A case of infantile rhabdomyofibrosarcoma with immunohistochemical, electronmicroscopical, and genetic analyses

CASE STUDIES REFERENCES 1. Enzinger FM, WeissSW (eds): Soft tissue tumors (ed 3). St Louis, MO, CVMosby,1995, pp 431-466 2. Crozat A, Aman P, Mandahl N, et al: Fusion of CHOP to a novel RNA-bindingprotein in human myxoidliposarcoma.Nature 363:640-644, 1993 3. Mentzel T, Fletcher CDM: Lipomatous turnours of soft tissues: An update. VirchowsArch 427:353-363, 1995 4. Dei Tos AP, Doglioni C, Piccinin S, et al: Molecularabnormalities of the p53 pathwayin dedifferentiated liposarcoma.J Pathol 181:8-13, 1997 5. Dei Tos AP, Piccinin S, Doglioni C, et al: Molecular aberrations of the G1-S checkpoint in myxoid and round cell liposarcoma.AmJ Pathol 151:15311539, 1997 6. Pilotti S, Della Torre G, LavarinoC, et al: Distinctmdm2/p53 expression patterns in liposarcoma subgroups: implications for different pathogenetic mechanisms.J Pathol 181:14-24, 1997 7. Schneider-Stock R, Walter H, Rys J, et al: No correlation of c-myc overexpressionand p53 mutations in liposarcomas.VirchowsArch 433:315-321, 1998 8. Schneider-StockR, Walter H, Radig K, et al: MDM2 amplification and loss of heterozygosityat Rb and p53 genes: No simultaneous alterations in the oncogenesisof liposarcomas.J Cancer Res Clin Oncol 124:532-540, 1998 9. Kim NW, Piatyszek MA, Prowse KR: Specific association of human tetomerase activitywith immortal cellsand cancer. Science 266:2011-2015, 1994

10. Schneider-StockR, Epplen JT, Walter H, et al: Telomeric lengths and telomerase activityin liposarcomas.Mol Carcinogen 24:144-151, 1999 11. SambrookJ, FritzschEF, Maniatis T: Molecular Cloning:A Laboratory Manual. Cold Spring Harbor, NewYork, Cold Spring Harbor Laboratory Press, 1989 12. Schneider-StockR, Radig K, Oda Y, et al: P53 gene mutations in soft-tissue sarcomas-correlationswith p53 immunohistochemistry and DNA ploidy.J Cancer Res Clin Onco1123:211-218, 1997 13. Midelman F, ed: 1SCN:An international systemof human cytogenetic nomenclature. Basel,Switzerland,S. Karger, 1985 14. Schneider-StockR, RysJ, Walter H, et al: A rare chimeric TLS/FUSCHOP transcript in a patient with multiple liposarcomas:A case report. Cancer Genet Cytogenet 111: 1-4, 1999 15. Knight JC, Renwick PJ, Dal Cin P, et al: Translocation t(12;16) (ql 3;p11) in myxoid liposarcoma and round cell liposarcoma: Molecular and cytogeneticanalysis.Cancer Res 55:24-27, 1995 16. Sato K, Miyahara M, Saito T, et ah C-myc mRNA overexpression is associated with lymph node metastasis in colorectal cancer. Eur J Cancer 30:1113-1117, 1994 17. FinleyGC, SchulzNT, Hill SA, et al: Expressionof the myc gene family in different stagesof human colorectal cancer. Oncogene 4:963-971, 1989 18. Isfort RJ, Cody DB, Lovell G, et al: Analysis of oncogenes, tumor suppressor genes, autocrine growth factor production, and differentiation state of human osteosarcomacell lines. Mol Carcinog 14:170-178, 1995

A CASE OF INFANTILE RHABDOMYOFIBROSARCOMA WITH IMMUNOHISTOCHEMICAL, ELECTRONMICROSCOPICAL, AND GENETIC ANALYSES HIROSHI MIKI, MD, SHOJI KOBAYASHI, MD, YOSHIO KUSHIDA, MD, MAKIKO SASAKI, MD, REIJI HAS& MD, EIICmRO HmAKAWA, MD, KAORU OGURA, MD, AND MASAm OHMORI, MD

