked Grade I GVHD, al-though, as expected, inflammatory cells were totally absent. Production ofa large xylocaine intradermal wheal (8 to 10 mm in diameter) producedartifactual edema of the dermal-epidermal junction and some intercellularedema 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 thediagnosis of GVHD vs cytotoxic effects in only 31% of cases; two of threeagreed in another 51%. Three different diagnoses were given in 14% ofcases. Observer 2 had a greater tendency to diagnose GVHD than theother two observers but also made the highest number of false-positivediagnoses in the cytotoxic group. Observer 1 made no false-positivediagnoses but made the lowest number of positive diagnoses in the GVHDgroup. Thus, although the three observers had different thresholds for thediagnosis of GVHD and cytotoxicity effects, these differences conferred nosignificant increase in diagnostic acumen to any of the observers. Thestudy provided an internal control for difference in threshold, whichdemonstrated rather emphatically the overlap of findings for the twoinjury types.Diagnoses

The diagnoses given by the three pathologists are listed in Tables 6 and7. A diagnosis of "GVHD Grade I" is considered diagnostic in Table 6."Grade I GVHD" diagnoses are ignored in Table 7; only diagnoses ofGrade II or higher are included. As can be seen, we were unable toseparate the two groups effectively regardless of the "grade threshold."The diagnostic categories of "GVHD," "cytotoxic effects," and "GVHDand cytotoxic effects" have approximately equal distributions in eachgroup.Histologic Criteria

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

ACUTE GVH DISEASEVol. 89, No. 3December 1977

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

GVHD group Cytotoxic group

No. of No. ofDiagnosis diagnoses % diagnoses %

GVHD 41 42 9 18Cytotoxicity effects 16 16 13 27Both or either 19 19 14 28Neither 22 23 13 27

Total 98 49

toxic drug administration, as has been suggested by Koss." We consis-tentlv used these three criteria for diagnosing cvtotoxic agent effects in theblind study. However, e could find little difference between the cvto-toxic group and the GVHD group with respect to these same threecriteria.The five criteria wve thought would distinguish the GVHD group were

basal cell vacuolization, epidermal inflammatory infiltrates, eosinophilicbodies, pyknosis, and cell dropout. However, the cvtotoxic group did notdiffer from the GVHD group with respect to these criteria either. Weexpressed these tw-o groups of criteria separately as an "'atvpia score" (orchemotherapy score) and a "GVHD score." Atvpia score was the average

grade of the three atypia criteria. GVHD score was the average grade ofthe five GVHD criteria. When these scores for each biopsy specimen were

plotted against each other on a scatter diagram, thex tended to scatteralmost at random with a correlation coefficient of 0.49. The markedsimilaritv in skin histology of allografted and autografted patients atcomparable postgrafting intervals (26 and 30 days) is illustrated in Figures1 and 2.

Table 7-Tabulation of Diagnoses in Initial Blind Study: High Threshold (Diagnosis Not Madeof GVHD With Less Than Grade II Level Changes)

GVHD group Cytotoxic group

No.of No.ofDiagnosis diagnoses % diagnoses %

GVHD 29 30 7 14Cytotoxicity effects 24 24 18 37Both or either 11 1 1 9 18Neither 34 35 15 31

Total 98 49

627


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

Day after last dose of cytotoxic agent

0-14 15 or later

Recipients of marrow from identical 4/13 2/9 (positive biopsies Days 18 and 19)twins

Recipients of conventional chemo- 1/13 1/4 (positive biopsy Day 15)therapy for various malignancies

Recipients of own stored marrow 1/2 6/6 (range, Days 15-31)(autografts)

Individual criteria thought to be useful in the diagnosis of GVHD wereseparately analyzed. Eosinophilic bodies were most frequent in the auto-grafted group, which is a group receiving a high drug dosage. Eosino-philic bodies were also present in 3 of 5 identical twins treated only withCY (120 mg/kg) and 1000 rad TBI. The allografts were not distinguishedfrom the syngeneic grafts. Epidermal inflammatory infiltrates of mono-nuclear cells were most frequent and extensive in the autografts, least intwins, and intermediate in the GVHD group. They were by no means aconstant or marked feature in the GVHD group and, therefore, were of nodistinguishing value. Minimal to moderate dermal perivascular mono-nuclear cell infiltrates were present in virtually all of the biopsy specimensand, therefore, conferred no discriminatory power.

