J. clin. Path., 1973, 26, 463-469

Benign sinus histiocytosis with massivelymphadenopathy: transient immunologicaldefects in a child with mediastinal involvementD. M. 0. BECROFT, M. R. DIX, J. C. GILLMAN, BETH J. L. MacGREGOR,AND R. L. SHAW

From the Princess Mary Hospital for Children and The Department of Pathology, University of AucklandSchool of Medicine, Auckland, New Zealand

SYNOPSIS A 16-month-old boy presented with cervical lymphadenopathy and a mediastinal masscausing tracheal displacement. Treatment of what at first was suspected to be a malignant neoplasmwas limited to low-dose irradiation of the mediastinum and biopsy excision of the cervical lymphnodes. There has been no recurrence of disease in the subsequent four years.The pathological features and clinical course correspond to a benign disease first described in

1969 in which there is massive proliferation of histiocytes in lymph node sinuses. No infectious causewas identified. Consistent but eventually reversible defects in lymphocyte response to phyto-haemagglutinin and in augmentation of nitro-blue tetrazolium reduction by neutrophils duringphagocytosis were demonstrated in the patient and in his monozygous twin. These defects incellular immune function are believed to be important in the pathogenesis of the histiocytosis.

In 1969 Rosai and Dorfman introduced the term'sinus histiocytosis with massive lymphadenopathy'to describe what they believed to be a newly recog-nized clinico-pathological entity which could bedistinguished from 'malignant reticuloendotheliosis'.Their four cases, all children, had illnesses character-ized by massive cervical lymphadenopathy, fever,leucocytosis, and hypergammaglobulinaemia. Thecourse was benign, though lymphadenopathy wasprolonged. Pathologically the enlarged nodes showeda dilatation of subcapsular and medullary sinuses bynon-neoplastic histiocytes, in parts progressing tototal effacement of the architecture. The histiocytesoften had intracytoplasmic lymphocytes ('emperi-polesis').

Recently Rosai and Dorfman (1972) reported on30 further referred cases with similar histologicalappearances. In the majority the onset was in thefirst decade of life, the sex incidence was equal, andthe distribution was world wide and multiracial.Extracervical lymph nodes and other tissues wereinvolved in some cases. The authors suggested that,although the aetiology was unknown, the diseasemight be the expression of an abnormal immuno-logical response.Received for publication 9 May 1973.

In this report we will describe the demonstrationof transient deficiencies in cellular immune functionsin a case from New Zealand mentioned previouslyby Rosai and Dorfman (1972).

Case Report

A 16-month-old Caucasian child presented in March1969 with a painless swelling of the left side of theneck for seven weeks. There had been mild pyrexiainitially but no other symptoms. He appeared ahealthy child of weight 11P4 kg and height 76 cm.There was a firm, nodular mass 4 cm in diameterbelow the angle of the jaw on the left. The liver andspleen were just palpable. A chest radiograph (figIA) showed widening of the superior mediastinumbilaterally and a left-sided paravertebral massextending down to the eleventh thoracic vertebra.The lower trachea was displaced to the right. Theskeleton was normal. There was a mild iron-deficiency anaemia. The white blood count was9300/cmm (53% neutrophils, 39% lymphocytes,3% eosinophils, 4% monocytes, and 1 % Turk cells).The bone marrow was normal. The tuberculin testwas negative.A mass of enlarged left upper deep cervical lymph

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Fig. 1A The patient's chest radiograph at presentationaged 16 months showing widening of the mediastinumand a left paravertebral mass.

Fig lB Normal appearances three and a halfyears later.

nodes was excised. Radiotherapy was directed to themediastinal mass, but because the histologicaldiagnosis was uncertain this was limited to a trialdose of 1000 rads. The mediastinal mass declinedvery slowly until after nine months the chest radio-graph was normal. However, five months afterradiotherapy was completed the left cervicallymphadenopathy recurred. The peripheral bloodand bone marrow were normal. The enlarged leftjugular, submaxillary, and posterior triangle lymphnodes were excised but no other treatment was

given. Four years after his presentation he is welland there has been no further recurrence of lympha-denopathy or of mediastinal lesions (fig 1B).

An apparently identical twin has been observedover the same time and has shown no clinical,radiological, or haematological abnormalities. Thereis no other relevant family history.

