Problems in the Identification of Fatty Tumors by Histochemical Procedures*

WILLIAM E. DELANEY, M.D.

Dr. Francis W. Baldwin Laboratories, St. Vincent’s Hospital and Medical Center of New York, and the Department of Pathology of the New York University School of Medicine, New York, NY 10011

For decades scientists and physicians have qualitative and quantitative chemical dif been perplexed by the relation of lipids to ferences in lipid content between areas of tumors for many reasons, the least of which neoplastic necrosis and areas of viable neo is the fact that most carcinogens are lipid- plastic cell growth have been interpreted as soluble. Basically, three problems have evidence that the lipid content in neoplasm blocked the acquisition of new knowledge is at least in part integral, rather than de of the lipid histochemistry of tumors; first, generative. controversy exists as to the role of de Certainly in the case of tumors which con generative process in the occurrence of tain large amounts of triglycerides in vac lipids in tumors; second, the peculiar uoles which ocoupy the greatest part of the physicochemical properties of lipids in volume of the neoplastic cell, there is no tissues make the task of qualitative and controversy that the lipid in these fatty quantitative analysis difficult; and third, tumors is integral, rather than degenerative. there is a lack of specific histochemical The phrase “fatty tumors” refers to lesions procedures for lipids. derived from adipose tissue, which may be defined as that specialized connective tissue Distinction Between Adipose and adapted to store triglycerides and other Non-adipose Tumors lipids as a mechanism of either insulation and energy storage (white adipose tissue) The pathogenesis of lipid accumulation in or heat producing activity (brown adipose tumors has been the subject of much de tissue). Neoplasms arising from adipose bate. Evidence for and against the role of tissue may be benign or malignant and are degeneration in the occurrence of lipid in classified as to brown or white fat type as tumors has been summarized by Wolman.43 in table I. The pathological features of Physical differences in neoplastic cell and these tumors have been reviewed in other stromal cell lipid in the same tumor and publications10’15 and are not within the scope of this paper. * Presented at the Applied Seminar on the Clinioal Pathology of the Lipids, November, 1971. Lipid is also present in tumor cells as a 217 218 DELANEY

TABLE I terol metabolism in neoplastic epithelial T u m o r s o f A d ip o s e T is s u e cells.37 Benign Malignant Problems in Lipid Histochemistry Hibernoma Malignant hibernoma (?) ( “fetal lipoma” ) The usual problems in histochemistry granular cell lipoma) are accentuated by the peculiar physical Lipoma Liposarcoma and chemical features of lipids in a basi Lipomatosis Myxoid ( “embryonal lipoma” ) cally aqueous environment, which are out- Lipoblastoma Non-myxoid Benign mesenchy Malignant mesenchy moma moma TABLE II H u m a n N o n -a d ip o s e T u m o r s R e p o r t e d t o C o n t a in L i p i d ®

