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The structure and growth of epidermanl lesions of Parophrys vetulus : a light and electron microscopic… Majack, Richard Allen 1975

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THE STRUCTURE AND GROWTH OF EPIDERMAL LESIONS OF PARQPHRYS VETULUS; A LIGHT AND ELECTRON MICROSCOPIC STUDY by RICHARD ALLEN MAJACK B . S c , U n i v e r s i t y o f I l l i n o i s , 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f : Zoology We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1975 In present ing th is thes is in p a r t i a l fu l f i lment o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It i s understood that copying or p u b l i c a t i o n of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my writ ten pe rm i ss i on . Department of The Univers i ty of B r i t i s h Columbia 20 75 Wesbrook Place Vancouver, Canada V6T 1W5 Date Q ± 8 iq-frD ABSTRACT N a t u r a l h i s t o r y , l i g h t m i c r o s c o p i c , and e l e c t r o n m i c r o s c o p i c s t u d i e s were made o f epidermal l e s i o n s a f f l i c t i n g j u v e n i l e lemon s o l e (Parophrys v e t u l u s ) i n the S t r a i t o f Georgia, near Vancouver, B r i t i s h Columbia, Canada. Emphasis was p l a c e d on the p r e s e n t a t i o n o f n a t u r a l h i s t o r y and h i s t o -l o g i c a l evidence f o r the p r o g r e s s i v e growth of these l e s i o n s from an i n i t i a l m o r p h o l o g i c a l form (the a n g i o e p i t h e l i a l nodule) to a mature and m o r p h o l o g i c a l l y d i f f e r e n t form (the epidermal p a p i l l o m a - l i k e l e s i o n ) . The study o f the h i s t o l o g -i c a l and c y t o l o g i c a l aspects of the growth o f the l e s i o n s n e c e s s a r i l y i n c o r p o r a t e d c l a r i f i c a t i o n of the nature o f u n i d e n t i f i e d epidermal and stromal c e l l types which compose much o f the mass o f the tumour. I t was noted t h a t the h i s t o l o g i c a l appearance o f the f l a t f i s h l e s i o n s d i f f e r s s i g n i f i c a n t l y from t h a t o f any known f i s h epidermal d i s e a s e , i n c l u d i n g those c h a r a c t e r i z e d by c e l l u l a r hypertrophy and c e l l u l a r h y p e r p l a s i a or neo-p l a s i a . N a t u r a l h i s t o r y s t u d i e s showed t h a t a n g i o e p i t h e l i a l nodules o c c u r r e d predominantly on younger f i s h w h i l e epidermal p a p i l l o m a - l i k e l e s i o n s were u s u a l l y found on l a r g e r f i s h , i n d i c a t i n g a p r o g r e s s i o n from the former to the l a t t e r . T h i s assumption was supported by the presence of m o r p h o l o g i c a l and h i s t o l o g i c a l i n t e r m e d i a t e s . The growth of the l e s i o n s was c h a r a c t e r i z e d by the g r a d u a l t r a n s f o r m a t i o n o f normal epidermal c e l l s i n t o " X - c e l l s " : o v o i d , h y p e r t r o p h i e d c e l l s w i t h e n l a r g e d n u c l e i , prominent n u c l e o l i , and n e c r o t i c i i i c ytoplasm. These " X - c e l l " types completely dominate the mature l e s i o n and are e n c l o s e d , or supported, by abnormal epidermal c e l l s termed "enveloping c e l l s " . I t was found t h a t the p a t t e r n o f s u b c e l l u l a r n e c r o s i s i n f l a t f i s h tumour c e l l s d i f f e r s from n e c r o s i s caused by n o n - s p e c i f i c l e t h a l i n j u r i e s t o c e l l s . T h i s o b s e r v a t i o n suggests t h a t the sub-c e l l u l a r changes observed may be a r e s u l t o f c e l l u l a r t r a n s f o r m a t i o n r a t h e r than a n o n - s p e c i f i c l e t h a l i n j u r y to the c e l l s . Other n a t u r a l h i s t o r y s t u d i e s i n d i c a t e d a d e c l i n e i n the number of tumours per tumourous f i s h and a d e c l i n e i n the p r e v a l e n c e of l e s i o n s as f i s h s i z e i n c r e a s e s . These r e s u l t s i n d i c a t e t h a t e i t h e r the d i s e a s e d f i s h are s e l e c -t i v e l y removed from the p o p u l a t i o n or t h a t the tumours necrose and are l o s t by the h o s t . Both p o s s i b i l i t i e s were d i s c u s s e d . No evidence was found to support the h y p o t h e s i s t h a t " X - c e l l s " are p a r a s i t i c p r otozoans. The presence o f v i r u s - l i k e p a r t i c l e s i n the cytoplasm o f the e n v e l o p i n g c e l l s o f mature l e s i o n s was commonly observed. The r e l a t i o n -s h i p between these v i r u s - l i k e p a r t i c l e s and the d i s e a s e process, i f any, i s unknown. i v TABLE OF CONTENTS Page INTRODUCTION 1 General I n t r o d u c t i o n 1 L i f e H i s t o r y Information 7 MATERIALS AND METHODS 9 C o l l e c t i o n Procedures and Data A n a l y s i s 9 L i g h t Microscopy 14 Transmission E l e c t r o n Microscopy 14 Scanning E l e c t r o n Microscopy 15 RESULTS 16 N a t u r a l H i s t o r y Data 16 Epidermal L e s i o n s i n Other P l e u r o n e c t i d s 21 Normal Epidermis, L i g h t Microscopy 21 Normal Epidermis, E l e c t r o n Microscopy 26 Tumour H i s t o l o g y , L i g h t Microscopy 28 A n g i o e p i t h e l i a l Nodules 28 AEN/EP T r a n s i t i o n Forms. . 33 Epidermal P a p i l l o m a - l i k e L e s i o n s . . 36 Tumour H i s t o l o g y , E l e c t r o n Microscopy 46 Stroma 46 AEN Epidermis 53 EP Epidermis 59 V i r u s - l i k e P a r t i c l e s . 74 DISCUSSION 77 Normal Epidermis 77 V Page B i o l o g y o f the Epidermal L e s i o n s 78 LITERATURE CITED 99 v i LIST OF FIGURES Fi g u r e Page 1. E x t e r n a l morphology o f AEN on j u v e n i l e lemon s o l e 12 2. E x t e r n a l morphology o f AEN/EP t r a n s i t i o n form on j u v e n i l e lemon s o l e 12 3. E x t e r n a l morphology o f E P - l i k e l e s i o n on j u v e n i l e lemon s o l e 12 4. Low-power l i g h t micrograph o f AEN 12 5. Low-power l i g h t micrograph o f AEN/EP t r a n s i -t i o n form 12 6. Low-power l i g h t micrograph o f E P - l i k e l e s i o n . 12 7. 1 The r e l a t i o n s h i p o f tumour prev a l e n c e and f i s h s i z e 18 8. The r e l a t i o n s h i p o f numbers o f tumours per f i s h and f i s h s i z e 20 9. The r e l a t i o n s h i p o f tumour types and f i s h s i z e 22a 10. L i g h t micrograph o f normal epidermis 25 11. E l e c t r o n micrograph o f normal e p i d e r m i s . . . . 25 12. E l e c t r o n micrograph o f normal e p i d e r m i s . . . . 25 13. Scanning e l e c t r o n micrograph o f normal e p i d e r m i s . 25 14. L i g h t micrograph o f stroma o f AEN 30 15. L i g h t micrograph of stroma o f AEN 30 16. C r o s s - s e c t i o n o f lemon s o l e b e a r i n g AEN w i t h i n v a s i v e q u a l i t i e s 30 v i i F i g u r e Page 17. Higher m a g n i f i c a t i o n l i g h t micrograph o f l e s i o n shown i n F i g u r e 16 30 18. L i g h t micrograph o f epidermal component o f AEN 35 19. L i g h t micrograph o f AEN 35 20. Low-power l i g h t micrograph o f AEN/EP t r a n s i -t i o n form 35 21. L i g h t micrograph o f epidermal h y p e r p l a s t i c stage o f AEN/EP t r a n s i t i o n form 35 22. Higher m a g n i f i c a t i o n l i g h t micrograph o f t i s s u e shown i n Fig u r e 21 38 23. L i g h t micrograph o f AEN/EP t r a n s i t i o n form . . 38 24. L i g h t micrograph of AEN/EP t r a n s i t i o n form . . 38 25. L i g h t micrograph o f E P - l i k e l e s i o n 38 26. L i g h t micrograph o f E P - l i k e l e s i o n , showing stromal and epidermal c h a r a c t e r i s t i c s . . . . 41 27. L i g h t micrograph o f epidermal component o f E P - l i k e l e s i o n 41 28. L i g h t micrograph o f E P - l i k e l e s i o n 41 29. L i g h t micrograph o f s u p e r f i c i a l a r e a o f EP-l i k e l e s i o n 41 30. Higher m a g n i f i c a t i o n l i g h t micrograph o f t i s s u e shown on Fi g u r e 26 45 31. L i g h t micrograph o f E P - l i k e l e s i o n , showing g e n e r a l t i s s u e n e c r o s i s 45 32. L i g h t micrograph o f E P - l i k e l e s i o n , showing n e c r o t i c m a t e r i a l 4 5 v i i i F i g u r e Page 33. L i g h t micrograph of E P - l i k e l e s i o n , showing n e c r o t i c m a t e r i a l 45 34. E l e c t r o n micrograph o f basement membrane area o f E P - l i k e l e s i o n 48 35. E l e c t r o n micrograph o f A EN stroma 48 36. E l e c t r o n micrograph o f AEN stroma 50 37. E l e c t r o n micrograph o f stromal " X - c e l l s " i n AEN 50 38. E l e c t r o n micrograph o f basement membrane area o f E P - l i k e l e s i o n , showing a non-ovoid stromal " X - c e l l " and epidermal " X - c e l l s " 55 39. E l e c t r o n micrograph of AEN ep i d e r m i s , showing the e a r l i e s t r e l a t i o n s h i p between normal epidermal c e l l s and " X - c e l l s " 55 40. E l e c t r o n micrograph of epidermal component of l a r g e AEN 58 41. E l e c t r o n micrograph o f epidermal component o f l a r g e AEN 58 42. E l e c t r o n micrograph o f AEN ep i d e r m i s , showing tr a n s f o r m i n g epidermal c e l l 61 43. E l e c t r o n micrograph o f l a r g e AEN ep i d e r m i s , showing d e v e l o p i n g envelope c e l l 61 44. E l e c t r o n micrograph o f s u p e r f i c i a l c e l l l a y e r s o f E P - l i k e l e s i o n 64 45. E l e c t r o n micrograph o f s u p e r f i c i a l c e l l l a y e r s o f E P - l i k e l e s i o n 64 46. E l e c t r o n micrograph o f deeper p o r t i o n o f EP-l i k e l e s i o n 67 i x F i g u r e Page 47. E l e c t r o n microgram o f deeper p o r t i o n o f EP-l i k e l e s i o n 67 48. E l e c t r o n microgram o f epidermal component o f E P - l i k e l e s i o n 70 49. E l e c t r o n microgram o f p o r t i o n o f cytoplasm o f a type A g i a n t c e l l found i n an E P - l i k e l e s i o n 70 50. E l e c t r o n micrograph o f nucleus and p o r t i o n of cytoplasm o f epidermal " X - c e l l " found i n E P - l i k e l e s i o n 73 51. Low-power e l e c t r o n micrograph o f EP-type l e s i o n , showing the c h a r a c t e r i s t i c s o f types A and B " X - c e l l s " 73 52. E l e c t r o n micrograph of v i r u s - l i k e p a r t i c l e s i n the cytoplasm o f an e n v e l o p i n g c e l l . . . 76 53. E l e c t r o n micrograph o f v i r u s - l i k e p a r t i c l e s i n the cytoplasm o f an e n v e l o p i n g c e l l . . . 76 54. E l e c t r o n micrograph o f v i r u s - l i k e p a r t i c l e s i n the i n t e r c e l l u l a r space o f an AEN . . . . 76 55. Summary o f the growth and s t r u c t u r e o f f l a t -f i s h epidermal l e s i o n s 82 LIST OF ABBREVIATIONS USED IN TEXT angioepithelial nodule epidermal papilloma-like lesion x i LIST OP ABBREVIATIONS USED IN FIGURES A type A X - c e l l AEN a n g i o e p i t h e l i a l nodule B type B X - c e l l BC b a s a l c e l l BM basement membrane Cap c a p i l l a r y Con c o n n e c t i v e t i s s u e c f c y t o p l a s m i c f i l a m e n t s D c e l l u l a r remains ( d e b r i s ) des desmosome EC epidermal c e l l Env e n v e l o p i n g c e l l Ep epidermis EP epidermal p a p i l l o m a - l i k e l e s i o n e r endoplasmic r e t i c u l u m F f i b r o b l a s t g g l y c o c a l y x ger g r a n u l a r endoplasmic r e t i c u l u m Gran g r a n u l o c y t e Gu guanidophore IC c e l l o f the int e r m e d i a t e l a y e r L lymphocyte M macrophage Mel melanocyte mit mitochondria MC mucous-secreting c e l l Mus muscle Mf m i c r o f i l a m e n t s mt microtubule nc n u c l e o l u s ne n u c l e a r envelope nu nucleus np n u c l e a r pore pm plasma membrane RBC r e d blood c e l l r i b ribosomes S stroma l X - c e l l SC s u p e r f i c i a l c e l l Sc s c a l e S i n s i n u s o i d SMC smooth muscle c e l l S t r stroma Trans t r a n s i t i o n form between AEN and EP vac, vacuole VLP v i r u s - l i k e p a r t i c l e X X - c e l l , type A or B x i i i ACKNOWLEDGEMENTS I g r a t e f u l l y wish t o acknowledge the c o o p e r a t i o n o f : Dr. A. B. Acton, my s u p e r v i s o r , f o r h i s p a t i e n c e and c r i t i c a l r e v i s i o n o f t h i s t h e s i s ; Drs. J . R. Adams and W. S. Hoar, f o r t h e i r c r i t i c a l r e a d i ngs o f the t h e s i s , and Mr. L. L. Veto, f o r h i s e x c e l l e n t and i n v a l u a b l e t e c h n i c a l knowledge. 1 INTRODUCTION General i n t r o d u c t i o n Since 1922 (Harold and Innes, 1922), epidermal s k i n tumors o f unknown a e t i o l o g y , diagnosed e i t h e r as epidermal p a p i l l o m a s (Wellings e_t a l . , 1964) or as h y p e r p l a s t i c epidermal d i s e a s e ( N i g r e l l i et; a l . , 1965), have been r e p o r t e d i n the lemon s o l e and o t h e r s p e c i e s o f the order P l e u r o n e c t i f o r m e s ( f a m i l i e s Bothidae and P l e u r o n e c t i d a e ) , e s p e c i a l l y along the P a i f i c c o a s t o f North America. S e v e r a l r e p o r t s i n d i c a t e r e l a -t i v e l y h i g h i n c i d e n c e s of the d i s e a s e , o f t e n i n a p a r t i c u l a r s p e c i e s and geographic l o c a t i o n . I t has been noted t h a t tumor i n c i d e n c e s i n some f l a t f i s h are a t t h e i r h i g h e s t i n the f i r s t year o f l i f e ; no tumors have been r e p o r t e d i n f l a t f i s h over f o u r years o f age (see Mawdesley-Thomas, 1972). P a c i s (1932) and Good (1940) r e p o r t e d d i s e a s e i n c i d e n c e s o f approximately 5% i n lemon s o l e from Puget Sound, Washington. Ketchen (1953) d e s c r i b e d s i m i l a r l e s i o n s i n a sand s o l e ( P s e t t i c h t h y s  m e l a n o s t i c t u s ) p o p u l a t i o n from Hecate S t r a i t , B r i t i s h Columbia. Incidences o f 6.4% were r e p o r t e d i n the f l a t h e a d s o l e (Hippo- g l o s s o i d e s elassodon) from the San Juan I s l a n d s , Washington; lemon s o l e , sand s o l e , and r e x s o l e (Glyptocephalus z a c h i r u s ) were a l s o a f f l i c t e d i n t h i s a r e a (Wellings e t a l . , 1964; Chuinard e t a l . , 1964; W e l l i n g s et_ aa., 1965). Young (1964), i n a study o f marine l i f e i n the San F r a n c i s c o a r e a , found epidermal l e s i o n s on the dover s o l e (Microstomus p a c i f i c u s ) . 2 Sand s o l e and rock s o l e ( L e p i d o p s e t t a b i l i n e a t a ) from Hecate S t r a i t were r e p o r t e d t o be a f f l i c t e d w i t h p a p i l l o m a - l i k e l e s i o n s ; the authors c o n s i d e r e d these l e s i o n s t o be a hyper-p l a s t i c epidermal d i s e a s e ( N i g r e l l i e t a l . , 1965). Levings (1967) r e p o r t e d t h a t j u v e n i l e rock s o l e i n the G u l f o f A l a s k a and B r i s t o l Bay were a f f e c t e d by t u m o r - l i k e s k i n growth w i t h a 10% i n c i d e n c e . S t u d i e s i n San F r a n c i s c o Bay by Cooper and K e l l e r (1969) i n d i c a t e t h a t lemon s o l e t h e r e are a f f l i c t e d w i t h the d i s e a s e w i t h i n c i d e n c e s o f 3 - 28%, depending on the time of the year and geographic l o c a t i o n . Two a d d i t i o n a l s p e c i e s , the s t a r r y f l o u n d e r ( P l a t i c h t h y s s t e l l a t u s ) and the b u t t e r s o l e ( I s o p s e t t a i s o l e p i s ) , were found t o be d i s e a s e d i n Puget Sound (McArn e t a l . , 1968; McArn and W e l l i n g s , 1971). M i l l e r and W e l l i n g s (1971) d e s c r i b e d the n a t u r a l h i s t o r y a spects o f the d i s e a s e i n the f l a t h e a d s o l e , i n c l u d i n g t umorigenesis, tumor i n c i d e n c e s , and growth and s u r v i v a l r a t e s . Recent Puget Sound s t u d i e s r e v e a l e d t h a t up t o 9% o f j u v e n i l e lemon s o l e sampled were a f f l i c t e d w i t h the d i s e a s e ( A n g e l l and M i l l e r , 1974). A r e p o r t o£ a southern C a l i f o r n i a study o f dover s o l e i n d i c a t e d tumor f r e q u e n c i e s o f 9% (Mearns and Sherwood, 1974). The epidermal tumors o f f l a t f i s h have been h i s t o -l o g i c a l l y d e s c r i b e d , t o some e x t e n t , f o r most s p e c i e s . Two h i s t o l o g i c a l l y and m o r p h o l o g i c a l l y d i s t i n c t tumor types were r e p o r t e d i n the f l a t h e a d s o l e (Wellings et_ a l , . , 1964). These were termed the a n g i o e p i t h e l i a l nodule ("AEN", s m a l l . 