Pathology

GROSSEach surgical specimen consisted of a mass ofadherent lymph nodes individually measuring up to3 cm in diameter. The larger nodes had firm, pale-yellow cut surfaces.

MICROSCOPICThe smaller nodes and parts of the larger werenormal or showed only non-specific sinus andfollicular hyperplasia. Elsewhere there was a variableexpansion of sinuses by large histiocytic cells whichin the largest nodes had replaced all structuresexcept for infrequent lymphoid follicles and rem-nants of medullary cords (fig 2). Most histiocyticcells were large and had abundant, palely eosino-philic homogeneous cytoplasm. Nuclei werecentrallyplaced, round, and occasionally large and hyper-chromatic, but mitoses were rare. In some areashistiocytes with densely eosinophilic cytoplasm wereclosely packed in an epithelial-like pattern (fig 3).Elsewhere, histiocytes were intermingled withlymphocytes, their cytoplasmic borders were scal-loped, and the cytoplasm was often vacuolated.There was minimal erythrophagocytosis, but astriking feature in these areas was the presence ofmany intact lymphocytic nuclei within the histio-cytic cytoplasm, the lymphocytes often ringing thecentrally placed nucleus (fig 4). A few cells hadfoamy cytoplasm but only small amounts ofsudanophilic lipid were demonstrated in frozensections and no mucopolysaccharide was detectedwith Alcian blue. The cytoplasm did not stain withperiodic-acid Schiff. There were no eosinophils, andneutrophils were confined to a few minute sub-capsular foci of necrosis in the first specimen. Theresidual lymphoid tissue contained numerous plasmacells.

Investigations into Aetiology and Pathogenesis

INFECTIONSNo serum antibodies to rubella or toxoplasmosiswere detected initially and no antibodies to Brucellawere present two years later. No organisms wereidentified in histological sections of the lymph nodesusing Gram, periodic-acid-Schiff, and Ziehl-Neelsenstains, nor in sections stained with rhodamine-auramine for fluorescing mycobacteria. No bacteria,

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Benign sinus histiocytosis with massive lymphadenopathy

Fig 2 A lymph node at low magnification showing Fig 3 Histiocytes filling a distended lymph node sinus.extensive replacement ofnormal structure by palely- Some nuclear pleomorphism and an epithelial-likestaining cells. A few lymphoid follicles persist. H and E appearance are shown H and E (x 400).( x 70).

Fig 4 A distendedlymph node sinus inwhich many histiocytescontain phagocytosedlymphocytes(emperipolesis),H and E ( x 400).

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viruses, or other specific cytoplasmic structures weredetected by electron microscopy of formalin-fixedmaterial from the second specimen (Rosai, 1970).Tissue from both biopsy specimens was cultured formycobacteria with negative results. No aerobic oranaerobic bacteria or fungi were isolated from thesecond specimen after prolonged culture. Attempts toestablish a tissue culture from the second specimenwere unsuccessful.

HUMORAL IMMUNITYThe patient's serum immunoglobulin levels estimatedby radial diffusion during his first hospital admissionwere: IgG 12 5 mg/ml, IgM 2-0 mg/ml, and IgA 1-4mg/ml. Three years later serum levels were: IgG9 9 mg/ml, IgM 0-9 mg/ml, and IgA 1-2 mg/ml. Theserum B1-A globulin (C3 complement component)also estimated by radial diffusion was normal at102 mp/100 ml. The patient's serum generated anormal chemotactic stimulus when tested by methodsgiven below.

CELLULAR IMMUNITYWhenever possible studies were made simultaneouslyon cells from the patient and his twin. The twinswere found to be identical for all blood group,serum, and histocompatibility antigens tested, givingan estimated probability (p) of dizygosity of < 0-003.

TESTS OF PHAGOCYTIC FUNCTIONIn September 1972 the patient's neutrophils hadnormal random mobility when studied by the methodof Harris (1953).Chemotaxis was studied in a modified Boyden

chamber using methods based on those of DeMeoand Andersen (1972). The chemotactic stimulus wasgenerated by incubation of Zymosan with eitherhomologous or control sera. The chemotacticresponse was assessed by counting the neutrophilstraversing a standard area of a 3 ,u pore-sizeMillipore filter. These studies, in September 1972,showed a normal response by the patient's cells anda normal stimulus generated by his serum.