reflection of functions other than storage or Epithelial heat production. The fact that all human Adenocarcinoma of breast, stomach, colon, rec tum, pancreas, gallbladder, ovary, kidney, cells possess membrane systems which are prostate and thyroid lipid in character and that cells metabolize Carcinoma of lung (poorly differentiated) Squamous cell carcinoma of skin, mouth, pharynx, fatty acids within their mitochondria and esophagus and uterine cervix microsomes13 is reason enough to explain Granulosa-theca cell tumor of ovary the appearance of fat in neoplastic lesions Seminoma of testis Malignant melanomat of non-adipose tissue. A wide variety Adrenal cortical adenoma (table II) of epithelial and non-epithelial Parathyroid adenoma Warthin tumor of parotid tumors have been reported to contain Lipid cell tumors of the ovary lipid.3’ 8’ n ’ 13’18’ 22>45 Identification of these Mesodermal lesions as non-adipose in origin is not or Sarcoma botryoides of uterus and urinary bladder dinarily difficult because of the distinctive Alveolar soft part sarcoma morphological features of the lesions in Leiomyosarcoma J Rhabdomyosarcoma! volved. However, in poorly differentiated Granular cell myoblastoma lesions it may be very difficult to distinguish Osteogenic sarcoma Chondrosarcoma adipose from non-adipose tumors on struc Chordoma tural grounds alone and the presence of Giant cell tumor of bone Meningioma lipid (usually by means of physical dye solu Burkitt’s lymphoma bility) may be very difficult to demon Reticulum cell sarcoma! Hodgkin’s disease (R-S cells) strate; indeed, it is claimed that some lipo- Acute myeloid leukemia sarcomas may not store lipid15 while fat Letterer-Siwe disease may be present in sarcomas of non-adipose Plasma cell myeloma origin.13 The problem is even more difficult * Adapted from Apifel and Baker, 3 Chang, at the ultrastructural level where morpho Speece and Russel, 8 Dobrogorski and Braun- logical and chemical distinctions are ob stein, 11 Elizalde and Korman, 13 Fisher, 18 Hadjio- loff, Tzarnowchanov and Georgieva, 22 and scured. Akin to the changes consistently Wright.45 observed in human hepatomas, an animal f Specifically denied by certain of the quoted model is available to study the changes in papers. | Reported only in a single publication (Elizalde the feedback system for control of choles and Korman) . 13 IDENTIFICATION OF FATTY TUMORS BY HISTOCHEMICAL PROCEDURES 219 lined in table III. By far the bigger of mended that fixation be employed to pre these factors are the physical factors con vent the displacement of hydrophobic lipids cerned with not only maintaining the and to aid in localization of tissue lipids in localization of lipids in their natural posi situ. Formaldehyde is not the fixative of tion in the cell, but also insuring that it choice because of loss of polar lipids, but has not been altered from its physiological rather the formol-calcium solution of Baker4 condition; should either of these factors not which probably acts by stabilizing free be met, the substantive nature of the reac fatty acids and phospholipids. By thin layer tions would be open to question. Com chromatography, it has been shown that plicating the problem is the fact that the water extracts most polar lipids from un lipids themselves have different physical fixed sections but not from formol-caloium features, the most important of which pro fixed sections.33 Sodium ions may also be duces a division30 into hydrophilic types used to solubilize the less hydrophilic polar (such as phospholipids especially, but also lipids.44 Acrolein has also been recom acidic and neutral glycolipids) and hydro- mended as a good fixative for lipid histo phobic varieties (notably triglycerides, chemistry particularly in preventing loss of waxes and cholesterol esters), based on the polar lipids but produces some morpho presence or absence of a polar structure.1 logical disruption.16 Artefact formation may Although the time-honored method of be a problem in lipid histochemistry of avoiding problems of fixation is performance neural substances, evidenced by “myelin of frozen sectioning prior to lipid histo- bud” structures of phospholipid origin, due chemical reactions, it is generally recom- to miscibility factors. Some polar lipids are lost on prolonged formalin fixation due to

TABLE III slow hydrolysis of phosphoglycerides to P r o b l e m s i n t h e H istochemical I dentification water soluble substances but cholesterol, o f T u m o r s o f A d ip o s e T is s u e phosphoinositides and sphingolipids are not

I. PHYSICAL hydrolyzed.21 A. Permanent localization of lipids in physio The classical lipid fixative is osmium logical condition—Substantivity of reactions tetroxide, which acts by binding unsaturated 1. Fixation Problems lipids; triglycerides are then densely or a. Displacement of hydrophilic or hydro phobic lipids lightly osmiophilic, depending on their b. Artefact formation degree of unsaturated fatty acid content. c. Special E.M. fixative problems Osmium tetroxide preserves hydrophilic 2. Diffusion Problems a. Lipid—lipid phospholipids but in a condition unsuit b. Lipid—solute able for histochemical studies.5 However, c. Lipid—solvent osmium tetroxide and potassium perman 3. Embedding Problems ganate do not prevent neutral lipid extrac B. Permeability factors 1. Polarity and miscibility tion during embedding for electron micros 2. Melting point copy, even though most phospholipids are 3. Masking not lost.27 Glutaraldehyde fixation followed II. CHEMICAL by osmication24 or formol-calcium fixation A. Specificity of reactions with post-chromation7 have been recom B. Recommended routine methods mended for electron microscopy. 220 DELANEY

