HISTOCHEMICAL STUDY OF ACID PHOSPHATASE ISOENZYME I N LEUKEMIC RETICULOENDOTHELIOSIS

I. KATAYAMA, MD, C. Y. Lr, MD,” AND L. T. YAM, MD

Histochemical study of the tartrate-resistant acid phosphatase isoenzyme was made on frozen sections of surgical specimens from leukemic reticuloendotheli- osis. The tumor cells of this disease were found to have strong tartrate-resistant enzyme activity. Control studies on preparations of malignant lymphoma, other hematologic disorders, neoplasms of nonhematopoietic organs and normal tissues showed very little isoenzyme activity. Exceptions were found in speci- mens involved by Hodgkin’s disease and in an adrenal of one patient with aldosteronism. It was proposed that the histochemical demonstration of the isoenzyme in the bone marrow, liver, or spleen can be used as a diagnostic criterion for leukemic reticuloendotheliosis, thus differentiating it from lym- phosarcoma and reticulum cell sarcoma.

EUKEMIC RETICULOENDOTHELIOSIS &RE) IS L a disease entity characterized by chronic course, anemia, frequent leukopenia, massive splenomegaly, and the presence of “reticulum cells” in peripheral blood and bone marrow. The diagnosis rests on identification of the characteristic mononuclear cells in the blood or bone marrow.6.‘~9JO The disease had been fairly well documented in the hematologic references, while recognition in the pathology literature has not been definitive; histopatho- logic descriptions of the entity have been sporadic and mostly brief.4-7.9JOJ8

Recently, a tartrate-resistant acid phospha- tase isoenzyme was shown to be specific for the reticulum cells of LRE, and it is now used as a cytochemical marker for diagnosis of this disease.12Jo However, such cytochemical stud- ies can be exceedingly difficult in patients with severe leukopenia and packed bone mar-

. From the Department of Pathology, University Hos- pital and Boston University School of Medicine, Boston, Mass. (Dr. Katayama and from the Blood Re-

Dital and Tufts Universitv School of Medicine. Boston. search Laboratory, New Eng 1 and Medical Center Hos-

Mass. @rs. Li and Yam).’ Supported, in part, by U. S. Public Health Service

Grants AM12444, GRS 611, and Atomic Energy Com- mission Contract Number AT(3O-1) 3808.

+ Present address: Department of Hematology, Tri- Service General Hospital, Taipei, Taiwan, Republic of China.

Address for reprints: I Katayama, MD. Department of Pathol y, University Hospital, Boston, Mass. 02118.

The auzors are grateful to Dr. M. Hayashi for ad- vice in the histochemistry. and to Drs. W. H. Crosby and R. Cotran for their critical evaluation of the man- uscript. They also thank Mr. John Hacunda for pho- tography and Miss Sheila Gill for typing the manu- script.

Received for publication June 4, 1971.

row resulting in “dry tap,” a situation encoun- tered so frequently in LRE. In such circum- stances, histochemical demonstration of the tartrate-resistant acid phosphatase isoenzyme in biopsy specimens may be the only alterna- tive approach toward a definite diagnosis.

Our purpose here is to present our data on the histochemical study of 7 patients with LRE, to evaluate the isoenzyme as a histo- chemical diagnostic criterion, to review briefly the literature on LRE as a clincopathologic entity, and to demonstrate, like others, that a diagnosis of LRE is of more than academic in- terest since it carries a more favorable prog- nosis and requires different clinical manage- ment than other leukemias and malignant lymphomas.10

MATERIALS AND METHODS

Ten surgical specimens of LRE from 7 pa- tients were the primary subject of this histo- chemical investigation (group 1, Table 1). All patients presented with the typical signs and symptoms of LRE, and the diagnosis was con- firmed by hematologic, cytochemical, and bio- chemical examinations. A clinical study in- volving 5 of these 7 patients will be reported elsewhere.

