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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.Sc,  University  of Illinois,  1973  A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  t h e Department of : Zoology  We a c c e p t t h i s required  THE  t h e s i s as conforming  t o the  standard  UNIVERSITY OF BRITISH COLUMBIA September, 1975  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  further  for  freely  of  the  requirements  B r i t i s h Columbia, I agree  available  for  this  this  thesis for  financial  The  gain s h a l l  of  U n i v e r s i t y of B r i t i s h Columbia  20 75 Wesbrook Place Vancouver, Canada V6T 1W5  Date  Q ± 8  iq-frD  or  i s understood that c o p y i n g or p u b l i c a t i o n  w r i t ten pe rm i ss i on .  Department  that  thesis  s c h o l a r l y purposes may be granted by the Head of my Department It  for  reference and study.  t h a t p e r m i s s i o n for e x t e n s i v e copying o f  by h i s r e p r e s e n t a t i v e s . of  fulfilment  the U n i v e r s i t y of  s h a l l make it  agree  in p a r t i a l  not  be allowed without my  ABSTRACT  Natural microscopic  history,  was  (Parophrys  on  evidence  f r o m an  initial  and  morphological  cytological  I t was flatfish  plasia.  and  the  noted  (the  The  stromal  t h a t the  disease,  hypertrophy  study  a progression  a s s u m p t i o n was  supported  and  histological  was  c h a r a c t e r i z e d by cells  cell  form  o f the  (the  histolog-  of  t y p e s w h i c h compose  and  histological  cellular  from the by  angioepithelial  larger  former t o the  the presence of The  any  neo-  younger f i s h w h i l e on  of  characterized  h y p e r p l a s i a or  s t u d i e s showed t h a t on  appearance  from t h a t o f  i n c l u d i n g those  intermediates.  into  histolesions  o f the nature  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 f o u n d  with enlarged  Emphasis  angioepithelial  significantly  nodules occurred predominantly  epidermal  of  tumour.  Natural history  indicating  Strait  of the growth o f the l e s i o n s  lesions differs  known f i s h e p i d e r m a l cellular  i n the  morphologically different  aspects  epidermal  much o f t h e mass o f  by  form  incorporated c l a r i f i c a t i o n  unidentified  lesions afflicting  Columbia, Canada.  papilloma-like lesion).  necessarily  electron  f o r the p r o g r e s s i v e growth o f these  n o d u l e ) t o a m a t u r e and epidermal  and  t h e 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  logical  the  vetulus)  near Vancouver, B r i t i s h  placed  ical  microscopic,  s t u d i e s were made o f e p i d e r m a l  j u v e n i l e lemon s o l e Georgia,  light  epidermal fish,  latter.  This  morphological  growth of the l e s i o n s  the g r a d u a l  transformation  "X-cells":  ovoid, hypertrophied  n u c l e i , prominent n u c l e o l i ,  and  of  normal  necrotic  cells  iii cytoplasm.  These  "X-cell"  m a t u r e l e s i o n and epidermal c e l l s  types c o m p l e t e l y dominate  a r e e n c l o s e d , o r s u p p o r t e d , by  termed  "enveloping c e l l s " .  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 cells  differs  from n e c r o s i s c a u s e d  injuries  to c e l l s .  cellular  c h a n g e s o b s e r v e d may  be  found  in flatfish  by n o n - s p e c i f i c  a result  than a n o n - s p e c i f i c  of  tumour  lethal  that the  sub-  cellular  lethal  injury  to  cells. Other n a t u r a l h i s t o r y  in  abnormal  I t was  This observation suggests  transformation rather the  necrosis  the  t h e number o f tumours p e r  studies  indicated  tumourous f i s h and  i n the prevalence of l e s i o n s  as  results  the d i s e a s e d f i s h are  tively  indicate  that either  removed from  a r e l o s t by  discussed.  No  the h o s t .  e v i d e n c e was  found  particles  increases.  These  selec-  tumours were  to support the h y p o t h e s i s The  commonly o b s e r v e d .  s h i p between t h e s e v i r u s - l i k e i f any,  decline  presence  of  i n the c y t o p l a s m o f the e n v e l o p i n g  o f mature l e s i o n s was  process,  decline  Both p o s s i b i l i t i e s  " X - c e l l s " are p a r a s i t i c protozoans.  virus-like cells  size  the p o p u l a t i o n o r t h a t t h e  n e c r o s e and  that  fish  a  a  i s unknown.  particles  and  The  relation-  the d i s e a s e  iv  TABLE OF CONTENTS Page INTRODUCTION  1  General Introduction  1  Life  7  History  Information  MATERIALS AND METHODS  9  C o l l e c t i o n Procedures  and Data  Analysis  9  Light Microscopy  14  Transmission Electron Microscopy  14  Scanning  15  E l e c t r o n Microscopy  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 E p i d e r m i s , L i g h t M i c r o s c o p y  21  Normal E p i d e r m i s , E l e c t r o n M i c r o s c o p y  26  Tumour H i s t o l o g y , L i g h t M i c r o s c o p y  28  A n g i o e p i t h e l i a l Nodules  28  AEN/EP  33  Transition  Forms. .  Epidermal Papilloma-like Lesions  . .  Tumour H i s t o l o g y , E l e c t r o n M i c r o s c o p y  36 46  Stroma  46  AEN E p i d e r m i s  53  EP  59  Epidermis  Virus-like  Particles  DISCUSSION Normal E p i d e r m i s  .  74 77 77  V  Page Biology o f the Epidermal Lesions LITERATURE CITED  78 99  vi  L I S T OF FIGURES Figure  Page  1.  External  morphology  o f AEN on j u v e n i l e  lemon  sole 2.  12  External  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 3.  External  morphology  juvenile  of EP-like  12 l e s i o n on  lemon s o l e  12  4.  Low-power l i g h t m i c r o g r a p h o f AEN  5.  Low-power l i g h t m i c r o g r a p h o f AEN/EP tion  6. 7.  1  form  12 lesion  .  12  The r e l a t i o n s h i p o f tumour p r e v a l e n c e a n d size  18  The r e l a t i o n s h i p o f numbers o f tumours p e r fish  9.  transi-  Low-power l i g h t m i c r o g r a p h o f E P - l i k e  fish 8.  12  and f i s h  size  20  The r e l a t i o n s h i p o f tumour t y p e s a n d  fish  size  22a  10.  L i g h t micrograph o f normal e p i d e r m i s  25  11.  E l e c t r o n micrograph o f normal  epidermis. . . .  25  12.  E l e c t r o n micrograph o f normal  epidermis. . . .  25  13.  Scanning e l e c t r o n micrograph o f normal epidermis.  25  14.  L i g h t micrograph o f stroma  o f AEN  30  15.  L i g h t micrograph o f 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 invasive  qualities  30  vii Figure 17.  Page Higher magnification lesion  18.  l i g h t micrograph o f  shown i n F i g u r e  16  30  L i g h t m i c r o g r a p h o f e p i d e r m a l component o f AEN  35  19.  L i g h t m i c r o g r a p h o f AEN  20.  Low-power l i g h t m i c r o g r a p h o f AEN/EP tion  21.  35 transi-  form  35  L i g h t micrograph o f epidermal  hyperplastic  s t a g e o f AEN/EP t r a n s i t i o n f o r m 22.  Higher magnification tissue  35  l i g h t micrograph o f  shown i n F i g u r e  21  38  23.  L i g h t m i c r o g r a p h o f AEN/EP t r a n s i t i o n f o r m  . .  38  24.  L i g h t m i c r o g r a p h o f AEN/EP t r a n s i t i o n f o r m  . .  38  25.  L i g h t micrograph o f E P - l i k e  lesion  26.  L i g h t micrograph o f E P - l i k e  l e s i o n , showing  s t r o m a l and e p i d e r m a l c h a r a c t e r i s t i c s . . . . 27.  lesion  41  28.  L i g h t micrograph o f E P - l i k e  29.  L i g h t micrograph o f s u p e r f i c i a l like  41  area o f EP41  Higher magnification  l i g h t micrograph o f t i s s u e  26  L i g h t micrograph o f EP-like general  32.  lesion  lesion  shown on F i g u r e 31.  41  L i g h t m i c r o g r a p h o f e p i d e r m a l component o f EP-like  30.  38  45 l e s i o n , showing  tissue necrosis  L i g h t micrograph o f E P - l i k e necrotic material  45 l e s i o n , showing 4  5  viii Figure 33.  Page L i g h t micrograph o f E P - l i k e  lesion,  showing  necrotic material 34.  45  E l e c t r o n m i c r o g r a p h o f basement membrane a r e a of EP-like  lesion  48  35.  E l e c t r o n m i c r o g r a p h o f A EN s t r o m a  48  36.  E l e c t r o n m i c r o g r a p h o f AEN s t r o m a  50  37.  E l e c t r o n micrograph o f stromal " X - c e l l s "  38.  E l e c t r o n m i c r o g r a p h o f b a s e m e n t membrane a r e a of EP-like  lesion,  showing  i n AEN  a non-ovoid stromal  " X - c e l l " and e p i d e r m a l " X - c e l l s " 39.  e a r l i e s t r e l a t i o n s h i p between  epidermal c e l l s 40.  55  E l e c t r o n m i c r o g r a p h o f AEN e p i d e r m i s , the  showing  normal  and " X - c e l l s "  55  E l e c t r o n m i c r o g r a p h o f e p i d e r m a l component o f l a r g e AEN  41.  58  E l e c t r o n m i c r o g r a p h o f e p i d e r m a l component of  42.  l a r g e AEN  58  E l e c t r o n m i c r o g r a p h o f AEN e p i d e r m i s ,  showing  transforming epidermal c e l l 43.  E l e c t r o n micrograph o f large showing  44.  AEN e p i d e r m i s ,  E l e c t r o n micrograph o f s u p e r f i c i a l  61 cell  lesion  E l e c t r o n micrograph o f s u p e r f i c i a l of EP-like  46.  61  developing envelope c e l l  layers of EP-like 45.  50  64 cell  lesion  E l e c t r o n micrograph o f deeper p o r t i o n like  lesion  layers 64  o f EP67  ix Figure 47.  Page E l e c t r o n microgram o f deeper like  48.  of  EP-  lesion  67  E l e c t r o n microgram o f epidermal of EP-like  49.  portion  component  lesion  70  E l e c t r o n microgram o f p o r t i o n a type A g i a n t  cell  found  o f cytoplasm  i n an  of  EP-like  lesion 50.  70  E l e c t r o n micrograph  o f n u c l e u s and p o r t i o n  cytoplasm of epidermal EP-like 51.  52.  EP-type  A and  "X-cells"  E l e c t r o n micrograph  of  of v i r u s - l i k e  particles  of virus-like  the  of virus-like  i n t e r c e l l u l a r space  Summary o f t h e g r o w t h a n d f i s h epidermal l e s i o n s  . . .  76  particles  t h e c y t o p l a s m o f an e n v e l o p i n g c e l l  E l e c t r o n micrograph  types 73  the c y t o p l a s m o f an e n v e l o p i n g c e l l  E l e c t r o n micrograph  in 55.  of  showing the c h a r a c t e r i s t i c s  in 54.  73  lesion, B  in  lesion  Low-power e l e c t r o n m i c r o g r a p h  in 53.  " X - c e l l " found  of  . . .  76  particles  o f an AEN  structure  of  . . . .  76  flat82  L I S T OF ABBREVIATIONS USED IN TEXT  a n g i o e p i t h e l i a l nodule epidermal papilloma-like lesion  xi  L I S T OP ABBREVIATIONS USED I N FIGURES  A  type A  X-cell  AEN  angioepithelial  B  type  BC  basal  BM  basement  Cap  capillary  Con  connective  cf  cytoplasmic  D  cellular  des  desmosome  EC  epidermal  Env  enveloping  Ep  epidermis  nodule  B X-cell cell membrane  tissue filaments  remains  (debris)  cell cell  EP  epidermal  papilloma-like lesion  er  endoplasmic r e t i c u l u m  F  fibroblast  g  glycocalyx  ger  granular endoplasmic r e t i c u l u m  Gran  granulocyte  Gu  guanidophore  IC  cell  L  lymphocyte  M  macrophage  Mel  melanocyte  mit  mitochondria  of the intermediate  layer  MC  mucous-secreting  Mus  muscle  Mf  microfilaments  mt  microtubule  nc  nucleolus  ne  nuclear  nu  nucleus  np  nuclear  pm  plasma  RBC  red  rib  ribosomes  S  stromal  SC  superficial  Sc  scale  Sin  sinusoid  SMC  smooth m u s c l e  Str  stroma  Trans  transition  vac,  vacuole  VLP  virus-like  X  X-cell,  cell  envelope  pore membrane  blood  cell  X-cell cell  cell  f o r m b e t w e e n AEN  particle  type A or B  and  EP  xiii  ACKNOWLEDGEMENTS  I g r a t e f u l l y w i s h t o acknowledge t h e c o o p e r a t i o n of: Dr. and  critical  A . B. A c t o n , my s u p e r v i s o r , revision  Drs.  of this  invaluable  thesis;  J . R. Adams a n d W. S. H o a r ,  readings o f the thesis, and  f o r h i s patience  technical  a n d Mr. L . L . V e t o , knowledge.  for their for his  critical  excellent  1  INTRODUCTION  General  introduction  Since  1922  ( H a r o l d and  Innes, 1922), e p i d e r m a l  tumors o f unknown a e t i o l o g y , d i a g n o s e d e i t h e r a s papillomas disease  ( W e l l i n g s e_t a l . , 1964)  other  s p e c i e s o f the  ( f a m i l i e s B o t h i d a e and  coast o f North America.  s p e c i e s and  geographic l o c a t i o n .  incidences  5%  Good  disease,  i n some f l a t f i s h a r e  of l i f e ;  four years  o f the  no  often  o f age  (see Mawdesley-Thomas, 1 9 7 2 ) . incidences  melanostictus) Incidences  l e s i o n s i n a sand s o l e  population  o f 6.4%  of  i n the  g l o s s o i d e s e l a s s o d o n ) from t h e  San  lemon s o l e , s a n d s o l e , and  sole  were a l s o a f f l i c t e d Chuinard  epidermal  i n t h i s area  e t a l . , 1964;  a study  Wellings  o f marine l i f e l e s i o n s on  rex  the  i n the  particular  i n the  dover s o l e  first over  Pacis  (1932)  Ketchen  British  flathead sole  (1953)  (Glyptocephalus  1965).  (Hippo-  zachirus) 1964;  Young  Francisco area, (Microstomus  Columbia.  Washington;  (Wellings e t a l . ,  San  tumor  approximately  Juan I s l a n d s ,  et_ aa.,  the  (Psettichthys  from H e c a t e S t r a i t ,  were r e p o r t e d  along  in flatfish  i n lemon s o l e f r o m P u g e t Sound, W a s h i n g t o n . similar  the  indicate rela-  in a  at their highest  disease  in  been n o t e d t h a t  t u m o r s have b e e n r e p o r t e d  (1940) r e p o r t e d  described  in  especially  I t has  epidermal  Pleuronectiformes  Several reports  incidences  and  hyperplastic  order  Pleuronectidae),  t i v e l y high  year  epidermal  ( N i g r e l l i et; a l . , 1 9 6 5 ) , h a v e b e e n r e p o r t e d  l e m o n s o l e and  Paific  o r as  skin  (1964), found  pacificus).  