EXPERIMENTAL AND MOLECULAR PATHOLOOY 47,69-75 (1987)

Giant Mitochondria in Acute Lymphocytic Leukemia MJTSUOKI EGUCHI,~,~ YOSHIHIKO IwAMA,~ FUTAHA OCHIAI,~

KOHJI ISHIKAWA,~ HITOSHI SAKAKIBARA,’ HISASHI SAKAMAKI,~ AND TOSHIHARU FURUKAWA~

‘Second Department of Pediatrics and 2Third Department of Internal Medicine, Dokkyo University School of Medicine, Mibu-machi, Tochigi-ken, 321-02 Japan

Received July 7, 1986, and in revisedform January 5, 1987

Giant mitochondria were observed in 2 cases among 28 cases of ALL by electron micros- copy. The cristae of the giant mitochondria in the leukemic cells were irregularly arranged, decreased in number, and formed concentric circles. Several morphological abnormalities were also observed in the normal mitochondria. Morphometric analysis of the mitochondria in the 2 patients disclosed that the sizes of mitochondria were well distributed from small to large and thus, the mitochondria could not be divided into different populations. Also, there were no clear differences in the distribution of shape between normal and giant mitochon- dria. These results suggest that the giant mitochondria were derived from normal mitochon- dria. Since they were observed before the initial treatment, they did not developed as a result of drug action. 0 1987 Academic Press, Inc.

INTRODUCTION

Grossly enlarged mitochondria have been reported in a variety of tissues under physiologic and pathologic conditions, and the term “giant mitochondria” has been employed to describe them. In the reports on pathologic conditions, the giant mitochondria were associated mainly with alcohol-induced liver disorders, mitochondrial myopathy, and drug intoxication (Albring et al., 1973; Hayder et al., 1971; Koch et al., 1976; Wagner and Rafael, 1977). The relation between neoplastic cells and abnormal mitochondrial metabolism has been discussed, and several ultrastructural studies have disclosed morphological abnormality in- cluding enlargement of mitochondria in neoplastic cells (Djaldetti, 1982; Firkin and Clark-Walker, 1979; Graham and Bemier, 1975; Horoupian, 1980; Tandler and Erlandson, 1983; Watanabe et al., 1976; White et al., 1974; Wilkie et al., 1983; Zafer et al., 1982). However, little is known about giant mitochondria in leukemic cells (Bessis and Breton-Gorius, 1969; Huhn et al., 1984; Schumacher et al., 1975). In the present study, we observed giant mitochondria in two patients with ALL. These mitochondria exhibited very unusual cristae formation, which has not been described in leukemic cells.

MATERIALS AND METHODS

Bone marrow aspirants from 28 patients with ALL (1 year old to 72 years old) were fixed with glutaraldehyde and osmium tetroxide before initial chemotherapy and embedded in epoxy resin for electron microscopy. Giant mitochondria with abnormal cristae were observed in two patients and were quantitatively analyzed on electron microscopic photographs. The cross-sectional area and circumfer- ence of mitochondria in 20 cell profiles in each case, totally about 200 mitochon-

3 To whom reprint requests should be addressed at The Second Department of Pediatrics, Dokkyo University, School of Medicine, Mibu-machi, Tochigi-ken 321-02 Japan.

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0014-4800/87 $3.00 Copyright 6 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

70 EGUCHI ET AL.

dria per patient, were measured by a computer-aided image analyzer with a digi- tizer and were compared with mitochondria in normal lymphocytes.

The mitochondrial shape index was calculated by the formula: circumference*/ area (Eguchi et al., 1977). A completely circular cross section has an index of (21rR)*/nR* = 47~ = 12.56. This is the theoretical minimal index, The mitochon- drial shape index increases as the shape becomes irregular or elongated.

The distribution of RNA was determined by the RNase-gold complex method of Bendayan (1981) to differentiate the giant mitochondria from the analogous cytoplasmic organelle, which is RNA positive.