3 hemorrhagic nodules) and the epidermal papilloma' 1' ("EP", l a r g e r , more d i f f u s e , g r a n u l a r l e s i o n s ranging up t o 60 mm i n d i a m e t e r ) . Morphologic t r a n s i t i o n s between the AEN and the EP were a l s o found, suggesting a p r o g r e s s i o n from an i n i t i a l AEN t o the mature EP (Wellings ,et a l ^ . , 1964). W e l l i n g s and Chuinard (1964) r e p o r t e d p r o g r e s s i v e growth of the tumor i n the l a b o r a t o r y . A t h i r d and uncommon morphologic type, termed the a n g i o e p i t h e l i a l polyp ("AEP"), was d e s c r i b e d i n the sand s o l e (Wellings e t a l . , 1964); the authors a l s o demonstrated the h i s t o l o g i c a l s i m i l a r i t i e s o f the lemon s o l e , rex s o l e , and f l a t h e a d s o l e tumors. H i s t o l o g i c a l d e s c r i p t i o n s o f sand s o l e l e s i o n s ( N i g r e l l i e t a l . , 1964) showed the mature p a p i l l o m a - l i k e l e s i o n s to be i d e n t i c a l t o the o t h e r s d e s c r i b e d . The author a l s o d e s c r i b e d n e c r o t i c and r e g r e s s i v e tendencies w i t h i n the tumor. McArn and h i s c o l l e a g u e s (1968) and McArn and W e l l i n g s (1971) r e p o r t e d t h a t the g r o s s morphology and h i s t o l o g y o f the lemon s o l e , s t a r r y f l o u n d e r , and f l a t h e a d s o l e l e s i o n s are i d e n t i c a l . The d e f i n i t e p r o g r e s s i o n o f the d i s e a s e from one m o r p h o l o g i c a l type (AEN) to another (EP) was !(N0TE: "Papilloma", i n a s t r i c t sense, r e f e r s to a benign neoplasm. Sinc e , i n t h i s case, the t r u e n e o p l a s t i c nature o f the l e s i o n i s d o u b t f u l ( N i g r e l l i e t a l . , 1965; W e l l i n g s , 1969A; Mawdesley-Thomas, 1972), a more c o r r e c t term would be " p a p i l l o m a - l i k e l e s i o n " (see W i l l i s , 1967). For the sake o f c l a r i t y and c o n t i n u i t y , the term "epidermal p a p i l l o m a " w i l l be used i n t h i s paper to d e s c r i b e the mature l e s i o n . A l s o , the term "tumor", i n t h i s r e p o r t , i s used f r e e l y i n r e f e r r i n g t o any h y p e r p l a s t i c o r n e o p l a s t i c growth.) 4 p o s t u l a t e d on the presence o f m o r p h o l o g i c a l t r a n s i t i o n a l s t a g e s , the c o r r e l a t i o n between tumor type and s i z e o f f i s h ( s m a l l e r f l a t f i s h had mostly AENs; l a r g e r f i s h had mainly EPs), and the l a b o r a t o r y o b s e r v a t i o n s o f d i s e a s e p r o g r e s s i o n (McArn e t a l . , 1968). I t should be noted t h a t o t h e r f i s h e x h i b i t i n g epidermal p a p i l l o m a s a l s o show the same s e r i e s o f e v e n t s : the i n i t i a l appearance o f an a n g i o e p i t h e l i a l - t y p e l e s i o n which p r o g r e s s e s i n t o a p a p i l l o m a (Lucke, 1938; a l s o see W e l l i n g s , 1969B). S e v e r a l Japanese r e p o r t s , although r a t h e r u n c l e a r , seem to i n d i c a t e t h a t two s p e c i e s o f f l o u n d e r (Kimura §_t a l . , 1967; Honma and Kon, 1968) and s e v e r a l g o b i i d f i s h e s (Oota, 1952; Imai and F u j i w a r a , 1959) may be a f f l i c t e d w i t h h i s t o l o g i c a l l y s i m i l a r p a p i l l o m a - l i k e tumors. Because the epidermal l e s i o n s o f the v a r i o u s s p e c i e s d e s c r i b e d show i d e n t i c a l s t r u c t u r e and morphology, i t i s g e n e r a l l y concluded t h a t the u n d e r l y i n g mechanisms o f the d i s e a s e are s i m i l a r , i f not i d e n t i c a l . Although the p r o g r e s s i o n from AENs to EPs i s s t r o n g l y suggested by a v a i l a b l e evidence, no h i s t o l o g i c a l p r o g r e s s i o n s t u d i e s have been done t o d a t e . H i s t o l o g i c a l l y the f l a t f i s h tumors are unique, as shown by i n i t i a l l i g h t m i c r o s c o p i c s t u d i e s (Wellings e t a l . , 1964; N i g r e l l i e t a l . ( 1965; W e l l i n g s e t aJL_., 1965; McArn e t a l . , 1968; McArn and W e l l i n g s , 1971). E l e c t r o n m i c r o s c o p i c s t u d i e s showed marked mor p h o l o g i c a l d i f f e r e n c e s between normal e p i t h e l i a l c e l l s and e p i t h e l i a l c e l l s found i n f l a t -head s o l e epidermal p a p i l l o m a s (Wellings e t a l . , 1967; Brooks e t a l . , 1969). U n i d e n t i f i e d c e l l types, termed " X - c e l l s " 5 (Brooks et: a l . , 1969) were d e s c r i b e d from the stromal and epidermal components o f the l e s i o n s . The nature o f these c e l l s was not determined; the authors p o s t u l a t e d the c e l l s t o be e i t h e r p a r a s i t i c protozoans o r transformed f i s h c e l l s . Comparisons between e p i t h e l i a l tumors o f f l a t f i s h and other epidermal d i s e a s e s known i n f i s h e s are i n e v i t a b l e . Such comparisons may be u s e f u l , e s p e c i a l l y when u s i n g h i s t o -l o g i c a l s t r u c t u r e as a d i a g n o s t i c b a s i s . The s t r u c t u r e o f the f l a t f i s h tumors i s u n l i k e h i s t o l o g i c d e s c r i p t i o n s o f c e l l u l a r h y p e r t r o p h i c d i s e a s e s o f f i s h e p i d e r m i s , such as e p i t h e l i o c y s t i s d i s e a s e (Hoffman e t a l . , 1969; Wolke e t a l . , 1970), lymphocystis d i s e a s e (see reviews by Weissenberg, 1965; and N i g r e l l i and R u g g i e r i , 1965), and m i c r o s p o r i d i a n i n f e c t i o n s (Sprague, 1968; Weissenberg, 1968). These l e s i o n s are c h a r a c t e r i z e d by e x c e s s i v e c e l l u l a r hypertrophy, u s u a l l y accompanied by evidence o f the i n i t i a t i n g organism. The f l a t f i s h tumors a l s o d i f f e r from h i s t o l o g i c a l d e s c r i p t i o n s o f c e l l u l a r p r o l i f e r a t i v e l e s i o n s o f f i s h e p i d e r m i s . Pro-l i f e r a t i v e l e s i o n s i n c l u d e f i s h pox (e.g., Mawdesley-Thomas and Bucke, 1967), h y p e r p l a s t i c epidermal d i s e a s e s (Smith, 1935; N i g r e l l i , 1948; Walker, 1969a and 1969b), p r o l i f e r a t i o n s due t o i n f e c t i o n s ( N i g r e l l i and Smith, 1940), epidermal p a p i l l o m a s (Coates, Cox, and Smith, 1938; Lumann and Mann, 1956; R u s s e l l and K o t i n , 1957; Deys, 1969; Steeves, 1969; Koops e t a l . , 1970), and epidermoid carcinomas (Lucke and Schlumberger, 1941; Tavolga, 1951; Arnowitz e t al _ . , 1951; S t o l k , 1953, 1956, 1958, and 1960). These p r o l i f e r a t i v e 6 r e a c t i o n s may or may not show a p a p i l l o m a t o u s growth s t r u c -t u r e . The h y p e r p l a s t i c or n e o p l a s t i c c e l l s tend to r e t a i n the form and f u n c t i o n o f the c e l l type of o r i g i n . G e n e r a l l y , the c e l l s tend t o r e t a i n t h e i r normal s t a i n i n g a b i l i t y w i t h many s t a i n s . U s u a l l y no n e c r o t i c o r c e l l u l a r d egenerative processes are apparent. Most hyper-p l a s t i c or n e o p l a s t i c growths tend t o e x h i b i t the same h i s t o l o g i c p a t t e r n d e s c r i b e d by Lucke and Schlumberger (1949) f o r epidermal p a p i l l o m a s . I t can be noted, by u s i n g h i s t o -l o g i c a l comparisons, t h a t the epidermal tumors found on f l a t f i s h d i f f e r s t r u c t u r a l l y from other d e s c r i b e d epidermal l e s i o n s o f f i s h e s . R e c e n t l y i t has been noted t h a t t h i s d i s e a s e i s q u i t e common i n j u v e n i l e lemon s o l e i n the S t r a i t o f G e o r g i a , near Vancouver, B r i t i s h Columbia. S i m i l a r l e s i o n s were a l s o found i n s m a l l numbers or i s o l a t e d cases o f s e v e r a l other s p e c i e s (sand s o l e , rock s o l e , s p e c k l e d sanddab, re x s o l e , s t a r r y f l o u n d e r , and f l a t h e a d s o l e ) . N a t u r a l h i s t o r y d a t a , l i g h t microscopy, and e l e c t r o n microscopy were u t i l i z e d i n the study o f t h i s d i s e a s e , as m a n i f e s t i n t h i s p o p u l a t i o n o f j u v e n i l e lemon s o l e . I n s o f a r as the nature o f a d i s e a s e must be known befo r e i t s a e t i o l o g y and consequences can be m e a n i n g f u l l y examined, emphasis was p l a c e d on the c l a r i f i c a t i o n o f two u n r e s o l v e d aspects o f the nature o f these l e s i o n s : 1) the h i s t o g e n e t i c r e l a t i o n s h i p between AENs and EPs, and 2) the nature o f the u n i d e n t i f i e d " X - c e l l " type which dominates the t i s s u e o f the mature l e s i o n . 7 The primary o b j e c t i v e s o f t h i s t h e s i s a r e : 1) t o p r e s e n t n a t u r a l h i s t o r y and h i s t o l o g i c a l evidence f o r , and d e s c r i p t i o n s o f , the p r o g r e s s i v e growth o f these l e s i o n s , and 2) to c l a r i f y , h i s t o l o g i c a l l y and u l t r a s t r u c t u r a l l y , the nature o f the u n i d e n t i f i e d " X - c e l l " type and t h e i r r e l a t i o n -s h i p t o the h i s t o l o g i c a l growth and s t r u c t u r e o f the l e s i o n s . H o p e f u l l y t h i s type of study o f these epidermal l e s i o n s and t h e i r unique h i s t o l o g y w i l l h e l p t o c l a r i f y the nature, a e t i o l o g y , and consequences of the d i s e a s e . L i f e H i s t o r y I n f o r m a t i o n The p r e v a l e n c e o f any d i s e a s e o c c u r r i n g i n a n a t u r a l p o p u l a t i o n ( e s p e c i a l l y i n f e c t i o u s d i s e a s e s ) g e n e r a l l y i s i n t i m a t e l y a s s o c i a t e d w i t h the n a t u r a l h i s t o r y o f the s p e c i e s a f f e c t e d . T h i s seems t o be the case w i t h f l a t f i s h e pidermal l e s i o n s . For t h i s reason, a s h o r t s y n o p s i s of the l i f e h i s t o r y o f the lemon s o l e f o l l o w s . The lemon s o l e (Parophrys v e t u l u s G i r a r d , 1854) i s a member o f the t e l e o s t f a m i l y P l e u r o n e c t i d a e ( P l e u r o -n e c t i f o r m e s ; " f l a t f i s h " ) . The n a t u r a l h i s t o r y o f t h i s s p e c i e s has been w e l l documented (see Hart, 1973); however, l i t t l e i s known about the h a b i t s o f the j u v e n i l e f i s h . In B r i t i s h Columbia, spawning may occur from January t o March ( T a y l o r , 1946). Lemon s o l e l a r v a e , l i k e other f l a t f i s h , are symmetri-c a l and p e l a g i c f o r s i x t o ten weeks bef o r e they undergo 8 metamorphosis and s e t t l e t o the bottom. In southern B r i t i s h Columbia metamorphosing l a r v a e are found on the beaches i n m i d - A p r i l ; when metamorphosis i s complete the f i s h g e n e r a l l y measure around 10 mm. Growth i s r a p i d (23 mm per month) i n the s p r i n g and summer months o f the f i r s t year o f l i f e ; they r e a c h 100 mm t o t a l l e n g t h by August. Lemon s o l e average 125 mm a t the end o f one year o f l i f e ; a f t e r two years they average 200 mm (Ketchen, 1947; F o r r e s t e r , 1969). Growth i s extremely slow d u r i n g f a l l and w i n t e r months (Ketchen, 1947; Manzer, 1951; Smith and N i t s o s , 1969; van Cleve and e l -sayed, 1969; F o r r e s t e r , 1969a); other s t u d i e s have shown t h a t i n mid-winter lemon s o l e undergo p e r i o d s o f semi-h i b e r n a t i o n and no food i n t a k e . There i s a d e f i n i t e y e a r l y movement o f a d u l t f i s h i n t o deeper waters i n the w i n t e r and a r e t u r n i n t o shallow waters i n the s p r i n g . L i k e most f l a t -f i s h , j u v e n i l e lemon s o l e tend t o congregate on c e r t a i n "nursery grounds" where they remain u n t i l mature (Rae, 1965) . S t u d i e s o f lemon s o l e m i g r a t o r y h a b i t s have shown t h a t , as a r u l e , e x t e n s i v e m i g r a t i o n s are not undertaken and p o p u l a t i o n s g e n e r a l l y remain d i s t i n c t (Ketchen and F o r r e s t e r , 1955; F o r r e s t e r , 1969b). Lemon s o l e on the B r i t i s h Columbia c o a s t are c h a r a c t e r i s t i c a l l y found i n v a r i o u s i s o l a t e d p o p u l a t i o n s a t the heads o f many i n l e t s (Hart, 1973). 9 MATERIALS AND METHODS C o l l e c t i o n Procedures and Data A n a l y s i s Normal and tumour-bearing lemon s o l e were c o l l e c t e d r e g u l a r l y from a sampling area near the mouth of the North Arm o f the Fr a s e r R i v e r , near Vancouver, B r i t i s h Columbia. Other areas i n the S t r a i t o f Georgia were sampled i r r e g u l a r l y . F l a t f i s h were c o l l e c t e d a t depths o f 1 - 20 fathoms, w i t h e i t h e r an o t t e r t r a w l or a door t r a w l towed a t slow speed. A l l f l a t f i s h under 180 mm t o t a l l e n g t h were removed and taken to the l a b o r a t o r y ; l a r g e r f l a t f i s h were r e l e a s e d a f t e r being examined and r e c o r d e d . Those f l a t f i s h removed t o the l a b o r a t o r y were e i t h e r kept i n tanks o r k i l l e d and pr e s e r v e d i n 10% f o r m a l i n . A l l f i s h were examined f o r evidence o f epidermal l e s i o n s and separated i n t o normal and tumor-b e a r i n g groups. A l l f l a t f i s h were i n d i v i d u a l l y d e s c r i b e d by s p e c i e s , t o t a l l e n g t h s , and weights. In a d d i t i o n , draw-ings were made o f each tumor-bearing f l a t f i s h , i l l u s t r a t i n g the p o s i t i o n , r e l a t i v e s i z e , c o l o r a t i o n , and g e n e r a l macro-s c o p i c d e s c r i p t i o n o f each l e s i o n . Epidermal l e s i o n s found on f l a t f i s h are o f two b a s i c morphologic types w i t h a continuum of i n t e r m e d i a t e s . The tumors can be c l a s s i f i e d m o r p h o l o g i c a l l y as a n g i o e p i t h e l i a l nodules (AENs), epidermal p a p i l l o m a s (EPs), and AEN/EP t r a n s i t i o n forms, as p r e v i o u s l y d e s c r i b e d (Wellings e t a l . , 1964). These c l a s s i f i c a t i o n s w i l l be used i n d e s c r i p t i o n s o f tumors s t u d i e d h i s t o l o g i c a l l y ; each type has d i s t i n c t i v e morphologic and h i s t o l o g i c c h a r a c t e r i s t i c s , as shown i n F i g u r e 1 - 6 . Tumors c l a s s i f i e d ( m o r p h o l o g i c a l l y ) as AENs were s m a l l , h e m i s p h e r i c a l , w h i t e - t o - r e d , smooth-surfaced nodules l o c a t e d anywhere on the body; the diameters o f the nodules v a r i e d from 1 - 5 mm. Those tumors c l a s s i f i e d as AEN/EP t r a n s i t i o n stages showed c h a r a c t e r i s t i c s o f both l e s i o n t y p e s . They g e n e r a l l y were l a r g e (3 - 7 mm i n diameter) h e m i s p h e r i c a l growths w i t h spreading, p l a q u e - l i k e edges, or w i t h a rough, g r a n u l a r s u r f a c e ( i n d i c a t i v e o f t h i c k e n i n g and i n f o l d i n g o f the e p i d e r m i s ) . Epidermal p a p i l l o m a type tumors were g e n e r a l l y l a r g e ( c o v e r i n g up to o n e - h a l f o f one s i d e of some f i s h ) , r a i s e d , g r a n u l a r , g r a y - t o - b l a c k l e s i o n s w i t h spreading, d i f f u s e edges; many n o n - d i f f u s e , c l e a r l y d e f i n e d epidermal p a p i l l o m a s were a l s o noted, a l t h o u g h i n s m a l l e r numbers. The s e s s i l e p a p i l l o m a s (with b r o a d l y based, graded edges) and the pedunculated p a p i l l o m a s (with a narrow base and w e l l - d e f i n e d edges) showed i d e n t i c a l h i s t o l o g i c a l s t r u c -t u r e and f o r t h a t reason were grouped as one type. Both m o r p h o l o g i c a l types of EP l e s i o n s e x h i b i t the s o - c a l l e d " c a u l i f l o w e r - l i k e " appearance shown by other epidermal p a p i l l o m a s i n other f i s h e s . The g r a d i n g o f epidermal l e s i o n s on the b a s i s o f e x t e r n a l morphology can be s u b j e c t i v e , e s p e c i a l l y when c o n s i d e r i n g an i n f i n i t e v a r i e t y o f i n t e r m e d i a t e s between the two b a s i c tumor t y p e s . C o r r e c t l y , any f l a t f i s h epidermal l e s i o n w i t h spreading, p l a q u e - l i k e edges or w i t h a g r a n u l a r -11 PLATE 1 F i g u r e s 1-6 F i g . 