Fresh surgical materials from 80 patients were used for control studies. These patients were categorized into 4 groups (groups II-V, Table 1) as follows:

Malignant lymphoma (group ZZ): This group included 10 patients with lymphosar- coma, 2 with reticulum cell sarcoma, and 9

157

158 CANCER January 1972 VOl. 29

TABLE 1. Number of Patients and Tissue Studied for Acid Phosphatase

Tissue Examined

Group Diagnosis No. of patients Lymph Bone

(No. of specimens) Spleen node marrow Miscellaneous

I Leukemic reticuloendotheliosis 7 Ly mphosarcoma 10

I1 Reticulum cell sarcoma Hodgkin’s disease

2 9

111 Normal tissue 18 IV Other hematologic disorders 25 V Neoplasms of non-hematopoietic

organs 16 TOTAL 87

* Buffy coat of blood.

with Hodgkin’s disease. In 3 patients (3 spleen and 1 lymph node) with known Hodgkin’s dis ease, the tissues were free of diagnostic histo- pathologic changes. In one case of known Hodgkin’s disease (1 spleen and 1 lymph node), the lymph node was positive while the spleen was negative for the histologic evidence of the disease.

Normal tissue (group ZZZ): Twenty-three specimens were obtained from 18 patients with miscellaneous clinical diagnoses. Speci- mens examined were two each of spleen, stom- ach, breast, uterus, ovary, adrenal, small intes- tine, and colon, and one each of kidney, urinary bladder, seminal vesicle, prostate, bone marrow, thymus, and skin. Clinical diag- noses were gastrointestinal hemorrhage, gastric ulcer, carcinoma of stomach, carcinoma of pancreas, Crohn’s disease, diverticulosis coli, fibrocystic disease of breast (2 cases), carci- noma of breast (2 cases), chronic glomerulone- phritis (2 cases), carcinoma of bladder, leiomy- omata uteri, ovarian cyst, menometrorrhagia, osteoporosis, and myasthenia gravis.

Hematologic disorders other than LRE and malignant lymphoma (group IV): This group consisted of 11 patients with various anemias, 5 with hypersplenism of undetermined origin, 3 with myelofibrosis with myeloid metaplasia, 2 with idiopathic thrombocytopenic purpura, 2 with polycythemia Vera, 1 with Di- Guglielmo’s disease, and 1 with thrombotic thrombocytopenic purpura.

Neoplasms other than LRE and malignant lymphoma (group V): This group included 2 patients with carcinoma of lung, and one pa- tient each with carcinoma of breast, stomach, pancreas, colon and prostate, and one each with benign cystic teratoma of ovary, synovial sarcoma of leg, malignant mesothelioma of

(16) 0 1 0 15

(104) 32 14 21 37

pleura, plural fibroma of lung, mixed tumor of parotid, glioblastoma multiforme, malig- nant mixed mullerian tumor of uterus, hy- perplasia of adrenal cortex, and squamous cell carcinoma of lung metastatic in a lymph node.

The histochemical examination for demon- stration of the acid phosphatase isoenzymz was performed on all 104 specimens. In addi- tion, histologic studies with hematoxylin and eosin and reticulum stains were carried out on bone marrows and spleens from cases of LRE.

Histochemical method: Thin tissue slices (about 5 x 5 x 2 mm) from surgical speci- mens were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 containing 1 drop of 1% CaC1, per 10 ml of the fixative, at 4C for 2 hours. Bone marrow biopsies were decalcified by 0.5 M disodium ethylenedia- mine tetracetate (EDTA).13 Blood was pro- cessed as a solidified disc of the bulTy coat.’ After fixation, the tissue slices were washed and then stored at 4C in a 0.2 M sucrose solu- tion in 0.1 M cacodylate buffer (pH 7.4) for periods ranging from overnight to 9 months.16 They were embedded in gelatin,14 and cxyo- stat-frozen sections were cut at a thickness of 8 p and mounted on glass slides. From each specimen, two slides were made. They were immersed in 50y0 acetone for 5 minutes at -15C and washed in cold distilled water, then the slides were incubated in the staining solution A or B (described below) for 30 min- utes at room temperature. Following incuba- tion, the slides were washed in distilled water briefly and then immersed in 70% ethyl alcohol for 30 minutes and again washed in distilled water. The sections were then coun- terstained with Celestine Blue* and mounted with a modified gum syrup.”