2 Sand s o l e  and rock s o l e  S t r a i t were r e p o r t e d lesions;  (Lepidopsetta  t o be a f f l i c t e d w i t h  (1967) r e p o r t e d Bristol  that  Studies  (1969) i n d i c a t e  with the disease with the  butter  the starry  sole  incidences  (Isopsetta  i n P u g e t Sound Miller  Bay b y C o o p e r a n d  there are a f f l i c t e d  o f 3 - 28%, d e p e n d i n g on  and W e l l i n g s  tumorigenesis,  (1971) d e s c r i b e d  sole,  history  including  and g r o w t h a n d s u r v i v a l  revealed  that  A report  indicated  tumor  1971).  rates.  up t o 9% o f j u v e n i l e (Angell  o£ a s o u t h e r n C a l i f o r n i a frequencies  study  o f 9% (Mearns and  1974). The  e p i d e r m a l t u m o r s o f f l a t f i s h have b e e n  l o g i c a l l y described, histologically reported  the n a t u r a l  i n the flathead  tumor i n c i d e n c e s ,  1974).  o f dover s o l e Sherwood,  s t e l l a t u s ) and t h e  s a m p l e d were a f f l i c t e d w i t h t h e d i s e a s e  Miller,  additional  i s o l e p i s ) , were f o u n d t o b e d i s e a s e d  R e c e n t P u g e t Sound s t u d i e s lemon s o l e  (Platichthys  Two  (McArn e t a l . , 1968; McArn a n d W e l l i n g s ,  aspects o f the disease  and  lemon s o l e  flounder  o f Alaska  by t u m o r - l i k e s k i n growth w i t h  time o f t h e y e a r and g e o g r a p h i c l o c a t i o n .  species,  Levings  i n the Gulf  i n San F r a n c i s c o  that  Hecate  t o be a h y p e r -  e t a l . , 1965).  j u v e n i l e rock sole  Bay were a f f e c t e d  a 10% incidence. Keller  (Nigrelli  from  papilloma-like  the authors considered these l e s i o n s  p l a s t i c epidermal disease  and  bilineata)  t o some e x t e n t ,  and m o r p h o l o g i c a l l y  i n the flathead  sole  histo-  f o r most s p e c i e s .  distinct  (Wellings  were t e r m e d t h e a n g i o e p i t h e l i a l n o d u l e  Two  tumor t y p e s were  et_ a l , . , ("AEN",  1964). small.  These  3  h e m o r r h a g i c n o d u l e s ) and  the  e p i d e r m a l papilloma' ' 1  l a r g e r , more d i f f u s e , g r a n u l a r in  l e s i o n s ranging  EP were a l s o t o the  Chuinard the  found, suggesting  m a t u r e EP  laboratory.  sand s o l e  (Wellings  s o l e , and  mm  the  tumor.  Wellings  (1971) r e p o r t e d  identical  o f the  and  tumor  in  type, in  also lemon  sole,  t h a t the  showed t h e  t o the  others  regressive  tendencies McArn  g r o s s morphology  lemon s o l e , s t a r r y f l o u n d e r ,  sole l e s i o n s are  identical.  disease  morphological type  and  and  flathead  d e f i n i t e progression (AEN)  mature  described.  (1968) and  h i s t o l o g y o f the  f r o m one  Wellings  authors  h i s colleagues  The  initial  Histological descriptions  n e c r o t i c and  McArn and  the  described  ( N i g r e l l i e t a l . , 1964)  author a l s o described  within  ("AEP"), was  similarities  and  from an  uncommon m o r p h o l o g i c  s o l e tumors.  l e s i o n s t o be  AEN  growth o f the  e t a l . , 1964); the  histological  flathead  papilloma-like  and  and  a n g i o e p i t h e l i a l polyp  o f sand s o l e l e s i o n s  The  progressive  A third  demonstrated the rex  a progression  ( W e l l i n g s ,et a l ^ . , 1964).  (1964) r e p o r t e d  termed the the  t o 60  diameter). M o r p h o l o g i c t r a n s i t i o n s between the  AEN  up  ("EP",  to another  of (EP)  the was  !(N0TE: "Papilloma", i n a s t r i c t sense, r e f e r s to a benign neoplasm. S i n c e , i n t h i s case, the t r u e n e o p l a s t i c n a t u r e o f t h e 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, 1 9 7 2 ) , a more c o r r e c t t e r m w o u l d 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 , 1 9 6 7 ) . For the s a k e o f c l a r i t y and c o n t i n u i t y , t h e t e r m " e p i d e r m a l p a p i l l o m a " w i l l be u s e d i n t h i s p a p e r t o d e s c r i b e t h e m a t u r e l e s i o n . A l s o , t h e t e r m "tumor", i n t h i s r e p o r t , i s u s e d 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 g r o w t h . )  4 p o s t u l a t e d on stages,  the  (smaller EPs),  the presence o f m o r p h o l o g i c a l  c o r r e l a t i o n b e t w e e n tumor t y p e  flatfish  and  had  mostly  exhibiting events:  epidermal  the  I t should papillomas  initial  1969B).  rather unclear,  (Oota,  identical  noted  1952;  a l s o show t h e  into a papilloma  Honma and  I m a i and  progression  s t r u c t u r e and  if  not  is  s t r o n g l y suggested  Kon,  (Lucke,  1938;  Fujiwara,  and  1959)  Although by  al.,  Nigrelli  be  1968;  McArn and  (  1965;  flatfish  Wellings  Wellings,  1971).  cells  head s o l e e p i d e r m a l et  a l . , 1969).  and  concluded  similar,  f r o m AENs t o no  histological  tumors a r e unique,  as  (Wellings e t a l . ,  e t aJL_., 1965;  McArn e t  Electron microscopic  cells  found  in  ( W e l l i n g s e t a l . , 1967;  Unidentified cell  EPs  d i f f e r e n c e s between  epithelial  papillomas  show  date.  studies  s t u d i e s showed marked m o r p h o l o g i c a l normal e p i t h e l i a l  afflicted Because  i t i s generally  the p r o g r e s s i o n  l i g h t microscopic  et a l .  flounder  several gobiid  may  a v a i l a b l e evidence,  the  also  various species described  morphology,  Histologically  1964;  of  although  species of  1968)  p r o g r e s s i o n s t u d i e s have b e e n done t o  initial  fish  same s e r i e s  t h a t t h e u n d e r l y i n g mechanisms o f t h e d i s e a s e a r e  shown by  fish  mainly  s i m i l a r p a p i l l o m a - l i k e tumors.  l e s i o n s o f the  identical.  of  that other  seem t o i n d i c a t e t h a t two  with h i s t o l o g i c a l l y the epidermal  be  of disease  S e v e r a l Japanese r e p o r t s ,  (Kimura §_t a l . , 1967; fishes  size  a p p e a r a n c e 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 progresses see W e l l i n g s ,  and  AENs; l a r g e r f i s h had  the l a b o r a t o r y observations  (McArn e t a l . , 1 9 6 8 ) .  transitional  types,  termed  flatBrooks  "X-cells"  5 ( B r o o k s et: a l . , 1969) were d e s c r i b e d f r o m t h e s t r o m a l a n d e p i d e r m a l components o f t h e l e s i o n s . cells to  was n o t d e t e r m i n e d ;  be e i t h e r  The n a t u r e o f t h e s e  the authors p o s t u l a t e d the c e l l s  p a r a s i t i c protozoans  or transformed  Comparisons between e p i t h e l i a l and  fish  cells.  tumors o f f l a t f i s h  o t h e r e p i d e r m a l d i s e a s e s known i n f i s h e s a r e i n e v i t a b l e .  Such comparisons logical  may b e u s e f u l ,  e s p e c i a l l y when u s i n g  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 .  histo-  The s t r u c t u r e o f  t h e 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 cellular  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  epitheliocystis disease 1970), 1965;  lymphocystis disease and N i g r e l l i  infections are  (Sprague,  1965),  1968; W e i s s e n b e r g ,  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  flatfish  cellular  proliferative lesions  and B u c k e , 1 9 6 7 ) , 1935;  Nigrelli,  1956;  organism.  R u s s e l l and K o t i n ,  usually  descriptions Pro-  f i s h p o x ( e . g . , Mawdesley-Thomas  hyperplastic epidermal diseases  (Nigrelli  lesions  The  lesions o f f i s h epidermis.  include  Koops e t a l . , 1 9 7 0 ) ,  Stolk,  These  hypertrophy,  from h i s t o l o g i c a l  and Smith,  ( C o a t e s , Cox, a n d S m i t h ,  Schlumberger,  1968).  1948; W a l k e r , 1969a a n d 1 9 6 9 b ) ,  to infections  papillomas  Weissenberg,  and m i c r o s p o r i d i a n  of the i n i t i a t i n g  tumors a l s o d i f f e r  liferative  due  (see reviews by  and R u g g i e r i ,  accompanied by e v i d e n c e  of  (Hoffman e t a l . , 1969; Wolke e t a l . ,  1940),  (Smith,  proliferations  epidermal  1938; Lumann a n d Mann,  1957; Deys, 1969; S t e e v e s , 1969;  and e p i d e r m o i d c a r c i n o m a s  ( L u c k e and  1941; T a v o l g a , 1951; A r n o w i t z e t a l _ . , 1951;  1953, 1956, 1958, a n d 1 9 6 0 ) .  These  proliferative  6 r e a c t i o n s may ture. the  The  o r may  n o t show a p a p i l l o m a t o u s g r o w t h  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  form and  f u n c t i o n o f the c e l l  Generally,  the c e l l s  type of  plastic  flatfish differ lesions of  that  the e p i d e r m a l  structurally  from  near  Vancouver, B r i t i s h  starry  t h e same  by u s i n g  Columbia.  histo-  tumors found  f l o u n d e r , and  flathead  and  that i n the  Similar  isolated  (sand s o l e , r o c k s o l e ,  l i g h t microscopy,  of juvenile  lemon  this  on  disease i s  S t r a i t of Georgia, l e s i o n s were  cases of s e v e r a l  sole).  sole,  Natural history  e l e c t r o n microscopy  data,  were u t i l i z e d  in  i n this population  sole.  I n s o f a r a s t h e n a t u r e o f a d i s e a s e must be i t s a e t i o l o g y and  e x a m i n e d , e m p h a s i s was  c o n s e q u e n c e s c a n be  p l a c e d on  unresolved aspects o f the nature  o f the u n i d e n t i f i e d  t h e t i s s u e o f the mature  meaningfully  o f these  lesions: EPs,  " X - c e l l " type which  lesion.  known  the c l a r i f i c a t i o n o f  h i s t o g e n e t i c r e l a t i o n s h i p b e t w e e n AENs a n d nature  also  other  s p e c k l e d sanddab, r e x  the s t u d y o f t h i s d i s e a s e , as m a n i f e s t  before  (1949)  other described epidermal  lemon s o l e  i n s m a l l numbers o r  species  hyper-  fishes.  common i n j u v e n i l e  found  or  Schlumberger  I t c a n be n o t e d ,  R e c e n t l y i t has been noted quite  Most  or n e o p l a s t i c growths tend t o e x h i b i t  comparisons,  normal  U s u a l l y no n e c r o t i c  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 b y L u c k e and  logical  their  degenerative processes are apparent.  for epidermal papillomas.  retain  origin.  tend to r e t a i n  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 . cellular  tend to  struc-  and  two  1)  the  2)  the  dominates  7  evidence of  The  primary objectives  of this thesis are:  1)  to present natural  h i s t o r y and h i s t o l o g i c a l  f o r , and d e s c r i p t i o n s  o f , the progressive  t h e s e l e s i o n s , and 2)  the  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 ,  nature o f the u n i d e n t i f i e d  ship  " X - c e l l " type and t h e i r r e l a t i o n -  t o t h e h i s t o l o g i c a l growth and s t r u c t u r e Hopefully  nature, aetiology,  Life History  The  affected.  is  is  been w e l l  seems t o be t h e c a s e w i t h  For t h i s reason, a short  lemon s o l e  "flatfish"). documented  cal  and p e l a g i c  flatfish  synopsis of  Girard,  1854)  (Pleuro-  The n a t u r a l h i s t o r y o f t h i s (see Hart,  larvae,  1 9 7 3 ) ; however,  o f the juvenile  C o l u m b i a , s p a w n i n g may o c c u r Lemon s o l e  generally  follows.  (Parophrys v e t u l u s  known a b o u t t h e h a b i t s  1946).  in a  h i s t o r y o f the  a member o f t h e 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  nectiformes; has  This  with the n a t u r a l  h i s t o r y o f t h e lemon s o l e The  occurring  (especially infectious diseases)  intimately associated  life  t o c l a r i f y the  and c o n s e q u e n c e s o f t h e d i s e a s e .  p r e v a l e n c e o f any d i s e a s e  epidermal l e s i o n s . the  epidermal  Information  natural population  species  of the l e s i o n s .  t h i s type o f study o f these  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  is  growth  fish.  other  little  In B r i t i s h  from J a n u a r y t o March  like  species  (Taylor,  f l a t f i s h , a r e symmetri-  f o r s i x t o t e n weeks b e f o r e  they undergo  8 metamorphosis  and  settle  Columbia metamorphosing  t o the bottom.  l a r v a e a r e f o u n d on t h e b e a c h e s  m i d - A p r i l ; when m e t a m o r p h o s i s measure a r o u n d the  spring  10 mm.  i s complete  Growth i s r a p i d  total  l e n g t h by August.  a t t h e e n d o f one y e a r o f l i f e ;  a v e r a g e 200 mm  ( K e t c h e n , 1947;  extremely slow d u r i n g Manzer, 1951; s a y e d , 1969; that  fall  Forrester,  1969a);  movement o f a d u l t into  food fish  intake.  j u v e n i l e lemon s o l e  p e r month) i n they 125  two y e a r s t h e y 1969).  Growth i s  (Ketchen,  1947;  v a n C l e v e and e l -  o t h e r s t u d i e s h a v e shown periods of semi-  There  is a definite  i n t o deeper waters  shallow waters  generally  Lemon s o l e a v e r a g e  Forrester,  1969;  in  year of l i f e ;  and w i n t e r months  S m i t h and N i t s o s ,  h i b e r n a t i o n and no  fish,  (23 mm  after  i n m i d - w i n t e r lemon s o l e u n d e r g o  a return  the f i s h  and summer months o f t h e f i r s t  r e a c h 100 mm mm  In southern B r i t i s h  i n t h e w i n t e r and  i n the s p r i n g .  