Because lipids tend to be mutually is seen in the higher melting point lipids soluble in each other, failure to retain into which reactants penetrate relatively localization because of diffusion can be a poorly. For fatty acid containing lipids, the problem in older specimens. Solubility fac melting point is related to the degree of tors also affect the phase of exposure of the unsaturation.1 A third and less well under tissue to reactants with diffusion of tissue stood factor involved in permeation of lipid into either solutes or solvents. Any of reagents into lipids is concerned with pro these diffusion effects may also result in tein binding; such bound lipids will be un changes in the physical properties of free- detectable (masked) by the usual methods, or protein bound-lipid. It is a major aim of will resist extraction by organic solvents the controlled chromation technic to pre and require dissociation by hydration, heat vent the dissolution of lipids by fat solvents or proteolytic enzymes in order for sub in preparation for routine embedding. sequent demonstration by lipid stains to be Physical displacement of globules of lipid effected.6 from fresh or fixed tissue frozen sections Chemical factors involved in lipid histo makes it necessary to process test and con chemistry are related to the above discussed trol sections in different vehicles and solu diffusion principles. Granting that the lipid tions so as to prevent cross contamination. can be preserved in its natural state within In addition, massive loss of fat globules the tissue and that the reactants can be from frozen sections with resultant collapse brought to it without altering the nature of of structure makes this procedure one of the the lipid, several major problems remain. least rewarding technically in diagnostic Methods based mainly on physical solu pathology. Infiltration and embedding in bility characteristics of dyes in lipids are polyethylene glycols (Carbowax) to avoid not specific for substances but rather iden frozen section artefacts is attended by the tify classes of lipids. Cross reacting or com same fixation problems as discussed pre peting substances may be located adjacent viously.46 to each other, leading to reduced specificity. Factors affecting the penetrance of In addition, the methods thus far employed reagents and dyes into tissue lipids are re are intrinsically far less specific than those lated to physical features determining used in comparable biochemical proce permeability of the lipid structures. The dures. Lastly, procedures used in lipid polarity and miscibility of lipids in an histochemistry are not quantitative. The aqueous medium is due to ionized basic last difficulty can be somewhat overcome, and phosphoryl groups.1 This ionized state however, by comparative extraction chem is related to the essentially aqueous nature ical analysis, using tissue blocks adjacent to of most tissue, reflected in the concomitant the ones processed by sectioning for micros need for aqueous histochemical reagents copy. and dyes. At the same time the permeabil A consensus of recommended procedures ity of the lipid membrane system must be for lipid histochemistry, including fixatives kept intact, and the latter status is related and sectioning methods, is presented in to the cholesterol content of the mem table IV, compiled from standard sources.2, branes.17 Another factor affecting pen 29 ,32 it j S to be noted that most proce etrance through closer packing of molecules dures are not specific and that many lipids IDENTIFICATION OF FATTY TUMORS BY HISTOCHEMICAL PROCEDURES 221

TABLE IV

Recommended lipid Recommended fixative Positively reacting Non-reacting histochemical reaction and sectioning lipids lipids