1 E x t e r n a l morphology of tumour type d e s i g n a t e d as a n g i o e p i -t h e l i a l nodules (AEN); on j u v e n i l e lemon s o l e . X 3/4. F i g . 2 E x t e r n a l morphology o f tumour type d e s i g n a t e d as AEN/EP t r a n s i t i o n forms (Tra n s ) ; on j u v e n i l e lemon s o l e . X 3/4. F i g . 3 E x t e r n a l morphology o f tumour type d e s i g n a t e d as E P - l i k e (EP) l e s i o n s ; on j u v e n i l e lemon s o l e . X 3/4. F i g . 4 T y p i c a l h i s t o l o g i c a l s t r u c t u r e o f AEN tumour s t a g e . Epidermis appears normal. Hematoxylin/eosiri. _ Approx. X 3.5. F i g . 5 T y p i c a l h i s t o l o g i c a l s t r u c t u r e o f AEN/CP t r a n s i t i o n forms. Epidermis i s s l i g h t l y t h i c k e n e d and i n f o l d e d ; s p r e a d i n g edges are apparent. Hematoxylin/eosin. Approx. X 3. F i g . 6 T y p i c a l h i s t o l o g i c a l s t r u c t u r e o f EP tumour stage. The epidermis i s h i g h l y t h i c k e n e d and i n f o l d e d . Hematoxylin/ e o s i n . Approx. X 3. 13 appearing epidermis should be termed an EP; h i s t o l o g i c a l l y , l e s i o n s a t these stages show more s i m i l a r i t i e s t o EPs than to AENs (see M i l l e r , 1969; M i l l e r and W e l l i n g s , 1971). For t h i s reason, o n l y the AEN and EP c l a s s i f i c a t i o n s were used i n c o m p i l a t i o n of the data comparing r e l a t i v e occurrences of tumour types ( i n F i g u r e 9 ) . Tumour n a t u r a l h i s t o r y data i n t h i s r e p o r t are presented as a f u n c t i o n o f f i s h s i z e ; the data are p r e s e n t e d i n v t e r m s o f 5 mm s i z e groups ( t o t a l l e n g t h s ) . The r e l a t i o n -s h i p between f i s h s i z e and age was not determined by o t o l i t h or s c a l e r e a d i n g s . I n t e r p r e t a t i o n o f age and s i z e r e l a t i o n -s h i p s were based on n a t u r a l h i s t o r y and growth r a t e data from pr e v i o u s s t u d i e s o f lemon s o l e growth (see i n t r o d u c t i o n f o r r e f e r e n c e s ) ; s i m i l a r growth p a t t e r n s can r e a s o n a b l y be assumed i n the lemon s o l e p o p u l a t i o n s t u d i e d . The a n a l y s i s of percentages o f tumour-bearing f i s h per s i z e group was based on 3896 lemon s o l e under two years of age (under 180 mm t o t a l l e n g t h ) , the t o t a l j u v e n i l e lemon s o l e c a t c h from the North Arm sampling s i t e d u r i n g the p e r i o d from September 1973 to February 1975. Other analyses o f the n a t u r a l h i s t o r y o f the l e s i o n s ( i . e . , average number o f l e s i o n s per f i s h , and r e l a t i v e i n c i d e n c e s o f AENs and EPs) were based on a sample o f 510 tumour-bearing f i s h c o l l e c t e d d u r i n g the p e r i o d from September 1973 to June 1974. 14 L i g h t Microscopy For l i g h t m i c r o s c o p i c s t u d i e s , normal s k i n and tumorous t i s s u e s were f i x e d i n 10% f o r m a l i n or Carnoy's s o l u t i o n , embedded i n p a r a f f i n , and s e c t i o n e d a t 5-15 microns. S e c t i o n s were s t a i n e d w i t h hematoxylin and e o s i n , t o l u i d i n e b l u e , or by the Feulgen r e a c t i o n ( c o u n t e r s t a i n e d w i t h f a s t g r e e n ) . H i s t o l o g i c a l s t u d i e s were based on s e c -t i o n s from 134 tumors o f v a r i o u s types from 38 lemon s o l e . Transmission E l e c t r o n Microscopy For u l t r a s t r u c t u r a l s t u d i e s , normal s k i n and tumorous t i s s u e s were removed from l i v e f i s h a n e s t h e t i z e d w i t h t r i c a i n e methanesulphonate (MS-222). These t i s s u e s were c u t i n t o 1 mm cubes and f i x e d f o r 1-2 hours i n 2.5% g l u t a r a l d e h y d e ( S a b a t i n i et, a l _ . , 1963) b u f f e r e d a t pH 7.4 wi t h a phosphate b u f f e r ( K a r l s s o n and S c h u l t z , 1965). The specimens were p o s t - f i x e d i n 1% osmium t e t r o x i d e f o r 1 hour, dehydrated i n a graded s e r i e s o f methanols, and embedded i n Epon ( L u f t , 1961). The bl o c k s were polymerized a t 60°C f o r 20 hours. Thin s e c t i o n s were c u t on a R e i c h e r t 0mU2 u l t r a -microtome or on a S o r v a l l MT-2 ultrataicrotome. The s e c t i o n s were s t a i n e d w i t h u r a n y l a c e t a t e (as i n S j o s t r a n d , 1967) and l e a d c i t r a t e (Reynolds, 1963) and observed w i t h a H i t a c h i HS-7S e l e c t r o n microscope. Thick (1 micron) s e c t i o n s were a l s o c u t from Epon-embedded m a t e r i a l , s t a i n e d w i t h t o l u i d i n e b l u e , and u t i l i z e d i n l i g h t m i c r o s c o p i c s t u d i e s . Scanning E l e c t r o n Microscopy For s t u d i e s o f the s u r f a c e s t r u c t u r e o f normal f l a t f i s h epidermal c e l l s , s m a l l (1 cm) squares o f lemon s o l e s k i n were removed, f i x e d i n 10% f o r m a l i n , dehydrated, coated w i t h a t h i n l a y e r o f g o l d , and examined w i t h a Cambridge Stereoscan scanning e l e c t r o n microscope. 16 RESULTS N a t u r a l H i s t o r y Data During t h i s study, a t o t a l o f 3896 lemon s o l e were c o l l e c t e d from the North Arm sampling s i t e . A l l f i s h were under 180 mm i n t o t a l l e n g t h and were t e n t a t i v e l y i d e n t i f i e d as being i n the f i r s t two years o f l i f e . A t o t a l o f 1164 lemon s o l e were found t o be tumour-bearing. These f i g u r e s i n d i c a t e a cumulative tumour prev a l e n c e o f 29.9% f o r the p e r i o d from September 1973 t o February 1975. However, the percentages o f f l a t f i s h b e a r i n g l e s i o n s v a r i e d monthly and showed a y e a r l y c y c l e . Tumourous f i s h appeared i n August or September, showed a peak p r e v a l e n c e i n November ( a p p r o x i -mately 63%), and showed d e c l i n i n g f r e g u e n c i e s t h e r e a f t e r t o a low i n June, J u l y , and August (approximately 5 % ) . F i g u r e 7 shows the r e l a t i o n s h i p between frequency o f tumour-bearing f i s h and f i s h l e n g t h . A d r a s t i c i n c r e a s e i n tumour prev a l e n c e i s noted between the 50-54 mm s i z e group and the 75-79 mm group. A f t e r t h i s i n c r e a s e (to a maximum percentage i n c i d e n c e o f 49.0%), f u r t h e r s i z e i n c r e a s e s are d i r e c t l y c o r r e l a t e d w i t h s t e a d i l y d e c r e a s i n g tumour percentages u n t i l the zero p o i n t i s reached i n the 160-164 mm group. The r e l a t i o n s h i p between f i s h s i z e and the number of tumours found on each f i s h i s shown i n F i g u r e 8. The histogram shows t h a t the average number o f tumors per f i s h FIGURE 7 The r e l a t i o n s h i p o f tumour prevalence and f i s h s i z e . Histogram i l l u s t r a t i n g the r e l a t i o n s h i p between the percentage o f tumour-bearing f i s h and f i s h l e n g t h , showing a decrease i n tumour prevalence as t o t a l l e n g t h i n c r e a s e s . . Data based on the a n a l y s i s o f 3896 lemon s o l e sampled from one area i n the S t r a i t o f Georgia from September 1973 t o February 1975. 18 Starting points of 5 mm size groups ( f i s h t o t a l lengths) FIGURE 8 The relationship of numbers of tumours per fish and fish size. Histogram illustrating the relationship between the average number of tumours per tumour-bearing fish and fish length, showing a gradual decrease in the number of tumours per fish as total length increases. Data based on the analysis of 510 tumour-bearing lemon sole. 20 xi co •H «H CO o u o E 15 co o s -p o 10 . 5 -CU cu > -p-o VJ1 o 0> o o oo o vO O O -» O O o O -p-o vn o ON o -o o Starting points of 5 mm size groups ( f i s h t o t a l length) decreases, w i t h i n c r e a s i n g f i s h l e n g t h s , from an average o f 11.5 i n the 55-59 mm group to 2.0 i n the 150-154 mm group. The r e l a t i v e i n c i d e n c e s o f a n g i o e p i t h e l i a l nodules and epidermal papillomas w i t h r e l a t i o n to f i s h s i z e are i l l u s t r a t e d i n F i g u r e 9. The data show a decrease i n the r e l a t i v e number o f AENs w i t h i n c r e a s i n g f i s h l e n g t h , w i t h a r e c i p r o c a l r i s e i n the frequency o f EPs. No AENs were found on f i s h l a r g e r than 120 mm t o t a l l e n g t h . Epidermal L e s i o n s i n Other Species S c a t t e r e d i n s t a n c e s o f epidermal l e s i o n s i n other s p e c i e s o f f l a t f i s h were a l s o observed i n the S t r a i t o f Georgia sampling a r e a s . H i s t o l o g i c a l examination o f the tumourous t i s s u e s showed them t o be i d e n t i c a l with the lemon s o l e tumours. These l e s i o n s i n c l u d e one AEN on one s t a r r y f l o u n d e r ; EPs on one sp e c k l e d sanddab ( C i t h a r i c h t h y s  stigmaeus) and one f l a t h e a d s o l e ; and AENs, AEN/EP t r a n s i -t i o n a l forms, and EPs on many sand s o l e . Normal Epidermis; L i g h t Microscopy The morphology o f the normal s k i n o f the lemon s o l e i s s i m i l a r to p r e v i o u s d e s c r i p t i o n s o f t e l e o s t epidermis (van Oosten, 1957; Andrew, 1959; L a g l e r e_t a l _ . , 1962; Romer, 1962; Hyman, 1962; P a t t and P a t t , 1969). The s k i n FIGURE 9 The relationship of tumour types and f i s h s i z e . Histogram i l l u s t r a t i n g the r e l a t i v e prevalences of AEN and EP tumour types as related to f i s h length. A steady drop i n AEN frequency and a r e c i p r o c a l r i s e i n EP frequency are shown as t o t a l length increases. Data based on the analysis of 2965 tumours from 510 lemon sole. 22a 23 consists of a simple squamous layer of epidermal cells overlying a connective tissue dermis (Figure 10) . The epidermis consists of 5-7 layers of ovoid, basophilic cells ; the exact number of layers varies with location on the body. Generally, the back and head regions are the thickest; the fin web epidermis is thinnest. A l l cells , from basal to superficial layers, are nucleate; no nucleoli are resolvable. Some flattening of the epidermal cells can be noticed in the most superficial cells ; these cells also tend to be more basophilic. Darkly-staining mucus-secreting cells in various stages of maturation are interposed between the epidermal cells ; the smallest mucus cells are generally located near the basal layers, while more mature cells can be observed near, and often opening to, the surface. Occasionally small, darkly-staining cells can be observed scattered throughout the epidermis, usually in the inter-mediate ce l l layers. These cells are tentatively identified as lymphocytes or resident macrophages (histiocytes). The dermis of scale-bearing skin is composed of two layers: a superficial scale-bearing region of loosely arranged collagen fibers, fibroblasts, and pigment cells (stratum spongiosum), and a second, scale-free region of dense layers of collagen fibers (stratum compactum), which forms the boundary between the dermis and the subcutaneous tissues. PLATE 2 Fi g u r e s 10-13 F i g . 10 Normal s k i n from the mid-side o f a j u v e n i l e lemon s o l e . Epidermis i s f i v e t o seven l a y e r s t h i c k and i n t e r s p e r s e d w i t h mucous-secreting c e l l s . Hematoxylin/eosin. X 160. F i g . 11 Normal lemon s o l e e p i d e r m i s , i l l u s t r a t i n g c e l l s o f the b a s a l and i n t e r m e d i a t e l a y e r s . X 4520. F i g . 12 Normal lemon s o l e e p i d e r m i s , i l l u s t r a t i n g the r e l a t i o n s h i p o f mucous and epidermal c e l l s ; mucous c e l l i s a p p a r e n t l y ready t o open to the s u r f a c e . M i c r o v i l l i - l i k e p l e a t s on s u p e r f i c i a l c e l l s are not apparent i n t h i s s e c t i o n . X 4520. F i g . 13 Scanning e l e c t r o n micrograph o f lemon s o l e e p i d e r m i s , showing c h a r a c t e r i s t i c a l l y arranged s u r f a c e p l e a t s . X 9500. 26 Normal Epidermis/ E l e c t r o n Microscopy The u l t r a s t r u c t u r a l morphology o f the lemon s o l e epidermis corresponds to e l e c t r o n m i c r o s c o p i c d e s c r i p t i o n s of the epidermis o f other t e l e o s t s (Hendrickson and M a t o l t s y , 1968; W e l l i n g s and Brown, 1969; Brown and W e l l i n g s , 1970; M e r r i l e e s , 1974). The epidermis i s composed o f two b a s i c c e l l t y p e s : squamous epidermal c e l l s and mucus-s e c r e t i n g c e l l s . Mucus c e l l s , as mentioned above, can be found randomly i n t e r p o s e d between epidermal c e l l s i n a l l l a y e r s . The epidermal c e l l s can be c o n s i d e r e d to form three d i s t i n c t l a y e r s : o v o i d b a s a l c e l l s , which immediately o v e r l i e the basement membrane; int e r m e d i a t e c e l l s , which o v e r l i e the b a s a l l a y e r and may occur as 3-5 c e l l l a y e r s ; and f l a t t e n e d s u p e r f i c i a l c e l l s , which compose the o u t e r -most l a y e r of the epidermis ( F i g u r e s 11 and 12). Epidermal c e l l s i n a l l l a y e r s can be c h a r a c t e r i z e d by the presence o f two zones: the p e r i n u c l e a r zone, which c o n t a i n s the nucleus and a l l c y t o p l a s m i c o r g a n e l l e s ; and the p e r i p h e r a l zone, which c o n t a i n s many c y t o p l a s m i c f i l a -ments and ribosomes (Figure 11). N u c l e i o f squamous c e l l s are i r r e g u l a r l y shaped, o f t e n w i t h i n d e n t a t i o n s . N u c l e o l i were r a r e l y observed. Cytoplasmic o r g a n e l l e s , i n the p e r i n u c l e a r zone o f a l l c e l l s , i n c l u d e mitochondria ( u s u a l l y w i t h t r a n s v e r s e c r i s t a e ) , G o l g i apparatus, g r a n u l a r and a g r a n u l a r endoplasmic r e t i c u l u m , and ribosomes o f 15.0 nm diameter. 27 D i f f e r e n c e s i n c e l l u l a r morphology can be seen i n the t r a n s i t i o n from b a s a l t o s u p e r f i c i a l l a y e r s . These changes i n c l u d e : a g e n e r a l f l a t t e n i n g o f the c e l l s , a r e d u c t i o n i n the complexity o f c e l l - t o - c e l l i n t e r d i g i t a t i o n s , an i n c r e a s e i n the number o f desmosomes and other j u n c t i o n a l - complexes, and an i n c r e a s e i n the number o f c y t o p l a s m i c f i l a m e n t s . The f r e e s u r f a c e o f the s u p e r f i c i a l c e l l s i s c h a r a c t e r i z e d by s h o r t , broad, s u r f a c e p l e a t s resembling m i c r o v i l l i i n c r o s s - s e c t i o n (Figure 1 3 ). Often the s u r f a c e i s covered w i t h an amorphous or filamentous extraneous c o a t i n g . Mucus c e l l s are found s c a t t e r e d throughout the epidermis a t v a r y i n g stages of m a t u r i t y . Immature c e l l s are g e n e r a l l y found i n the deeper l a y e r s , w h i l e mature, d i s t e n d e d c e l l s are found i n the more s u p e r f i c i a l epidermal l a y e r s , o f t e n seen opening to the s u r f a c e . The mucus c e l l cytoplasm c o n t a i n s abundant g r a n u l a r endoplasmic r e t i c u l u m . The mucus d r o p l e t s compose a l a r g e area o f the c e n t r a l cytoplasm; most o f t e n the c y t o p l a s m i c o r g a n e l l e s are compressed i n t o a p e r i p h e r a l p r o t o p l a s m i c r i m a t the c e l l edges. The mucus d r o p l e t s tend to be f i n e l y f i b r i l l a r i n appearance and are surrounded by a s i n g l e a g r a n u l a r l i m i t i n g membrane (Figure 12). D i r e c t l y u n d e r l y i n g the b a s a l c e l l l a y e r i s a basement membrane composed o f a f i n e f i b r i l l a r m a t e r i a l . A l a y e r o f dermal c o l l a g e n l i e s beneath the basement membrane. U n d e r l y i n g , and p a r a l l e l t o , these l a y e r s , are the v a r i o u s 28 pigment-containing c e l l s . Melanophores are the commonest pigm e n t - c o n t a i n i n g c e l l s and are c h a r a c t e r i z e d by numerous, dense melanin granules i n the cytoplasm. I n t e r s p e r s e d among the melanophores are i r i d o p h o r e s (guanidophores) which are c h a r a c t e r i z e d by numerous, l a r g e , empty c l e f t s i n the cytoplasm; i n l i f e , these c e l l s c o n t a i n p l a t e l i k e guanine c r y s t a l s which are leached out d u r i n g specimen p r e p a r a t i o n . Tumor H i s t o l o g y : L i g h t Microscopy Three morphologic v a r i a t i o n s o f the f l a t f i s h epidermal l e s i o n s can be d e s c r i b e d , each w i t h a c h a r a c t e r -i s t i c h i s t o l o g i c a l appearance (Fi g u r e s 1-6). These tumor types have p r e v i o u s l y been desi g n a t e d as a n g i o e p i t h e l i a l nodules (AENs), epidermal p a p i l l o m a s (EPs), and AEN/EP t r a n s i t i o n forms (Wellings e t a l . , 1964). The g r o s s mor-p h o l o g i c appearance o f each type has been g i v e n above; h i s t o l o g i c a l d e s c r i p t i o n s are g i v e n below. A n g i o e p i t h e l i a l Nodules The stromal component o f the AEN v a r i e s from angiomous t o fibromous; o f t e n a s i n g l e AEN shows c h a r a c t e r -i s t i c s o f each (Figure 14). The fibromous stroma l type i s c h a r a c t e r i z e d by a h y p e r p l a s i a o f dermal f i b r o b l a s t s iwith an accompanying i n c r e a s e i n c o l l a g e n f i b e r s . The hyper-p l a s i a can be seen t o o r i g i n a t e , i n s m a l l AENs, i n the stratum compactum, i n the stratum spongiosum, or i n the t i s s u e area d i r e c t l y below the stratum compactum. The PLATE 3 F i g u r e s 14-17 F i g . 