L i k e most  t e n d t o c o n g r e g a t e on  " n u r s e r y g r o u n d s " where t h e y r e m a i n u n t i l  yearly  flat-  certain  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 h a v e 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 a r e n o t u n d e r t a k e n and  populations  g e n e r a l l y remain d i s t i n c t  1955;  Forrester, are at  1969b).  Forrester,  Lemon s o l e on t h e B r i t i s h C o l u m b i a  characteristically t h e heads  ( K e t c h e n and  found  o f many i n l e t s  in various (Hart,  isolated  1973).  coast  populations  9 MATERIALS AND METHODS  Collection  P r o c e d u r e s and Data  Analysis  Normal and tumour-bearing regularly Arm  lemon s o l e were  f r o m a s a m p l i n g a r e a n e a r t h e mouth o f t h e N o r t h  o f the Fraser  R i v e r , near Vancouver,  Other areas i n t h e S t r a i t  Columbia.  trawl  o r a door  speed.  under  180 mm  taken t o the laboratory;  irregularly.  a t d e p t h s o f 1 - 20 f a t h o m s ,  w i t h e i t h e r an o t t e r A l l flatfish  British  o f G e o r g i a were s a m p l e d  F l a t f i s h were c o l l e c t e d  and  collected  trawl  total  larger  towed a t s l o w  l e n g t h were removed  f l a t f i s h were  a f t e r b e i n g examined and r e c o r d e d .  Those  flatfish  released removed  t o t h e l a b o r a t o r y were e i t h e r k e p t i n tanks o r k i l l e d and preserved  i n 10% formalin.  A l l f i s h were e x a m i n e d  o f epidermal l e s i o n s and s e p a r a t e d i n t o normal bearing groups. by  species,  total  i n g s were made o f e a c h t u m o r - b e a r i n g the p o s i t i o n ,  relative  size,  s c o p i c d e s c r i p t i o n o f each Epidermal b a s i c morphologic The  draw-  illustrating  and g e n e r a l m a c r o -  lesions  found on f l a t f i s h  types w i t h a continuum  a r e o f two  of intermediates.  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  (AENs), e p i d e r m a l p a p i l l o m a s ( E P s ) , and AEN/EP  transition 1964).  In addition,  flatfish,  coloration,  described  lesion.  tumors c a n be c l a s s i f i e d  nodules  and tumor-  A l l f l a t f i s h were i n d i v i d u a l l y l e n g t h s , and w e i g h t s .  f o r evidence  forms, as p r e v i o u s l y d e s c r i b e d  These  classifications will  be u s e d  (Wellings e t a l . , i n descriptions  o f tumors s t u d i e d h i s t o l o g i c a l l y ; morphologic Figure  and  1-6.  e a c h t y p e has  histologic characteristics, Tumors c l a s s i f i e d  l o c a t e d anywhere on  nodules  varied  from  AEN/EP t r a n s i t i o n lesion  types.  as  shown i n  ( m o r p h o l o g i c a l l y ) as  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 , nodules  distinctive  smooth-surfaced  t h e body; t h e d i a m e t e r s  1 - 5 mm.  AENs  of  the  Those tumors c l a s s i f i e d  s t a g e s showed c h a r a c t e r i s t i c s  They g e n e r a l l y were l a r g e  of  (3 - 7 mm  as  both in  diameter)  h e m i s p h e r i c a l growths w i t h s p r e a d i n g , p l a q u e - l i k e edges, with  a rough, g r a n u l a r s u r f a c e  infolding  o f the e p i d e r m i s ) .  were g e n e r a l l y l a r g e some f i s h ) ,  raised,  spreading, d i f f u s e  ( i n d i c a t i v e o f t h i c k e n i n g and Epidermal  ( c o v e r i n g up  t o o n e - h a l f o f one  e d g e s ; many n o n - d i f f u s e , c l e a r l y  epidermal  p a p i l l o m a s were a l s o n o t e d ,  numbers.  The  e d g e s ) and and  papilloma type  granular, gray-to-black lesions  sessile  papillomas  the p e d u n c u l a t e d  although  defined  i n smaller  types  o f EP  lesions exhibit  one the  " c a u l i f l o w e r - l i k e " a p p e a r a n c e shown b y o t h e r papillomas  i n other  e x t e r n a l m o r p h o l o g y c a n be infinite  b a s i c tumor t y p e s .  lesion with  type.  struc-  Both  so-called epidermal  fishes.  The g r a d i n g o f e p i d e r m a l  c o n s i d e r i n g an  graded  (with a narrow base  f o r t h a t r e a s o n were g r o u p e d as  morphological  two  with  (with b r o a d l y based,  papillomas  tumors  side of  w e l l - d e f i n e d e d g e s ) showed i d e n t i c a l h i s t o l o g i c a l  t u r e and  or  l e s i o n s on  the b a s i s o f  s u b j e c t i v e , e s p e c i a l l y when  variety  o f i n t e r m e d i a t e s between  Correctly,  any  flatfish  the  epidermal  s p r e a d i n g , p l a q u e - l i k e edges o r 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 Fig.  1  External thelial  Fig.  m o r p h o l o g y o f tumour t y p e d e s i g n a t e d nodules  m o r p h o l o g y o f tumour t y p e  transition  forms  (Trans);  designated  on j u v e n i l e  m o r p h o l o g y o f tumour t y p e  (EP)  l e s i o n s ; on j u v e n i l e  Fig.  4  Typical  a s AEN/EP  lemon s o l e .  designated  lemon s o l e .  h i s t o l o g i c a l structure  appears normal.  X 3/4.  as  EP-like  X 3/4.  o f AEN tumour s t a g e .  Hematoxylin/eosiri. _  Epidermis  A p p r o x . X 3.5.  5  Typical  h i s t o l o g i c a l structure  Epidermis  is slightly  edges a r e apparent.  Fig.  X 3/4.  3  External  Fig.  lemon s o l e .  2  External  Fig.  (AEN); o n j u v e n i l e  as a n g i o e p i -  o f AEN/CP t r a n s i t i o n  t h i c k e n e d and i n f o l d e d ; Hematoxylin/eosin.  forms.  spreading  A p p r o x . X 3.  6  Typical  h i s t o l o g i c a l structure  epidermis eosin.  i s highly  o f EP tumour s t a g e .  t h i c k e n e d and i n f o l d e d .  A p p r o x . X 3.  The  Hematoxylin/  13 appearing epidermis should  be  t e r m e d an  EP;  histologically,  l e s i o n s a t t h e s e s t a g e s show more s i m i l a r i t i e s t o EPs t o AENs this  (see M i l l e r ,  reason, only  in compilation tumour t y p e s  1969;  the  Miller  AEN  o f the  and  EP  and  Wellings,  in terms of v  a function of 5 mm  size  and  the  age  was  not  s h i p s were b a s e d on  n a t u r a l h i s t o r y and  for  references);  assumed i n the The per  size  o f age  b a s e d on mm  total  length),  N o r t h Arm  of  n a t u r a l h i s t o r y o f the  during  the  and  relation-  size  (see  can  otolith  relationdata  introduction  reasonably  f i s h , and  u n d e r two  total  lesions  tumour-bearing  f r o m September 1973  to  June  years  the  Other  ( i . e . , average  incidences  fish  j u v e n i l e lemon  s i t e during  t o F e b r u a r y 1975.  a sample o f 510  period  the  sampling  relative  be  studied.  3896 lemon s o l e  f r o m September 1973  were b a s e d on  The  a n a l y s i s of percentages of tumour-bearing  from the  of l e s i o n s per  presented  growth r a t e  growth p a t t e r n s  period the  data are  lemon s o l e g r o w t h  lemon s o l e p o p u l a t i o n  (under 180  sole catch  of  similar  g r o u p was  of  are  d e t e r m i n e d by  I n t e r p r e t a t i o n o f age  studies  report  (total lengths).  or s c a l e r e a d i n g s .  from p r e v i o u s  used  occurrences  in this  fish size;  groups  s h i p between f i s h s i z e  For  9).  Tumour n a t u r a l h i s t o r y d a t a p r e s e n t e d as  1971).  c l a s s i f i c a t i o n s were  d a t a comparing r e l a t i v e  (in Figure  than  analyses number  o f AENs and fish  EPs)  collected  1974.  14 Light  Microscopy  For  l i g h t microscopic  tumorous t i s s u e s were f i x e d  s t u d i e s , normal s k i n and  i n 10% f o r m a l i n or Carnoy's  solution,  embedded  in paraffin,  microns.  Sections  were s t a i n e d w i t h  toluidine blue, with  t y p e s f r o m 38 lemon s o l e .  ultrastructural  s t u d i e s , n o r m a l s k i n and  tumorous t i s s u e s were removed f r o m l i v e t r i c a i n e methanesulphonate  were c u t i n t o 1 mm glutaraldehyde with  ( S a b a t i n i et, a l _ . ,  1963) b u f f e r e d  ( K a r l s s o n and S c h u l t z ,  Thin  The b l o c k s  were s t a i n e d w i t h lead citrate  were p o l y m e r i z e d  uranyl  ultrataicrotome.  acetate  7.4  1965).  The  0mU2 u l t r a The  sections 1967)  ( R e y n o l d s , 1963) and o b s e r v e d w i t h  were a l s o c u t f r o m Epon-embedded blue,  a t 60°C f o r  (as i n S j o s t r a n d ,  H i t a c h i HS-7S e l e c t r o n m i c r o s c o p e .  toluidine  a t pH  t e t r o x i d e f o r 1 hour,  s e c t i o n s were c u t on a R e i c h e r t  m i c r o t o m e o r on a S o r v a l l MT-2  and  These t i s s u e s  i n a g r a d e d s e r i e s o f m e t h a n o l s , a n d embedded i n  ( L u f t , 1961).  20 h o u r s .  anesthetized  f o r 1-2 h o u r s i n 2.5%  s p e c i m e n s were p o s t - f i x e d i n 1 % osmium  Epon  fish  (MS-222).  c u b e s and f i x e d  a phosphate b u f f e r  dehydrated  sec-  E l e c t r o n Microscopy  For  with  (counterstained  H i s t o l o g i c a l s t u d i e s were b a s e d o n  f r o m 134 tumors o f v a r i o u s  Transmission  a t 5-15  h e m a t o x y l i n and e o s i n ,  o r by the F e u l g e n r e a c t i o n  fast green).  tions  and s e c t i o n e d  and u t i l i z e d  Thick  a  (1 m i c r o n )  material, stained  i n l i g h t microscopic  sections  with studies.  Scanning E l e c t r o n  For flatfish sole  Microscopy  studies  o f the s u r f a c e s t r u c t u r e o f normal  epidermal c e l l s ,  s k i n were removed,  coated with a t h i n  layer  s m a l l (1 cm)  fixed  i n 10%  s q u a r e s o f lemon  formalin,  dehydrated,  o f g o l d , and e x a m i n e d w i t h a  Cambridge S t e r e o s c a n s c a n n i n g e l e c t r o n  microscope.  16  RESULTS  Natural History  Data  During t h i s study, a t o t a l collected  from  u n d e r 180 mm  t h e N o r t h Arm s a m p l i n g  in total  o f 3896 lemon s o l e were site.  l e n g t h a n d were t e n t a t i v e l y  as b e i n g i n t h e f i r s t  two y e a r s o f l i f e .  lemon s o l e were f o u n d  t o be t u m o u r - b e a r i n g .  indicate period  A l l f i s h were  A total  identified o f 1164  These  figures  a c u m u l a t i v e tumour p r e v a l e n c e o f 2 9 . 9 % f o r t h e  from  percentages  September 1973 t o F e b r u a r y 1 9 7 5 . of flatfish  However, t h e  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 a p p e a r e d  i n August  o r September, showed a peak p r e v a l e n c e i n November mately  6 3 % ) , and showed d e c l i n i n g  a l o w i n June,  July,  Figure  and August  freguencies thereafter to  (approximately 5 % ) .  7 shows t h e r e l a t i o n s h i p between  o f tumour-bearing  fish  (approxi-  and f i s h  length.  frequency  A drastic increase  i n tumour p r e v a l e n c e i s n o t e d b e t w e e n t h e 50-54 mm group  and t h e 75-79 mm  maximum p e r c e n t a g e are d i r e c t l y percentages mm  group.  After  i n c i d e n c e o f 49.0%),  correlated with steadily until  this  the z e r o p o i n t  size  increase (to a  further size increases  d e c r e a s i n g tumour  i s reached  i n t h e 160-164  group. The  r e l a t i o n s h i p between f i s h  o f t u m o u r s f o u n d on e a c h  fish  size  and t h e number  i s shown i n F i g u r e 8.  h i s t o g r a m shows t h a t t h e a v e r a g e  number  The  o f tumors p e r f i s h  FIGURE 7 The r e l a t i o n s h i p  o f tumour p r e v a l e n c e and f i s h  Histogram the  percentage  showing  illustrating  o f tumour-bearing  a d e c r e a s e i n tumour  the r e l a t i o n s h i p fish  and f i s h  Data based  sole  from one a r e a i n t h e S t r a i t  September  1973  between  length,  p r e v a l e n c e as t o t a l  increases.. sampled  size.  length  on t h e a n a l y s i s o f 3896 lemon  t o February  1975.  of Georgia  from  18  S t a r t i n g p o i n t s of 5 mm  s i z e groups ( f i s h t o t a l  lengths)  FIGURE 8  The relationship of numbers of tumours per f i s h and f i s h size. Histogram i l l u s t r a t i n g the relationship between the average number of tumours per tumour-bearing f i s h and f i s h length, showing a gradual decrease i n the number of tumours per f i s h as t o t a l length increases.  Data based  on the analysis of 510 tumour-bearing lemon sole.  20  xi co •H «H CO  o  u  15  o E  co  10 .  o s -p o  5 -  CU cu >  -p-  o  VJ1  o  0>  o  o  oo  o  vO O  S t a r t i n g p o i n t s of 5 mm  vn o o s i z e groups ( f i s h t o t a l O O  -» O  o  O  -p-  -o o o length) ON  decreases, with  increasing  o f 11.5 i n t h e 55-59 mm  fish  l e n g t h s , from  an  average  g r o u p t o 2.0 i n t h e 150-154  mm  group. The and  epidermal  illustrated relative  relative  papillomas with r e l a t i o n  i n F i g u r e 9.  number  on f i s h  Epidermal  The d a t a  o f AENs w i t h  a reciprocal rise found  incidences of angioepithelial  Lesions  than  increasing  120 mm  i n Other  Scattered  sampling  total  s o l e tumours.  Histological  These l e s i o n s  The sole  examination  o f the  Light  t h e lemon  sole;  starry  (Citharichthys  and AENs, AEN/EP  transi-  sole.  Microscopy  o f t h e lemon  to previous d e s c r i p t i o n s o f t e l e o s t  ( v a n O o s t e n , 1957; Andrew, 1959; L a g l e r e_t a l _ . , Romer,  i n other  i n the S t r a i t of  morphology o f the normal s k i n  i s similar  lesions  i n c l u d e one AEN on one  a n d E P s o n many s a n d  Normal E p i d e r m i s ;  No AENs were  showed them t o be i d e n t i c a l w i t h  s t i g m a e u s ) a n d one f l a t h e a d forms,  length, with  Species  f l o u n d e r ; EPs on one s p e c k l e d s a n d d a b  tional  i n the  length.  instances of epidermal  areas.  