A case of infantile rhabdomyofibrosarcoma arising on the buttocks of a 15-month-old boy is reported Mth histological, immunohistochemical, electronmicroscopical, and cytogenetic findings. Histological examination showed a proliferation of spindle-shaped ceils in a fasciculated pattern, with occasional rounded rhabdomyoblastic cells with abundant eosinophillc cytoplasm. Immunohistochemically, the tumor cells expressed desmin and MyoD1 but were only weakly positive for myoglobin. No clear rhabdomyoblastic features were observed by electronmieroscopic examination. Chromosome analysis showed a clone of 46, XY, der(2)t(2;ll)(q37;q13), different from any karyotypic abnormality in the original report of this neoplasm. Loss of heterozygosity at 11p15.5, the most frequent genetic alteration in embryonal rhabdomyosarcoma, was not detected. The low degree of striated muscle differentiation and tumor localization supported the

diagnosis of infantile rhabdomyofibrosarcoma rather than spindlecell rhabdomyosarcoma in this case. The present case has been uneventful as of 25 months after surgery. The rather long recurrencefree period, which has not been reported in previous cases, may be attributable to chemotherapy-induced rhabdoid differentiation of the tumor cells. HuM PATHOL 30:1519-1522. Copyright © 1999 by W.B. Saunders Company Key words: infantile rhabdomyofibrosarcoma, MyoD1, spindlecell rhabdomyosarcoma. Abbreviations: CT, computed tomography; MRI, magnetic resonance imaging; H & E, hematoxylin and eosin; PAS, periodic acid-Schiff reaction; d-PAS, diastase digestion; PCR, polymerase chain reaction.

Infantile r h a b d o m y o f i b r o s a r c o m a is a fairly n e w clinicopathological entity p r o p o s e d by L u n d g r e n et al, 1 who rep o r t e d 3 cases. To date, only 1 a d d i t i o n a l case has b e e n r e p o r t e d , in the G e r m a n literature. 2 Infantile r h a b d o m y o f i b r o s a r c o m a is c o m p o s e d mainly o f s p i n d l e - s h a p e d cells resembling infantile fibrosarcoma, b u t ultrastructural a n d i m m u n o h i s t o c h e m i c a l e v i d e n c e o f striated muscle d i f f e r e n t i a t i o n in infantile r h a b d o m y o f i b r o s a r c o m a distinguishes it f r o m infantile fibrosarcoma. Also, it is controversial w h e t h e r infantile r h a b d o m y o f i b r o s a r c o m a is d i f f e r e n t f r o m spindle-cell r h a b d o m y o s a r c o m a , a subtype o f e m b r y o n a l r h a b d o m y o s a r c o m a w h i c h shows a similar histological p a t t e r n c o m p o s e d mainly o f e l o n g a t e d s p i n d l e cells, b u t differs in a n a t o m i c a l distribution, d e g r e e o f m y o g e n i c differentiation, a n d prognosis. 3 H e r e we

r e p o r t a case o f infantile r h a b d o m y o f i b r o s a r c o m a in a 15m o n t h - o l d boy with histological, i m m u n o h i s t o c h e m i c a l , elect r o n microscopical, a n d g e n e t i c findings a n d discuss differe n c e s b e t w e e n infantile r h a b d o m y o f i b r o s a r c o m a a n d spindlecell r h a b d o m y o s a r c o m a .

From the Departments of Pathology and Pediatric Surgery, Kagawa Medical University, Miki, Kagawa, Japan. Address correspondence and reprint requests to Hiroshi Miki, MD, Department of Pathology, Kagawa Medical University, 1750-1 Ikenobe, Miki, Kita, Kagawa 7610793,Japan. Copyright © 1999 by W.B. Saunders Company

0046-8177/99/3012-0023510.00/0

CLINICAL SUMMARY A 15-month-old boy p r e s e n t e d at t h e Kagawa Medical University H o s p i t a l , J a p a n with a painless mass o n the buttocks that h a d b e e n n o t i c e d 4 m o n t h s b e f o r e a n d h a d rapidly i n c r e a s e d in size over that period. C o m p u t e d t o m o g r a p h y (CT) a n d m a g n e t i c r e s o n a n c e i m a g i n g (MRI) s h o w e d a well d e m a r c a t e d mass o f 7 c m in diameter, mainly involving the r i g h t gluteus muscle a n d e x t e n d i n g into the pelvis. Results o f l a b o r a t o r y tests were within n o r m a l range, i n c l u d i n g t u m o r m a r k e r s such as CEA, CA19-9, AFP, a n d NSE. Histological e x a m i n a t i o n o f the biopsy s p e c i m e n s h o w e d a p r o l i f e r a t i o n o f s p i n d l e - s h a p e d cells, a n d infantile f i b r o s a r c o m a o r m a l i g n a n t m y o g e n i c t u m o r was suspected. C h e m o t h e r a p y consisting o f carboplatin, vincristine, a c t i n o m y c i n D, c y c l o p h o s p h a m i d e , a n d t h e r a r u b i c i n was a d m i n i s t e r e d , b u t t h e size o f the t u m o r