Staining solution A was made up by mixing

No. 1 LEUKEMIC RETICULOENDOTHELIOSIS 0 Katayama et al. 159

the following: 1. 0.4 ml of pararosanilin solu- tion (1 g pararosanilin hydrochloride in 20 ml distilled water and 5 ml concentrated hydro- chloric acid); 2. 0.4 ml of 4y0 sodium nitrite solution; 3. 10 ml of 0.2 M acetate buffer PH 5.2, and 4. 10 mg naphthol AS-BI phosphoric acid (No. N-2125, Sigma Chemical Co., St. Louis, Mo.) dissolved in 2.4 ml 0.022 M so- dium bicarbonate solution and 2.6 ml distilled water. The mixture was adjusted to pH 5.2 with 1 N NaOH, brought to final volume of 20 ml with distilled water, and then filtered by a No.1 Whatman filter paper.2 Staining so- lution B for demonstration of tartrate-resist- ant isoenzyme was made by adding 75 mg L (+) tartaric acid to a 10-ml aliquot of solution A to make a tartrate concentration 0.05 M and adjusting pH to 5.2 by concentrated NaOH.11

RESULTS

Results of the histochemical study of acid phosphatase in LRE and controls are summa- rized in Table 2.

Leukemic reticuloendotheliosis (group I, Table 2): Preparations of spleen and bone marrow showed strong acid phosphatase activ- ity evidenced by dense red staining of the cy- toplasm of all tumor cells (Fig. 1A). With the addition of tartrate, the enzyme activity was equally intense or even stronger (Fig. IB). In- travascular tumor cells in the sinuosids dem-

onstrated an identical reactivity (with or with- out tartrate) to that seen in infiltrating tumor cells in the red pulp. The flattened endothe- lial cells lining the sinusoids showed much weaker reaction than the tumor cells. Enzyme activity was not significantly affected by stor- age in 4C for a long period of time, and was still demonstrable more than 8 months after the tissue was fixed and stored in 0.2 M su- crose in cacodylate buffer (Figs. lA, B). The buffy coat of blood solidified by the fixative and processed like solid tissue specimens showed an intense tartrate-resistant acid phos- phatase reaction in LRE cells, while neutro- phils lost enzyme activity after the addition of tartrate.

Malignant lymphoma (group ZZ): In lym- phosarcoma (both lymphocytic and lympho- blastic types), acid phosphatase activity in the tumor cells was weak and observed only in some of the cells (Fig. 2A). The reaction was completely inhibited by tartrate (Fig. 2B). En- zyme activity diminished upon storage. In one spleen involved by lymphosarcoma, the en- zyme activity became negative after storage for 8 months. In reticulum cell sarcoma, a weak acid phosphatase activity was seen in some re- ticulum cells (Fig. 3A), which was partially tartrate-resistant (Fig. 3B). In Hodgkin's dis- ease, 5 specimens with no definite histologic involvement of Hodgkin's disease exhibited moderate acid phosphatase activity, which was inhibited by tartrate. On the other hand, acid

TABLE 2. Results of Acid Phosphatase Activity ~~ ~~~

Activity* Activity* without with

Group Diagnosis tartrate tartrate

I Leukemic reticuloendotheliosis + + + + + + + 4- Ly mphosarcoma +-++ 0

Hodgkin's disease +-+++ O N + + + I1 Reticulum cell sarcoma +-++ 0-+

(focal)

I11 Normal tissue

Remarks

All tumor cells showed strong isoenzyme activity. Activity in Hodgkin's disease was not uniform in all cells. Fre uent Sternberg-Reed cells an! large mono- nuclear cells showed no reaction with or without tartrate.

+-+++ 0 Partial tartrate-resistant activity was observed in occasional isolated mesenchymal cells.

IV Other hematologic disorders +-+++ 0 Partial tartrate-resistant activity was observed in occasional isolated mesenchymal cells.

V Neoplasms of non-hematopoietic organs +-+++ o** **Exception: One adrenal of a patient

with aldosteronism demonstrated isoenzyme activity in all cells of the cortex.

* Acid phosphatase activity was graded; ++++maximal; +++intense; ++moderate; +slight; 0 no noticeable activity.