Quantitative nitro-blue tetrazolium (NBT) testswere performed using the method of Baehner andNathan (1968) in which the intracellular reductionof NBT by peripheral blood phagocytes is measuredspectrophotometrically, with and without phagocyticstimulation by exposure to latex particles. The'resting' and 'stimulated' levels expressed as opticaldensities and the differences (A 0 D) obtained instudies on the patient and his twin between Nov-ember 1971 and September 1972 are presented infigure 5. The time relationship with the results ofother studies are indicated in the table. Theresting levels were normal or near normal on all

.400

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a .100-

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A C.G.D.* patino Twin

1

I

Rehtn Phagocytosing

Fig 5 Quantitative NBT tests on leucocytes. Restingand stimulated levels for individual tests are linked andthe difference (a OD) is shown separately. Verticalcolumns indicate the mean levels ± 2 SD.for 11 normalcontrols studied concurrently. The means offive tests ontwo children with chronic granulomatous disease (CGD)are also shown.

occasions but the patient's cells on the first threeoccasions and the twin's cells on one occasionfailed to show normal stimulation during observedphagocytosis of latex. The difference between theresponses in the twins and in controls is significant(p < 0-001). Further studies six months later gavean intermediate level of response in the patientand a normal response in his twin and after afurther three months a normal response was obtainedwith the patient's cells. Levels of NBT reductionwere also estimated by the cytochemical method ofHicks and Bennett (1971) which differs from theabove in that cyanide-sensitive metabolic pathwaysare not inhibited and reduction is assessed by acytochemical scoring of neutrophils, either 'resting'or after latex stimulation. Resting scores in bothchildren were significantly higher than those ob-tained in 38 control subjects (p < 0-0005) and theirmother, while the augmentation during latexstimulation was significantly less (p < 0-0025 forpatient, < 0-01 for twin) than in eight controls.The bactericidal capacity of peripheral blood

phagocytes against the Oxford staphylococcus wasstudied by the method of Quie, White, Holmes, andGood (1967) and the results given in the table arewithin normal limits.

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Subject Date PHA Response" NBT Tests Bactericidal"Tests

Spectrophotometric Cytochemical Score

0. D." Resting Stimuated Difference

Patient Nov 1971 50 (74-6)' 0 030 94'Dec 1971 0-6 5-1)Feb 1972 7-0 (37-6) 0-033June 1972 0060 33 31 -2July 1972 59 5 (106-6)Sept 1972 88-1 (657) 0-144 16 23 +7

Twin Dec 1971 0-9 (5-1)Feb 1972 4-8 (37-6) 0 014June 1972 0190 21 25 +4July 1972 111(106 6) 88 %'

Mother June 1972 7 57 +50Controls(mean SD) 0182 4 0-029 6 3 + 2-5 59+ 11 53 15

(n =11) (n = 38) (n =8) (n =8)

Table Tests on blood leucocytes'Tritiated thymidine uptake (cpm per 103 lymphocytes). Unstimulated levels < 1-0 in all experiments."Concurrent control values in parentheses'Difference between resting and stimulated levels'Percentage of bacteria killed in two hours.

TESTS OF LYMPHOCYTE RESPONSIVENESSThe response of blood lymphocytes to phyto-haemagglutinin (PHA) stimulation was assessed bya method based on that of Fitzgerald (1972).Lymphocytes from whole plasma, or after partialseparation from other leucocytes on a nylon column,were exposed to a range of concentrations of PHAand the response was measured by the uptake oftritiated thymidine. The results given in the table areof the maximal stimulation obtained, this usuallyoccurring at PHA concentrations of either 20 or200 ,tg/ml. The results largely parallel those of theNBT test, with deficient responses from specimensobtained from the patient and his twin beforeFebruary 1971, while more recent studies have givennormal results.