Case Report

Case I. A 13-year-old boy was admitted because of bilateral knee joint pain and anemia. On admission, he showed generalized lymphadenopathy. The liver and the spleen were not enlarged. Examination of the blood revealed RBC of 169 x 104/~1, hemoglobin 5.1 g/dl, hematocrit 16%, platelet 1.3 x 104/pl and WBC 34,000/~1 with 78% blast. May-Giemsa staining of the blasts revealed pink inclu- sions in the cytoplasm. The inclusions presumably corresponded to the giant mi- tochondria because the giant mitochondria was the only cell organelle of equal size by electron microscopic observation. The blasts were negative for peroxi- dase, PAS, and T- or B-cell markers. He was diagnosed as having nonB-nonT type of ALL. Induction therapy with vincristine, prednisolone, cyclophospha- mide, and adriamycin was successful. He was in complete remission for 8 months and then relapsed.

Case 2. A 6-year-old boy was admitted because of a 4-day history of abdominal pain and general fatigue. On admission, he showed hepatomegaly and generalized small lymph node swelling. Routine hematological data were as follows: RBC of 509 x 104/tJ, hemoglobin 13.2 g/d& hematocrit 40.6%, reticulocyte l.l%, platelet 123,000/~1, WBC 13,OOO/pl with 15% band forms, 52% segmented polymorpho- nuclear cells, 21% lymphocytes, 4% monocytes, 3% eosinophils, and 4% blasts. Bone marrow examination showed marked blastosis (cell count 11.0 x 104/p,l with 88% blasts). The blasts had pink inclusions in the cytoplasm by May- Giemsa staining, and were negative for peroxidase, acid phosphatase, and PAS but positive for surface IgM. The diagnosis of B-cell ALL was established. In- duction therapy with cyclophosphamide, vincristine, and methotrexate was suc- cessful, and he has been in complete remission for 1 year.

RESULTS

Case 1. The nuclei of blasts displayed round to irregular forms with dispersed chromatin. Mitochondria were the predominant cell organelles in the scant cyto- plasm (Fig. 1). The size of the mitochondria varied. In a single cell profile, some of them seemed to be normal while others were several times larger than normal. In the giant mitochondria most of the cristae formed concentric circles (Figs. 2 and 3), while some of them partially lacked the cristae (Fig. 4). An increased number of cristae, which is known to be the result of fusion or suppressed mitosis of mitochondria (Wakabayashi et al., 1979; Wakabayashi, 1982), was not ob- served in the giant mitochondria.

Normal-size mitochondria occasionally exhibited irregularly arranged cristae but did not display the concentric circles. Dense granules were not observed in

GIANT MITOCHONDRIA IN ALL 71

FIG. 1. A leukemic lymphoblast from case 1. Cristae of giant mitochondria exhibit concentric circles (arrow). Reduced cristae are observed in normal and giant mitochondria (arrowheads). x 8500.

FIG. 2. Higher magnification of mitochondria with concentric circles in case 1. x 19,600. FIG. 3. Double membrane structure is clearly observed in a giant mitochondrion and nearby small

mitochondria. Case 1. x 28,700.

72 EGUCHI ET AL.

mitochondria. The giant mitochondria were mainly round or oval, and only a few of them were irregularly shaped (Fig. 2).

The RNase-gold complex method showed almost no RNA in the giant mito- chondria as in normal mitochondria (Iwama et al., 1984) (Fig. 5). This differen- tiates giant mitochondria with concentric circles from analogous cytoplasmic or- ganelles such as coiled rough endoplasmic reticulum or ribosome lamellar com- plex, which are rich in RNA.

Quantitative analysis disclosed that the sizes of the mitochondria were well distributed from small to large, hence they could not be divided into different populations (Fig. 8). There also seemed to be no difference in the shape index between them.

Case 2. Blast cells had round or indented nuclei with poorly condensed chro- matin. The cytoplasm was scant. Normal and enlarged mitochondria coexisted in a single cell profile. Normal-size mitochondria showed regularly arranged cristae but the enlarged mitochondria showed a decreased number of cristae or irregu- larly formed cristae (Figs. 6 and 7). Cristae with concentric circles were not fre- quently observed compared with Case 1.