FIG. 1. Spleen of LRE, stained for acid phosphatase without (A, left) and with (B, right) tartrate. Strong reaction is demonstrated in both A and B. This specimen had been stored in sucrose solution for over 8 months ( ~ 5 4 0 ) . FIG. 2. Lymph node of lymphosarcoma, stained for acid phosphatase without (A, left) and with (B, right) tartrate. Activity Seen in scattered cells (A). is entirely suppressed by tartrate (B), 'x450. FIG. 3. Lymph node of reticulum cell sarcoma, stamed for acid phosphatase without (A, [eft) and with (B, right) tartrate. Positive reaction in some reticulum cells (A) is reduced but not completely inhibited by tartrate (B), ~ 5 4 0 . FIG. 4. Spleen of idiopathic thrombocytopenic purpura, stained for acid phosphatase without (A, left) and with (B, right) tartrate. Strong activity in the red pulp (A) is entirely inhibited by tartrate (B). Most specimens in groups 111-V react similarly ( ~ 4 5 0 ) .

No. 1 LEUKEMIC RETICU LOE NDOTHELIOSIS * Ka tay a m a e t a 1. 161

phosphatase activity in 8 specimens with histo- logic involvement was increased and tartrate resistant, and, in some areas, i t was compara- ble to that seen in LRE. However, while the enzyme activity in LRE was evenly distributed in all tumor cells, it was focally distributed among only some of the cells in Hodgkin’s dis- ease: many Sternberg-Reed cells and occa- sional monoculear cells were entirely negative for the enzyme reaction both with and with- out tartrate.

Normal tissue (group 111): All 23 specimens from 18 patients showed acid phosphatase re- action of slight-to-moderate intensity in both parenchymal and stromal cells. This activity was completely and uniformly suppressed by tartrate. Exceptional were the occasional iso- lated mesenchymal cells which displayed acid phosphatase activity with partial resistance to tartrate.

Hematologic disorders and neoplastic dis- eases other than LRE and malignant lym- phoma (groups ZV and V): In these patients, acid phosphatase activity was normal and tar- trate sensitive (e.g., idiopathic thrombocyto- penic purpura, Figs. 4A, B). An exception was one adrenal cortex from a case of aldosteron- ism, which demonstrated diffuse and strong tartrate-resistant enzyme activity.

Histologic studies of LRE: The histologic picture was quite characteristic. The spleen showed loss of the basic architecture by diffuse proliferation of tumor cells (Fig. 5), which were composed of medium-sized polyhedral cells with vesicular nuclei, prominent nu- cleoli, and poorly stained cytoplasm. Mitoses were rare, and cytologic anaplasia was vir- tually absent (Fig. 6). Erythrophagocytosis, hemorrhage, and necrosis were not observed. Reticulum stains revealed prominent reticu- lin fibers around individual tumor cells. Sec- tions of the bone marrow displayed nearly complete replacement of the marrow spaces by similar mononuclear cells as those seen in the spleen (Fig. 7). Characteristically, the tumor cells in the bone marrow were not as closely packed as in infiltrates of chronic lymphatic leukemia or lymphosarcoma.

DISCUSSION

Leukemic reticuloendotheliosis is cBarac- terized clinically by insidious onset, vague systemic symptoms, marked splenomegaly, some hepatomegaly, and insignificant super- ficial lymphadenopathy.509 Most patients are

able to perform normally in their usual oc- cupations until infection or hemorrhage de- velops in the later stage,g although a rapidly fatal course was recorded in approximately one quarter of several large series.5Jo The disease most commonly involves males in the 4th to 6th decade, with incidence of 2% in all leukemias in one series.5 The disease is probably not so rare as might be assumed generally. For a more detailed clinical pic- ture, there are excellent re~iews.~.~-QJO Hema- tologically, anemia is constant while leuko- cyte count is variable. Diagnosis is based on demonstration in the smears of blood or bone marrow of characteristic mononuclear cells which are considered to be reticulum cells by most workers.5JOJ8

Despite its fairly wide recognition in the hematologic literature, LRE as a pathologic entity is still to be differentiated from malig- nant lymphoma, Schilling’s monocytic leu- kemia, histiocytic leukemia,3 and reticulum cell sarcoma with circulating reticulum cells.20 The pathology of LRE described by others 4-7.9~10~18 and reviewed here (Figs. 5-7) is char- acterized by primary major involvement of spleen and bone marrow, diffuse infiltration by monotonous “reticulum cells” with prac- tically no mitoses or cytologic anaplasia, and prominence of “perimonocellular” reticulin fibers.4 Duhamel et al.6 consider the entity a form of lymphosarcoma with predominant localization in the bone marrow. A histologic diagnosis of malignant lymphoma has been made in some cases of LRE.7 Indeed, on a pure morphological basis, the most important and difficult differential diagnosis of LRE is from lymphosarcoma and reticulum cell sar- coma. This aspect is discussed critically by Boiron et a1.,4 who propose several very help- ful morphological criteria, none of which, however, qualifies as pathognomonic by itself.