tumourous t i s s u e s  fish  o f EPs.  s p e c i e s o f f l a t f i s h were a l s o o b s e r v e d Georgia  size are  show a d e c r e a s e  i n the frequency  larger  to f i s h  nodules  1962; Hyman, 1962; P a t t a n d P a t t , 1 9 6 9 ) .  epidermis  1962; The  skin  FIGURE 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 . Histogram  i l l u s t r a t i n g the r e l a t i v e p r e v a l e n c e s  of AEN and EP tumour types as r e l a t e d t o f i s h l e n g t h .  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 l e n g t h  increases.  Data  based on the a n a l y s i s o f 2965 tumours from 510 lemon s o l e .  22a  23  consists of a simple squamous layer of epidermal cells overlying a connective tissue dermis (Figure 1 0 ) .  The  epidermis consists of 5-7 layers of ovoid, basophilic c e l l s ; 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 c e l l s , from basal to  superficial layers, are nucleate; no nucleoli are resolvable. Some flattening of the epidermal cells can be noticed in the most superficial c e l l s ; these cells also tend to be more basophilic.  Darkly-staining mucus-secreting cells in  various stages of maturation are interposed between the epidermal c e l l s ; 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 intermediate c e 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 F i g u r e s 10-13 Fig.  10  Normal s k i n Epidermis  from  i s five  the mid-side t o seven  with mucous-secreting Fig.  o f a j u v e n i l e lemon  layers  cells.  t h i c k and  Fig.  11  and i n t e r m e d i a t e l a y e r s .  X  o f the  12  o f mucous and e p i d e r m a l c e l l s ; t o open t o t h e s u r f a c e .  on s u p e r f i c i a l c e l l s X  cells  4520.  N o r m a l 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  ready  interspersed  H e m a t o x y l i n / e o s i n . X 160.  N o r m a l 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 basal  sole.  the r e l a t i o n s h i p  mucous c e l l  i s apparently  Microvilli-like  are not apparent  i n this  pleats  section.  4520.  F i g . 13 Scanning  e l e c t r o n micrograph  o f lemon s o l e  showing c h a r a c t e r i s t i c a l l y a r r a n g e d  surface  epidermis, pleats.  X  9500.  26 Normal E p i d e r m i s / E l e c t r o n  The  Microscopy  u l t r a s t r u c t u r a l m o r p h o l o g y o f t h e lemon  epidermis corresponds  to electron microscopic  of the epidermis o f other t e l e o s t s Matoltsy, 1970;  1968;  Merrilees,  basic c e l l secreting be  W e l l i n g s and 1974).  types: cells.  The  epidermis  squamous e p i d e r m a l Mucus c e l l s ,  descriptions  (Hendrickson  Brown, 1969;  The  epidermal  three d i s t i n c t  cells  layers:  and  Brown and W e l l i n g s ,  i s composed o f cells  and  c a n be  which  overlie  t h e basement membrane; i n t e r m e d i a t e c e l l s ,  overlie  the b a s a l l a y e r  and  flattened  most l a y e r  and may  superficial  by t h e p r e s e n c e  cells  o f two  the p e r i p h e r a l  are  which layers; outer-  12). characterized  t h e p e r i n u c l e a r zone, which  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  shaped, o f t e n w i t h Cytoplasmic  zone o f a l l c e l l s ,  with transverse cristae), agranular endoplasmic diameter.  cell  i n a l l l a y e r s c a n be  (Figure 11).  were r a r e l y o b s e r v e d . perinuclear  immediately  z o n e , w h i c h c o n t a i n s many c y t o p l a s m i c  ribosomes  irregularly  ina l l  form  w h i c h compose t h e  ( F i g u r e s 11 and  zones:  c o n t a i n s t h e n u c l e u s and  ments and  cells,  of the e p i d e r m i s Epidermal  o c c u r a s 3-5  can  cells  considered to  ovoid basal c e l l s ,  two  mucus-  as mentioned above,  found randomly i n t e r p o s e d between e p i d e r m a l  layers.  sole  fila-  N u c l e i o f squamous indentations.  cells  Nucleoli  o r g a n e l l e s , i n the  include mitochondria  G o l g i apparatus,  r e t i c u l u m , and  granular  ribosomes  (usually and  o f 15.0  nm  27 Differences i n the t r a n s i t i o n  from b a s a l  changes i n c l u d e :  a general  reduction an -  in cellular  m o r p h o l o g y c a n be  to superficial  i n t h e number o f  The f r e e s u r f a c e  characterized microvilli  by s h o r t ,  (Figure  pleats  13).  a  junctional  cytoplasmic  of the s u p e r f i c i a l  broad, surface  i n cross-section  These  interdigitations,  i n t h e number o f desmosomes a n d o t h e r  complexes, and an i n c r e a s e filaments.  layers.  f l a t t e n i n g o f the c e l l s ,  i n the complexity o f c e l l - t o - c e l l  increase  seen  cells is  resembling  Often the surface  i s c o v e r e d w i t h a n amorphous o r f i l a m e n t o u s  extraneous  coating. Mucus c e l l s epidermis a t varying are  generally  distended layers,  found  cells  often  a r e found s c a t t e r e d stages of maturity.  are found  seen opening t o the s u r f a c e .  The  compose a l a r g e a r e a  mucus d r o p l e t s  c y t o p l a s m ; most o f t e n  the cytoplasmic  epidermal  The mucus  endoplasmic  reticulum.  organelles are rim a t the c e l l  t e n d t o be f i n e l y  fibrillar in  a p p e a r a n c e and a r e s u r r o u n d e d by a s i n g l e a g r a n u l a r membrane  cell  o f the c e n t r a l  into a peripheral protoplasmic  The mucus d r o p l e t s  cells  mature,  i n t h e more s u p e r f i c i a l  abundant g r a n u l a r  edges.  Immature  i n the deeper l a y e r s , w h i l e  cytoplasm contains  compressed  throughout the  limiting  (Figure 12). Directly  basement membrane  underlying  composed o f a f i n e  A l a y e r o f dermal c o l l a g e n Underlying,  the b a s a l  and p a r a l l e l  lies  cell  layer  fibrillar  is a  material.  b e n e a t h t h e b a s e m e n t membrane.  t o , these l a y e r s , are the various  28 pigment-containing  cells.  pigment-containing  cells  dense melanin granules  Melanophores are and  i n the  among t h e m e l a n o p h o r e s a r e are  c h a r a c t e r i z e d by  cytoplasm;  in life,  c r y s t a l s which are  Tumor H i s t o l o g y :  are  the  c h a r a c t e r i z e d by  cytoplasm.  iridophores  (guanidophores) which  numerous, l a r g e , empty c l e f t s these  cells  leached  contain platelike  out during  specimen  l e s i o n s can  be  described,  appearance  each with  histological  types  have p r e v i o u s l y b e e n d e s i g n a t e d (AENs), e p i d e r m a l  transition phologic  forms  (Figures 1-6).  papillomas  has  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 Angioepithelial stromal  o f each  an  accompanying  plasia stratum tissue  can  be  a  character-  These tumor  The  AEN/EP gross  been g i v e n  mor-  above;  below.  component o f t h e AEN  The  increase  varies shows  from character-  fibromous stromal  a h y p e r p l a s i a o f dermal  type  in collagen fibers.  stratum  d i r e c t l y below the  The  spongiosum, or  stratum  is  f i b r o b l a s t s iwith hyper-  s e e n t o o r i g i n a t e , i n s m a l l AENs, i n  compactum, i n the area  flatfish  Nodules  (Figure 14).  c h a r a c t e r i z e d by  guanine  preparation.  ( E P s ) , and  angiomous t o f i b r o m o u s ; o f t e n a s i n g l e AEN istics  the  as a n g i o e p i t h e l i a l  (Wellings e t a l . , 1964).  appearance o f each type  The  in  L i g h t Microscopy  istic  nodules  numerous,  Interspersed  Three morphologic v a r i a t i o n s o f the epidermal  commonest  compactum.  in  the the The  PLATE 3 Figures Fig.  14-17  14  Stroma o f AEN,  showing  Hematoxylin/eosin. X Fig.  characteristics.  160.  15  Stroma o f AEN, cells". Fig.  fibroma-like  s h o w i n g p a c k e d s i n u s o i d s and s t r o m a l  Toluidine blue,  1 u section.  640.  16  C r o s s s e c t i o n o f lemon  sole bearing  AEN  i n v a s i o n o f m u s c l e t i s s u e b y t h e AEN i s normal. Fig.  X  "X-  Hematoxylin/eosin.  X  lesion,  stroma.  showing  Epidermis  5.  17  Higher m a g n i f i c a t i o n Hematoxylin/eosin.  of lesion X  160.  shown i n F i g u r e  16.  angiomous s t r o m a l t y p e sinusoids;  c o n s i s t s o f many c a p i l l a r i e s  u s u a l l y these  of b l o o d c e l l lymphocytes,  packing. and  v a s c u l a r b e d s show a h i g h This packing  of red blood  g r a n u l o c y t e s becomes e s p e c i a l l y  and degree  cells, 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  cell  type, randomly d i s p e r s e d  t h e s t r o m a l component among t h e these  lesions.  unidentified  These c e l l s ,  stromal c e l l  staining characteristics dermal c e l l  types  f i b r o b l a s t s and  round  hereafter referred  type, d i f f e r from  nucleus able.  The and  t o as  appear  similar  a p p e a r as l i g h t l y  i n hematoxylin/eosin  coloring  and  and  a slightly  granularity of  o f t e n make t h e two  staining,  observed  were commonly s e e n  angiomous.. and  in  darker  the  cells  transparent, nucleoli.  w i t h i n blood v e s s e l s but fibromous  m o s t o f t e n s i n g l y , b u t c a n be  to four c e l l s .  diameter  indistinguish-  vacuolated c e l l s with  T h e s e c e l l s were n e v e r  t y p e s A and  and  s e c t i o n s as  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  cell  and  the  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 t h e  o f two  the  epidermal,  type are g e n e r a l l y twice  the cytoplasm  were o b s e r v e d  in  i n morphology  normal b l o o d ,  c e l l s with a pale red cytoplasm  nucleus.  i s found  (Figure 15).  Cells of this of  fibroblasts,  throughout  These s t r o m a l  B o f the epidermis  areas.  found  "X-cells"  in  They  "cords"  differ  from  ( d e s c r i b e d below) i n s i z e  The t e r m " X - c e l l " i s u s e d c o l l e c t i v e l y i n r e f e r r i n g t o any o f t h e u n i d e n t i f i e d , g i a n t c e l l t y p e s . This includes s t r o m a l " X - c e l l s " , t y p e A " X - c e l l s " , and t y p e B " X - c e l l s " .  32 and  staining characteristics.  component was  seen t o  often penetrating (Figures  16  and  The  I n some c a s e s ,  invade the u n d e r l y i n g  e p i d e r m a l component o f t h e  tend  characteristics.  cell  Toluidine blue  appears to begin superficial  appearance is  This i n the  i s seen.  hyperplastic  stage  cell  these c e l l s  type observed  in the an  the  the  i s the  increase  i n the  normal  cell such  epidermal  basal  t o the  normal epidermis  basophilic  (identified  They a r e  appear  c h a r c t e r i z e d by  nucleusrcytoplasm  ratio;  of a darkly-staining, central nucleolus;  the  as  degrees  appearance o f a d i f f e r e n t c e l l first  of  layer  Accompanying v a r y i n g  e p i d e r m a l component; t h e s e c e l l s  basement membrane.  epidermis;  hyperplasia  more o f t e n no  j u s t above t h e  the  contacts  i n mucus  appear i d e n t i c a l  i n the  in  l a y e r s o f the  epidermal  loosen-  a p p e a r a n c e o f a l a r g e number  lymphocytes or macrophages). of hyperplasia  described  A l s o c h a r a c t e r i s t i c o f the i s the  to a  to r e t a i n a r e l a t i v e l y  (Figure 19);  small, darkly-staining c e l l s (Figure 19);  normal s t a i n i n g  of c e l l u l a r  intermediate  Often  from  i n i t i a l AENs, a l l  a subsequent i n c r e a s e  secretions  varies  s p a c e s , e v i d e n t l y due  l a y e r s tend  a c c o m p a n i e d by  increase  fibers  s e c t i o n s show a marked  loosening  (Figure 18).  numbers and  AEN  membrane i n t e r d i g i t a t i o n s  normal epidermis.  the  In  to r e t a i n t h e i r  in intercellular  o f the  masses,  17).  epidermal c e l l s  ing  stromal  muscle  d e e p among i n d i v i d u a l m u s c l e  normal to s l i g h t l y h y p e r p l a s t i c .  increase  the  type along  enlargement; appearance  l o s s of s t a i n i n g  33 w i t h Feulgen's  reagent;  n u c l e u s and c y t o p l a s m eosin sections. diameter,  will  "giant c e l l " identical B  and l o s s o f b a s o p h i l i a .  s t a i n a pale pink color  This c e l l  characteristics,  ( a s d e s c r i b e d i n EP l e s i o n s ,  The forms AEN,  t o a s one o f t h e t u m o r ' s  types; i t i s t e n t a t i v e l y  AEN/EP T r a n s i t i o n  i n hematoxylin/  type, because o f i t s i n c r e a s e d  h e r e a f t e r be r e f e r r e d  staining  Both  identified,  due t o  a s an " X - c e l l " o f type  below).  Forms  s t r o m a l component o f t h e AEN/EP  ( F i g u r e s 20 a n d 21) r e m a i n s  identical  transition  to that o f the  d e s c r i b e d above. The  epidermal  component o f t h e 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 b y 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 thickening,  t h e e p i d e r m i s becomes i n f o l d e d , b o t h  initial  s t r o m a l g r o w t h a n d o u t w a r d s from  (Figure  20).  of  Some o f t h e o b s e r v e d  the i n i t i a l  focus  hyperplastic reactions  the epidermis reach considerable proportions without  further cytological reactions  (i.e.,  changes and resemble  the c e l l s  r e t a i n normal s t a i n i n g (Figures  21 a n d 2 2 ) .  epidermis)  occur  hyperplastic  c h a r a c t e r i s t i c s and p o l a r i t y ) Most o f t e n ,  i n c r e a s i n g numbers o f t h e ( t y p e B; d e s c r i b e d i n t h e  occur w i t h epidermal  When t h e s e c e l l s  typical  show l i t t l e p l e o m o r p h i s m a n d  ovoid, pale-staining giant c e l l s AEN  over the  infoldings  (Figure 23).  