Red Sudan dyes Formol-calcium Unsaturated triglycerides Crystaline hydrophobic e.g. Oil Red O Frozen section Unsaturated cholesterol lipids esters Phospholipid Unsaturated fatty acids beta-lipoprotein Sudan Black B F ormol-calcium Phospholipids Frozen section Gly colipids Unsaturated triglycerides Unsaturated cholesterol esters Unsaturated free fatty acids Fettrot 7B (Ciba) Formol-calcium Permanent lipid stain Frozen section (others fade) Osmium tetroxide F ormol-calcium CIS unsaturated fatty Cholesterol Paraffin section acids (blocked by pre oxidation with per- formic acid) Osmium tetroxide- F ormol-calcium Non-polar hydrophobic alpha naphthylamine Paraffin section unsaturated lipids, (OTAN) black—polar hydro philic unsaturated lipids orange red Copper/rubeanic acid Formol-calcium Free fatty acids with EDTA pretreat- Frozen section ment22 Acid hematein (1 ) Controlled chromation Phospholipid Paraffin section ( 2 ) F ormol-calcium Phospholipid ( esp. Frozen section mitochondrial) Copper phthalocyanin Dichromate Lipoprotein Sphingomyelin Cold frozen section Phospholipids except sphingomyelin Perchloric acid— F ormol-calcium Cholesterol naphthoquinone (PAN) Frozen section Cholesterol esters Periodic acid—Schiff F ormol-calcium Cerebroside (PAS)—diastase Paraffin section Ganglioside Phospholipid Ethylene bonds of lipids Sphingomyelin Alpha-ketol steroids do not react in most procedures. It is to cover slip.41 All the recommended reactions be also noted that certain classical reac were chosen because of the ease of use in tions, such as the Lieberman-Burchardt the routine laboratory and their applicabil reaction for cholesterol, are omitted because ity to fixed tissue and to previously em of technical difficulties inherent in the pro bedded specimens. Care should be taken cedures, such as gas production under the in interpreting the results without controls 222 DELANEY

T A B L E V ture (2:1 v/v) or by the same mixture C h e m is t r y o f B e n ig n A d ip o s e T u m o r s diluted 93:6 with water.1 The use of acetone