14 Stroma o f AEN, showing f i b r o m a - l i k e c h a r a c t e r i s t i c s . Hematoxylin/eosin. X 160. F i g . 15 Stroma o f AEN, showing packed s i n u s o i d s and stroma l "X-c e l l s " . T o l u i d i n e b l u e , 1 u s e c t i o n . X 640. F i g . 16 Cross s e c t i o n o f lemon s o l e b e a r i n g AEN l e s i o n , showing i n v a s i o n o f muscle t i s s u e by the AEN stroma. Epidermis i s normal. Hematoxylin/eosin. X 5. F i g . 17 Higher m a g n i f i c a t i o n o f l e s i o n shown i n F i g u r e 16. Hematoxylin/eosin. X 160. angiomous stromal type c o n s i s t s o f many c a p i l l a r i e s and s i n u s o i d s ; u s u a l l y these v a s c u l a r beds show a h i g h degree of b l o o d c e l l p a c k i n g . T h i s p a c k i n g o f r e d b l o o d c e l l s , lymphocytes, and g r a n u l o c y t e s becomes e s p e c i a l l y apparent i n t o l u i d i n e b l u e - s t a i n e d s e c t i o n s o f Epon-embedded m a t e r i a l (Figure 15). A new c e l l type, randomly d i s p e r s e d throughout the s t r o m a l component among the f i b r o b l a s t s , i s found i n these l e s i o n s . These c e l l s , h e r e a f t e r r e f e r r e d t o as the u n i d e n t i f i e d stromal c e l l type, d i f f e r i n morphology and s t a i n i n g c h a r a c t e r i s t i c s from normal blood, epidermal, and dermal c e l l types (Figure 15). C e l l s o f t h i s type are g e n e r a l l y twice the diameter o f f i b r o b l a s t s and appear i n hematoxylin/eosin s e c t i o n s as round c e l l s w i t h a p a l e r e d cytoplasm and a s l i g h t l y darker n u c l e u s . The s i m i l a r c o l o r i n g and g r a n u l a r i t y o f the nucleus and the cytoplasm o f t e n make the two i n d i s t i n g u i s h -a b l e . I n t o l u i d i n e b l u e , Epon-embedded s e c t i o n s the c e l l s appear as l i g h t l y s t a i n i n g , v a c u o l a t e d c e l l s w i t h t r a n s p a r e n t , n o n - s t a i n i n g n u c l e i s w i t h prominent, d a r k l y s t a i n i n g n u c l e o l i . These c e l l s were never observed w i t h i n b l o o d v e s s e l s but were commonly seen i n angiomous.. and fibromous a r e a s . They were observed most o f t e n s i n g l y , but can be found i n "cords" o f two to four c e l l s . These stroma l " X - c e l l s " d i f f e r from c e l l types A and B of the epidermis ( d e s c r i b e d below) i n s i z e The term " X - c e l l " i s used c o l l e c t i v e l y i n r e f e r r i n g to any o f the u n i d e n t i f i e d , g i a n t c e l l t y p es. T h i s i n c l u d e s s t r o m a l " X - c e l l s " , type A " X - c e l l s " , and type B " X - c e l l s " . 32 and s t a i n i n g c h a r a c t e r i s t i c s . In some cases, the s t r o m a l component was seen t o invade the u n d e r l y i n g muscle masses, o f t e n p e n e t r a t i n g deep among i n d i v i d u a l muscle f i b e r s ( Figures 16 and 17). The epidermal component of the AEN v a r i e s from normal to s l i g h t l y h y p e r p l a s t i c . In i n i t i a l AENs, a l l epidermal c e l l s tend to r e t a i n t h e i r normal s t a i n i n g c h a r a c t e r i s t i c s . T o l u i d i n e blue s e c t i o n s show a marked inc r e a s e i n i n t e r c e l l u l a r spaces, e v i d e n t l y due t o a l o o s e n -ing o f the c e l l membrane i n t e r d i g i t a t i o n s d e s c r i b e d i n the normal e p i d e r m i s . T h i s l o o s e n i n g o f c e l l u l a r c o n t a c t s appears to begin i n the intermediate l a y e r s o f the e p i d e r m i s ; the s u p e r f i c i a l l a y e r s tend to r e t a i n a r e l a t i v e l y normal appearance (Figure 18). Often the epidermal h y p e r p l a s i a i s accompanied by a subsequent i n c r e a s e i n mucus c e l l numbers and s e c r e t i o n s (Figure 19); more o f t e n no such i n c r e a s e i s seen. A l s o c h a r a c t e r i s t i c o f the epidermal h y p e r p l a s t i c stage i s the appearance o f a l a r g e number o f s m a l l , d a r k l y - s t a i n i n g c e l l s j u s t above the b a s a l l a y e r (Figure 19); these c e l l s appear i d e n t i c a l t o the b a s o p h i l i c c e l l type observed i n the normal epidermis ( i d e n t i f i e d as lymphocytes or macrophages). Accompanying v a r y i n g degrees o f h y p e r p l a s i a i s the appearance o f a d i f f e r e n t c e l l type i n the epidermal component; these c e l l s f i r s t appear along the basement membrane. They are c h a r c t e r i z e d by enlargement; an i n c r e a s e i n the n u c l e u s r c y t o p l a s m r a t i o ; the appearance o f a d a r k l y - s t a i n i n g , c e n t r a l n u c l e o l u s ; l o s s of s t a i n i n g 33 w i t h Feulgen's reagent; and l o s s o f b a s o p h i l i a . Both nucleus and cytoplasm s t a i n a p a l e pink c o l o r i n hematoxylin/ e o s i n s e c t i o n s . T h i s c e l l type, because o f i t s i n c r e a s e d diameter, w i l l h e r e a f t e r be r e f e r r e d t o as one o f the tumor's " g i a n t c e l l " t y p e s ; i t i s t e n t a t i v e l y i d e n t i f i e d , due to i d e n t i c a l s t a i n i n g c h a r a c t e r i s t i c s , as an " X - c e l l " o f type B (as d e s c r i b e d i n EP l e s i o n s , below). AEN/EP T r a n s i t i o n Forms The stromal component of the AEN/EP t r a n s i t i o n forms ( F i g u r e s 20 and 21) remains i d e n t i c a l t o t h a t o f the AEN, d e s c r i b e d above. The epidermal component o f the t r a n s i t i o n forms i s c h a r a c t e r i z e d by t h i c k e n i n g due t o h y p e r p l a s i a ; w i t h i n c r e a s e d t h i c k e n i n g , the epidermis becomes i n f o l d e d , both over the i n i t i a l stromal growth and outwards from the i n i t i a l focus (Figure 20). Some of the observed h y p e r p l a s t i c r e a c t i o n s o f the epidermis r e a c h c o n s i d e r a b l e p r o p o r t i o n s without f u r t h e r c y t o l o g i c a l changes and resemble t y p i c a l h y p e r p l a s t i c r e a c t i o n s ( i . e . , the c e l l s show l i t t l e pleomorphism and r e t a i n normal s t a i n i n g c h a r a c t e r i s t i c s and p o l a r i t y ) ( Figures 21 and 22). Most o f t e n , i n c r e a s i n g numbers o f the ov o i d , p a l e - s t a i n i n g g i a n t c e l l s (type B; d e s c r i b e d i n the AEN epidermis) occur w i t h epidermal i n f o l d i n g s (Figure 23). When these c e l l s occur i n l a r g e numbers, they tend t o show i n c r e a s i n g volume changes and d e c r e a s i n g s t a i n a b i l i t y w i t h hematoxylin and Feulgen's reagent ( F i g u r e s 24 and 25); most PLATE 4 F i g u r e s 18-21 F i g . 18 Epidermal component o f AEN. Note the i n c r e a s e i n mucous c e l l numbers, the i n c r e a s e i n i n t e r c e l l u l a r space i n b a s a l and i n t e r m e d i a t e l a y e r s , and the apparent v a c u o l a t i o n o f c e l l s i n the b a s a l l a y e r (arrow). These v a c u o l a t e d c e l l s a p p a r e n t l y are type B " X - c e l l s " . T o l u i d i n e b l u e , 1 /a s e c t i o n . X 640. F i g . 19 S e c t i o n o f AEN, showing stromal and epidermal components of the t i s s u e . Note the accumulation o f d a r k l y - s t a i n i n g c e l l s j u s t above the b a s a l l a y e r . Hematoxylin/eosin. X 160. F i g . 20 S e c t i o n through AEN/EP t r a n s i t i o n form l e s i o n . Hematoxylin/ e o s i n . X 5. F i g . 21 I n i t i a l h y p e r p l a s t i c stage o f AEN/EP t r a n s i t i o n a l form e p i d e r m i s . Mucous c e l l s are absent. Hematoxylin/eosin. X 160. 36 o f these c e l l s can be c l a s s i f i e d as type A c e l l s (see d e s c r i p t i o n s below). Epidermal P a p i l l o m a - l i k e L e s i o n s The stromal p o r t i o n o f the p a p i l l o m a - l i k e l e s i o n appears i d e n t i c a l to the stromal component o f the AEN and AEN/EP t r a n s i t i o n s t a g e s . The elements o f the stroma i n c l u d e chromatophores, f i b r o b l a s t s , c o l l a g e n f i b e r s , s i n u -s o i d s , and c a p i l l a r i e s (Figure 26). The v a s c u l a r elements o f t h i s branching stroma tend t o be s m a l l e r i n s i z e and show no packing o f b l o o d c e l l s . S t r u c t u r e s resembling a l t e r e d c a p i l l a r i e s can be found i n the stroma o f the p a p i l l o m a -l i k e l e s i o n s ; these s t r u c t u r e s presumably r e p r e s e n t c o l l a p s e d or c l o s e d c a p i l l a r y channels (Figure 26). Numerous u n i d e n t i f i e d c e l l s o f the same type, i d e n t i c a l t o those p r e v i o u s l y d e s c r i b e d i n the AEN stroma, can be observed i n the t h i n s t a l k s o f the stroma o f the pa p i l l o m a ( F i g u r e 26). The dominant components o f the p a p i l l o m a epidermis are e n l a r g e d , o v o i d , " g i a n t " c e l l types (Figure 27). The epidermis i s composed o f th r e e c e l l types, each w i t h d i s t i n c -t i v e s t a i n i n g c h a r a c t e r i s t i c s and morphology. These c e l l types have been c l a s s i f i e d a c c o r d i n g l y as type A ( l a r g e g i a n t c e l l s ) , type B (smaller g i a n t c e l l s ) , and type C ( s u p e r f i c i a l and "enveloping" c e l l s ) . S t a i n i n g c h a r a c t e r i s t i c s were determined by stu d y i n g paraffin-embedded s e c t i o n s s t a i n e d w i t h hematoxylin and eos i n : : 37 PLATE 5 F i g u r e s 22-25 F i g . 22 Higher m a g n i f i c a t i o n o f t i s s u e shown i n F i g u r e 20. Note the homogeneity o f the h y p e r p l a s t i c epidermal c e l l s . The average diameter o f these c e l l s i s around 8 / i , the s i z e o f c e l l s i n the normal e p i d e r m i s . Hematoxylin/eosin. X 640. F i g . 23 S e c t i o n through AEN/EP t r a n s i t i o n a l form, showing the i n i t i a l o ccurrence o f type B c e l l s ( l i g h t l y s t a i n e d ) along the basement membrane; upper epidermal c e l l s r e t a i n t h e i r normal appearance. Feulgen's, f a s t green c o u n t e r s t a i n . X 160. F i g . 24 S e c t i o n through AEN/EP t r a n s i t i o n a l stage, showing i n c r e a s e d numbers o f l a r g e , l i g h t l y s t a i n i n g c e l l s i n the e p i d e r m i s . Larger, more c e n t r a l " X - c e l l s " show s t a i n i n g c h a r a c t e r i s t i c s o f type A c e l l s ; type B c e l l s can a l s o be r e c o g n i z e d . Hematoxylin/eosin. X 160. F i g . 25 S e c t i o n through EP stage l e s i o n . Note the extreme s i z e d i f f e r e n c e between the s u p e r f i c i a l c e l l s (above) and the type A " X - c e l l s " (below). Type A c e l l s do not s t a i n w i t h the Feulgen r e a c t i o n . Feulgen's, f a s t green c o u n t e r s t a i n . X 640. 38 39 Type A. The cytoplasm o f type A c e l l s s t a i n s l i g h t r e d t o v i o l e t and i s e v e n l y g r a i n e d . The nucleus seldom appears s t a i n e d ; the n u c l e o l u s , however, s t a i n s d a r k l y . . The c e l l s o f type A are seldom found near the basement membrane or near the tumor s u r f a c e ; g e n e r a l l y they are found i n the intermediate l a y e r s of the epidermal f o l d s . Type B. Type B c e l l s are c h a r a c t e r i z e d by a pa l e pink t o l i g h t r e d cytoplasm, w i t h a dark v i o l e t n u c l e u s . These c e l l s are always found i n s m a l l groups along the base-ment membrane; r a r e l y they can be found i n the intermediate l a y e r s o f the epidermal i n f o l d i n g s . Type C. The cytoplasm o f type C c e l l s s t a i n s v i o l e t ; the n u c l e i s t a i n b l u e . F r e q u e n t l y the n u c l e i are swollen and the chromatin appears g r a n u l a r . C e l l s o f t h i s type occur i n the s u p e r f i c i a l l a y e r s : o f the tumor; they a l s o appear t o f i l l i n the spaces between the much l a r g e r g i a n t c e l l s ( c e l l types A and B). For t h i s reason, both the n u c l e i and the cytoplasm o f these c e l l s appear h e t e r o -morphic. The morphologic c h a r a c t e r i s t i c s o f the v a r i o u s c e l l types found i n the p a p i l l o m a - l i k e l e s i o n s were d e t e r -mined from a study o f s e c t i o n s o f Epon-embedded m a t e r i a l s t a i n e d w i t h t o l u i d i n e b l u e : Type A: Type A c e l l s are c h a r a c t e r i s t i c a l l y e n l a r g e d , i r r e g u l a r l y shaped c e l l s which can re a c h a diameter o f nine times t h a t of normal epidermal c e l l s ( F i g u r e s 26, 28 and 29). The cytoplasm g e n e r a l l y s t a i n s l i g h t l y w i t h 40 PLATE 6 F i g u r e s 26-29 F i g . 26 S e c t i o n through EP-type l e s i o n , showing c h a r a c t e r i s t i c s o f stromal and epidermal components. Note a l t e r e d c a p i l l a r y s t r u c t u r e (arrow). The average diameter o f the type A "X-c e l l s " shown i s 23 / i ; compare w i t h F i g u r e 21. T o l u i d i n e b l u e , 1 u s e c t i o n . X 640. F i g . 27 Epidermal component o f EP-type l e s i o n , showing the complete dominance o f the t i s s u e by " X - c e l l " t y p e s . The s t r o m a l component serves o n l y as a s u p p o r t i n g framework f o r the e p i d e r m i s . Note the abundance o f s m a l l , d a r k l y s t a i n i n g c e l l s near the c e n t e r o f the i n f o l d i n g s ; these c e l l s have been t e n t a t i v e l y i d e n t i f i e d as macrophages (arrows). Hema-t o x y l i n / e o s i n . X 160. F i g . 28 S e c t i o n through EP-type. l e s i o n , showing s t r u c t u r e s b e l i e v e d t o be c o l l a p s e d " X - c e l l s " (arrows). T o l u i d i n e b l u e , 1 jx s e c t i o n . X 640. F i g . 29 S e c t i o n through s u p e r f i c i a l area o f EP-type l e s i o n , showing the c h a r a c t e r i s t i c s t r u c t u r e o f the outermost ( s u p e r f i c i a l ) c e l l s . T o l u i d i n e b l u e , 1 ja s e c t i o n . X 640. 