i n l a r g e numbers, t h e y t e n d t o show  i n c r e a s i n g volume c h a n g e s a n d d e c r e a s i n g s t a i n a b i l i t y hematoxylin  and F e u l g e n ' s  reagent  ( F i g u r e s 24 a n d 2 5 ) ;  with most  PLATE 4 Figures Fig.  18-21  18  E p i d e r m a l component o f AEN. cell and  intermediate layers, i n the b a s a l l a y e r  apparently are type B  and  space  i n basal  the apparent v a c u o l a t i o n o f  (arrow).  "X-cells".  These v a c u o l a t e d  cells  T o l u i d i n e b l u e , 1 /a  section.  640.  Fig.  19  S e c t i o n o f AEN,  s h o w i n g s t r o m a l and  of  Note the a c c u m u l a t i o n  the t i s s u e .  cells Fig.  j u s t above t h e b a s a l l a y e r .  eosin. Fig.  epidermal of  components  darkly-staining  Hematoxylin/eosin.  X  160.  20  S e c t i o n t h r o u g h AEN/EP t r a n s i t i o n X  form  lesion.  Hematoxylin/  5.  21  Initial  hyperplastic  epidermis. X  i n c r e a s e i n mucous  numbers, t h e i n c r e a s e i n i n t e r c e l l u l a r  cells  X  Note t h e  160.  s t a g e o f AEN/EP t r a n s i t i o n a l  Mucous c e l l s  are absent.  form  Hematoxylin/eosin.  36 o f these c e l l s descriptions  c a n be c l a s s i f i e d  below).  Epidermal The appears  Papilloma-like Lesions 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  identical  AEN/EP t r a n s i t i o n  t o t h e s t r o m a l component o f t h e AEN a n d stages.  i n c l u d e chromatophores, soids,  and c a p i l l a r i e s  c a n be f o u n d  lesions;  Structures resembling  i n t h e stroma  channels  i n the  thin  collapsed  (Figure 26). cells  o f t h e same  type,  s t a l k s o f t h e stroma  stroma, o f the  d o m i n a n t components o f t h e p a p i l l o m a  are enlarged, ovoid,  tive  o f the papilloma-  (Figure 26). The  epidermis  a n d show  altered  t o t h o s e p r e v i o u s l y d e s c r i b e d i n t h e AEN  c a n be o b s e r v e d  sinu-  elements  t e n d t o be s m a l l e r i n s i z e  Numerous u n i d e n t i f i e d  papilloma  The v a s c u l a r  these s t r u c t u r e s presumably r e p r e s e n t  or c l o s e d c a p i l l a r y  identical  o f t h e stroma  collagen fibers,  (Figure 26).  of blood c e l l s .  capillaries like  The e l e m e n t s  fibroblasts,  o f t h i s b r a n c h i n g stroma no p a c k i n g  as type A c e l l s (see  "giant" c e l l  types  i s composed o f t h r e e c e l l  staining  giant c e l l s ) , (superficial  (Figure 27).  types, each with  c h a r a c t e r i s t i c s and morphology.  t y p e s have been c l a s s i f i e d type B  with hematoxylin  These  a c c o r d i n g l y as type A  (smaller giant c e l l s ) ,  and " e n v e l o p i n g " c e l l s ) .  were d e t e r m i n e d  The distinccell  (large  and t y p e C  Staining  by s t u d y i n g paraffin-embedded and eos i n : :  epidermis  characteristics  sections stained  37  PLATE 5 F i g u r e s 22-25 Fig.  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 2 0 .  the homogeneity o f the h y p e r p l a s t i c epidermal average of cells  diameter  o f these c e l l s  i s around  i n the normal e p i d e r m i s .  Note  cells.  8 /i,  The  the size  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  occurrence o f type B c e l l s  (lightly  form,  stained) along the  basement membrane; u p p e r e p i d e r m a l c e l l s normal appearance.  Feulgen's,  showing t h e i n i t i a l  retain  their  f a s t green c o u n t e r s t a i n .  X 160.  Fig.  24  Section  through  AEN/EP t r a n s i t i o n a l  numbers o f l a r g e ,  lightly  L a r g e r , more c e n t r a l o f type  A cells;  staining  cells  i n the epidermis.  " X - c e l l s " show s t a i n i n g  type B c e l l s  Hematoxylin/eosin.  s t a g e , showing i n c r e a s e d  characteristics  c a n a l s o be r e c o g n i z e d .  X 160.  F i g . 25 S e c t i o n through  EP s t a g e l e s i o n .  d i f f e r e n c e between t h e s u p e r f i c i a l type A  "X-cells"  (below).  the Feulgen r e a c t i o n . X 640.  Note t h e extreme cells  Type A c e l l s  Feulgen's,  size  (above) a n d t h e do n o t s t a i n  with  f a s t green c o u n t e r s t a i n .  38  39 Type A .  The c y t o p l a s m o f t y p e A c e l l s  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 . appears cells  stained;  seldom  found near  l a y e r s of the epidermal  Type B. pink to l i g h t These c e l l s  Type B c e l l s  ment membrane; r a r e l y  found  a r e c h a r a c t e r i z e d by a p a l e  Type C.  i n s m a l l groups  t h e y c a n be f o u n d  layers o f the epidermal  nucleus.  along the base-  i n the intermediate  infoldings.  The c y t o p l a s m o f t y p e C c e l l s  the n u c l e i s t a i n blue.  i n the s u p e r f i c i a l  a l s o appear  to f i l l  giant cells  (cell  stains  Frequently the n u c l e i are  s w o l l e n and t h e c h r o m a t i n a p p e a r s type occur  i n the  folds.  r e d cytoplasm, w i t h a dark v i o l e t  a r e always  The  t h e b a s e m e n t membrane  t h e tumor s u r f a c e ; g e n e r a l l y t h e y a r e f o u n d  intermediate  violet;  The n u c l e u s  light  t h e n u c l e o l u s , however, s t a i n s d a r k l y . .  o f type A a r e seldom  or near  stains  granular.  Cells of this  l a y e r s : o f t h e tumor;  i n t h e s p a c e s b e t w e e n t h e much  t y p e s A and B ) .  For t h i s  the n u c l e i and t h e cytoplasm o f t h e s e c e l l s  they larger  reason, appear  both hetero-  morphic. The cell  types  morphologic  found  c h a r a c t e r i s t i c s o f the various  i n the papilloma-like  l e s i o n s were d e t e r -  m i n e d f r o m a s t u d y o f s e c t i o n s o f Epon-embedded stained with toluidine Type A: enlarged,  blue:  Type A c e l l s  irregularly  shaped  o f n i n e times t h a t o f normal and  29).  material  are c h a r a c t e r i s t i c a l l y  cells  which c a n r e a c h a  epidermal c e l l s  The c y t o p l a s m g e n e r a l l y  stains  diameter  ( F i g u r e s 26, 28  lightly  with  40 PLATE 6 F i g u r e s 26-29 Fig.  26  S e c t i o n through s t r o m a l and structure cells" blue, Fig.  EP-type l e s i o n ,  epidermal  (arrow).  components.  The  shown i s 23 / i ; 1 u section.  showing c h a r a c t e r i s t i c s  average diameter  compare w i t h  X  Note a l t e r e d  F i g u r e 21.  component o f E P - t y p e l e s i o n ,  type  A  "X-  Toluidine  epidermis. cells  Note t h e  toxylin/eosin.  identified X  The  complete  stromal  framework f o r t h e  abundance o f s m a l l , d a r k l y  near the c e n t e r o f the  been t e n t a t i v e l y  showing the  " X - c e l l " types.  component s e r v e s o n l y as a s u p p o r t i n g  infoldings;  these  as macrophages  staining cells  (arrows).  have Hema-  160.  28  Section be  through  EP-type. l e s i o n ,  collapsed "X-cells"  section. Fig.  o f the  640.  dominance o f t h e t i s s u e b y  to  capillary  27  Epidermal  Fig.  of  X  showing s t r u c t u r e s b e l i e v e d  (arrows).  Toluidine blue,  1 jx  640.  29  S e c t i o n through  superficial  the c h a r a c t e r i s t i c cells.  area o f EP-type l e s i o n ,  s t r u c t u r e o f the outermost  T o l u i d i n e b l u e , 1 ja  section.  X  640.  showing  (superficial)  41  toluidine  blue and appears h e a v i l y g r a i n e d .  cytoplasm  o f these  cytoplasmic The  appears vacuolated;  i n c l u s i o n s c a n be o b s e r v e d  diameter  o f the c e l l s  of the epidermal  tends  infolding  never s t a i n s with enlarged.  giant cells  A central,  dark  i n the larger  cells.  t o i n c r e a s e as the c e n t e r  i s approached.  toluidine  Often the  The  nucleus  b l u e and i s a l w a y s g r e a t l y  darkly staining nucleolus i s often  observed. Type B:  Cell  o f type A c e l l s : diameter  type  B appears as a s m a l l e r v e r s i o n  t h e c e l l s a r e somewhat e n l a r g e d  o f normal epidermal  cells);  (twice the  a r e rounded; possess  e n l a r g e d , round, n o n - s t a i n i n g nucleus  with  adarkly-staining,  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 26).  These c e l l s ,  i n groups along of the epidermal  tissue  This c e l l  type,  l a y e r s o f t h e tumorous  as w e l l as i n t e r s p e r s e d between t h e g i a n t c e l l s o f  cells  (Figure 26).  appear  with neighboring  I n t h e most s u p e r f i c i a l  f l a t t e n e d , possess cells  cerned,  probably  of this  cell  connections  ( w i t h much i n t e r c e l l u l a r  s p a c e ) , and  cells  No n u c l e i c a n be  due t o t h e i n t e n s e s t a i n i n g  type.  layers  finger-like  stain darkly with toluidine blue.  giant  a t the periphery  as p r e v i o u s l y mentioned,  i n t h e most s u p e r f i c i a l  t y p e s A and B the  (i.e.,  found  infoldings).  Type C: c a n be f o u n d  (Figure  a s m e n t i o n e d a b o v e , a r e most o f t e n  t h e b a s e m e n t membrane  an  The c e l l s  of the epidermis  found  dis-  characteristics  i n t e r s p e r s e d among t h e  are irregularly  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  "filler"  between the  lightly  stained giant  be  referred to hereafter  no  n u c l e i can  be  resolved  i n t e n s i t y o f the  be  "enveloping  hence, they cells".  i n these c e l l s  will  Again,  because of  the  staining.  Often, can  as  cells;  i n EP-type l e s i o n s , evidences of  necrosis  observed. In t o l u i d i n e blue  identified  as  sections,  collapsed giant cells  c e n t r a l areas o f the  epidermal  structures tentatively  can  be  found  infoldings  in  (Figure  the  28).  T h e s e s t r u c t u r e s c o n s i s t o f a d e n s e mass o f h e a v i l y material  surrounded by  a c l e a r p e r i p h e r a l zone.  to represent  individual cellular  necrosis  be  can  observed  necrosis.  i n some t u m o r s  granular  This  General  (Figure  tissue  31).  Most  often  (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  found  i n the  lumens f o r m e d by  i n f o l d i n g epidermis  (Figure  32).  This  necrotic material  o f remnants o f g i a n t c e l l s , darkly (Figure and  the  can  identified  by  s t a i n i n g n u c l e o l i inside enlarged, 33).  scanty  c y t o p l a s m can  interspersed epidermal  Lymphocytes w i t h s m a l l ,  between t h e  infoldings  be  (Figure  30).  seen to  the  be  consist  presence  transparent  of nuclei  darkly-staining nuclei  found, o f t e n  giant cells  be  seems  i n large  a t the  center  numbers, of  the  44 PLATE 7 Figure 30-33 Fig. 30 Higher magnification of section through EP-type lesion shown in Figure 26, illustrating abundant, darkly staining c e l l s , 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 infolding 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 tion  form,  and  s t r o m a l component o f t h e AEN, t h e EP w e r e s e e n t o b e  t h e AEN/EP  identical  i n structure.  For t h i s reason 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 w i l l be stroma  d e s c r i b e d o n l y once. i s composed o f t h e  identical scopic  fibers,  and  tissues  Electron microscopically,  following elements:  c a p i l l a r i e s , white blood c e l l s , collagen  transi-  macrophages,  an u n i d e n t i f i e d c e l l  chromatophores, fibroblasts,  type  t o t h a t d e s c r i b e d i n the stroma  the  evidently  of light  micro-  sections. Numerous f i b r o b l a s t s  o u t t h e s t r o m a l component. t h e y c a n be n o t e d oriented  scattered  When l o c a t e d n e x t  they are heteromorphic;  f i b r o b l a s t p r o c e s s e s c a n be  c a n be  f i b e r s , measuring  observed extending  36 and  a p p r o x i m a t e l y 100  elements  o f the  34).  narrow  found o r i e n t e d a t v a r i o u s a n g l e s  i n t e r s p e r s e d between the c e l l u l a r ( F i g u r e s 35,  often  (Figure  spaces, o f t e n surrounding neighboring  Collagen i n diameter,  through-  t o the e p i d e r m i s ,  i n much t h e same manner as c h r o m a t o p h o r e s  the i n t e r s t i t i a l  into cells. nm  and  stroma  37).  Capillaries  c a n be o b s e r v e d  They g e n e r a l l y a r e s m a l l exhibit  found  l y i n g p a r a l l e l w i t h t h e basement membrane,  Deeper i n the stroma arm-like  c a n be  regularly  (one r e d b l o o d c e l l  i n the  diameter)  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 t h e  p e r i p h e r y , e s p e c i a l l y toward  t h e lumen.  The  cytoplasm  stroma. and cell of  PLATE 8 F i g u r e s 34-35  Fig.  34  Electron  micrograph  epidermal  l e s i o n , showing a  fibroblast, lesion's cells").  o f b a s e m e n t membrane a r e a o f m a t u r e longitudinally-oriented  t h e b a s e m e n t membrane, a n d t h e c e l l s  epidermal  component  (Enveloping c e l l s  o f the  a n d "X-  X 10500.  F i g . 35  -=  P o r t i o n o f t h e s t r o m a l component o f a n a n g i o e p i t h e l i a l nodule,  s h o w i n g some t y p i c a l e l e m e n t s o f t h e AEN s t r o m a :  fibroblasts, granulocytes, structures.  large deposits o f collagenous stromal X 10500.  tissue,  " X - c e l l s " , and abnormal c a p i l l a r y  PLATE 9 Figures  36-37  F i g . 36 Portion o f stromal  component o f a n a n g i o e p i t h e l i a l  illustrating  some f e a t u r e s t y p i c a l  lymphocytes,  fibroblasts,  c o l l a g e n , and " X - c e l l s " . Fig.  nodule,  o f t h e AEN s t r o m a :  c a p i l l a r i e s , macrophages, X 10500.  