Additional Studies

Satellitosis Study. Satellitosis of mononuclear cells around degeneratingepidermal cells was found in 14 of 33 (42%) of the GVHD patients'specimens and 8 of 16 (50%) of the cytotoxic patients' specimens. If thisdesignation is limited to satellitosis of mononuclear cells around an anu-cleate red or eosinophilic mass, then only 4 of 33 (12%) of the GVHDgroup and 4 of 16 (25% ) of the cytotoxic group demonstrated this feature.

Additional Epidermal Toxicity Studies. Results of the expanded studyof drug and irradiation skin toxicity are summarized in Table 8. Seventeenbiopsy specimens were from patients receiving chemotherapy for dis-orders such as leukemia and testicular carcinoma. Only two of thesespecimens showed epidermal changes mimicking Grade II GVHD;changes in one of these were noted on Day 15. Specimens from 6 recipi-ents of marrow from an identical twin showed Grade II changes: 2 of thesewere as late as Days 18 and 19. Patients receiving their own stored marrow


showed the most extensive and long-lasting changes. Grade II changes inseven of eight specimens from this group were noted as late as Dav 31.The chemotherapy regimens in this group were generallv more extensiveand were usuallv preceded by long-term maintenance chemotherapy. Thelargest subgroup of biopsy specimens available from patients homogenouswith regard to preconditioning regimen includes specimens from identicaltwins treated with CY (60 mg/kg X 2) plus 1000 rad TBI. In this group,four specimens exhibited changes consistent with Grade II GVHD (Davs7, 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 fromDay 7 to Day 19, but only approximately one-half of the patients on whombiopsies were performed during this period had such changes. The 10negative specimens were obtained between Day 8 and Day 28.

Serial Biopsies After Syngeneic and Autologous Grafting. All identicaltwin and autograft cases with serial skin specimens were reviewed (Table4). Twenty-two specimens from 6 identical twin marrow recipients and 3autologous marrow recipients were available. All of the data were consis-tent with epidermal damage, peaking at 1 to 2 weeks and resolving by 3 to4 weeks. All 3 autografted patients showed Grade II to III changes whichbegan to recede after 3 weeks. All of these patients had at least occasionalindividual necrotic epidermal cells on one or more specimens. Five ofthem 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 negativefor convincing skin biopsy changes beyond the 14th day. Only one biopsyspecimen (of nine) from this group, taken before Day 29, showed Grade Ichanges, and none of the 24 biopsies available after Day 28 in this groupshowed epidermal changes of Grade I. In contrast, before Day 29, threeof eight biopsv specimens from patients with GVHD syndrome had GradeII changes, three had Grade I changes, and two had neither. After Dav29, 8 of 12 specimens from the GVHD group had Grade II changes. Itappears that histologic Grade II changes in most allografted patientsbevond Week 3 to Week 4 are verv likelv to be associated with the clinicalsyndrome of GVDH.

Additional Cincal Observations

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

630 SALE ETAL. American Journalof Pathology

These are very similar to changes reported with bleomycin and long-termhydroxyurea.'5- ' Patients with chronic myelogenous leukemia who havebeen on long-term busulfan therapy seem to have a higher incidence ofskin complications whether they receive allografts, autografts, or twingrafts, suggesting that long-term chemotherapy predisposes skin to suchdamage. Data obtained from examination of biopsy specimens from thesepatients must be interpreted cautiously with regard to GVHD.

DiscussionDirect dose-related cytopathic effects of cytotoxic agents on epithelial

surfaces include those on skin.'4 The hyperplastic epidermis of patientswith psoriasis is sensitive to low-dose methotrexate or hydroxyurea, asmanifested by individual cell necrosis indistinguishable from that seen inpatients with GVHD.'6"8," Normal skin undergoes similar damage duringtherapy with bleomycin,' 520'2' adriamycin,22 radiation coupled with acti-

23,24 1nomycin D or adriamycin, and long-term hydroxyurea."7 Our biopsieson autografted patients produced evidence of marked acute cytotoxicityeffects mimicking those attributed to GVHD in allografted patients. Weexpected to be able to distinguish the two by using criteria such ascytologic atypia (to identify cytotoxic effects) and eosinophilic bodies,satellitosis, and epidermal inflammatory infiltrates (to identify GVHD).Instead, the two proved histologically indistinguishable when subjected todouble-blind analysis. The two complexes of histologic variables which weexpected to be separable and specific (one for GVHD and the other forchemotherapy and irradiation damage) segregated together. This sug-gested a) that the two injurious stimuli produce nearly identical effectsand b) that in the allografted patients with GVHD the effects of the twoinjuries are additive. The relative nonspecificity of dyskeratosis (in-distinguishable from eosinophilic body formation) was not unexpected.Satellitosis was equally present in patients with GVHD damage and thosewith cytotoxic damage. Thus, one must factor time after the drug regimenand grafting into the diagnostic equation. Cytologic atypia of mild tomoderate degree is not specific for cytotoxic therapy; it is probably afeature of regeneration after injury. The artifact study dictates specialcaution in interpretation of milder changes, which could be mimicked bylocal anesthesia or osmotic damage. The histologic threshold for diagnosiswas therefore raised by our study.