41 t o l u i d i n e blue and appears h e a v i l y g r a i n e d . Often the cytoplasm o f these g i a n t c e l l s appears v a c u o l a t e d ; dark c y t o p l a s m i c i n c l u s i o n s can be observed i n the l a r g e r c e l l s . The diameter o f the c e l l s tends t o i n c r e a s e as the c e n t e r o f the epidermal i n f o l d i n g i s approached. The nucleus never s t a i n s w i t h t o l u i d i n e blue and i s always g r e a t l y e n l a r g e d . A c e n t r a l , d a r k l y s t a i n i n g n u c l e o l u s i s o f t e n observed. Type B: C e l l type B appears as a s m a l l e r v e r s i o n o f type A c e l l s : the c e l l s a r e somewhat e n l a r g e d (twice the diameter o f normal epidermal c e l l s ) ; are rounded; possess an en l a r g e d , round, n o n - s t a i n i n g nucleus w i t h a d a r k l y - s t a i n i n g , c e n t r a l n u c l e o l u s ; and appear e x c e s s i v e l y v a c u o l a t e d (Figure 26). These c e l l s , as mentioned above, are most o f t e n found i n groups along the basement membrane ( i . e . , a t the p e r i p h e r y o f the epidermal i n f o l d i n g s ) . Type C: T h i s c e l l type, as p r e v i o u s l y mentioned, can be found i n the most s u p e r f i c i a l l a y e r s o f the tumorous t i s s u e as w e l l as i n t e r s p e r s e d between the g i a n t c e l l s o f types A and B (Figure 26). In the most s u p e r f i c i a l l a y e r s the c e l l s appear f l a t t e n e d , possess f i n g e r - l i k e c onnections w i t h n e i g h b o r i n g c e l l s (with much i n t e r c e l l u l a r space), and s t a i n d a r k l y w i t h t o l u i d i n e b l u e . No n u c l e i can be d i s -cerned, probably due to the i n t e n s e s t a i n i n g c h a r a c t e r i s t i c s o f t h i s c e l l type. The c e l l s found i n t e r s p e r s e d among the g i a n t c e l l s of the epidermis are i r r e g u l a r l y shaped and appear i n t o l u i d i n e b l u e s e c t i o n s as a d a r k l y s t a i n e d " f i l l e r " between the l i g h t l y s t a i n e d g i a n t c e l l s ; hence, they w i l l be r e f e r r e d t o h e r e a f t e r as "enveloping c e l l s " . Again, no n u c l e i can be r e s o l v e d i n these c e l l s because of the i n t e n s i t y o f the s t a i n i n g . Often, i n EP-type l e s i o n s , evidences o f n e c r o s i s can be observed. In t o l u i d i n e b l u e s e c t i o n s , s t r u c t u r e s t e n t a t i v e l y i d e n t i f i e d as c o l l a p s e d g i a n t c e l l s can be found i n the c e n t r a l areas o f the epidermal i n f o l d i n g s (Figure 28). These s t r u c t u r e s c o n s i s t o f a dense mass o f h e a v i l y g r a n u l a r m a t e r i a l surrounded by a c l e a r p e r i p h e r a l zone. T h i s seems to r e p r e s e n t i n d i v i d u a l c e l l u l a r n e c r o s i s . General t i s s u e n e c r o s i s can be observed i n some tumors (Figure 31). Most o f t e n ( i . e . , most c o n s p i c u o u s l y ) , n e c r o t i c t i s s u e can be found i n the lumens formed by the i n f o l d i n g epidermis (Figure 32). T h i s n e c r o t i c m a t e r i a l can be seen t o c o n s i s t o f remnants o f g i a n t c e l l s , i d e n t i f i e d by the presence o f d a r k l y s t a i n i n g n u c l e o l i i n s i d e e n l a r g e d , t r a n s p a r e n t n u c l e i (Figure 33). Lymphocytes w i t h s m a l l , d a r k l y - s t a i n i n g n u c l e i and s c a n t y cytoplasm can be found, o f t e n i n l a r g e numbers, i n t e r s p e r s e d between the g i a n t c e l l s a t the c e n t e r o f the epidermal i n f o l d i n g s (Figure 30). 44 PLATE 7 Figure 30-33 Fig. 30 Higher magnification of section through EP-type lesion shown in Figure 26, illustrating abundant, darkly staining cells , presumed to be macrophages, found in the central areas of epidermal infoldings. Hematoxylin/eosin. X 640. Fig. 31 Section through EP-type lesion which shows evidence of general tissue necrosis. Hematoxylin/eosin. X 160. Fig. 32 Section through EP-type lesion, showing large amounts of evidently necrotic material in a lumen formed by the infold-ing epidermis. Hematoxylin/eosin. X 160. Fig. 33 Illustration of necrotic material found in an epidermal lumen, showing the presence of structures similar to remnants of "X-cells". Toluidine blue, 1 p. section. X 640. 46 Tumour H i s t o l o g y , E l e c t r o n Microscopy  Stroma The stromal component o f the AEN, the AEN/EP t r a n s i -t i o n form, and the EP were seen t o be i d e n t i c a l i n s t r u c t u r e . For t h i s r e a s on the u l t r a s t r u e t u r e o f the s t r o m a l t i s s u e s w i l l be d e s c r i b e d o n l y once. E l e c t r o n m i c r o s c o p i c a l l y , the stroma i s composed o f the f o l l o w i n g elements: chromatophores, c a p i l l a r i e s , white b l o o d c e l l s , macrophages, f i b r o b l a s t s , c o l l a g e n f i b e r s , and an u n i d e n t i f i e d c e l l type e v i d e n t l y i d e n t i c a l t o t h a t d e s c r i b e d i n the stroma o f l i g h t m i c r o -s c o p i c s e c t i o n s . Numerous f i b r o b l a s t s can be found s c a t t e r e d through-out the s t r o m a l component. When l o c a t e d next to the e p i d e r m i s , they can be noted l y i n g p a r a l l e l w i t h the basement membrane, o r i e n t e d i n much the same manner as chromatophores (Figure 34). Deeper i n the stroma they are heteromorphic; o f t e n narrow ar m - l i k e f i b r o b l a s t p rocesses can be observed extending i n t o the i n t e r s t i t i a l spaces, o f t e n surrounding n e i g h b o r i n g c e l l s . C o l l a g e n f i b e r s , measuring approximately 100 nm i n diameter, can be found o r i e n t e d a t v a r i o u s angles and i n t e r s p e r s e d between the c e l l u l a r elements o f the stroma ( F i g u r e s 35, 36 and 37). C a p i l l a r i e s can be observed r e g u l a r l y i n the stroma. They g e n e r a l l y are s m a l l (one r e d b l o o d c e l l diameter) and e x h i b i t a l a r g e number o f p i n o c y t o t i c v e s i c l e s a t the c e l l p e r i p h e r y , e s p e c i a l l y toward the lumen. The cytoplasm o f PLATE 8 F i g u r e s 34-35 F i g . 34 E l e c t r o n micrograph o f basement membrane ar e a o f mature epidermal l e s i o n , showing a l o n g i t u d i n a l l y - o r i e n t e d f i b r o b l a s t , the basement membrane, and the c e l l s o f the l e s i o n ' s epidermal component (Enveloping c e l l s and "X-c e l l s " ) . X 10500. F i g . 35 -= P o r t i o n o f the stromal component o f an a n g i o e p i t h e l i a l nodule, showing some t y p i c a l elements o f the AEN stroma: f i b r o b l a s t s , l a r g e d e p o s i t s o f co l l a g e n o u s t i s s u e , g r a n u l o c y t e s , stromal " X - c e l l s " , and abnormal c a p i l l a r y s t r u c t u r e s . X 10500. PLATE 9 F i g u r e s 36-37 F i g . 36 P o r t i o n o f stromal component o f an a n g i o e p i t h e l i a l nodule, i l l u s t r a t i n g some f e a t u r e s t y p i c a l o f the AEN stroma: lymphocytes, f i b r o b l a s t s , c a p i l l a r i e s , macrophages, c o l l a g e n , and " X - c e l l s " . X 10500. F i g . 37 I l l u s t r a t i o n o f some f e a t u r e s o f the stromal " X - c e l l s " . Note the e x c e s s i v e v a c u o l a t i o n and l a c k o f r e c o g n i z a b l e c y t o p l a s m i c o r g a n e l l e s . X 21000. 51 the endothelial cells tends to be quite electron dense; generally, mitochondria and granular endoplasmic reticulum can be observed. Often smooth muscle cells can be noted in close conjunction with the capillary (Figure 35). Altered capillaries, morphologically similar to those described in toluidine blue stained, Epon-embedded sections, can be observed (Figure 35). These appear as infolded endothelial cel ls . Cells interpreted as macrophages are common in the stroma. These cells are characterized by a large, horseshoe-shaped nucleus with a periphery of condensed chromatin. The nucleoplasm is relatively electron-lucent. The cytoplasm contains many large mitochondria and membrane-bound, electron-dense structures of various sizes and shapes; these were interpeted as digestive vacuoles and residual bodies. The cel l membrane tends to be irregular in outline and forms many finger-like processes (Figure 36). Many small lymphocytes (Figure 36) can be observed randomly dispersed throughout the stroma. These cells are characterized by a scanty cytoplasm and an ovoid nucleus with a large amount of condensed chromatin, especially at the nuclear periphery. The cytoplasm commonly contains a few mitochondria and a few grandular endoplasmic reticulum lamellae. Another white blood cel l type, interpreted as a member of the granulocyte series, was also observed. (Figure 35). These granulocytes contain a number of dense cytoplasmic granules. The nucleus i s commonly d i s p l a c e d t o the edge o f the c e l l ; most mitochondria tend t o be l o c a t e d i n the p e r i n u c l e a r r e g i o n . An u n i d e n t i f i e d stromal c e l l type, s i m i l a r i n most asp e c t s to the u n i d e n t i f i e d c e l l types (to be d e s c r i b e d ) i n the epidermal component, can be found i n t e r s p e r s e d a t random between f i b r o b l a s t s . These c e l l s can be c o n s i d e r e d to be the same u n i d e n t i f i e d stromal c e l l s d e s c r i b e d by l i g h t m i c r o -scopy (F i g u r e s 15 and 26); they appear s l i g h t l y e n l a r g e d or may r e t a i n the s i z e o f normal f i b r o b l a s t s . They are most o f t e n observed s i n g l y , b u t may occur i n p a i r s or l a r g e r groups. The c e l l s tend t o be round or o v o i d and possess no i n t e r c e l l u l a r connections or c e l l - t o - c e l l c o n t a c t o f any s o r t . G e n e r a l l y the c e l l s are e n c l o s e d by the reaches o f f i b r o b l a s t "arms" or p a r t i a l l y surrounded by c e l l s i n t e r p r e t e d as macrophages ( F i g u r e s 36 and 37). The nucleus i s u s u a l l y round w i t h d i s c r e t e clumps o f chromatin s c a t t e r e d randomly throughout the nucleus; most o f t e n a c e n t r a l , e n l a r g e d n u c l e o l u s i s p r e s e n t . The nucleus i s surrounded by a t y p i c a l double-membraned s t r u c t u r e . O c c a s i o n a l l y , normal-appearing g r a n u l a r endoplasmic r e t i c u l u m can be observed i n the c e l l s ' cytoplasm. Otherwise, no t y p i c a l c e l l u l a r o r g a n e l l e s can be found. The cytoplasm o f these c e l l s appears h e a v i l y g r a n u l a t e d and i s r e l a t i v e l y e l e c t r o n - d e n s e . Large, double membraned vacu o l e s are common i n the cytoplasm; these vacuoles e i t h e r appear empty, appear to c o n t a i n an amorphous substance, or appear t o c o n t a i n membrane remnants which 53 sometimes resemble m i t o c h o n d r i a l c r i s t a e ( F i g u r e s 35, 36 and 37). Some o f these c e l l s c o n t a i n l a r g e numbers of extremely e l e c t r o n - d e n s e , l a r g e i n c l u s i o n s s i m i l a r to melanin g r a n u l e s ( F i g u r e s 36 and 37). I t i s d i f f i c u l t to determine whether or not these p a r t i c l e s are bounded by membrane s t r u c t u r e s . Some u n i d e n t i f i e d stromal c e l l s c o n t a i n what appear t o be c r y s t a l l i n e i n c l u s i o n s : spindle-shaped, e l e c t r o n - l u c e n t elements g e n e r a l l y surrounded by an area o f e l e c t r o n d e n s i t y (Figure 37). O c c a s i o n a l l y , non-ovoid c e l l s (Figure 38) ( i . e . , e l o n gated c e l l s o r i e n t e d p a r a l l e l t o the basement membrane, s i m i l a r t o chromatophores or f i b r o b l a s t s ) appear e x c e s s i v e l y v a c u o l a t e d i n much the same manner as the rounded u n i d e n t i -f i e d c e l l t y p e s . The i r r e g u l a r l y shaped vacuoles are common throughout the cytoplasm; they u s u a l l y appear empty o r p a r t i a l l y f i l l e d w i t h an amorphous substance o r membrane fragments. AEN Epidermis The epidermal component o f the a n g i o e p i t h e l i a l nodule tumor stage c o n s i s t s o f normal e p i t h e l i a l c e l l s , c e l l s of an unknown type ( " X - c e l l s " ; g i a n t c e l l s ) , and many in t e r m e d i a t e s and v a r i a t i o n s . The u n i d e n t i f i e d g i a n t c e l l type can be observed i n t e r s p e r s e d among normal epidermal c e l l s (Figure 39). The epidermal c e l l s appear as i n normal e p i d e r m i s ; i n t e r d i g i t a t i o n s between n e i g h b o r i n g c e l l s remain complex; mitochondria, f r e e ribosomes, and n u c l e i appear as normal. Interposed between (and seemingly not d i s r u p t i n g ) 54 PLATE 10 F i g u r e s 38-39 F i g . 38 Basement membrane area o f an EP-type l e s i o n . A non-ovoid stromal " X - c e l l " i s shown l y i n g p a r a l l e l t o the basement membrane ( r i g h t ) . Note the e x c e s s i v e v a c u o l a t i o n and resem-blance t o stromal f i b r o b l a s t s (Figure 34), i n d i c a t i n g a probable morphogenetic r e l a t i o n s h i p between f i b r o b l a s t s and s t r o m a l " X - c e l l s " . Two s m a l l epidermal " X - c e l l s " , encompassed by an envel o p i n g c e l l , l i e above the basement membrane ( l e f t ) . X 16200. F i g . 39 Epidermis o f AEN tumour stage, showing the r e l a t i o n s h i p between the i n i t i a l " X - c e l l " types t o normal epidermal c e l l s . Note the normal-appearing plasma membrane i n t e r -d i g i t a t i o n s between n e i g h b o r i n g epidermal c e l l s . X 16200. 5 6 the epidermal c e l l s are small, rounded, vacuolated c e l l s with enlarged nuclei and central n u c l e o l i . The c e l l mem-branes of t h i s new c e l l type are c l o s e l y apposed to the outer membranes of the neighboring epidermal c e l l s . No desmosomes, f i n g e r - l i k e processes, or other forms of c e l l u l a r contacts are v i s i b l e . Nuclei of c e l l s of t h i s type are round and enlarged; surrounded by an even, double membrane; contain discrete clumps of chromatin scattered at random throughout the nucleus; and contain large, c e n t r a l , dense n u c l e o l i which appear to be composed of a heavily granular substance. Nuclear pores are v i s i b l e i n some c e l l s ; they appear at regular i n t e r v a l s , measure 100 nm i n diameter, and may be associated with microtubules (Figure 40). The cytoplasm can appear extremely lucent and often seems to contain l i t t l e ground substance. Large, ovoid vacuoles are common in the cytoplasm; often they are founded by a double membrane, while i n other cases the membranes seem incomplete. Again, as i n the analogous unid e n t i f i e d stromal c e l l type, dark electron-dense granules are present, usually without evidence of a surrounding membrane structure. These unidentified c e l l s of the AEN epidermis appear i n various stages of enlargement, cyto-plasmic densities, and vacuolation (Figures 40 and 41). This unidentified giant c e l l type can be found, as described above, with extremely lucent cytoplasmic sub-stance. U l t r a s t r u c t u r a l l y s i m i l a r c e l l s can also be found which tend to be extremely dense and heavily stained (Figure 41) . PLATE 11 F i g u r e s 40-41 F i g . 40 Epidermis o f l a r g e AEN-type l e s i o n . X 21000. F i g . 41 Epidermis o f l a r g e AEN-type l e s i o n . U n i d e n t i f i e d c e l l types (type B " X - c e l l s " ) are found i n v a r y i n g stages o f " b a l l i n g up"; the d e n s i t i e s o f these c e l l s v a r i e s from extremely l i g h t t o dark. S e v e r a l epidermal c e l l s have r e t a i n e d t h e i r normal appearance. X 21000. 59 Normal-appearing epidermal c e l l s can a l s o appear densely s t a i n e d ; these "dark" c e l l s possess abnormal n u c l e i and l a r g e c y t o p l a s m i c vacuoles (Figure 4 0 ) . Those epidermal c e l l s w i t h normal o r g a n e l l e u l t r a s t r u c t u r e g e n e r a l l y have l o s t the p e r i n u c l e a r / p e r i p h e r a l c y t o p l a s m i c d i v i s i o n which normally c h a r a c t e r i z e them and appear as elongated, r a t h e r than o v o i d , c e l l s . These "normal" epidermal c e l l s tend t o r e t a i n desmosomes and other j u n c t i o n a l complexes but l o s e a l l f i n g e r - l i k e i n t e r d i g i t a t i o n s . C e r t a i n o f these normal-appearing appear i n the process o f l o s i n g t h e i r normal c y t o p l a s m i c d e n s i t y and o r g a n e l l e s (Figure 4 2 ) . Other epidermal c e l l s i n c r e a s e t h e i r c y t o p l a s m i c d e n i s t y and appear as p r e c u r s o r s of the "enveloping c e l l s " o f the mature l e s i o n . These c e l l s c o n t a i n many mit o c h o n d r i a and f r e e ribosomes, many c y t o p l a s m i c f i l a m e n t s which extend a c r o s s the c e l l a long the p e r i p h e r y , and many desmosomes (some o f which appear g r e a t l y lengthened) a t the t i p s o f the c e l l u l a r "arms" (see l a t e r d e s c r i p t i o n s o f e n v e l o p i n g c e l l s o f the EP). O c c a s i o n a l l y these c e l l s c o n t a i n d i l a t e d endoplasmic r e t i c u l u m (Figure 4 3 ) . EP - epidermis The epidermal component o f the mature p a p i l l o m a -l i k e l e s i o n , as d e s c r i b e d by l i g h t microscopy ( F i g u r e s 26-29), i s composed o f two g e n e r a l t i s s u e areas and three b a s i c c e l l t y p e s . The s u p e r f i c i a l l a y e r s o f the tumor are composed o f r e l a t i v e l y normal e p i t h e l i a l c e l l s (type C) w i t h l a r g e i n t e r c e l l u l a r spaces. These same normal-appearing epidermal 60 PLATE 12 F i g u r e s 42-43 F i g . 