The present study represents a histochemi- cal approach to the problem. The findings in- dicate a distinctly different reactivity of acid phosphatase in LRE (Figs. lA, B), lymphosar- coma (Figs. 2+4, B), and reticulum cell sar- coma (Figs. 3A,*B). The result helps in, the differential diagnosis of the three diseased and also serves as additional evidence for the path- ologic +distinction. Bf LRE from malignant lymphom&

In clinical practice, LRE is frequently con- fused with other hematologic disorders, such as hypersplenism of diverse etiologies, myelofi- brosis with myeloid metaplasia, lymphosar-

162 Januury 1972 Vol. 29

FIG. 5 (top). Spleen from a case of LRE, showing diffuse monotonous infiltration of tumor cells with erosion of the trabecula. Neither mitosis nor cytologic anaplasia is evident (H and E, ~510) . FIG. 6 {bottom). Magnified view of Fig. 5, showing the detail of tumor cells with vesicular nuclei, prominent nucleoli, and polyhedral poorly delineated cytoplasm. Compare the size of the tumor cell nuclei with an eosinophil in the center (H and E, ~1900) .

No. 1 LEUKEMIC RETICULOENDOTHELIOSIS - Kutuyama et al. 163

FIG. 7. Bone marrow from a case of LRE, showing diffuse infiltration of the tumor cells in the marrow space. The characteristic tumor cells are not as closely packed as in infiltrates of lyrnpho- sarcoma or chronic lymphatic leukemia (H and E, ~ 9 0 0 ) .

coma, and chronic lymphatic leukemia. In the diagnostic work-up of such cases, pancyto- penia and difficulty in bone marrow aspira- tion frequently necessitate biopsy of bone marrow, liver, or spleen. Here acid phospha- tase isoenzyme histochemistry has a most prac- tical application. In 2 of the 7 patients in this series, the diagnosis of LRE was firmly estab- lished by the histochemical study of the bone marrow biopsies.

The presence of tartrate-resistant isoenzyme activity in specimens involved by Hodgkin's disease deserves special consideration. While isoenzyme activity is demonstrable in all cells of LRE, it is seen in some but not all cells among the polymorphous cellular infiltrates of Hodgkin's disease. Whether such isoenzyme- positive cells (7 histiocytes) of Hodgkin's dis- ease are very close to, if not identical with, the tumor cells of LRE is unclear. In tissues other than LRE, Hodgkin's disease and one adrenal with cortical hyperplasia, tartrate-resistant acid phosphatase activity is minimal or ab- sent. When it is present, i t is seen in mesen- chymal cells, such as macrophages in the

lumen of adenocarcinoma and histiocy tes in the lamina propria of the intestine. If these cells are to be related to the cells of LRE or Hodgkin's disease, then the tumor cells in LRE may be considered as histiocytes with maturation arrest at a very early stage. Fur- ther histochemical investigation into these problems may lead to elucidation of the histo- genesis and interrelationship of LRE, malig- nant histiocytosis and malignant lymphoma.

Of the three adrenals in this series, one is that of cortical hyperplasia showing strong is- oenzyme activity. The other two are normal adrenal glands removed for palliation of carci- noma of breast, which are entirely free of the isoenzyme reaction. The very limited number of adrenal specimens in this series precludes any generalization except that presence of tar- trate-resistant isoenzyme in the hyperplastic adrenal on one hand and LRE and Hodgkin's disease on the other suggests an analogy to Reagan isoenzyme, a placental alkaline phos phatase isoenzyme produced ectopically in various tumors.16

164 CANCER January 1972 Vol. 29

Thus, it would appear that the tartrate-re- tissues of a variety of diseases that it can be sistant acid phosphatase isoenzyme is not pres- used as a pathognomonic criterion for the di- ent exclusively in the tumor cells of LRE. agnosis of LRE, a disease warranting a recog- Nevertheless, its presence is so infrequent in nition as a clinicopathologic entity.

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