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did not reduce remarkably. Then, surgical resection of the tumor was performed, accompanied by intraoperative irradiation (20 Gy). Although no further chemotherapy or radiotherapy was given after the operation, there has been no evidence of local recurrence or distant metastasis for 25 months. MATERIALS AND METHODS For light microscopy, samples were fixed in 10% buffered formalin and e m b e d d e d in paraffin, then stained with hematoxylin and eosin (HE), periodic acid-Schiff reaction (PAS) with diastase digestion (d-PAS), Azan Mallory for collagen fiber, and silver impregnation for reticulin fiber. For immunohistochemistry, paraffin-embedded thin sections were prepared and streptavidin biotin was applied using a SAB-PO kit (Nichirei Co, Tokyo, Japan). Antibodies used were the following: anti-myoglobin (DAKO, Glostrup, Denmark), anfi-desmin (DAKO), anti-vimentin (DAKO), anti~smooth muscle actin (DAKO), anti-S-100 protein (DAKO), anti-neuron specific enolase (DAKO), anti-factor VIII (DAKO), anti-CD34 (Novocastra, Newcastle, UK), and anti-MyoD1 (DAKO). For electron microscopy, small pieces of formalin-fixed specimens were re-fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide solution, and then e m b e d d e d in Epon 812. After staining with uranyl acetate and lead citrate, ultrathin sections were observed with a JEOL JEM-200 CX electron microscope (Jeol Ltd, Tokyo,Japan). For chromosome analysis, cultured cells from the tumor were treated with colcemide at 500 n g / m L and were analyzed with the Giemsa-banding technique. For analysis of loss of heterozygosity at 11p15.5, tumor cells and the surrounding normal cells were separately collected by microdissection from formalin-fixed and paraffne m b e d d e d tissue. DNA was obtained by standard proteinase K digestion and phenol-chloroform extraction. Microsatellite markers, DllS922, DllS932, and DllS988, (Research Genetics, Huntsville, AL) were used. Polymerase chain reaction (PCR) mixture was prepared using Taq DNA polymerase according to the manufacturer's instructions (Perkin Elmer, Foster, CA). The conditions for PCR were 10 cycles at 95 °C for 50 seconds, 55°C for 40 seconds, and 72°C for 120 seconds, 25 cycles at 95°C for 40 seconds, 55°C for 30 seconds, and 72°C for 60 seconds. PCR products were electrophoresed on a 10% to 20% gradient polyacrylamide gel and visualized with silver staining (Dalichikagaku, Tokyo,Japan). PATHOLOGICAL FINDINGS

Biopsy specimen The biopsy specimen showed a uniform proliferation of spindle-shaped cells with interstitial collagen (Fig 1), and myxoid matrix was observed in some areas. Mitotic activities were about 10 per 10 high power fields in highly cellular areas. Rhabdomyoblastic cells were not found in the biopsy specimen. Immunohistochemically, desmin was positive in many tumor cells, but myoglobin was negative in this specimen.

Surgical specimen Macroscopically, the tumor was 7.0 × 5.5 cm in size and well circumscribed. The cut surface was white and showed massive necrosis. Microscopically, the tumor was dominated by a highly cellular spindle-cell component arranged in a fasciculated pattern. In most of the fascicular areas, abundant collagen was found between the tumor cells. In some of the highly cellular areas, tumor cells were more primitive; that is, r o u n d or oval with sparse, poorly outlined, cytoplasmic borders. In some areas, a prominent myxoid matrix and vascular rich areas were observed. Spindle-shaped tumor cells had a