42 P o r t i o n o f cytoplasm o f a normal epidermal c e l l o c c u r r i n g i n an AEN-stage l e s i o n . T h i s p a r t o f the cytoplasm i s "sandwiched" between two " X - c e l l " types (top and bottom). Note the g e n e r a l d i s o r g a n i z a t i o n , l a c k o f c y t o p l a s m i c ground substance, and l o s s o f r e c o g n i z a b l e o r g a n e l l e s i n the c y t o -plasm. X 19000. F i g . 43 Epidermis o f l a r g e AEN-type l e s i o n , i l l u s t r a t i n g an e n v e l o p -i n g c©ll d e v e l o p i n g among s e v e r a l u n i d e n t i f i e d " X - c e l l " t y p e s . X 16200. 61 62 c e l l s are also present i n the lower portion of the epidermal infoldings, where they compose a "matrix" i n which the c h a r a c t e r i s t i c giant c e l l s (types A and B) are embedded. The s u p e r f i c i a l c e l l s comprise the top three-to-four layers of the tumor. C h a r a c t e r i s t i c a l l y , they appear as very dense, flattened c e l l s with numerous f i n g e r l i k e c e l l u l a r extensions which connect them, often with desmosbmes, to neighboring c e l l s (Figures 44 and 4 5 ) . The e x t r a c e l l u l a r spaces are most often empty, but occasionally c e l l u l a r debris, resembling the remnants of a necrotic c e l l , can be found (Figure 4 5 ) . The most s u p e r f i c i a l c e l l s possess m i c r o v i l l i - l i k e p l e a t s , often coated with a f i b r i l l a r substance, s i m i l a r to those found i n normal epidermis (Figure 4 4 ). These c e l l s contain numerous mitochondria and r e t a i n close c e l l - t o - c e l l attachments i n the horizontal plane (Figure 4 5 ) . Quite often the endoplasmic reticulum of these c e l l s i s d i l a t e d and f i l l e d with an unknown, electron-dense, often heavily granular, substance (Figures 44 and 4 5 ) . Nuclei of these s u p e r f i c i a l c e l l s s t a i n evenly dark, with l i t t l e condensed chromatin. Overall, the n u c l e i , cytoplasm, and organelles of these c e l l s s t a i n with such even i n t e n s i t y that discrimina-t i o n between c e l l u l a r elements i s often impossible. Interspersed among the s u p e r f i c i a l epidermal c e l l s small lymphocytes and h i s t i o c y t e s can be found. The lympho-cytes are characterized by a large, chromatin-rich nucleus and a scanty cytoplasm, which commonly contains a few lamellae 63 PLATE 13 F i g u r e s 44-45 F i g . 44 S u p e r f i c i a l c e l l l ayers o f a mature EP-type l e s i o n . In t h i s case, the s e c t i o n i l l u s t r a t e s a p o r t i o n o f one " i n - f o l d " o f the e p i d e r m i s . X 13300. F i g . 45 S u p e r f i c i a l c e l l l a y e r s o f a mature EP-type l e s i o n , showing the c h a r a c t e r i s t i c s t e l l a t e epidermal c e l l s , macrophages, and i n t e r c e l l u l a r spaces o c c a s i o n a l l y f i l l e d w i t h what appears t o be c e l l u l a r d e b r i s . The e n v e l o p i n g and " X - c e l l " types l i e i n the deeper p o r t i o n o f the l e s i o n (to the r i g h t ) . X 13300. 65 o f g r a n u l a r endoplasmic r e t i c u l u m (Figure 4 5 ) . H i s t i o c y t e s (Figure 45) are c h a r a c t e r i z e d by a l a r g e , horseshoe-shaped nucleus w i t h condensed p e r i p h e r a l chromatin. M i t o c h o n d r i a , endoplasmic r e t i c u l u m , G o l g i apparatus, r e s i d u a l b o d i e s , and d i g e s t i v e v a c u o l e s c o n s t i t u t e the m a j o r i t y o f c y t o p l a s m i c elements. The deeper p o r t i o n s o f the epidermal i n f o l d i n g s are composed p r i m a r i l y o f g i a n t c e l l s enveloped by the arms o f a l t e r e d , heteromorphic epidermal c e l l s o f normal c y t o -p l a s m i c u l t r a s t r u c t u r e . These e n v e l o p i n g c e l l s are c h a r a c t e r -i z e d by a homogenous, e l e c t r o n dense nucleoplasm, which sometimes shows a s m a l l degree o f condensed chromatin. Grandular endoplasmic r e t i c u l u m , m i t o c h o n d r i a , and many f r e e ribosomes o f t e n occur i n the p e r i n u c l e a r a r e a . The "arms" of these c e l l s c o n t a i n many f r e e ribosomes and c y t o -p l a s m i c f i l a m e n t s , which u s u a l l y run p a r a l l e l w i t h the a x i s o f the arm. (Fig u r e s 46 and 4 7 ) . Numerous desmosomal connec-t i o n s occur between a d j o i n i n g e n v e l o p i n g c e l l s , e s p e c i a l l y a t the t i p s o f the cy t o p l a s m i c arms. No desmosomal or other j u n c t i o n a l complexes were ever observed between the enve l o p i n g c e l l s and the g i a n t c e l l s . Small lymphocytes can o c c a s i o n a l l y be found i n t e r s p e r s e d among the g i a n t and env e l o p i n g c e l l types (Figure 4 7 ) . The g i a n t c e l l s of the tumor ( c e l l types A and B as d e s c r i b e d l i g h t m i c r o s c o p i c a l l y ) are m o r p h o l o g i c a l l y i d e n t i c a l i n most a s p e c t s . U l t r a s t r u c t u r a l d i s t i n c t i o n between the two c e l l types i s based on s i z e d i f f e r e n c e s and on the presence o f dense c y t o p l a s m i c i n c l u s i o n s . 66 PLATE 14 F i g u r e s 46-47 F i g . 46 Deeper p o r t i o n o f mature EP-type l e s i o n , showing the c h a r a c t e r i s t i c h i s t o l o g i c arrangement of g i a n t c e l l types ( " X - c e l l s " ) and env e l o p i n g c e l l s . X 14500. F i g . 47 Deeper p o r t i o n o f mature EP-type l e s i o n (near the basement membrane), i l l u s t r a t i n g the occurrence o f s m a l l lymphocytes i n t e r s p e r s e d o c c a s i o n a l l y among the " X - c e l l " and e n v e l o p i n g c e l l t y p e s . X 10500. 67 68 Apart from these two d i f f e r e n c e s (to be d e s c r i b e d below), g i a n t c e l l s i n the epidermal tumor component show the same p a t h o l o g i c c h a r a c t e r i s t i c s . The nucleus i s e n l a r g e d and ovo i d , w i t h randomly d i s p e r s e d , d i s c r e t e clumps of condensed chromatin; a c e n t r a l dense n u c l e o l u s o f eve n l y , t h i c k l y g r a n u l a r m a t e r i a l can o f t e n r e a c h a s i z e o f o n e - h a l f the diameter o f the n u c l e u s . The nucleus i s surrounded by a double membrane. Nuclear pores o f 100nm diameter are commonly p r e s e n t a t r e g u l a r i n t e r v a l s ( F igure 4 6 ) . The cy t o p l a s m i c ground substance v a r i e s from dense to t h i n l y g r a n u l a r . Normal endoplasmic r e t i c u l u m l a m e l l a e and mit o -c h o n d r i a are p r e s e n t i n few cases (Figure 4 8 ) . G e n e r a l l y the o n l y d i s t i n g u i s h a b l e c y t o p l a s m i c elements p r e s e n t are membrane- or non-membrane-bound dense i n c l u s i o n s o f i r r e g u l a r s i z e and shape; and l a r g e , ovoid-to-round vacuoles i d e n t i c a l t o those d e s c r i b e d i n the stromal and AEN u n i d e n t i f i e d c e l l t y p e s . Often these vacuoles can appear elongated or h o r s e -shoe shaped and may appear s i m i l a r t o extremely d i l a t e d endo-p l a s m i c r e t i c u l u m l a m e l l a e (Figure 4 8 ) . In other cases the presence o f c r i s t a e - l i k e membrane fragments l o c a t e d a t the p e r i p h e r y o f the vacuo l e s i s su g g e s t i v e o f degenerative or d i l a t e d m itochondria (Figure 4 9 ) . O c c a s i o n a l l y bundles o f m i c r o f i l a m e n t s , arranged i n a p a r a l l e l a r r a y and bound by a s i n g l e membrane, can be observed i n the cytoplasm (Figure 4 9 ) . The plasma membrane i s f r e e from i n t e r c e l l u l a r c o n n e c t i o n s and, i n g e n e r a l , i s smooth or s l i g h t l y i r r e g u l a r . O c c a s i o n a l l y g i a n t c e l l s can be found w i t h plasma membrane i n f o l d i n g s 69 PLATE 15 F i g u r e s 48-49 F i g . 48 Epidermal component o f mature EP-type l e s i o n , showing e n v e l o p i n g and " X - c e l l " t y p e s . Note the normal endoplasmic r e t i c u l u m and horseshoe- or elongate-shaped v a c u o l e s . X 10500. F i g . 49 P o r t i o n o f cytoplasm of a type A g i a n t c e l l . The cytoplasm o f two enveloping c e l l s l i e s on e i t h e r side* o f the type A c e l l . Note the e x t e n s i v e plasma membrane i n v a g i n a t i o n s . X 22000. 71 (Fi g u r e s 46 and 4 9 ) . S e v e r a l i n s t a n c e s o f n u c l e a r membrane " p r o l i f e r a t i o n " ( i . e . , s e v e r a l l a y e r s o f a g r a n u l a r endo-pl a s m i c r e t i c u l u m c l o s e l y apposed to the n u c l e a r envelope) were noted i n these c e l l s (Figure 50). Many " X - c e l l " types a l s o c o n t a i n e d c y t o p l a s m i c "membrane whorls" (F i g u r e s 38, 49, and 51). L i g h t microscopy showed the presence o f two types of epidermal g i a n t c e l l s ; type B c e l l s u s u a l l y were l o c a t e d i n groups a l o n g the basement membrane, w h i l e type A c e l l s were g e n e r a l l y found i n the more c e n t r a l areas o f the epidermal i n f o l d i n g s . U l t r a s t r u c t u r a l l y , d i f f e r e n c e s can be noted between those g i a n t c e l l s l o c a t e d a l o n g the basement membrane and those g i a n t c e l l s l o c a t e d i n the i n n e r tumor p o r t i o n s . U l t r a s t r u c t u r a l l y , those g i a n t c e l l s n e a r e s t the basement membrane are o v o i d and range up t o twice the diameter o f normal epidermal c e l l s . They tend to c o n t a i n extremely l a r g e c y t o p l a s m i c vacuoles arranged i n a c i r c u l a r f a s h i o n about the c e n t r a l l y l o c a t e d nucleus (F i g u r e s 38,and 5 1 ) . These c e l l s are s i m i l a r i n s i z e and morphology t o the u n i d e n t i -f i e d g i a n t c e l l types found i n the stroma and i n the AEN e p i d e r m i s . Deeper i n t o the epidermal i n f o l d i n g s , the g i a n t c e l l s tend t o be much l a r g e r (up t o nine times the diameter o f normal epidermal c e l l s ) ; they tend t o be heteromorphic r a t h e r than o v o i d i n shape; and they tend t o possess l a r g e r numbers o f dense c y t o p l a s m i c i n c l u s i o n s ( F i g u r e s 46 and 51). The e l e c t r o n - d e n s e i n c l u s i o n s u s u a l l y do not appear t o be 72 PLATE 16 F i g u r e s 50-51 F i g . 50 Nucleus and p o r t i o n o f cytoplasm o f an epidermal " X - c e l l " , i l l u s t r a t i n g a p r o l i f e r a t i o n o f the n u c l e a r envelope which o c c a s i o n a l l y was observed. Note the i n d i s c r i m i n a t i v e l y clumped chromatin. X 22000. F i g . 51 Low-power micrograph o f epidermal p o r t i o n o f a mature EP-type l e s i o n , i l l u s t r a t i n g the c h a r a c t e r i s t i c s o f type A and type B g i a n t c e l l s " X - c e l l s " . X 9500. 74 membrane-enclosed. With i n c r e a s i n g c e l l s i z e , the number o f vacuoles i n these l a r g e r c e l l s seems to i n c r e a s e , w h i l e the average diameter o f the vacuoles seems t o decrease. V i r u s - l i k e P a r t i c l e s Q u i t e f r e q u e n t l y s m a l l v i r u s - l i k e p a r t i c l e s o f unknown composition or o r i g i n are p r e s e n t i n the cytoplasm o f the e n v e l o p i n g c e l l s and s u p e r f i c i a l c e l l s o f the p a p i l l o m a -l i k e l e s i o n s ( F i g u r e s 52 and 53). In one i n s t a n c e they were observed i n the i n t e r c e l l u l a r space o f the epidermis o f an a n g i o e p i t h e l i a l nodule ( F i g u r e 54). These p a r t i c l e s appear round or hexagonal and measure 30 nm i n diameter. The p a r t i c l e s were never observed i n t y p i c a l v i r u s c r y s t a l l i n e a r r a y s ; most o f t e n they were found i n s m a l l groups or randomly s c a t t e r e d throughout the cytoplasm o f the e n v e l o p i n g or super-f i c i a l c e l l s . 75 PLATE 17 F i g u r e s 52-54 F i g u r e 52 P o r t i o n o f cytoplasm o f an e n v e l o p i n g c e l l found i n a mature EP-type l e s i o n , showing the presence o f d a r k l y -s t a i n i n g v i r u s - l i k e p a r t i c l e s . The p a r t i c l e s c o n s i s t e n t l y measure 30 nm i n diameter. X 30000. F i g . 53 V i r u s - l i k e p a r t i c l e s i n the cytoplasm o f an e n v e l o p i n g c e l l l y i n g next t o the basement membrane. X 56000. F i g . 54 V i r u s - l i k e p a r t i c l e s found i n the i n t e r c e l l u l a r space o f an AEN-type l e s i o n . X 14400. 77 DISCUSSION Normal Epidermis Normal epidermal s t r u c t u r e i n t e l e o s t s has been w e l l d e s c r i b e d both l i g h t m i c r o s c o p i c a l l y (van Oosten, 1957; Andrew, 1959; L a g l e r et^ al _ . , 1962; Romer, 1962; Hyman, 1962; P a t t and P a t t , 1969) and u l t r a s t r u c t u r a l l y (Hendrickson and M a t o l t s y , 1968; M e r r i l e e s , 1974). R e c e n t l y the b i o l o g y o f the s k i n o f the f l a t h e a d s o l e , a f l a t f i s h o f t e n a f f l i c t e d w i t h epidermal p a p i l l o m a s , has been s t u d i e d i n some d e t a i l . E l e c t r o n m i c r o s c o p i c s t u d i e s o f the s c a l e s (Brown and W e l l i n g s , 1969) , o f the l a r v a l s k i n (Wellings and Brown, 1969), and o f the s k i n o f two-to-three year o l d f i s h (Brown and W e l l i n g s , 1970) have been p r e s e n t e d . L i g h t and e l e c t r o n m i c r o s c o p i c s t u d i e s of the s k i n of the lemon s o l e showed l i t t l e v a r i a t i o n from p r e v i o u s d e s c r i p t i o n s o f t e l e o s t e p i d e r m i s . S t r u c t u r e s i n t e r p r e t e d i n p r e v i o u s r e p o r t s as broad, s h o r t m i c r o v i l l i on the s u r f a c e of s u p e r f i c i a l c e l l s were shown by scanning e l e c t r o n m i c r o -scopy to be c r o s s - s e c t i o n s o f numerous s u r f a c e p l e a t s , o f t e n arranged i n c o n c e n t r i c p a t t e r n s (Figure 13). T h i s f i n g e r p r i n t -l i k e c e l l u l a r topography i s s i m i l a r to t h a t r e p o r t e d i n other t e l e o s t s p e c i e s (Jones e t a l . , 1966; Luse and K r e j s a , 1969; M e r r i l e e s , 1974). U l t r a s t r u c t u r a l o b s e r v a t i o n s o f lemon s o l e guanophores correspond t o p r e v i o u s d e s c r i p t i o n s (Seto-g u i t , 1967); j u n c t i o n a l complexes i n the epidermis were t y p i c a l 78 o f those found i n amphibian s k i n (Farquhar and Palade, 1965). In an a u t o r a d i o g r a p h i c study o f the growth o f f i s h e p i d e r m i s , Hendrickson (1967) found i n c o r p o r a t i o n o f thymidine i n a l l epidermal c e l l l a y e r s . T h i s i n d i c a t e s t h a t mitoses are not r e s t r i c t e d t o the b a s a l l a y e r (as they are i n other v e r t e b r a t e s ) , and t h a t a l l epidermal c e l l s p a r t i c i p a t e i n the maintenance o f the t i s s u e . T h i s fundamental d i f f e r e n c e must be c o n s i d e r e d when comparing t i s s u e r e a c t i o n s of f i s h e pidermis w i t h those o f o t h e r v e r t e b r a t e s . No evidence o f k e r a n t i n i z a -t i o n or other d i f f e r e n t i a t i v e processes i s observed i n the epidermis o f most f i s h ; i n s t e a d , the epidermis i s p r o t e c t e d by a v a r i a b l e t h i c k n e s s o f mucus s e c r e t e d by u n i c e l l u l a r s e c r e t o r y g l a n d s . Reduction or a l t e r a t i o n o f the mucus l a y e r can r e s u l t i n i n c r e a s e d s u s c e p t i b i l i t y o f the epidermal c e l l s to o u t s i d e agents (van Oosten, 1957). B i o l