Discussion

This child presented with cervical lymphadenopathyand radiological findings which are assumed to havebeen due to massive enlargement of mediastinallymph nodes by the same pathological process.Because of initial uncertainty as to the diagnosistreatment was limited to biopsy-excision of cervicallymph nodes and low-dose irradiation to themediastinum. Excision of a later recurrence was alsoconsidered incomplete. Therefore the subsequentcourse, without further recurrence, is consistent withan intrinsically benign disease and corresponds withthat of other cases described by Rosai and Dorfman(1972). The close histological resemblance has beenconfirmed (Rosai, 1970) and there seems little doubtthat this is the same clinico-pathological entity. The

cervical lymphadenopathy was less severe than inmany other cases, possibly because of early palliativesurgery. This case was one of nine described byRosai and Dorfman (1972) with perihilar ormediastinal masses, out of 21 in which chest radio-graphs were taken. One other had a paravertebralmass.The paravertebral mass in our patient caused

strong suspicion initially of a primary abdominalmalignancy and of neuroblastoma in particular.Neuroblastoma, other embryonic tumours, thecommon malignant lymphomas, and the storagediseases were excluded promptly by histologicalexamination of biopsy material. The identificationof the cells expanding the lymph nodes was com-plicated by the epithelial-like appearance of someareas, but once their histiocytic nature was appreci-ated the major diagnostic problem lay in theexclusion of the generalized and usually lethal formsof systemic histiocytic proliferation or 'reticulo-endothelioses' (Rappaport, 1966). Histiocytic medul-lary reticulosis and other malignant histiocytoses,histiocytosis 'X', and variously named familial formsof histiocytic or lymphohistiocytic proliferation(Berard, Cooper, Freireich, and Rabson, 1966;Miller, 1966) appeared unlikely on clinical andpathological grounds.The benign course, the initial fever and hyper-

immunoglobulinaemia, and the leucocytosis notedin other cases are consistent with an infectiousaetiology. Both patient and twin when apparentlywell had significantly elevated 'resting' levels ofNBT reduction as assessed by the cytochemicalmethod which is being applied widely as a test

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indicative of active bacterial infection (Lancet, 1971).However, the distribution of the lymphadenopathycould not be accounted for by a single inflammatoryfocus nor was such a focus demonstrated. Otherinvestigations excluded infections by several micro-organisms likely to cause chronic lymphadenopathy.

Infection remains the most likely primaryaetiology but a histiocytic reaction of this degree,without abscess or granuloma formation, would bean exceptional response except where cellularimmune reactions were impaired. Associated im-mune defects have been offered as an explanationfor extreme histiocytic reactions produced byidentifiable infections such as rubella (Claman,Suvatte, Githens, and Hathaway, 1970) and atypicalmycobacteria (Clinico-pathological Conference,1970). There may be striking histiocytic prolifer-ation in some children who have severe congenitaldefects in T-lymphocyte-mediated cellular immunityin association with thymic dysplasia (Becroft andDouglas, 1968). Widespread proliferation of pig-mented lipid-containing histiocytes occurs in chronicgranulomatous disease in which a metabolic defectin phagocytes leads to defective intracellularkilling of certain bacteria (Carson, Chadwick,Brubaker, Cleland, and Landing, 1965). Thus,histiocytic proliferation may be the result of dim-inished killing of phagocytosed microorganismsdue either to intrinsic cellular defects or to thenature of the organism. Progression to granulomaformation would be usual but this progressionwould tend to be inhibited by any associated defectin lymphocyte-mediated delayed-type hypersensi-tivity reactions.

Therefore there may be considerable pathogeneticsignificance in the finding in our patient's leucocytesof a combination of defective lymphocyte responseto PHA and diminished augmentation of NBTreduction during phagocytosis. Although theseresults were obtained when he appeared free ofdisease, each defect was confirmed on three separateoccasions over an eight-month period when con-current control values were generally satisfactory.The defects were transient and therefore are pre-sumed to be acquired rather than congenital. Adefective PHA response is widely accepted asindicating a defect in lymphocyte-mediated cellularimmunity. It is less certain whether the failure ofaugmentation of the NBT response over normal orelevated resting levels has the same functionalsignificance as the defects in both resting and stimu-lated levels observed in chronic granulomatousdisease (Baehner and Nathan 1968). Similartransient defects in augmentation of the NBTresponse in two recently described cases wereassociated with a transient increase in susceptibility

to bacterial infection (Douglas, Lahav, and Fuden-berg, 1970; Kretschmer, Lopez-Osuna, and Valen-zuela, 1972). In one case a bactericidal defect wasalso demonstrated in vitro (Douglas et al, 1970)whereas our patient's leucocytes had normalbactericidal activity against the single organismtested. A. child described recently (McCracken,Weinberg, and Carlin, 1972) resembled our patientin showing widespread histiocytic proliferation andacquired defects in both lymphocyte reactivity andin augmentation of the NBT response, but thedefects were progressive and the child died ofgranulomatous lung disease.The interpretation of the results in our patient