FIGURE 1. A uniform proliferation of spindle-shaped cells in a fasciculated pattern in a biopsy specimen (hematoxylin-eosin, magnification ×330). predominantly elongated nucleus with moderate pleomorphism. Occasionally, r o u n d e d rhabdomyoblastic tumor cells with abundant eosinophilic cytoplasm were found, which were not observed in the biopsy specimen (Fig 2). Spindle-shaped and r o u n d tumor cells had vesicular nuclei with 1 or 2 prominent nucleoli, and they lacked evident cross-striations. Mitotic activity ranged from 0 to 10 mitoses per 10 high power fields. PAS reaction showed cytoplasmic reactivity in the r o u n d e d cells and some of the spindle cells, and PAS-positive granules were eliminated by diastase digestion. Immunohistochemically, vimentin was positive in most tumor cells. Desmin was positive in the cytoplasm of the round cells and some of the spindle cells. MyoD1 was positive in most rhabdomyoblastic cells and some of the spindle cells, but myogiobin was only weakly positive in a few r o u n d cells. S-100 protein, neuron specific enolase, c~-smooth muscle actin, factor VIII, and CD34 were negative. Ultrastructurally, most tumor cells showed fibroblastic features; that is, elongated nuclei and cytoplasm containing abundant rough endoplasmic reficulum. The cells were surrounded by large amounts of collagen. Some of the tumor cells were less differentiated. Occasionally, large cytoplasm containing thick and thin filaments in a parallel arrangement forming a dense body was observed (Fig 3). Chromosome analysis showed a clone of 46, XY, der(2) t(2; 11) (q37;q13), which was different from any karyotypic abnormality described in the first report by Lundgren. 1 No loss of

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FIGURE 2. Mixed round rhabdomyoblastic tumor cells with abundant eosinophilic cytoplasm in a surgical specimen (hematoxylin-eosin, magnification ×660).

heterozygosity at 11p15.5 was detected in the tumor cells compared to normal control ceils. DISCUSSION Infantile rhabdomyofibrosarcoma is a very rare clinicopathological entity proposed by Lundgren in 2993, I and since then only one case has been added to the literature. 2 This tumor shows spindle cell proliferation with desmoplastic portions simulating infantile fibrosarcoma. Cross striation is not observed. Immunohistochemically, the tumor cells express vimentin and desmin but not myoglobin. Ultrastructurally, only a portion of the tumor cells in 2 of the 3 cases in the original report displayed the features of rhabdomyoblasts, with sarcomere-like structures formed by thin and thick filaments of actin and myosin types, and a distinct Z band was noted only in 1 of these 2 cases. Prognostically, 3 of the total of 4 patients reported before this current report experienced metastasis and died within 3 years of the primary operation; the 1 surviving patient has local recurrence. In summary, infantile rhabdomyofibrosarcoma can be defined as a childhood spindle-cell sarcoma with a low degree of rhabdoid differentiation and with aggressive clinical behavior. Spindle-cell rhabdomyosarcoma is a subtype of embryonal rhabdomyosarcoma, which is also histologically characterized by fasciculated arrangement of spindle cells, and it has been controversial whether infantile rhabdomyofibrosarcoma and spindle cell sarcoma are identical.4 However, differences 1521

have been pointed out between spindle-cell rhabdomyosarcoma and infantile rhabdomyofibrosarcoma in spite of the morphological similarity. First, a high degree of striated muscle differentiation is evident in spindle-cell rhabdomyosarcoma; that is, cross striation is usually observed.3,5.6 Immunoreactivity for myoglobin is uniformly and strongly shown s,5,6 and ultrastructurally, most tumor cells have both thick and thin filaments with Z-bands.6 Second, spindle cell rhabdomyosarcoma is marked by a low malignant potential. Cavazzana et al 3 reported that 16 of 17 patients were well and alive 24 to 101 months after diagnosis, which is in contrast to the poor prognosis in infantile rhabdomyofibrosarcoma. Third, a strong predilection for the paratesticular or head and neck areas of boys and young m e n has been observed in spindle-cell rhabdomyosarcoma, 3 whereas no such predilection has been noted in infantile rhabdomyofibrosarcoma, although the n u m b e r of cases is too limited to draw a conclusion. Cytogenetically, 2 of the 3 cases of infantile rhabdomyofibrosarcoma in the original report showed monosomy of chromosome 19 and 22 and other various abnormal karyot~]~es.1 The present case showed a karyotype of 46, XY, der(2)t(2;ll) (q37;q13), which was not present in the original report. It is also different from t(2;13)(q37;q14), the most common chromosomal abnormality in alveolar rhabdomyosarcoma. 7 Loss of heterozygosity at chromosome 11p15.5 has been reported to characterize the embryonal subtype of rhabdomyosarcoma, s However, our case showed no evidence of loss of heterozygosity at 11p15.5. Unfortunately, cytogenetic analysis thus far has not seemed to be helpful in differentiating among various types of rhabdomyosarcoma, including the spindle-cell type. Interestingly, in the present case, rhabdomyoblastic cells with round and ample eosinophilic cytoplasm were only found in the surgical specimen. One possible reason for this is that the chemotherapy may have induced differentiation of the tumor cells, as has been described in rhabdomyosarcoma. 9 Still, no clear cross striation was found even in the rhabdomyoblastic cells. Ultrastructurally, most tumor cells showed fibroblastic features and not rhabdomyoblastic features, l m m u n o histochemically, myoglobin was only weakly positive in a few rhabdomyoblastic cells; however, MyoD1 was clearly expressed in most rhabdomyoblastic cells and in a small n u m b e r of spindle cells. MyoD1 was also positive in the fourth case reported from Germany. MyoD1 is a DNA-binding nuclear protein that initiates muscle differentiation in mesenchymal stern cells at the