o g y o f Epidermal L e s i o n s Evidence f o r the p r o g r e s s i o n o f epidermal l e s i o n s from an i n i t i a l AEN stage t o a mature EP l e s i o n c o n s i s t s o f n a t u r a l h i s t o r y data, l a b o r a t o r y o b s e r v a t i o n s , the presence o f morphologic i n t e r m e d i a t e s , and h i s t o l o g i c a l s t u d i e s . The f r e q u e n c i e s o f occurrences o f AEN and EP stages o f lemon s o l e l e s i o n s were analyzed i n terms o f 5 mm-size groups. The r e s u l t s (Figure 9) suggest a p r o g r e s s i o n from i n i t i a l AENs to mature EPs: as f i s h s i z e i n c r e a s e s , the r e l a t i v e percentage o f AENs (as compared t o EPs) decreases 79 u n t i l no AENs are found (above 135 mm). The AEN t o EP t r a n s i t i o n o f the f l a t f i s h l e s i o n s was p o s t u l a t e d e a r l i e r on the b a s i s o f s i m i l a r evidence (McArn, 1968; McArn e_t a l . , 1968; McArn and W e l l i n g s , 1971). There appears t o be a tendency, i n any d i s e a s e d ^ p o p u l a t i o n , f o r AENs t o occur p r e d o m i n a t e l y e a r l y i n the year and on younger (smaller) f l a t f i s h ; EPs tend to occur almost e x c l u s i v e l y on o l d e r ( l a r g e r ) f l a t f i s h , a phenomenon b e s t e x p l a i n e d by p r o g r e s s i v e growths o f the l e s i o n s . AENs on f l a t f i s h kept under l a b o r a t o r y c o n d i t i o n s have been observed, i n s i g n i f i c a n t numbers, t o p r o g r e s s to t y p i c a l EP l e s i o n s (McArn, 1968; McArn e t a l . , 1968); p r o g r e s s i v e growths o f the tumours were a l s o r e p o r t e d by W e l l i n g s and Chuinard (1964). The occurrence o f morphologic t r a n s i t i o n a l stages between AENs and EPs has been d e s c r i b e d and a l s o seems to i n d i c a t e a p r o g r e s s i v e p r o c e s s (Wellings e t a l . , 1964; McArn, 1968; McArn and W e l l i n g s , 1971). The h i s t o l o g y ( l i g h t and e l e c t r o n m i c r o s c o p i c ) o f the tumour types found i n the lemon s o l e , and other s p e c i e s r e p o r t e d here, corresponds t o p r e v i o u s d e s c r i p t i o n s i n other s p e c i e s (see i n t r o d u c t i o n ) . Most r e p o r t s gave d e s c r i p t i o n s o f the AEN and EP stages o f tumour growth. U s u a l l y the presence o f morphologic i n t e r m e d i a t e s ( e x t e r n a l l y ) was noted, but no attempt was made to c o r r e l a t e h i s t o l o g i c a l s t r u c t u r e w i t h e x t e r n a l m o r p h o l o g i c a l s t r u c t u r e . H i s t o l o g i c a l examina-t i o n of the v a r i o u s tumour types (AENs, t r a n s i t i o n a l forms, 80 and EPs) shows: 1) a d i s t i n c t i v e h i s t o l o g i c a l s t r u c t u r e f o r each type ( i . e . , a d e f i n i t e c o r r e l a t i o n between e x t e r n a l morphology and i n t e r n a l s t r u c t u r e , as d e s c r i b e d above), and 2) a d e f i n i t e p a t t e r n , i n v o l v i n g a continuum o f i n t e r m e d i a t e forms, o f h i s t o m o r p h o l o g i c a l changes from the AEN t o EP stages. I t i s t h i s p a t t e r n t h a t w i l l be d e s c r i b e d below, w i t h a d i s c u s s i o n o f each stage, as a l i k e l y h i s t o l o g i c e x p l a n a t i o n o f the changes i n v o l v e d i n the p r o g r e s s i v e growth from an i n i t i a l l e s i o n to a mature, p a p i l l o m a - l i k e l e s i o n . Based on c o r r e l a t i o n s between h i s t o l o g i c a l and e x t e r n a l morphology (presented above), the f o l l o w i n g , i n s i m p l i f i e d form, r e p r e s e n t s what I b e l i e v e to be the p a t t e r n o f h i s t o l o g i c and c y t o l o g i c changes o c c u r r i n g i n the p r o g r e s -s i o n from i n i t i a l t o mature f l a t f i s h l e s i o n s (a more d e t a i l e d d e s c r i p t i o n f o l l o w s ; the stages are i l l u s t r a t e d d i a g r a m a t i c a l l y i n F i g u r e 55): 1) h y p e r p l a s i a o f dermal f i b r o b l a s t s , c a p i l l a r y growth, and an inflammatory-type response (F i g u r e s 14 and 15); 2) appearance o f u n i d e n t i f i e d stromal c e l l types ( " X - c e l l s " ) , p r o b a b l y from t r a n s f o r m a t i o n o f a dermal c e l l type (Figure 15; see d i s c u s s i o n o f u n i d e n t i f i e d c e l l t y p e s ) ; 3) h y p e r p l a s i a o f epidermal c e l l s and f o l d i n g o f the epidermis ( F i g u r e s 18, 20 and 21); 4) appearance o f u n i d e n t i f i e d g i a n t c e l l type (type B) along the basement membrane i n the epidermal component (Figure 23); 5) appearance of type A g i a n t c e l l s i n the i n t e r i o r 81 FIGURE 55 Summary o f the growth and s t r u c t u r e o f f l a t f i s h epidermal l e s i o n s . The p o s t u l a t e d growth, and s t r u c t u r e o f epidermal l e s i o n s found on j u v e n i l e f l a t f i s h . The numbered f i g u r e s are e x p l a i n e d on page 80 o f the t e x t . 82 83 o f the epidermal i n f o l d i n g s , p r o b a b l y from type B p r e c u r s o r s ( F i g u r e s 24 and 25;; see d i s c u s s i o n o f u n i d e n t i f i e d c e l l t y p e s ) ; 6) continued i n c r e a s e i n the numbers o f c e l l types A and B i n the epidermis u n t i l they dominate the t i s s u e ; t r a n s f o r m a t i o n o f other epidermal c e l l s i n t o e n v e l o p i n g and s u p e r f i c i a l c e l l s ( F i g u r e s 26-29); 7) n e c r o s i s o f g i a n t c e l l types (Figure 28); 8) consequent t i s s u e n e c r o s i s (?) (see d i s c u s s i o n o f n e c r o s i s o f t i s s u e s and death o f the h o s t f i s h ) ( F i g u r e s 31-33). The f i r s t obvious r e a c t i o n i n tumorigenesis i n the lemon s o l e s k i n appears t o be a p r o l i f e r a t i o n - o f dermal f i b r o -b l a s t s ; t h i s type o f h i s t o l o g i c a l s t r u c t u r e was found e x c l u s i v e l y i n the s m a l l e s t AENs s t u d i e d . T h i s i n i t i a l h y p e r p l a s i a e v i d e n t l y g i v e s r i s e t o the stromal component o f the l e s i o n ( F i g u r e 4 ) . T h i s f i b r o b l a s t h y p e r p l a s i a resembles a fibroma, b e i n g composed mainly o f f i b r o b l a s t s and c o l l a g e n f i b e r s . In some tumours many b l o o d v e s s e l s can be observed; u s u a l l y these show packing o f r e d bloo d c e l l s , i n d i c a t i n g s t a s i s ( F i g u r e 14 and 15). F i b r o s i s , o f t e n accompanied by an i n c r e a s e i n c a p i l l a r i e s , i s a common response o f f i s h epidermis t o v a r i o u s agent ( N i g r e l l i and Smith, 1938, 1940; N i g r e l l i , 1954). E l e c t r o n m i c r o s c o p i c -a l l y g r a n u l o c y t e s (see Weinreb, 1963; Andrews, 1959), macro-phages, and lymphocytes can be observed i n the stroma i n t e r -spersed among the f i b r o b l a s t s , c o l l a g e n , and c a p i l l a r i e s ( F i g u r e s 35 and 36). T h i s would i n d i c a t e some degree o f 84 inflammatory response, as suggested by Brooks and co-workers (1969). They suggested the AEN stroma t o be inflammatory i n nature on the b a s i s o f r e d b l o o d c e l l s , macrophages, lympho-c y t e s , plasma c e l l s , e o s i n o p h i l g r a n u l o c y t e s , and v a s c u l a r d i l a t a t i o n (with s t a s i s ) found e l e c t r o n m i c r o s c o p i c a l l y . In t h i s study, no e x t r a v a s c u l a r r e d b l o o d c e l l s or plasma c e l l s were observed i n the stroma o f the AENs s t u d i e d . The stromal component resembles a subacute inflammatory response p r e -dominated by a f i b r o b l a s t i c and a n g i o b l a s t i c p r o l i f e r a t i o n (see L a V i a and H i l l , 1971). N i g r e l l i (1954) has noted the occurrence o f macrophages, g r a n u l o c y t e s , and plasma c e l l s i n response t o p a r a s i t i c i n v a s i o n s o f f i s h . Presumably the inflammatory elements o f the stromal response r e g r e s s w i t h time, l e a v i n g the fibromous framework, w i t h v a s c u l a r i z a t i o n , as the s u p p o r t i n g stroma f o r the EP. The u n i d e n t i f i e d stromal c e l l type (stromal "X-c e l l s " ) can be found i n most, b u t seemingly not a l l , AENs. (Figure 1 5). They o f t e n occur i n extremely l a r g e numbers. I t i s not known e x a c t l y when the u n i d e n t i f i e d c e l l type i n i t i a l l y appears i n the l e s i o n s ; examination of the e a r l i e s t AEN stages r e v e a l e d no c e l l s o f t h i s t y pe. I t appears t h a t i n some AENs the i n i t i a l fibromatous r e a c t i o n may be i n v a s i v e i n nature (F i g u r e s 16 and 17). I n v a s i v e growth o f the stroma were never observed i n t r a n s i -t i o n a l or EP stages; e i t h e r the i n v a s i v e growth i s damaging enough t o be f a t a l t o the h o s t a t an e a r l y stage o f tumour growth, or the growths are s e l f - l i m i t i n g and/or r e g r e s s i v e . 85 Angiomous growths, as observed in another study (Wellings, et a l . , 1964), often show invasive q u a l i t i e s without being invasive i n nature. In studies of true epidermal papillomas i n other families of f i s h , other authors have reported i n i t i a l growths simi l a r to the an g i o e p i t h e l i a l nodule types described i n f l a t -f i s h . Lucke and Schlumberger (1941) found pre-papilloma lesions in other species to consist of an i n i t i a l hyperemia and p r o l i f e r a t i o n of dermal blood vessels. S c a r p e l l i (1969) observed similar AEN-type to EP-type growth t r a n s i t i o n s for papillomas of the slippery dick. I t has been suggested that t h i s could be a common pattern i n the growth of papilloma-l i k e growths i n f i s h (Wellings, 1969B). Some hyperplasia of the epidermal c e l l s , s imilar to those hyperplasias noted by N i g r e l l i (1954) as t y p i c a l responses to various p a r a s i t i c s t i m u l i i n other fishes, evidently follows a varying degree of i n i t i a l AEN stromal growth (Figure 21). As the epidermis thickens and begins i n f o l d i n g , the stromal component of the lesions becomes gradually reduced; eventually, i n the mature EP, i t consists of a branching series of narrow supporting "arms". Accompany-ing the epidermal thickening, and presumably following the hyperplasia, i s the appearance of an unide n t i f i e d c e l l type ("X-cells; giant c e l l s ) , described e a r l i e r as c e l l type B. Usually these c e l l s begin to accumulate along the basement membrane (Figure 23). Gradually, as the epidermis thickens and beings to i n f o l d , these c e l l s appear larger and more numerous (Figure 24). Staining becomes less and less intense (Figure 25). These larger c e l l s , usually found near the center of the epidermal info l d i n g s , e x h i b i t d i f f e r e n t s t a i n -ing c h a r a c t e r i s t i c s and were c a l l e d c e l l type A. Electron microscopically, c e l l types A and B were found to be morphol-o g i c a l l y i d e n t i c a l except for s i z e , degree of vacuolation, and amount of cytoplasmic inclusions (see discussion below). I t seems very l i k e l y , on the basis of u l t r a s t r u c t u r a l morphology and time and location of the appearance of the two c e l l types (type B c e l l s generally occur i n late AEN or t r a n s i t i o n a l forms along the basement membrane; type A c e l l s occur i n t r a n s i t i o n a l and EP stages i n the center of the infoldings and only, i t seems, af t e r the appearance of a large number of type B c e l l s ) , that type B c e l l s are precursors of type A c e l l s . Eventually, i n the mature le s i o n , these giant c e l l types dominate the epidermal component; they are found "embedded" in a matrix of enveloping c e l l s (Figure 26), which also are evidently morphologically changed epidermal c e l l s (Figure 43 and 46). In every mature l e s i o n studied h i s t o l o g i c a l l y , structures interpreted as collapsed giant c e l l s are found, usually near the very center of the epidermal infoldings (Figure 28). Generally, large numbers of c e l l s t e n t a t i v e l y i d e n t i f i e d as h i s t i o c y t e s are found i n the same central regions, indicative of much phagocytic a c t i v i t y (Figure 30). Several mature tumors showed general necrotic tendencies throughout the tissue (Figure 31). These observations, when considered with the general necrotic condition (to be d i s -cussed below) of the giant c e l l types composing the tissue, seem to indicate that necrosis i s the ultimate fate of the tumors. N i g r e l l i and his colleagues (1965) also reported evidence of necrosis within mature and sole epidermal l e s i o n s . The ultimate fate of the epidermal lesions or of the f l a t f i s h bearing these lesions remains uncertain. Previous studies on the natural h i s t o r y of t h i s disease, as well as the present study, indicate that usually no epidermal lesions are found on f l a t f i s h over 200 mm t o t a l length. Evidently cer t a i n factors are operating i n the removal of tumourous tissues from f i s h , or i n the selected removal of tumour-bearing f i s h from the population. When the frequencies of tumour-bearing lemon sole are expressed as percentages per 5 mm size group, a d e f i n i t e r i s e and f a l l of tumour prevalence i s noted as f i s h length increases. This phenomenon was noted in previous studies of the natural history of this disease (Good, 1940; N i g r e l l i et a l . , 1965; Wellings et a l . , 1965; McArn and Wellings, 1971; M i l l e r and Wellings, 1971; Mearns and Sherwood, 1974). An i n i t i a l increase i n the r e l a t i v e prevalence of tumour-bearing f i s h occurs u n t i l the 75-79 mm group, where the incidence approaches 50%. After t h i s peak, the gradual decrease i n tumour prevalence (to a zero point i n the 160— 164 mm group) indicates one (or both) of two things: 1) the lesions are regressing or are being sloughed-off the f i s h surface at a f a i r l y constant rate, and/or 2) tumour-bearing f i s h are being removed from the population at a much faster rate than are non-tumour-bearing f i s h . The main evidence that the lesions are rejected or l o s t (due to necrotic processes) i s the h i s t o l o g i c a l presence of c e l l u l a r and tissue necrosis, as observed i n t h i s study in lemon sole EPs and i n sand sole EPs by N i g r e l l i and h i s co-workers (1965). K e l l y (1971), i n a study of epidermal papillomas on lemon sole in Puget Sound, Washington, reported sloughing or r e j e c t i o n of tumors under laboratory conditions at a rate of 70% over a two month period. In other studies (Wellings et. a l . , 1964; M i l l e r and Wellings, 1971), tumors were reported to progress i n the laboratory; no evidence of necrosis or tumor regression was found. This contradictory evidence i s possibly due to s i z e differences, and consequently tumor maturity, of the f i s h selected for study. Mature tumors would seem more l i k e l y to show evidence of necrosis or regression than would young, progressive tumor stages. There are at l e a s t two instances of regression or r e j e c t i o n of tumor-like growths in other families of f i s h . Fish pox, a hyperplastic epidermal disease, appears to be s e l f - l i m i t i n g and i s probably controlled by temperature and/or the season. Fish pox lesions have been observed to undergo regression (Lucke and Schlumberger, 1948). N. Peters (personal communica-tion) has observed that European eels, a f f l i c t e d with c a u l i f l o w e r - l i k e papillomas, often r e j e c t tumorous t i s s u e s . Evidence that the drop i n tumor frequency with increasing s i z e i s caused by the death of tumor-bearing f i s h i s inconclusive. Analyses of the average number of lesions per f i s h with r e l a t i o n to size (Figure 8) shows an i r r e g u l a r , but d e f i n i t e , decline as f i s h length increases. This phenomenon was shown i n the present study of lemon sole as well as i n e a r l i e r studies (Wellings e t a l . , 1965; M i l l e r and Wellings, 1971) . Presumably, i f the tumors are not being sloughed-off or rejected, then the absence of lesions in f i s h over 180 mm in length