37  Illustration  o f some f e a t u r e s o f t h e s t r o m a l  Note t h e e x c e s s i v e cytoplasmic  "X-cells".  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  organelles.  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 c e l l s . 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 membranebound, electron-dense structures of various sizes and shapes; these were interpeted as digestive vacuoles and residual bodies.  The c e l 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 c e l 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 the c e l l ;  nucleus  i s commonly d i s p l a c e d  most m i t o c h o n d r i a t e n d t o be  t o t h e edge  located  of  i n the  perinuclear region. An u n i d e n t i f i e d  stromal c e l l  aspects to the u n i d e n t i f i e d  cell  t h e e p i d e r m a l component, c a n be  types found  These c e l l s  t h e same u n i d e n t i f i e d  stromal c e l l s  scopy  26); they appear  may  retain  the s i z e o f normal  o f t e n observed groups. no  The  cells  d e s c r i b e d by  tend t o be  occur round  as macrophages  ( F i g u r e s 36 and  round w i t h d i s c r e t e throughout nucleolus  The  g r a n u l a r endoplasmic  found.  surrounded  nucleus  The  Otherwise, The  g r a n u l a t e d and  no  substance,  or appear  of  interpreted  scattered  randomly  enlarged by a  typical  normal-appearing  observed  typical cellular  empty, a p p e a r  any  i s usually  i n the  cells'  o r g a n e l l e s can  appears  i s r e l a t i v e l y electron-dense.  appear  possess  nucleus  heavily  Large,  membraned v a c u o l e s a r e common i n t h e c y t o p l a s m ; vacuoles either  larger  by c e l l s  Occasionally,  cytoplasm o f these c e l l s  most  the reaches  i s surrounded  r e t i c u l u m c a n be  micro-  contact of  t h e n u c l e u s ; most o f t e n a c e n t r a l ,  double-membraned s t r u c t u r e .  cytoplasm.  or  o r o v o i d and  clumps o f c h r o m a t i n  i s present.  light  They a r e  in pairs  37).  be  s l i g h t l y enlarged or  connections or c e l l - t o - c e l l  "arms" o r p a r t i a l l y  a t random  considered to  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  fibroblast  be  c a n be  fibroblasts.  s i n g l y , b u t may  intercellular  sort.  and  i n most  ( t o be d e s c r i b e d ) i n interspersed  between f i b r o b l a s t s .  ( F i g u r e s 15  type, s i m i l a r  double  these  t o c o n t a i n an  amorphous  t o c o n t a i n membrane r e m n a n t s w h i c h  53 sometimes r e s e m b l e and  37).  mitochondrial cristae  Some o f t h e s e c e l l s  extremely  c o n t a i n l a r g e numbers o f  electron-dense, large  granules  ( F i g u r e s 36 a n d 3 7 ) .  ( F i g u r e s 35, 36  inclusions  similar  to melanin  I t i s d i f f i c u l t t o determine  whether o r n o t these p a r t i c l e s  a r e bounded b y membrane  structures.  stromal c e l l s  Some u n i d e n t i f i e d  a p p e a r t o be c r y s t a l l i n e  inclusions:  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  c o n t a i n what  spindle-shaped, by an a r e a o f  e l e c t r o n density (Figure 37). O c c a s i o n a l l y , non-ovoid elongated c e l l s similar  ( F i g u r e 38)  throughout partially  excessively  i n much t h e same manner a s t h e r o u n d e d  c e l l types.  unidenti-  The i r r e g u l a r l y s h a p e d v a c u o l e s a r e common  the cytoplasm; filled  (i.e.,  t o t h e basement membrane,  t o chromatophores o r f i b r o b l a s t s ) appear  vacuolated fied  oriented parallel  cells  t h e y u s u a l l y a p p e a r empty o r  w i t h an amorphous s u b s t a n c e  o r membrane  fragments. AEN  Epidermis The  nodule cells  epidermal  tumor s t a g e  component o f t h e a n g i o e p i t h e l i a l  consists  o f an unknown t y p e  o f normal e p i t h e l i a l  ("X-cells"; giant c e l l s ) ,  i n t e r m e d i a t e s and v a r i a t i o n s . t y p e c a n be o b s e r v e d cells  (Figure 39).  epidermis;  and many  The u n i d e n t i f i e d g i a n t  interspersed  among n o r m a l  The e p i d e r m a l c e l l s  f r e e ribosomes,  I n t e r p o s e d between  cell  epidermal  appear as i n normal  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  complex; m i t o c h o n d r i a , as n o r m a l .  cells,  and n u c l e i  (and s e e m i n g l y  remain  appear  not disrupting)  54  PLATE 10 F i g u r e s 38-39  Fig.  38  Basement membrane a r e a o f a n E P - t y p e l e s i o n . stromal  " X - c e l l " i s shown l y i n g p a r a l l e l t o t h e b a s e m e n t  membrane blance  (right).  t o stromal  probable and  N o t e t h e e x c e s s i v e v a c u o l a t i o n and r e s e m fibroblasts  (Figure 34), i n d i c a t i n g  morphogenetic r e l a t i o n s h i p  stromal  encompassed membrane  Fig.  A non-ovoid  "X-cells".  Two s m a l l e p i d e r m a l  by an e n v e l o p i n g  (left).  X  between  cell,  a  fibroblasts "X-cells",  l i e above t h e b a s e m e n t  16200.  39  Epidermis  o f AEN tumour  between t h e i n i t i a l cells.  stage,  showing the r e l a t i o n s h i p  " X - c e l l " types  t o normal  epidermal  N o t e t h e n o r m a l - a p p e a r i n g p l a s m a membrane  digitations  between n e i g h b o r i n g  epidermal  cells.  interX  16200.  56  the epidermal c e l l s a r e s m a l l , rounded, v a c u o l a t 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 and c e n t r a l n u c l e o l i .  The c e l l mem-  branes o f t h i s new c e l l type a r e c l o s e l y apposed t o the outer membranes o f the n e i g h b o r i n g epidermal c e l l s .  No  desmosomes, f i n g e r - l i k e p r o c e s s e s , or o t h e r forms o f cellular  c o n t a c t s are v i s i b l e .  N u c l e i o f c e l l s o f t h i s type  are round and e n l a r g e d ; surrounded  by an even, double  membrane; c o n t a i n d i s c r e t e clumps o f chromatin  scattered  a t random throughout the n u c l e u s ; and c o n t a i n l a r g e , c e n t r a l , dense n u c l e o l i which appear t o be composed o f a h e a v i l y g r a n u l a r substance.  Nuclear pores are v i s i b l e  i n some  c e l l s ; they appear a t r e g u l a r i n t e r v a l s , measure 100 nm i n diameter, and may be a s s o c i a t e d w i t h m i c r o t u b u l e s ( F i g u r e 40).  The cytoplasm can appear extremely l u c e n t and o f t e n  seems t o c o n t a i n l i t t l e ground substance.  Large, o v o i d  vacuoles are common i n the cytoplasm; o f t e n they a r e founded by a double membrane, w h i l e i n o t h e r cases the membranes seem incomplete.  Again, as i n the analogous  u n i d e n t i f i e d stromal c e l l type, dark e l e c t r o n - d e n s e g r a n u l e s are p r e s e n t , u s u a l l y without evidence o f a surrounding membrane s t r u c t u r e .  These u n i d e n t i f i e d c e l l s o f the AEN  epidermis appear i n v a r i o u s stages o f enlargement,  cyto-  p l a s m i c d e n s i t i e s , and v a c u o l a t i o n ( F i g u r e s 40 and 4 1 ) . T h i s u n i d e n t i f i e d g i a n t c e l l type can be found, as d e s c r i b e d above, w i t h extremely l u c e n t c y t o p l a s m i c substance.  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 a l s o be found  which tend t o be extremely dense and h e a v i l y s t a i n e d (Figure 41) .  PLATE 11 F i g u r e s 40-41  Fig.  40  Epidermis  Fig.  o f l a r g e AEN-type l e s i o n .  21000.  41  E p i d e r m i s o f l a r g e AEN-type types  X  (type B  "X-cells")  lesion.  are found  Unidentified  cell  i n varying stages o f  " b a l l i n g up"; t h e d e n s i t i e s o f t h e s e c e l l s v a r i e s  from  extremely  have  retained  light  to dark.  Several epidermal  t h e i r normal appearance.  X  21000.  cells  59 Normal-appearing epidermal densely and  s t a i n e d ; these  lost  can  possess  a l s o appear  abnormal  (Figure 40).  Those  nuclei  epidermal  n o r m a l 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  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  normally than  c h a r a c t e r i z e them and  ovoid, c e l l s .  These  r e t a i n desmosomes and finger-like  appearing  junctional  interdigitations.  appear  i n the process  cells  (Figure  along  cells  "arms"  o f the EP).  (see l a t e r  endoplasmic r e t i c u l u m EP  -  (Figure  normal-  cytoplasmic  "enveloping  cells"  mitochondria extend  many desmosomes  descriptions of  O c c a s i o n a l l y these  lose  f i l a m e n t s which  t h e p e r i p h e r y , and  to  42).  (some o f w h i c h a p p e a r g r e a t l y l e n g t h e n e d ) a t t h e the c e l l u l a r  tend  normal  c o n t a i n many  f r e e r i b o s o m e s , many c y t o p l a s m i c  across the c e l l  their  increase their  These c e l l s  rather  C e r t a i n o f these  appear as p r e c u r s o r s of the  o f the mature l e s i o n .  cells  which  complexes b u t  of losing  organelles  Other epidermal d e n i s t y and  appear as e l o n g a t e d ,  "normal" epidermal  other  c y t o p l a s m i c d e n s i t y and  and  cells  large cytoplasmic vacuoles  c e l l s with  all  "dark"  cells  cells  tips  of  enveloping  contain  dilated  43).  epidermis The  epidermal  component o f t h e m a t u r e  like  lesion,  as d e s c r i b e d b y  29),  i s composed o f two g e n e r a l t i s s u e a r e a s  cell  types.  The  superficial  l i g h t microscopy  l a y e r s o f the  of r e l a t i v e l y  normal e p i t h e l i a l  intercellular  spaces.  cells  papilloma-  (Figures and  three  tumor a r e  ( t y p e C) w i t h  These same n o r m a l - a p p e a r i n g  26basic  composed large  epidermal  60 PLATE 12 F i g u r e s 42-43  Fig.  42  Portion in  an  o f cytoplasm o f a normal epidermal c e l l  AEN-stage l e s i o n .  " s a n d w i c h e d " between two Note the  general  s u b s t a n c e , and plasm.  Fig.  X  T h i s p a r t o f the " X - c e l l " types  disorganization,  occurring  cytoplasm  ( t o p and  is  bottom).  lack of cytoplasmic  loss of recognizable  organelles  ground  i n the  cyto-  19000.  43  E p i d e r m i s 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 i n g c©ll d e v e l o p i n g types.  X  16200.  among s e v e r a l u n i d e n t i f i e d  an  envelop-  "X-cell"  61  62 c e l l s are a l s o p r e s e n t i n the lower p o r t i o n of the epidermal i n f o l d i n g s , where they compose a "matrix" i n which the characteristic 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 four l a y e r s o f the tumor.  the top t h r e e - t o -  C h a r a c t e r i s t i c a l l y , they appear  as very dense, f l a t t e n e d c e l l s w i t h numerous f i n g e r l i k e c e l l u l a r e x t e n s i o n s which connect them, o f t e n w i t h desmosbmes, to  (Figures 44 and 4 5 ) .  neighboring c e l l s  The  spaces are most o f t e n empty, but o c c a s i o n a l l y  extracellular cellular  d e b r i s , resembling the remnants o f a n e c r o t i c c e l l , found  can be  (Figure 4 5 ) . The most s u p e r f i c i a l c e l l s possess  p l e a t s , o f t e n coated w i t h a f i b r i l l a r those found i n normal epidermis  microvilli-like  substance, s i m i l a r t o  (Figure 4 4 ) .  These c e l l s  c o n t a i n numerous mitochondria and r e t a i n c l o s e attachments  i n the h o r i z o n t a l plane  o f t e n the endoplasmic  cell-to-cell  (Figure 4 5 ) .  r e t i c u l u m o f these c e l l s  Quite  is dilated  and f i l l e d w i t h an unknown, e l e c t r o n - d e n s e , o f t e n h e a v i l y g r a n u l a r , substance  (Figures 44 and 4 5 ) .  N u c l e i o f these  s u p e r f i c i a l c e l l s s t a i n e v e n l y dark, w i t h l i t t l e condensed chromatin. of  O v e r a l l , the n u c l e i , cytoplasm, and o r g a n e l l e s  these c e l l s s t a i n w i t h such even i n t e n s i t y t h a t d i s c r i m i n a -  t i o n between c e l l u l a r elements  i s often impossible.  I n t e r s p e r s e d among the s u p e r f i c i a l epidermal s m a l l lymphocytes  and h i s t i o c y t e s can be found.  The  cells  lympho-  c y t e s are c h a r a c t e r i z e d by a l a r g e , c h r o m a t i n - r i c h nucleus and a scanty cytoplasm, which commonly c o n t a i n s a few l a m e l l a e  63  PLATE 13 F i g u r e s 44-45  Fig.  44  Superficial case,  cell  the s e c t i o n  the e p i d e r m i s .  Fig.  layers  illustrates a portion  X  "in-fold" of  13300.  cell  l a y e r s o f a mature EP-type l e s i o n ,  the c h a r a c t e r i s t i c  s t e l l a t e epidermal  i n t e r c e l l u l a r spaces  appears types X  o f one  In t h i s  45  Superficial  and  o f a mature E P - t y p e l e s i o n .  occasionally  t o be c e l l u l a r d e b r i s .  l i e i n the deeper p o r t i o n  13300.  showing  c e l l s , macrophages, f i l l e d with  The e n v e l o p i n g and o f the l e s i o n  what "X-cell"  ( t o the r i g h t ) .  65 of granular  endoplasmic reticulum  ( F i g u r e 45) a r e c h a r a c t e r i z e d  (Figure 4 5 ) .  by a l a r g e ,  horseshoe-shaped  n u c l e u s w i t h condensed p e r i p h e r a l c h r o m a t i n . endoplasmic reticulum,  Histiocytes  Mitochondria,  G o l g i apparatus, r e s i d u a l bodies,  digestive vacuoles constitute the majority  of  and  cytoplasmic  elements. The are  deeper p o r t i o n s  o f the epidermal  composed p r i m a r i l y o f g i a n t  cells  infoldings  e n v e l o p e d b y t h e arms  of a l t e r e d , heteromorphic epidermal c e l l s  o f normal  plasmic  cells  ultrastructure.  