FIGURE 3. Large cytoplasm containing thick and thin filaments in a parallel arrangement with dense bodies (magnification x 14000).

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earliest stage of c o m m i t m e n t to a striated muscle phenotype. Its expression has proved a most sensitive m a r k e r for detection of myogenic differentiation, especially in cases with a low degree of differentiation. 1°'11 In the present case, even some spindle cells were positive for MyoD1, as well as most rhabdomyoblastic cells. Taken t o g e t h e r with the weak or lack of reaction with conventional striated muscle markers in the r e p o r t e d cases, this indicates that in infantile rhabdomyofibrosarcoma the t u m o r cells are at the early stage of striated muscle differentiation. Because of the a b o v e - m e n t i o n e d less evident striated muscle differentiation c o m p a r e d with typical spindle-cell r h a b d o m y o s a r c o m a and of the t u m o r location that is different f r o m the paratesticular or h e a d and neck region, we diagnosed this case as infantile rhabdomyofibrosarcoma. However, the lack of evidence of local r e c u r r e n c e or distant metastasis in this case is n o t in accordance with previous cases. This may be owing to the different timing of chemotherapy. In our case, c h e m o t h e r a p y was given preoperatively with successful induction of differentiation, with the appearance of rhabdomyoblastic cells. All of the previously r e p o r t e d cases were postoperatively treated. A m o n g those in the first report, 2 received t r e a t m e n t after metastatic or local recurrence, and 1 after i n c o m p l e t e removal of the primary t u m o r ) R e c u r r e n c e was not n o t e d at the time of postoperative t r e a t m e n t in the patient that was later reported, 2 b u t this patient died of t u m o r r e c u r r e n c e 3 years after surgery. A l t h o u g h the stage at diagnosis should be considered and f u r t h e r follow-up is r e q u i r e d to warrant a conclusion, the present case suggests the advantage of preoperative chemotherapy in infantile rhabdomyofibrosarcoma. O u r case also suggests that considering the m o r p h o l o g i cal similarity, infantile r h a b d o m y o f i b r o s a r c o m a and spindlecell r h a b d o m y o s a r c o m a may be within a s p e c t r u m of rhabdomyosarcoma of spindle-shaped cells, with a low degree of myogenic differentiation and p o o r prognosis at one e n d and a high degree of differentiation and favorable prognosis at the

other. T h e present case may fall between infantile rhabdomyofibrosarcoma and spindle-cell rhabdomyosarcoma, presenting a low d e g r e e of striated muscle differentiation but a g o o d prognosis, although it may have b e e n affected by the stage at diagnosis and preoperative chemotherapy. As the n u m b e r of cases of infantile r h a b d o m y o f i b r o s a r c o m a is very limited, further accumulation and analysis of similar cases are called for to d e t e r m i n e w h e t h e r infantile r h a b d o m y o f i b r o s a r c o m a is different f r o m spindle-cell rhabdomyosarcoma.