Since satellitosis can be observed in patients with syngeneic marrowgrafts, it seems risky to assume that such a finding in the allograftedpatient necessarily represents immune cell-mediated cytotoxicity. It mayrepresent the natural fate of injured epidermal cells regardless of the


cause. These findings underscore the fact that the mechanism of epider-mal cell injurv in cutaneous GVHD or GVHR is not understood.','Further studies are needed to examine serum factors active against iso-lated epidermal cells in vitro 2I and immunoglobulins or other moleculesdeposited 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 late-

onset GVHD (perhaps after Day 35 or 40), when effects of cytotoxic agentshave usually disappeared, and if artifacts are minimized by proper speci-men handling.

2. Eosinophilic bodies (dyskeratotic cells), with or without satellitosis,are a necessary, but not a sufficient, criterion for GVHD diagnosis, sincecvtotoxic agents alone can produce them. However, in a patient receivingonlv CY (60 mg/kg X 2) plus 1000 rad TBI, eosinophilic bodies areunusual beyond 19 days after the graft.

3. Persistence in obtaining two or more serial biopsy specimens isnecessarv in some cases for the diagnosis of GVHD. An equivocal biopsyshould be repeated in 2 to 3 days. The probability that epidermal damageis due to cytotoxic agents should be waning as the probability that thedamage is due to GVHD increases.

4. As a result of the nonspecificitv demonstrated by this study, aclinicopathologic correlation should be made bv the pathologist and theclinician, assessing rectal and liver biopsy data when available, as well asclinical data.The diagnosis of human GVHD is a complex clinicopathologic skill

requiring considerable experience. There as yet appears to be no patho-gnomonic single clinical or histologic feature in the acute phase. There-fore, there are no shortcuts to careful serial gross or microscopic observa-tions of skin or to consideration of all other available relevant data.

Referwecs1. Thomas ED, Storb R, Clift RA, Fefer A, Johnson FL, Neiman PE, Lerner KG,

Glucksberg H, Buckner CD: Bone-marrow Transplantation. N Engl J Med292:832-843, 895-902, 1975

2. Mathe G, Amiel JL, Schwarzenberg L: Bone Marrow Transplantation and Leuco-cyte Transfusions. (American Lecture Living Chemistry Series.) Springfield, Ill.,Charles C. Thomas, Publisher, 1971

3. Vries MJ de, Vos 0: Delayed mortality of radiation chimeras: A pathological andhematological studv. J Natl Cancer Inst 23:1403-1439, 1959

4. Vries MJ de, Crouch BG, Putten LM van, Bekkum DW van: Pathologic changes inirradiated monkeys treated with bone marrow. J Natl Cancer Inst 27:67-97, 1961

5. Glucksberg H, Storb R, Fefer A, Buckner CD, Neiman PE, Clift RA, Lemer KG,

632 SALE ETAL. American Journalof Pathology

Thomas ED: Clinical manifestations of graft-versus-host disease in human recipi-ents of marrow from HL-A-matched sibling donors. Transplantation 18:295-304,1974

6. Krueger GRF, Berard CW, DeLellis RA, Graw RG Jr, Yankee RA, Leventhal BG,Rogentine GN, Herzig GP, Halterman RH, Henderson ES: Graft-versus-host dis-ease. Morphologic variation and differential diagnosis in 8 cases of HL-A matchedbone marrow transplantation. Am J Pathol 63:179-202, 1971

7. Krueger GRF, Graw RG Jr, Rogentine GN, Darrow CC II, Neefe JR, LuetzelerJ: Pathology of modified graft-versus-host disease in bone marrow allograftedmonkeys treated with antilymphocyte serum. Blut 30:19-30, 1975

8. Slavin RE, Santos GW: The graft versus host reaction in man after bone marrowtransplantation: Pathology, pathogenesis, clinical features, and implication. ClinImmunol Immunopathol 1:472-498, 1973

9. Slavin RE, Woodruff JM: The pathology of bone marrow transplantation. Pathol-ogy Annual, Vol 9. Edited by SD Sommers. New York, Appleton-Century-Crofts,1974, pp 291-344

10. Woodruff JM, Eltringham JR, Casey HW: Early secondary disease in the Rhesusmonkey. I. A comparative histopathologic study. Lab Invest 20:499-511, 1969