These e n v e l o p i n g  are character-  i z e d b y a homogenous, e l e c t r o n d e n s e n u c l e o p l a s m , sometimes shows a s m a l l  degree o f condensed  Grandular endoplasmic r e t i c u l u m , f r e e ribosomes o f t e n occur "arms" o f t h e s e c e l l s plasmic  the tips  other  o f the cytoplasmic  cells  with the axis  Numerous desmosomal  enveloping arms.  connec-  cells, especially  No desmosomal o r  and t h e g i a n t c e l l s .  o c c a s i o n a l l y be f o u n d  enveloping tumor  The  j u n c t i o n a l c o m p l e x e s were e v e r o b s e r v e d b e t w e e n t h e  enveloping can  area.  many f r e e r i b o s o m e s a n d c y t o -  46 a n d 4 7 ) .  t i o n s o c c u r between a d j o i n i n g at  m i t o c h o n d r i a , and many  filaments, which u s u a l l y r u n p a r a l l e l  o f t h e arm. ( F i g u r e s  which  chromatin.  i n the perinuclear  contain  cyto-  c e l l types  interspersed  (Figure 4 7 ) .  identical  among t h e g i a n t a n d  light  i n most a s p e c t s .  d i s t i n c t i o n b e t w e e n t h e two c e l l t y p e s differences  lymphocytes  The g i a n t c e l l s  ( c e l l types A and B as d e s c r i b e d  are m o r p h o l o g i c a l l y  Small  of the  microscopically) Ultrastructural  i s b a s e d on s i z e  and on t h e p r e s e n c e o f d e n s e c y t o p l a s m i c  inclusions.  66 PLATE 14 F i g u r e s 46-47  Fig.  46  Deeper p o r t i o n  o f mature  characteristic  histologic  ("X-cells")  Fig.  EP-type l e s i o n , arrangement  and e n v e l o p i n g  cells.  X  showing the  of giant c e l l  types  14500.  47  Deeper  portion  o f mature  membrane), i l l u s t r a t i n g  EP-type l e s i o n  (near the  the occurrence o f small  interspersed  o c c a s i o n a l l y among t h e " X - c e l l " and  cell  X  types.  10500.  basement  lymphocytes enveloping  67  68 Apart  from  giant cells  t h e s e two d i f f e r e n c e s i n the epidermal  ( t o be d e s c r i b e d  tumor component show t h e same  pathologic characteristics.  The n u c l e u s  o v o i d , w i t h randomly d i s p e r s e d , d i s c r e t e chromatin;  double  o f the nucleus.  membrane.  The n u c l e u s  Nuclear pores  clumps o f condensed  c y t o p l a s m i c ground substance  varies  Normal e n d o p l a s m i c  o f one-half the  i s surrounded  ( F i g u r e 4 6 ) . The  from  dense t o t h i n l y  (Figure 4 8 ) . Generally  c y t o p l a s m i c elements  present are  membrane- o r non-membrane-bound d e n s e i n c l u s i o n s s i z e and shape; and l a r g e ,  by a  r e t i c u l u m l a m e l l a e and m i t o -  c h o n d r i a a r e p r e s e n t i n few c a s e s the o n l y d i s t i n g u i s h a b l e  thickly  o f 100nm d i a m e t e r a r e  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  granular.  i s e n l a r g e d and  a c e n t r a l dense n u c l e o l u s o f e v e n l y ,  granular m a t e r i a l can o f t e n reach a s i z e diameter  below),  ovoid-to-round  of irregular  vacuoles  identical  t o t h o s e d e s c r i b e d i n t h e s t r o m a l a n d AEN u n i d e n t i f i e d types.  Often these vacuoles can appear elongated o r h o r s e -  s h o e s h a p e d a n d may a p p e a r s i m i l a r plasmic reticulum lamellae presence  cell  of cristae-like  t o extremely  (Figure 4 8 ) .  dilated  endo-  In other cases the  membrane f r a g m e n t s  located a t the  periphery o f the vacuoles i s suggestive o f degenerative or d i l a t e d mitochondria microfilaments,  (Figure 4 9 ) .  arranged  O c c a s i o n a l l y bundles o f  in a parallel  a r r a y a n d bound b y a  s i n g l e membrane, c a n be o b s e r v e d  i n the cytoplasm  The  intercellular  and,  p l a s m a membrane  i s free  from  i n g e n e r a l , i s smooth o r s l i g h t l y  giant cells  connections  irregular.  c a n be f o u n d w i t h p l a s m a membrane  (Figure 4 9 ) .  Occasionally  infoldings  69  PLATE 15 F i g u r e s 48-49  Fig.  48  E p i d e r m a l component o f mature E P - t y p e e n v e l o p i n g and  "X-cell" types.  r e t i c u l u m and h o r s e s h o e -  Fig.  lesion,  Note the normal  or elongate-shaped  two  cell. X  endoplasmic  vacuoles.  X  10500.  49  P o r t i o n o f cytoplasm of a type A g i a n t c e l l . of  showing  enveloping c e l l s  lies  on e i t h e r  cytoplasm  side* o f t h e t y p e  N o t e t h e e x t e n s i v e p l a s m a membrane  22000.  The  invaginations.  A  71 ( F i g u r e s 46 a n d 4 9 ) . "proliferation"  (i.e.,  plasmic reticulum were n o t e d  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 several  l a y e r s o f a g r a n u l a r endo-  closely  apposed  to the nuclear  i n these c e l l s  (Figure  5 0 ) . Many  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 w h o r l s "  envelope)  "X-cell"  types  ( F i g u r e s 38, 4 9 , a n d  51). L i g h t microscopy of  epidermal g i a n t c e l l s ;  i n groups  type B c e l l s  infoldings.  found  cells  those g i a n t c e l l s  cells  d i f f e r e n c e s c a n be n o t e d  l o c a t e d a l o n g t h e b a s e m e n t membrane  located  Ultrastructurally,  i n t h e i n n e r tumor those g i a n t c e l l s  b a s e m e n t membrane a r e o v o i d a n d r a n g e normal  located  i n t h e more c e n t r a l a r e a s o f t h e e p i d e r m a l  Ultrastructurally,  between t h o s e g i a n t  of  u s u a l l y were  a l o n g t h e b a s e m e n t membrane, w h i l e t y p e A  were g e n e r a l l y  and  showed t h e p r e s e n c e o f two t y p e s  epidermal c e l l s .  portions.  nearest the  up t o t w i c e t h e d i a m e t e r  They t e n d t o c o n t a i n e x t r e m e l y  large cytoplasmic vacuoles arranged  in a circular  about  ( F i g u r e s 38,and 5 1 ) .  the c e n t r a l l y  These c e l l s fied  located nucleus  are s i m i l a r  giant c e l l  types  i n size  found  fashion  and m o r p h o l o g y t o t h e u n i d e n t i -  i n t h e stroma  a n d i n t h e AEN  epidermis. Deeper i n t o t h e e p i d e r m a l cells of  t e n d t o be much l a r g e r  normal  rather  epidermal c e l l s ) ;  infoldings,  (up t o n i n e  t h e y t e n d t o be  the diameter  heteromorphic  than o v o i d i n shape; and they t e n d t o p o s s e s s  numbers o f d e n s e c y t o p l a s m i c i n c l u s i o n s The  times  the g i a n t  larger  ( F i g u r e s 4 6 and 5 1 ) .  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 n o t a p p e a r  t o be  72 PLATE 16 Figures 50-51  Fig.  50  Nucleus  and  portion  of cytoplasm  o f an e p i d e r m a l  "X-cell",  illustrating  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  occasionally  was  observed.  clumped c h r o m a t i n .  Fig.  X  Note t h e  which  indiscriminatively  22000.  51  Low-power m i c r o g r a p h type l e s i o n ,  o f e p i d e r m a l p o r t i o n o f a mature  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  type B g i a n t c e l l s  "X-cells".  X  9500.  EP-  o f type A  and  74 membrane-enclosed. of  vacuoles  the average  With  increasing c e l l  size,  t h e number  i n t h e s e l a r g e r c e l l s seems t o i n c r e a s e , diameter  Virus-like Quite  while  o f t h e v a c u o l e s seems t o d e c r e a s e .  Particles frequently small v i r u s - l i k e p a r t i c l e s  of  unknown c o m p o s i t i o n o r o r i g i n a r e p r e s e n t i n t h e c y t o p l a s m of  t h e 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 t h e p a p i l l o m a -  like  lesions  observed  ( F i g u r e s 52 and 5 3 ) .  i n the i n t e r c e l l u l a r space  a n g i o e p i t h e l i a l nodule round  o r hexagonal  (Figure 54).  observed  throughout  cells.  These p a r t i c l e s i n diameter.  i n typical virus  a r r a y s ; most o f t e n t h e y were f o u n d  ficial  o f the epidermis o f an  and measure 30 nm  p a r t i c l e s were n e v e r  scattered  I n one i n s t a n c e t h e y were  appear  The  crystalline  i n s m a l l groups  o r randomly  the cytoplasm o f the enveloping or super-  75 PLATE 17 F i g u r e s 52-54  Figure  52  P o r t i o n o f c y t o p l a s m o f an e n v e l o p i n g c e l l mature EP-type  lesion,  showing t h e presence  staining virus-like particles. measure 30 nm  Fig.  i n diameter.  X  of darkly-  The p a r t i c l e s  consistently  30000.  53  Virus-like  particles  i n t h e c y t o p l a s m o f an e n v e l o p i n g  l y i n g n e x t t o t h e b a s e m e n t membrane.  Fig.  found i n a  X  cell  56000.  54  Virus-like  particles  an AEN-type l e s i o n .  found X  i n the i n t e r c e l l u l a r space o f  14400.  77  DISCUSSION  Normal  Epidermis  Normal e p i d e r m a l s t r u c t u r e well  described  Andrew, 1959; Patt  and  the  both l i g h t m i c r o s c o p i c a l l y Lagler  Patt,  Matoltsy,  1969)  1968;  et^ a l _ . , and  flathead  1974).  sole, a  with epidermal papillomas,  has  Electron microscopic  studies  1969) , o f t h e  skin  1970)  larval  1957;  Romer, 1962;  Hyman,  1962;  (Hendrickson  Recently  the  biology  f l a t f i s h often  been s t u d i e d  o f the  scales  (Wellings  and  of  afflicted  i n some  detail.  (Brown and  Wellings,  Brown, 1 9 6 9 ) ,  fish  and  (Brown and  and Wellings,  have b e e n p r e s e n t e d .  of the  lemon s o l e  descriptions of in previous  s c o p y t o be arranged  of the  Structures  c e l l s were shown by  interpreted the  scanning e l e c t r o n  o f numerous s u r f a c e  patterns  (Figure  13).  ( J o n e s e t a l . , 1966;  1974).  L u s e and  to previous  1967); j u n c t i o n a l complexes  i n the  microoften  This f i n g e r p r i n t in  Krejsa,  U l t r a s t r u c t u r a l observations  s o l e guanophores c o r r e s p o n d  surface  pleats,  topography i s s i m i l a r to that reported  species  skin  previous  as b r o a d , s h o r t m i c r o v i l l i on  in concentric  Merrilees,  studies  v a r i a t i o n from  t e l e o s t epidermis.  cross-sections  cellular  teleost  electron microscopic  showed l i t t l e  reports  of s u p e r f i c i a l  guit,  Oosten,  s k i n of two-to-three year o l d  L i g h t and  like  1962;  been  (van  ultrastructurally  Merrilees,  s k i n of the  o f the  i n t e l e o s t s has  of  other 1969;  lemon  descriptions  (Seto-  e p i d e r m i s were  typical  78 of  those  found i n amphibian  skin  (Farquhar and P a l a d e ,  1965). In  an a u t o r a d i o g r a p h i c s t u d y o f t h e growth o f f i s h  epidermis, Hendrickson in are  a l l epidermal c e l l not restricted  vertebrates),  (1967) f o u n d layers.  This indicates  t o the basal layer  that  mitoses  (as they a r e i n o t h e r  and t h a t a l l e p i d e r m a l c e l l s  maintenance o f t h e t i s s u e . be  i n c o r p o r a t i o n o f thymidine  participate  T h i s fundamental  difference  c o n s i d e r e d when c o m p a r i n g t i s s u e r e a c t i o n s o f f i s h  w i t h those o f o t h e r v e r t e b r a t e s . t i o n or other d i f f e r e n t i a t i v e e p i d e r m i s o f most f i s h ; by a v a r i a b l e  must  epidermis  No e v i d e n c e o f k e r a n t i n i z a -  processes  instead,  i n the  i s observed  the epidermis  i nthe  i sprotected  t h i c k n e s s o f mucus s e c r e t e d b y u n i c e l l u l a r  secretory glands.  R e d u c t i o n o r a l t e r a t i o n o f t h e mucus  layer  can r e s u l t  i n increased s u s c e p t i b i l i t y o f the epidermal  cells  t o o u t s i d e agents  Biology o f Epidermal Evidence  (van Oosten,  1957).  Lesions  f o r the progression o f epidermal  lesions  from an i n i t i a l  AEN s t a g e t o a m a t u r e EP l e s i o n c o n s i s t s o f  natural history  data, l a b o r a t o r y  of  morphologic The  of  lemon s o l e  groups. initial relative  observations, the presence  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  studies.  f r e q u e n c i e s o f o c c u r r e n c e s o f AEN a n d EP s t a g e s l e s i o n s were a n a l y z e d i n t e r m s o f 5 mm-size  The r e s u l t s  ( F i g u r e 9) s u g g e s t a p r o g r e s s i o n f r o m  AENs t o m a t u r e E P s : percentage  o f AENs  as f i s h  size  increases, the  ( a s compared t o E P s ) d e c r e a s e s  79  until  no AENs a r e  found  t r a n s i t i o n o f the  (above  flatfish  135  l e s i o n s was  on t h e b a s i s o f s i m i l a r e v i d e n c e 1968;  i n any  (larger)  EPs  flatfish,  to  i n t h e y e a r and  EP  postulated earlier McArn e_t a l . ,  appears  t o be  on y o u n g e r  a  occur  (smaller)  e x c l u s i v e l y on o l d e r  a phenomenon b e s t e x p l a i n e d b y p r o g r e s s i v e  lesions.  AENs on  flatfish  have been observed, t y p i c a l EP  There  tend to occur almost  growths o f the  AEN  d i s e a s e d ^ p o p u l a t i o n , f o r AENs t o  predominately e a r l y flatfish;  The  (McArn, 1968;  McArn and W e l l i n g s , 1 9 7 1 ) .  tendency,  mm).  lesions  kept under l a b o r a t o r y c o n d i t i o n s  i n s i g n i f i c a n t numbers, t o p r o g r e s s (McArn, 1968;  McArn e t a l . ,  1968);  p r o g r e s s i v e growths o f t h e tumours were a l s o r e p o r t e d Wellings  and  Chuinard  The  EPs  histology  t h e tumour t y p e s reported here, species  a l s o seems t o  (Wellings e t a l . ,  presence  found  ( l i g h t and  1964;  McArn,  and  corresponds  EP  was  Most r e p o r t s gave  intermediates  o f t h e v a r i o u s tumour t y p e s  i n other  descriptions  U s u a l l y the  ( e x t e r n a l l y ) was  made t o c o r r e l a t e h i s t o l o g i c a l  with external morphological structure.  