Lipom a Hibernoma to extract tissue lipids is not recommended because it differentially extracts hydro- Total lipid content Identical to nor Same as lipomas mal tissue (90% phobic lipids (triglycerides, fatty acids, triglycerides) cholesterol and its esters) while not affect Rate of fatty acid Increased 5X over ing polar lipids (such as phospholipids)12 synthesis normal tissue except in arteriosclerotic plaques, where Cholesterol Higher than lipoma Unsaponified fat Higher than lipoma even phospholipids are removed, accord Iron Absent Present ing to the OTAN reaction.14 The extract of lipid can be examined chromatographically in view of the proven variation in tissue to characterize the major fatty acid compo content of lipids as a result of drugs, exer sition.28 In lipomas it has been found not to cise, diet and other variables.26 vary significantly from one patient to another in a given geographical area; major Chemistry of Benign Adipose Tumors increases in fat turnover between lipomas and normal fat (increased in the former by In general, benign neoplasms char a factor of 5) have been found.19 Biochem acterized by the accumulation of large ical activities of lipomas have also been amounts of triglycerides have been studied investigated by studying the incorporation histochemically only by simple lipid stains of radioactive acetate in fatty acids of tissue such as the red sudan dyes, which react slices, with confirmation of the five-fold positively.9' 40 The paucity of cytoplasm in increase in turnover of fatty acids in lipomas adult fat cells accounts for the lack of versus normal subcutaneous fat.25 The ac reactivity by light microscopy with the more tivity was found in the microsomal frac complex fat stains.36 The total lipid content tions plus the supemate of fractionated of lipomas is identical to that of normal specimens. subcutaneous fat and is composed of more Of great interest in the study of lipomas than 90 percent triglycerides (table V). is the recently described association of type Hibernomas do have relatively abundant IV hyperlipoproteinemia and elevated blood stainable cytoplasm and their cholesterol triglyceride levels with lipomatosis and and unsaponified fat content is higher gout.20 There were no histochemical studies than that of lipomas, although the total lipid reported, although the fat was reported content is the same in the two lesions.9,31 normal by light microscopy. In addition, iron has been described in hibernomas but not in lipomas.35 The pig ment coloring brown fat has not been iden Histochemistry of Malignant Adipose tified although hibernomas lose their color Tumors on prolonged formalin fixation. Increased Aside from the infrequent malignant catecholamine activity has been described mesenchymoma (included here because of in brown fat.42 its content of neoplastic fat) and the rare Extraction of lipids from tissue is best (?non-existent?) malignant hibernoma, the performed by a chloroform/methanol mix malignant tumors of adipose tissue—lipo- IDENTIFICATION OF FATTY TUMORS BY HISTOCHEMICAL PROCEDURES 223 sarcomas—exhibit the same histochemical tissue promises to solve some of the need activity as lipomas. In general, they are for specificity of reactions lacking in most recognized by storage of hydrophobic lipids of the older techniques. Work performed in vacuolated malignant cells. This has been to date has not included study of lipids in identified on occasion as being composed of neoplasms by radioautography.38 sudanophilic lipid, phospholipid and choles terol.43 In addition, the occurrence of a Summary myxoid variety of liposarcoma is char Aside from adipose tissue neoplasms, the acterized by the intercellular (and some role of lipid deposition in tumors is pos times intracellular) occurrence of ground sibly still at least partially on a degenera substance rich in acid mucopolysaccharide; tive basis but evidence points to at least a the material is recognized by its positivity partial integral role of lipids in tumors. As with the Hale colloidal iron and (less so) a result of many problems concerned alcian blue stains for acid mucopoly mainly with the mixed hydrophobic and saccharide, which is digestible by bovine hydrophilic nature of tissue lipids and the testicular hyaluronidase.15 Some authors34 consequent difficulty in assuring the sub- have failed to recognize the characteristic stantivity and specificity of the reactions, ground substance material while others39 there are limited technics of use in the have identified it in foamy “X” cells which identification of fatty tumors by histochem are also PAS positive, as are the numerous istry. Assuming the eventual solution of the mast cells (the latter also toluidine blue numerous technical problems, the use of positive) present in this variety of liposar ultrastructural histochemistry may permit coma. The occurrence of glycolipid within greater localization and specificity of reac vacuolated neoplastic cells, sometimes sur tions but may also be associated with ob rounding an arginine rich core, has been fuscation of the ease of distinction of adi correlated with the presence of electron- pose tumors from lipid-containing non lucid (non osmiophilic) areas by electron adipose neoplasms by histochemistry. The microscopy of a liposarcoma.34 It appears use of radioautography in lipid histochem that the lipid content of liposarcomas is istry is a promising tool for development of much more complex than that of lipomas, greater specificity of reactions. according to the scanty amount of reported histochemical investigations. No reports of References extraction analysis of liposarcomas are to be 1. A d a m s , C. W. M.: Lipid histochemistry. Ad found in the American and English litera vances in Lipid Research, Vol. 7, Paoletti R. and Kritchevsky, D., eds., pp. 1-62, Academic ture. Press, New York, 1969.

2. A d a m s , C. W. M.: Vascular Histochemistry, Radioautography Yearbook Medical Publishers, Chicago, 1967. 3. A f f f e l , C. A ., a n d B a k e r , J. R.: Lipid drop With the addition of new techniques of lets in the cytoplasm of malignant cells. Cancer 17-. 176-184, 1964. tissue preparation which can improve the 4. B a k e r , J. R.: The histochemical recognition of preservation of tissue lipids in their natural lipine. Quart. J. Micr. Sci. 87:441-471, 1946. state, the use of radioautography for light 5. B a k e r , J. R.: Fixation in cytochemistry and electron-microscopy. J. Histochem. Cytochem. and electron microscopic study of lipids in 6:303-308, 1958. 224 DELANEY