must indicate that the disease i s f a t a l . I f t h i s i s the case, then heavily-infected f i s h would be expected to be removed from the population at a faster rate than others; t h i s would manifest i t s e l f in a decrease i n the average number:of tumors per f i s h as f i s h size increases. Part, i f not a l l , of this decrease can be explained by observations indicating that very often the spreading edges of many AENs merge to form a single large EP. Cooper and K e l l e r (1969), i n t h e i r study of lesions on lemon sole i n San Francisco Bay, presented evidence which indicated that the disease had no l e t h a l e f f e c t s on the population. M i l l e r and Wellings (1971), on the other hand, have shown a marked reduction i n growth rates of tumor-bearing flathead sole i n the second year of l i f e , as compared with normal f i s h ; t h i s indicates that the lesions have a d e t r i -mental e f f e c t on the health of the f i s h . Wellings and his co-workers (1964) found that i t could not be shown that the rate of disappearance of tumorous f i s h from the population exceeded that of normal f i s h . Although no d e f i n i t e statement can be made concerning the fate of the lesions or of f i s h bearing the lesions, i t seems c e r t a i n that some f l a t f i s h must die from the a f f l i c t i o n of EPs. Although no metastases or t r u l y invasive growths were observed, f a t a l i t i e s could be caused by i n d i r e c t e f f e c t s of the disease, of which there are many p o s s i b i l i t i e s . The p o s i t i o n of lesions probably has an obvious e f f e c t on the health of the f i s h , e s p e c i a l l y when they occur about the g i l l s , eyes, or mouth. Fin tumors could be expected to cause sluggishness and would decrease the f i s h ' s a b i l i t y to escape predators. Any d e b i l i t a t i n g e f f e c t on the health of the f l a t f i s h i s l i k e l y to increase the s u s c e p t i b i l i t y of the f i s h to other agents (e.g. b a c t e r i a l i n f e c t i o n s ) , predators, or harsh environmental conditions. Possibly, both factors ( f i s h death and tumour necrosis and loss) are at work i n the removal of numbers of tumour-bearing f l a t f i s h from the population. Conceivably, s u r v i v a l of tumour-bearing f i s h long enough for the tumours to mature, and necrose, i s r e q u i s i t e for the phenomenon of tissue necrosis and sloughing-off. The nature of the uni d e n t i f i e d c e l l types ("X-c e l l s " ) found i n the stroma and epidermis of f l a t f i s h skin lesions remains, to some extent, uncertain. Two hypotheses have been proposed (Brooks et^ al^., 1969): 1) the c e l l s are u n i c e l l u l a r p a r a s i t i c organisms, and 2) the c e l l s are trans-formed f i s h c e l l s . The uni d e n t i f i e d c e l l s were postulated to be u n i c e l l u l a r parasites because: 1) the morphology of the c e l l s i s similar, i n some ways, to protozoans, 2) the un i d e n t i f i e d c e l l s are often found, i n the stroma, i n cords of two to four, 3) the c e l l s are usually surrounded by a 91 50 nm wide c e l l coat, 4) the stroma shows evidence of an inflammatory response, 5) collagen and f i b r o b l a s t processes often e n c i r c l e the c e l l s , and 6) macrophages can be seen i n the process of engulfing the unidentified c e l l s . No c e l l coats were observed around the c e l l s in the present study. The c e l l s studied i n the lemon sole showed no r e a l resemblance to any described u n i c e l l u l a r organism (see P i t e l k a , 1963), although other workers reported a resemblance to a haplo-sporidian parasite of f i s h as described by Perkins i n 1968 (McArn e t a l . , 1968). The u n i d e n t i f i e d c e l l type of the lemon sole possessed no evidently functional cytoplasmic organelles; those c e l l s which did, appeared to be t r a n s i -tions between normal epidermal c e l l s and the new c e l l type. The i n i t i a l appearance of t h i s new c e l l type evidently occurs, i n the stroma, aft e r the angioblastic and f i b r o -b l a s t i c p r o l i f e r a t i o n and, i n the epidermis, a f t e r an i n i t i a l t y p i c a l hyperplasia. These observations suggest that these un i d e n t i f i e d c e l l s occur as a r e s u l t of, and not as a cause of, the tissue reactions c h a r a c t e r i s t i c of the disease. The inflammatory nature of the AEN stroma possibly could be e l i c i t e d by these c e l l s i f they underwent antigenic changes due to transformation or i n f e c t i o n , s i m i l a r to the phenomenon of virus-induced tumor antigens. C e l l u l a r hypertrophic reactions i n f i s h c e l l s of various species have been shown to be e l i c i t e d by several agents (Weissenberg, 1949); these agents include viruses ( N i g r e l l i and Ruggieri, 1965; Weissenberg, 1965), R i c k e t t s i a (Wolke, 1970), bacteria (Davis, 1953), fungi, and p a r a s i t i c protozoans ( N i g r e l l i and Smith, 1940; N i g r e l l i , 1948b; Sprague, 1968; Weissenberg, 1968; Lom, 1970; Trager, 1974). U l t r a -s t r u c t u r a l studies of the unidentified c e l l types i n f l a t f i s h tumors showed no s i m i l a r i t i e s i n morphology to any of the above agents, or to any cytopathological changes (except hypertrophy) e l i c i t e d by these agents. Histo l o g i c evidence presented i n t h i s thesis indicates that the unidentified stromal and epidermal c e l l s found i n f l a t f i s h lesions probably are transformed f i s h c e l l s , rather than protozoan parasites; the transformed c e l l s d i f f e r from the normal i n that they are extremely hypertrophied and show c h a r a c t e r i s t i c s of extreme and i r r e v e r s i b l e c e l l u l a r dmage. This conclusion i s based on: 1) the absence of any evidence ind i c a t i v e of protozoan or other p a r a s i t i c invasion, i n t r a c e l l u l a r or e x t r a c e l l u l a r , and 2) observations of a number of morphological intermediates between normal epidermal c e l l s and the giant c e l l types found i n the l e s i o n s . The following discussion i l l u s t r a t e s what I have concluded to be a l i k e l y progression of cytopathic events involved i n the transformation of normal c e l l s to giant c e l l types. Probably the f i r s t reaction of the epidermal c e l l s i s a loss of c e l l - t o - c e l l contact, which i n some cases appears to r e s u l t i n an increase i n i n t r a c e l l u l a r space and i n the formation of s t e l l a t e or " p r i c k l e " c e l l s , a condition often noted i n h i s t o l o g i c a l studies of hyperplastic tissues (Figure 44). Presumably the c e l l s then lose desmosomal contact and 93 " b a l l up" (Figure 41). Vacuolation of the c e l l apparently follows, as does loss of c e l l volume regulation. The increasingly severe vacuolation i s correlated with the d i s -appearance of a l l cytoplasmic organelles (Figures 41 and 42). Very often the vacuoles tend to be elongated, horseshoe-shaped structures which resemble d i l a t e d endoplasmic reticulum (Figure 48); other larger, more rounded vacuoles often contain membrane remnants and resemble d i l a t e d , degenerated mito-chondria. The matrix of such vacuoles i s clear; membranes resembling c r i s t a e tend to be extremely small, i r r e g u l a r , and located at the vacuole periphery (Figure 49). These observa-tions tend to suggest that these "mitochondria" (Wellings e t a l . , 1967; Brown et a l . , 1969) are non-functional, degenerative structures. The cytoplasmic vacuoles can be found bounded by either a single or a double (the more common) membrane, presumably depending on the i r o r i g i n (Figures 48 and 49). On the basis of the above observations, I believe these vacuoles to be the product of damaged and d i l a t e d mitochondria and endoplasmic reticulum. Often membrane "whorls" occur in the cytoplasm, another i n d i c a t i o n of c e l l u l a r damage (Figures 38 and 49). The cytoplasmic ground substance tends to become i r r e g u l a r l y amorphic and may become extremely l i g h t (Figure 46) . The n u c l e i of giant c e l l types i n mature lesions tend to be enlarged; chromatin i s always clumped into d i s c r e t e , random clumps, n u c l e o l i are enlarged, prominent, and consist exclu-s i v e l y of ribosome-like p a r t i c l e s (Figure 46). These observations tend to indicate degenerative processes; the 94 cytopathic changes described are changes to non-functionality and ultimate necrosis (Trump and Ginn, 1969; Trump and A r s t i l a , 1971). I t i s important to note here that the subcellular pattern of necrosis of f l a t f i s h " X - c e l l s " d i f f e r s from the pattern of necrosis found i n other c e l l s i n reaction to l e t h a l i n j u r i e s to the c e l l . In p a r t i c u l a r , necleolar d i s i n t e g r a t i o n and karyolysis, common changes i n necrotic c e l l s (Ginn et a l . , 1968), do not occur i n f l a t f i s h tumour c e l l s . This would suggest that the necrotic changes described i n f l a t f i s h c e l l s are not the r e s u l t of a non-specific l e t h a l injury to the c e l l (e.g. anoxia), but may instead be the r e s u l t of c e l l u l a r transformation ( i . e . , changes i n the genome) or of a s p e c i f i c subcellular injury caused by a s p e c i f i c a e t i o l o g i c a l agent. These changes are c h a r a c t e r i s t i c of both the hyper-trophic epidermal c e l l types as well as the analogous stromal c e l l type. C e l l s c l a s s i f i e d as type A giant c e l l s show these c h a r a c t e r i s t i c s ; however, they have undergone further hyper-trophy and vacuolation than have the smaller, type B, giant c e l l s . They are characterized by the appearance of large numbers of extremely electron-dense cytoplasmic inclusions of unknown o r i g i n and composition (Figure 46 and 51). Brooks and h i s co-workers (1969) indicated that i f these u n i d e n t i f i e d c e l l types were transformed f i s h c e l l s , then m o t i l i t y of such c e l l s must be required, since the c e l l s are found i n both stromal and epidermal components of the l e s i o n . This hypothesis need not be the case. Staining properties and u l t r a s t r u c t u r a l studies of the two c e l l types indicate c e r t a i n differences between the epidermal and stromal unknown c e l l s . These differences might a r i s e i f the o r i g i n of the c e l l types were d i f f e r e n t : a degenerative, necrotic disease process presumably would be capable of causing s i m i l a r cyto-pathic e f f e c t s i n stromal f i b r o b l a s t s , for instance, and in epidermal c e l l s . The stromal uni d e n t i f i e d c e l l type was never observed to undergo extreme c e l l u l a r hypertrophy to the extent that i t s analogous epidermal c e l l type can. The o r i g i n of the stromal c e l l type i s unknown; an obvious hypothesis might suggest an o r i g i n from f i b r o b l a s t s , but no concrete evidence whatsoever has been observed. There i s doubt as to whether the f l a t f i s h epidermal lesions should be considered as hyperplastic or neoplastic processes (Wellings e t a l . , 1968; Mawdesley-Thomas, 1972). There might also be doubt as to whether the lesions represent a hypertrophic process with compensatory hyperplasia of s u p e r f i c i a l and enveloping c e l l s , or whether the lesions are true hyperplastic or neoplastic reactions preceding a secondary, and i n c i d e n t a l , c e l l u l a r hypertrophy. Discussion or speculation without further studies would be inconclusive at best. Some workers (Wellings et a l . , 1965; M i l l e r and Wellings, 1971) believed the lesions to be true neoplasms. N i g r e l l i and h i s co-workers (1965) postulated that the lesions were hyperplastic i n nature. The difference between hyper-p l a s i a and neoplasia usually cannot be determined by h i s t o l o g i c a l studies, as noted in the case of f i s h pox (Schlumberger and Lucke, 1948). According to the d e f i n i t i o n of neoplastic hyperplasia (Prehn, 1972) as: "that form of hyperplasia which i s caused, at l e a s t i n part, by an i n t r i n s i c inheritable abnormality i n the involved c e l l s " , animal neo-plasms must be transferrable by the inoculation of l i v i n g neoplastic c e l l s . Transplantation studies done on f l a t f i s h tumors have a l l proved negative (Good, 1940; Chuinard and Wellings, 1964; McArn, 1968; Wellings, 1969). The aetiology of epidermal papilloma-like lesions a f f l i c t i n g P a c i f i c coast f l a t f i s h remains an enigma. Many factors may be involved i n the genesis of any disease process; probably t h i s multiple factor approach applies to f l a t f i s h l e s i o n s . Evidence of v i r u s - l i k e p a r t i c l e s present in the c e l l s of mature epidermal lesions of lemon sole was given above; the r e l a t i o n s h i p of these p a r t i c l e s to the disease process, i f any, has not been determined. Viruses have been proven to be the cause of one c e l l u l a r hypertrophic disease of f i s h epidermis, lympho-c y s t i s disease (Weissenberg, 1965; Wolf et a l . , 1966), and have been implicated as a probable cause of several other epidermal hyperplastic diseases i n f i s h . These l a t t e r include f i s h pox ( N i g r e l l i , 1952; Mawdesley-Thomas and Bucke, 1967), the cauliflower disease of eels (Koops e t a l . , 1970), and other hyperplastic reactions of f i s h epidermis (Walker, 1966 and 1968). Electron microscopic evidence of v i r u s - l i k e p a r t i c l e s has been given in t h i s report (Figures 52-54) as well as i n other reports of studies of f l a t f i s h epidermal lesions (Wellings et al_., 1964, 1965 and 1967; McArn, 1968; Ke l l y , 1971). The use of v i r a l techniques i n i s o l a t i o n or transmission of the virus has proved negative (Wellings §_t a l . , 1965; McArn, 1968; Wellings, 1969). From the evidence presented i n t h i s thesis, i t seems reasonable to conclude that: 1) e p i t h e l i a l tumours i n P a c i f i c coast f l a t f i s h progress from i n i t i a l a n g i o e p i t h e l i a l nodules to mature epidermal papilloma-like growths, and 2) t h i s progression i s manifest i n h i s t o l o g i c a l and c y t o l o g i c a l changes, as documented above. Other evidence presented i n t h i s thesis suggests that the uni d e n t i f i e d " X - c e l l s " which compose the mature tissue are transformed, necrotic f i s h epidermal c e l l s , rather than i n t r a - or e x t r a c e l l u l a r para-s i t i c protozoans (as has been hypothesized). Evidence for the progression of normal epidermal c e l l s into " X - c e l l s " i s p r i m a r i l y the presence of morphologic intermediates, the timing of appearance of the new c e l l type i n the tissue, and the lack of resemblance of the " X - c e l l s " to any known protozoan. Further work must be done to d e f i n i t e l y e s t a b l i s h t h i s f a c t . The f u l l b i o l o g i c a l nature of the lesions ( i . e . , whether the growths are the r e s u l t of hyperplastic, neo-p l a s t i c , or hypertrophic processes) i s unknown, and further work i s c e r t a i n l y needed and desirable. Autoradiographic studies of the various tumour stages would show where (by which c e l l s ) hyperplastic or neoplastic growth was occurring. Further, and more extensive, observations of tumour-bearing f i s h under laboratory conditions may be useful i n determining the hyperplastic or neoplastic nature of the l e s i o n s . The nature of f l a t f i s h tumours i s presently unclear and the similitude of these growths and true epidermal papillomas seems questionable. Certainly the histomorphology of the f l a t f i s h lesions d i f f e r s s u f f i c i e n t l y from other known papillomas, carcinomas, or other diseases of f i s h epidermis to warrant further study. 99 LITERATURE CITED Angell, C.L. and M i l l e r , B.S. (1974), Ecology of Tumor-bearing English Sole (Parophrys vetulus) i n Puget Sound, Washington. J . Fish Res. Bd. Can. (submitted). (Cited by Mearns and Sherwood, 1974). Andrew, W. (1959), A covering c a l l e d skin. In: A textbook of Comparative Histology. 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