11. Woodruff JM, Butcher WI, Hellerstein Li: Early secondary disease in the rhesusmonkey. II. Electron microscopy of changes in mucous membranes and externalepithelia as demonstrated in the tongue and lip. Lab Invest 27:85-98, 1972

12. Lerner KG, Kao GF, Storb R, Buckner CD, Clift RA, Thomas ED: Histopathologyof graft-vs-host reaction (GvHR) in human recipients of marrow from HL-A-matched sibling donors. Transplant Proc 6:367-371, 1974

13. 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 bychemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood49:511-533, 1977

14. Koss LG: A light and electron microscopic study of the effects of a single dose ofcyclophosphamide on various organs in the rat. I. The urinary bladder. Lab Invest16:44-65, 1967

15. Blum RH, Carter SK, Agre K: A clinical review of bleomycin.-A new anti-neoplastic agent. Cancer 31:903-914, 1973

16. Smith C, Gelfant S: Effects of methotrexate and hydroxyurea on psoriatic epi-dermis. Preferential cytotoxic effects on psoriatic epidermis. Arch Dermatol110:70-72, 1974

17. Kennedy BJ, Smith LR, Goltz RW: Skin changes secondary to hydroxyurea ther-apy. Arch Dermatol 111:183-187, 1975

18. Weinstein GD, Velasco J: Selective action of methotrexate on psoriatic epidermalcells. J Invest Dermatol 59:121-127, 1972

19. Singer RM, Gelfant S: Continuous inhibition of DNA synthesis in mouse earepidermis using hydroxyurea. Exp Cell Res 73:270-271, 1972

20. 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

21. Cohen IS, Mosher MB, O'Keefe EJ, Klaus SN, DeConti RC: Cutaneous toxicity ofbleomycin therapy. Arch Dermatol 107:553-555, 1973

22. Rudolph R, Stein RS, Pattillo RA: Skin ulcers due to adriamycin. Cancer38:1087-1094, 1976

23. Greco FA, Brereton HD, Kent H, Zimbler H, Merrill J, Johnson RE: Adriamycinand enhanced radiation reaction in normal esophagus and skin. Ann Intern Med85:294-298, 1976


24. D'Angio GJ. Farber S, Maddock CL: Potentiation of x-ray effects by actinomycinD. Radiology 73:175-177, 1959

25. Grebe SC, Streilein JW: Graft-versus-host reactions: A review. Adv Immunol22:119-221. 1976

26. Spielvogel RL, Ullman S, Goltz R': Skin changes in graft-vs-host disease. SouthMed J 69:1277-1281, 1976

27. Steinmuller D, WTunderlich JR: The use of freshly explanted mouse epidermal cellsfor the in vitro induction and detection of cell-mediated cvtotoxicity. Cell Immunol24:146-163, 1976

28. Ullman S, Spielvogel RL, Kersey JH. Goltz RWV: Immunoglobulins and com-plement in skin in graft-versus-host disease. Ann Intern Med 85:205. 197-6

29. Merritt CB, Mann DL. Rogentine GN Jr: Cytotoxic antibody for epithelial cells inhuman graft versus host disease. Nature 232:63839. 1971

AkuowednentsThe 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 FredHutchinson Cancer Research Center for general assistance. especially Charles Mlahan and RobertRaff.


[ Illustrations follow]

- ~~~~~~~~~~~~~~~~~~~~~~

Figure 1-Skin specimen from allografted patient with clinical GVHD syndrome 30 days afterallograft for acute T-cell lymphoblastic leukemia preconditioned with 1 ,3-bis(2-chloroethyl)-1-nitrosourea (400 mg/M2), CY (60 mg/kg x 2), and 1000 rad TBI. Note relatively sparseinfiltration of lymphocytes in upper dermis and lower epidermis, extensive basal cell vacu-olization, and degeneration with some individual cell necrosis. Small nuclei adjacent to dyingepidermal cells indicate satellitosis (arrow). There is also some epidermal hypertrophy, widen-ing of the granular layer, and hyperkeratosis, all nonspecific epidermal responses. (Hematox-ylin and eosin, x 250) Fgw 2-Skin specimen from patient with chronic myelogenousleukemia in blast crisis, who received his own stored bone marrow (autograft); biopsy wasperformed 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 cellvacuolization, and necrosis forming eosinophilic bodies, with lymphocyte nuclei in basal layeroccasionally adjacent to necrotic epidermal cells (arrow). Note strong similarities between theallografted patient's and autografted patient's skin histopathology. (Hematoxylin and eosin, x200)

a


[End of Article]

versus-host disease in man

Documents