6 . B e r e n b a u m , M. C.: The histochemistry of ( Launois-Bensaude adenolipomatosis ) with bound lipids. Quart. J. Micr. Sci. 99:231-242, gout and hyperlipoproteinemia. Amer. J . Med. 1958. 48:239-246, 1970. 7. C a s l e y -S m i t i i , J. R.: Some observations on the 21. H e s l i n g a , F. J . M ., a n d D e i e r k a u f , F. A .: electron microscopy of lipids. J. Roy. Micro The action of formaldehyde solutions on scop. Soc. 87:463-473, 1967. human brain lipids. J . Histochem. Cytochem. 10:704-709, 1962. 8 . C h a n g , J. P., S p e e c e , A. J., a n d R u s s e l , W. O.: Histochemical aspects of enzymes, lipids, 22. H a d j i o l o f f , A. I ., T zarnowchanov , P., polysaccharides, and nucleic acids in human a n d G e o r g i e v a , S.: Histochemie der lipid- melanomas. Pigment Cell Biology, Gordon, einschlüsse in den reticulohistocyten bei M., ed., pp. 359-370, Academic Press, New einem fall von Letterer-Siwescher kraulcheit, York, 1959. pp. 503-509 in V Symposium histologicum internationale. Histochemie et Cytochemie des 9. D a s G u p t a , T. K.: Tumors and tumor like conditions of the adipose tissue. Current Prob Lipides, Hadjioloff, A. I., ed., Sofia, 1966. lems in Surgery, Yearbook Medical Pub 23. H o l c z i n g e r , L.: Histochemischer Nachweis lishers, Chicago, 1970. freier Fettsäuren. Acta Histochem. 8:167-175, 1959. 10. D e l a n e y , W. E., N e a l o n , T. F., a n d P i e r u c c i , L.: The soft tissues. Management 24. I d e l m a n , S .: Conservation des lipides par les of the Patient with Cancer, Nealon, T. F., techniques utilisées en microscopie électro ed., pp. 907-968, W. B. Saunders Co., Phila nique. Histocheme 5:18-23, 1965. delphia, 1965. 25. J a c o b , M. I.: Fatty acid synthesis by adipose 11. D o b r o g o r s k i , O. J., a n d B r a u n s t e i n , H.: tissue of humans. Fed. Proc. 21:288, 1962. Histochemical study of staining lipid, gly 26. K h a n , B ., C o x , G . E., a n d A s d e l , K .: cogen, and mucin in human neoplasms. Amer. Cholesterol in human tissues. New considera J. Clin. Path. 40:435-443, 1963. tions. Arch. Path. 76:369-381, 1963. D u n n i g a n , 12. M. G.: The distribution of phos 27. K o r n , E. D ., a n d W e i s m a n , R . A.: Loss of pholipid within macrophages in human lipids during preparation of amoebae for atheromatous plaques. J. Atheroscler. Res. 4: electron microscopy. Biochim. Biophys. Acta 144-150, 1964. 116:309-316, 1966.

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38. S t e i n , O., a n d S t e i n , Y.: Light and electron istry of Lipids in Pathology by M. Wolman microscopic radioautography of lipids: Tech in Handbuch der Histochemie, pp. 515-525, niques and biological applications. Advances Vol. V, Graumann, W. and Neuman, K., eds., in Lipid Research, vol. 9, pp. 1-72, Paoletti, Gustav Fischer Verlag, Stuttgart, 1964. R. and Kritchevsky, D., eds., Academic Press, 44. W o l m a n , M., a n d W i e n e r , H.: Structure of New York, 1971. the myelin sheath as a function of concentra 39. T r e m b l a y , M., a n d B o n e n f o n t , J. L.: Em tion of ions. Biochim. Biophys. Acta 102:269- bryonal liposarcoma (myxoid liposarcoma). A 279, 1965. study of six cases. Canad. Med. Ass. J. 100: 45. W r i g h t , D. H.: Microscopic features, histo 281-285, 1969. chemistry, histogenesis and diagnosis. Bur- kitt’s Lymphoma, Burkitt, D. P., and Wright, 40. V e l l i o s , F., B a e z , J., a n d S c h u m a c h e r , H. D. H., pp. 82-102, Livengstone, London, 1970. B .: Lipoblastomatosis: a tumor of fetal fat different from hibernoma. Amer. J. Path. 34: 46. Z u g i b e , F. T.: Diagnostic Histochemistry, C. 1149-1159, 1958. V. Mosby, St. Louis, 1970.

Strange—is it not?—that of the myriads who Before us passed the door of Darkness through, Not one returns to tell us of the road Which to discover we must travel too.

Omar Khayyam. Rubaiyat.