REFERENCES 1. Lundgren L, AngervallL, Stenman G, et al: Infantile rhabdomyofibrosarcoma: A high-grade sarcoma distinguishable from infantile fibrosarcoma and rhabdomyosarcoma. HUMPATHOL24:785-795, 1993 2. Mentzel T, Mentzel HJ, Katenkamp D: Infantiles Rhabdomyofibrosarkom: Ein aggressiverTumorim Spektrum der Spindelzentumoren im Kindesalter. Pathologe 17:296-300, 1996 3. CavazzanaAO, Schmidt D, Ninfo V, et al: Spindle cell rhabdomyosarcoma: A prognostically favorable variant of rhabdomyosarcoma. Am J Surg Patho116:229-235, 1992 4. Rosai J: Soft tissues, in Gery L, Joiner P (eds): Ackerman's Surgical Pathology. St Louis, MO, Mosby, 1996, pp 2080-2087 5. Edel G, Wuisman P, Erlemann R: Spindle cell (leiomyomatous) rhabdomyosarcoma:A rare variant of embryonal rhabdomyosarcoma. Pathol Res Praet 189:102-107, 1993 6. Leuschner I, Newton WA, Schmidt D, et al: Spindle cell variants of embryonal rhabdomyosarcoma in the paratesticular region: A report of the intergroup rhabdomyosarcoma study,AmJ Surg Pathol 17:221-230, 1993 7. Enzinger FM, Weiss SW: Soft tissue tumors, in Gay SM, Gery L (eds): Rhabdomyosarcoma. St Louis, MO, Mosby, 1995, pp 539-577 8. ScrableH, Witte D, Shimada H, et al: Molecular differential pathology of rhabdomyosarcoma. Genes Chromosomes Cancer 1:23-35, 1989 9. Molenaar WM, OosterhuisJW, Kamps WA: Cytologic"differentiation" in childhood rhabdomyosarcomas following polyehemotherapy. Hu~ PATHOL 15:973-979, 1984 10. Dias P, Parham DM, Shapiro DN, et al: Myogenic regulatory protein (MyoD1) expression in childhood solid tumors: Diagnosticutilityin rhabdomyosarcoma. AmJ Pathol 137:1283-1291, 1990 11. Tallini G, Parham DM, Dias P, et al: Myogenic regulatory protein expression in adult soft tissue sarcomas. A sensitive and specific marker of skeletal muscle differentiation. AmJ Pathol 144:693-701, 1994

BOOK REVIEWS Organelle Diseases: Clinical Features, Diagnosis, Pathogenesis and Management. D.A. Applegarth, J.E. Dimmick, J.G. Hall. NewYork, NY, C h a p m a n & Hall Medical, 1997 $150.00. With this u n i q u e b o o k on organelle diseases, the editors outline the function and associated diseases of lysosomes, perioxosomes, and m i t o c h o n d r i a with m i n o r emphasis o n o t h e r subcellular organelles. With i m p r o v e d enzyme analysis, m o l e c u l a r studies, cytogenetic evaluation, and ultrastructural observation, diagnosis of disease secondary to subcellular dysfunction is possible and available in most major medical centers. It is now possible to provide prenatal diagnoses, d e t e r m i n e familial susceptibility, and even provide therapeutic and palliative t r e a t m e n t in several disorders associated with lysosomes, peroxisomes, and mitochondria. As n o t e d by the editors, this textbook deals with lysosomes, peroxisomes, and m i t o c h o n d r i a and only tangentially with o t h e r subcellular structures, w h e n these have specific features which are seen with abnormalities in lysosomal, peroxisomal, and m i t o c h o n d r i a l diseases. T h e b o o k is divided into the following parts: (a) Lysosomal Disorders; (b) Peroxisomal Disorders; (c) Mitochondrial Disorders; and (d) Practi-

cal Issues. T h e first 3 parts are divided into chapters which provide (a) the basic science, biology and pathobiology of the organelle and associated diseases, (b) clinical diagnostic features; (c) laboratory, biochemical, and m o l e c u l a r genetic aspects in diagnosis; and (d) pathology of the associated organelle diseases. T h e final part is divided into chapters presenting: (a) therapy for organelle disorders; (b) the use of family history, p e d i g r e e and genetic counseling; (c) physical examination; and (d) autopsy considerations in metabolic disease. Also i n c l u d e d are appendices dealing with D N A banking consent, various metabolic organizations' addresses and websites, and a very inclusive glossary of metabolic disease terms. T h e chapters are well illustrated with easy to follow tables, illustrations and diagrams of metabolic disease pathways, clinical p h o t o g r a p h s of affected patients, and a wide variety of anatomic pathology micrographs, including electronmicrographs. T h e quality of these are quite good, and the r e a d e r can readily identify the c o n c e p t that the chapter authors are presenting without re-reading the text or re-reviewing illustrative material. T h e b o o k is multi-authored by well recognized individuals in all aspects of m e t a b o l i c / o r g a n e l l e diseases. T h e

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