Morphometric observation revealed that the mitochondria could not be divided

FIG. 4. A racket-shaped mitochondrion possesses cristae in the handle portion. Most parts lack cristae formation. Case 1. x 29,400.

FIG. 5. The RNase-gold complex method on an unosmificated specimen. There are very few gold particles in the mitochondria (arrows). Numerous gold particles are observed in ribosome-rich cyto- plasm. Case 1. X31,500.

FIG. 6. Giant mitochondrion with sparse cristae in case 2. Arrow indicates a crista of the mitochon- drion. ~21,400.

FIG. 7. A mitochondrion with irregularly shaped cristae in case 2. x 25,900.

GIANT MITOCHONDRIA IN ALL 73

into two populations by size as in Case 1, and the shape index of the giant mito- chondria did not indicate increased irregularity (Fig. 8).

DISCUSSION Giant mitochondria should be differentiated from swollen mitochondria that are

fixation artifacts. Swollen mitochondria show a patchy electron lucent appear- ance, while the giant mitochondria do not; thus, the enlarged mitochondria in the present study were giant mitochondria, and not fixation artifacts.

Giant mitochondria have been observed in several pathological conditions (Albring et al., 1973; Dallman and Goodman, 1970; Dallman and Goodman, 1971; Hayder et al., 1971; Horoupian, 1980; Tandler, 1983; Wagner and Rafael, 1977; Wakabayashi, 1982). However, there are few reports on giant mitochondria in leukemic cells (Bessis and Breton-Gorius, 1969; Huhn et al., 1984; Schumacher et al., 1975) and little is known about their mechanism of development. Bessis and Breton-Gorius (1969) observed giant mitochondria in 1 out of 15 cases of AML. They classified the mitochondria into two different types: normal and giant.

shape Case 1

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FIG. 8. Distribution of mitochondria according to size and shape. The size of mitochondria repre- sents the area measured on electron micrographs. The shape index was calculated by the formula: circumference%rea. The mitochondrial shape index increases as the shape receding from a circle.

74 EGUCHI ET AL.

Similarly, we found giant mitochondria coexisting with normal-size mitochon- dria in a single cell profile. However, they could not be quantitatively classified into two groups, and qualitatively, some of normal size exhibited abnormal cristae arrangements resembling those seen in giant mitochondria. Therefore, the giant mitochondria were not independent of normal mitochondria, but originated from them.

Ghadially and Skinnder (1974) reported giant mitochondria in macrophages from leukemic patients even when the leukemic cell did not contain giant mito- chondria. As the giant mitochondria were observed after medication, it was con- cluded that the giant mitochondria in leukemia were the result of drug action rather than the disease itself (Bessis and Breton-Gorius, 1969; Ghadially and Skinnder, 1974). The present observations were made before the initial treatment; therefore, the action of drugs could not be involved in the development of the giant mitochondria.

Three possible mechanisms have been considered for the formation of giant mitochondria (Wakabayashi, 1982). The first is excess of cristae and/or matrical materials due to abnormal metabolism (Wakabayashi, 1982; Wilkie er al., 1983). The second is suppression of division of mitochondria (Albring et al., 1973; Wanger and Rafael, 1977). The third is fusion of existing mitochondria by the outer membrane (Asano et al., 1978; Wakabayashi er al., 1979). Constricted mi- tochondria, or mitochondria with an isolation membrane, are observed in the second or the third mechanism. These giant mitochondria probably had increased shape indexes. However, as shown in Fig. 8, the giant mitochondria possessed shape indexes similar to those of normal mitochondria, in other words, irregu- larity of giant mitochondria was not increased. Also the cristae were rather de- creased in some giant mitochondria. From these results, it is difficult to conclude whether the giant mitochondria in ALL were formed by suppression of division or by fusion. The giant mitochondria in. the present cases appear to have been formed by abnormal metabolism in the leukemic cells (Firkin and Clark-Walker, 1979; Zafer et al., 1982). This abnormality also produced concentric circles or a reduction of cristae, which was not observed in giant mitochondria in patients with alcohol-induced liver disorders or other diseases.

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