was further complicated by the similar findings inthe apparently unaffected monozygous twin. Therewas less repetition of abnormal results in the twin,but if these are accepted as valid then alternativepossibilities are that the twin had a subclinical formof the disease, that both had an inherited buttransient immune defect which predisposed to adisease precipitated in the patient by some additionalfactor, or that the defects and the disease wereunrelated. In our opinion there is strong likelihoodthat the defects were of significance in the patho-genesis of the histiocytic reaction, but the primaryaetiology and relative contribution of genetic andenvironmental factors remain in doubt.

References

Baehner, R. L., and Nathan, D. G. (1968). Quantitative nitrobluetetrazolium test in chronic granulomatous disease. New Engl. J.Med., 278, 971-976.

Becroft, D. M. O., and Douglas, R. (1968). Thymic alymphoplasia.monoclonal gammopathy, and Pneumocystis carinii pneu-monia in an infant. Arch. Dis. Childh., 43, 444-450.

Berard, C. W., Cooper, R. A., Freireich, E. J., and Rabson, A. S.(1966). Disseminated histiocytosis associated with atypicallymphoid cells (lymphohistiocytosis). Cancer (Philad.), 19,1429-1437.

Carson, M. J., Chadwick, D. L., Brubaker, C. A., Cleland, R. S.,and Landing, B. H. (1965). Thirteen boys with progressiveseptic granulomatosis. Pediatrics, 35, 405-412.

Claman, H. N., Suvatte, V., Githens, J. H., and Hathaway, W. E.(1970). Histiocytic reaction in dygammaglobulinemia andcongenital rubella. Pediatrics, 46, 89-96.

Clinico-pathological Conference (1970). Histiocytosis and infectionwith atypical mycobacteria. Amer. J. Med., 49, 830-840.

DeMeo, A. N., and Andersen, B. R. (1972). Defective chemotaxisassociated with a serum inhibitor in cirrhotic patients. NewEngl. J. Med., 286, 735-740.

Douglas, S. D., Lahav, M., and Fudenberg, H. H. (1970). A reversibleneutrophil bactericidal defect associated with a mixed cryo-globulin. Amer. J. Med., 49, 274-280.

Fitzgerald, M. G. (1972). A satisfactory quantitative test of lympho-cyte response to phytohaemagglutinin for the definition ofnormal control values and recognition of immunologicaldefects. J. clin. Path., 25, 163-168.

Harris, H. (1953). Chemotaxis of granulocytes. J. Path. Bact., 66,135-146.

Hicks, R. G., and Bennett, J. M. (1971). An improved cytochemicalmethod for nitroblue tetra-zolium reduction by neutrophils.Amer. J. med. Technol., 37, 226-229.

Kretschmer, R. R., L6pez-Osuna, M., and Valenzuela, R. H. (1972).Reversible neutrophil defect in atazia telangectasia. Pediatrics,50, 147-150.

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Lancet (1971). Editorial. Nitroblue tetrazolium, a routine test?Lancet, disease of childhood. J. clin. Invest., 46, 668-679.2,909-910. Rappaport, H. (1966). Tumors of the Hematopoietic System (Atlas of

McCracken, G. H., Weinberg, A. G., and Carlin, M. E. (1972). Tumor Pathology, Sect. 111, Fasc. 8). Armed Forces InstituteGranulomatous lung disese with progressive deterioration of of Pathology, Washington, D.C.phagocytic function. J. Pedlat., 81, 267-274. Rosai, J. (1970). Personal communication.

Miller, D. R. (1966). Familial reticuloendotheliosis: concurrence Rosai, J., and Dorfman, R. F. (1969). Sinushistiocytosis with massiveof disease in five siblings. Pediatrics, 38, 986-995. lymphadenopathy. Arch. Path., 87, 63-70.

Quie, P. G., White, J. G., Holmes, B., and Good, R. A. (1967). In Rosai, J., and Dorfman, R. F. (1972). Sinus histiocytosis with massivevitro bactericidal capacity of human polymorphonuclaer lymphadenopathy: a pseudolymphomatous benign disorder.leukocytes: diminished activity in chronic granulomatous Cancer (Philad.), 30, 1174-1188.

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