of  other species  to previous descriptions  s t a g e s o f tumour g r o w t h .  o f morphologic  b u t no a t t e m p t  electron microscopic)  i n t h e lemon s o l e , and  (see i n t r o d u c t i o n ) .  t h e AEN  tion  stages  McArn and W e l l i n g s , 1 9 7 1 ) . The  of  transitional  h a s b e e n d e s c r i b e d and  indicate a progressive process 1968;  by  (1964).  occurrence o f morphologic  b e t w e e n AENs and  to  noted,  structure  Histological  (AENs, t r a n s i t i o n a l  examinaforms,  80  and  EPs)  shows:  each type  (i.e.,  m o r p h o l o g y and 2)  1)  a distinctive  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  I t i s this pattern  c h a n g e s from that w i l l  with a d i s c u s s i o n of each stage,  f r o m an  o f the  initial  changes  form, r e p r e s e n t s  i n the  above), the  what I b e l i e v e  t o be  t o mature f l a t f i s h  Figure  follows;  the  g r o w t h , and  hyperplasia an  2)  3)  see  pattern progres-  diagramatically  (Figures  14  cell  o f a dermal  discussion of unidentified c e l l  (Figures  4)  (Figure  the  in  o f dermal f i b r o b l a s t s , c a p i l l a r y  from t r a n s f o r m a t i o n  hyperplasia  epidermis  B)  and  (a more d e t a i l e d  appearance o f u n i d e n t i f i e d stromal  ( F i g u r e 15;  (type  lesion.  i n the  illustrated  inflammatory-type response  ("X-cells"), probably  the  stages are  lesions  growth  55): 1)  type  below,  following,  sion  initial  EP  progressive  c y t o l o g i c changes o c c u r r i n g  in  to  described  o f h i s t o l o g i c and  description  AEN  a likely histologic  involved  (presented  and  intermediate  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  e x t e r n a l morphology  from  above),  l e s i o n t o a mature, p a p i l l o m a - l i k e  B a s e d on  simplified  as  be  the  for  external  i n v o l v i n g a continuum o f  forms, o f h i s t o m o r p h o l o g i c a l  explanation  structure  a d e f i n i t e c o r r e l a t i o n between  a d e f i n i t e pattern,  stages.  histological  18,  of epidermal c e l l s 20  and  and  the  15);  types cell types);  folding of  21);  appearance o f u n i d e n t i f i e d g i a n t c e l l  along  and  basement membrane i n t h e  type  epidermal  component  23); 5)  appearance of type A g i a n t c e l l s  i n the  interior  81  FIGURE 55 Summary o f t h e g r o w t h a n d s t r u c t u r e  of flatfish  epidermal  lesions.  The p o s t u l a t e d lesions are  f o u n d on j u v e n i l e  explained  g r o w t h , and s t r u c t u r e flatfish.  on page 80 o f t h e  text.  o f epidermal  The numbered  figures  82  83 of the epidermal i n f o l d i n g s , probably (Figures  from t y p e B  precursors  24 and 25;; s e e 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  types); 6)  continued  increase  A and B i n t h e e p i d e r m i s u n t i l transformation superficial  o f other  cells  i n t h e numbers o f c e l l  they dominate t h e t i s s u e ;  epidermal c e l l s  (Figures  types  i n t o enveloping  and  26-29);  7)  necrosis of giant c e l l  types  8)  consequent t i s s u e n e c r o s i s  (Figure 28);  (?) ( s e e 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  fish)  (Figures  31-33). The  first  obvious r e a c t i o n i n tumorigenesis  lemon s o l e s k i n a p p e a r s t o b e a p r o l i f e r a t i o n blasts; in  this  type o f h i s t o l o g i c a l  t h e s m a l l e s t AENs s t u d i e d .  gives This  rise  t o the stromal  This  initial  hyperplasia  resembles a fibroma,  cells,  indicating stasis  o f t e n accompanied by an i n c r e a s e  S m i t h , 1938, 1940; N i g r e l l i ,  a l l y granulocytes  1954).  being I n some  ( F i g u r e 14 a n d 1 5 ) .  in capillaries, i s agent  Electron  (Nigrelli  microscopic-  ( s e e W e i n r e b , 1963; Andrews, 1 9 5 9 ) , m a c r o -  p h a g e s , a n d l y m p h o c y t e s c a n be o b s e r v e d spersed  (Figure 4 ) .  fibers.  a common r e s p o n s e o f f i s h e p i d e r m i s t o v a r i o u s and  evidently  v e s s e l s c a n b e o b s e r v e d ; u s u a l l y t h e s e show  o f r e d blood  Fibrosis,  fibro-  s t r u c t u r e was f o u n d e x c l u s i v e l y  composed m a i n l y o f f i b r o b l a s t s a n d c o l l a g e n  packing  o f dermal  component o f t h e l e s i o n  fibroblast hyperplasia  tumours many b l o o d  -  i nthe  among t h e f i b r o b l a s t s ,  ( F i g u r e s 35 a n d 3 6 ) .  i n t h e stroma  inter-  c o l l a g e n , and c a p i l l a r i e s  This would  i n d i c a t e some d e g r e e o f  84 inflammatory (1969).  response,  as s u g g e s t e d by Brooks  They s u g g e s t e d  t h e AEN  stroma  t o be  n a t u r e on t h e b a s i s o f r e d b l o o d c e l l s , c y t e s , plasma c e l l s , dilatation this  inflammatory  macrophages,  dominated  i n the stroma  lympho-  or plasma  o f t h e AENs s t u d i e d .  a subacute  by a f i b r o b l a s t i c  ( s e e L a V i a and H i l l ,  inflammatory  response  and a n g i o b l a s t i c  1971).  Nigrelli  The  to parasitic  stromal pre-  proliferation  (1954) h a s  inflammatory  invasions of f i s h .  elements  time, l e a v i n g  the  noted  as the s u p p o r t i n g stroma The cells")  (Figure 15). It  the  stromal c e l l  i n most, b u t  i n the l e s i o n s ;  s t a g e s r e v e a l e d no I t appears  r e a c t i o n may  be  cells  that  invasive  o r EP  enough t o b e  type  (stromal  large  examination  of this  cell  i n nature  "XAENs.  numbers. type  of the  earliest  type.  i n some AENs t h e i n i t i a l  stages; either fatal  regress with  seemingly not a l l ,  ( F i g u r e s 16 and  I n v a s i v e g r o w t h o f t h e s t r o m a were n e v e r tional  the  EP.  They o f t e n o c c u r i n e x t r e m e l y  appears  in  vascularization,  i s n o t known e x a c t l y when t h e u n i d e n t i f i e d  initially AEN  found  framework, w i t h  f o r the  unidentified  c a n be  Presumably  o f the s t r o m a l response  fibromous  In  cells  o c c u r r e n c e o f m a c r o p h a g e s , g r a n u l o c y t e s , and p l a s m a c e l l s response  in  found e l e c t r o n m i c r o s c o p i c a l l y .  s t u d y , 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  component r e s e m b l e s  co-workers  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  (with s t a s i s )  were o b s e r v e d  and  observed  the i n v a s i v e growth  t o t h e h o s t a t an e a r l y  growth, o r the growths are s e l f - l i m i t i n g  fibromatous 17). in transi-  i s damaging  s t a g e o f tumour  and/or  regressive.  85  Angiomous growths, et  al.,  as observed i n another study ( W e l l i n g s ,  1964), o f t e n show i n v a s i v e q u a l i t i e s w i t h o u t b e i n g  invasive i n nature. In  s t u d i e s o f t r u e epidermal p a p i l l o m a s i n other  f a m i l i e s o f f i s h , other authors have r e p o r t e d i n i t i a l  growths  s i m i l a r t o the a n g i o e p i t h e l i a l nodule types d e s c r i b e d i n f l a t fish.  Lucke and Schlumberger  (1941) found p r e - p a p i l l o m a  l e s i o n s i n other s p e c i e s t o c o n s i s t o f an i n i t i a l and p r o l i f e r a t i o n o f dermal b l o o d v e s s e l s .  hyperemia  Scarpelli  (1969)  observed s i m i l a r AEN-type t o EP-type growth t r a n s i t i o n s f o r papillomas o f the s l i p p e r y d i c k . t h i s c o u l d be a common p a t t e r n l i k e growths i n f i s h  I t has been suggested  i n the growth o f p a p i l l o m a -  ( W e l l i n g s , 1969B).  Some h y p e r p l a s i a o f the epidermal c e l l s , to  that  those h y p e r p l a s i a s noted by N i g r e l l i  similar  (1954) as t y p i c a l  responses t o v a r i o u s p a r a s i t i c s t i m u l i i n other f i s h e s , e v i d e n t l y f o l l o w s a v a r y i n g degree o f i n i t i a l AEN stromal growth (Figure 21).  As the epidermis t h i c k e n s and begins  i n f o l d i n g , the stromal component o f t h e l e s i o n s becomes g r a d u a l l y reduced; e v e n t u a l l y , i n the mature EP, i t c o n s i s t s of ing  a branching s e r i e s o f narrow s u p p o r t i n g "arms". the epidermal t h i c k e n i n g , and presumably  h y p e r p l a s i a , i s the appearance  Accompany-  f o l l o w i n g the  o f an u n i d e n t i f i e d c e l l  type  ( " X - c e l l s ; g i a n t c e l l s ) , d e s c r i b e d e a r l i e r as c e l l type B. U s u a l l y these c e l l s b e g i n t o accumulate membrane (Figure 23).  a l o n g the basement  G r a d u a l l y , as the epidermis t h i c k e n s  and beings t o i n f o l d , these c e l l s appear  l a r g e r and more  numerous (Figure 24). (Figure 25).  S t a i n i n g becomes l e s s and l e s s i n t e n s e  These l a r g e r c e l l s , u s u a l l y found near the  c e n t e r of the epidermal i n f o l d i n g s , e x h i b i t d i f f e r e n t i n g 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.  stain-  Electron  m i c r o s c o p i c a l l y , c e l l types A and B were found to be morpholo g i c a l l y i d e n t i c a l except f o r s i z e , degree o f v a c u o l a t i o n , and amount o f c y t o p l a s m i c i n c l u s i o n s  (see d i s c u s s i o n  below).  I t seems v e r y l i k e l y , on the b a s i s o f u l t r a s t r u c t u r a l morphology and time and l o c a t i o n o f the appearance o f the two c e l l  types  (type B c e l l s g e n e r a l l y occur i n l a t e AEN or t r a n s i t i o n a l forms a l o n g 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 c e n t e r o f the  infoldings  and o n l y , i t seems, a f t e r the appearance of a l a r g e number o f type B c e l l s ) , cells.  t h a t type B c e l l s are p r e c u r s o r s of type A  E v e n t u a l l y , i n the mature l e s i o n , these g i a n t  cell  types dominate the epidermal component; they are found i n a matrix o f e n v e l o p i n g c e l l s  "embedded"  (Figure 26), which a l s o are  e v i d e n t l y m o r p h o l o g i c a l l y changed epidermal c e l l s  (Figure 43  and 4 6 ) . In every mature l e s i o n s t u d i e d h i s t o l o g i c a l l y , s t r u c t u r e s i n t e r p r e t e d as c o l l a p s e d g i a n t c e l l s are u s u a l l y near the very c e n t e r of the epidermal (Figure 28).  found,  infoldings  G e n e r a l l y , l a r g e numbers o f c e l l s  tentatively  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 c e n t r a l r e g i o n s , i n d i c a t i v e o f much p h a g o c y t i c a c t i v i t y S e v e r a l mature tumors showed g e n e r a l n e c r o t i c throughout  the t i s s u e  ( F i g u r e 31).  (Figure 30).  tendencies  These o b s e r v a t i o n s , when  considered  w i t h the g e n e r a l n e c r o t i c c o n d i t i o n  (to be  dis-  cussed below) o f the g i a n t c e l l types composing the t i s s u e , seem  to i n d i c a t e t h a t n e c r o s i s  tumors.  N i g r e l l i and  i s the u l t i m a t e  his colleagues  (1965) a l s o  evidence o f n e c r o s i s w i t h i n mature and The the  ultimate  f l a t f i s h bearing  fate of  the  reported  s o l e epidermal l e s i o n s .  f a t e of the epidermal l e s i o n s or o f  these l e s i o n s remains u n c e r t a i n .  Previous  s t u d i e s on the n a t u r a l h i s t o r y o f t h i s d i s e a s e , as w e l l the p r e s e n t are  as  study, i n d i c a t e t h a t u s u a l l y no epidermal l e s i o n s  found on f l a t f i s h over 200  c e r t a i n f a c t o r s are o p e r a t i n g  mm  t o t a l length.  Evidently  i n the removal of tumourous  t i s s u e s from f i s h , or i n the s e l e c t e d removal o f tumourbearing  f i s h from the When the  population.  frequencies  o f tumour-bearing lemon s o l e  are expressed as percentages per r i s e and  fall  increases.  5 mm  s i z e group, a d e f i n i t e  o f tumour prevalence i s noted as f i s h  T h i s phenomenon was  noted i n p r e v i o u s  o f the n a t u r a l h i s t o r y o f t h i s d i s e a s e e t a l . , 1965; 1971; An  Wellings  e t a l . , 1965;  M i l l e r and W e l l i n g s ,  initial  bearing  increase  1971;  f i s h occurs u n t i l the 75-79 mm  Wellings,  Sherwood, 1974).  group, where the  A f t e r t h i s peak, the  decrease i n tumour prevalence mm  Mearns and  Nigrelli  i n the r e l a t i v e prevalence o f tumour-  i n c i d e n c e approaches 50%.  164  studies  (Good, 1940;  McArn and  length  group) i n d i c a t e s one  (to a zero p o i n t i n the  (or both) o f two  l e s i o n s are r e g r e s s i n g or are being s u r f a c e a t a f a i r l y constant  gradual  things:  sloughed-off  the  160— 1)  the  fish  r a t e , and/or 2) tumour-bearing  f i s h are being removed from the p o p u l a t i o n r a t e than are non-tumour-bearing The lost  at a much f a s t e r  fish.  main evidence t h a t the l e s i o n s are r e j e c t e d or  (due t o n e c r o t i c 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  t i s s u e n e c r o s i s , as observed i n t h i s study  i n lemon s o l e EPs co-workers (1965).  and  i n sand s o l e EPs by N i g r e l l i  and  K e l l y (1971), i n a study o f epidermal  p a p i l l o m a s on lemon s o l e i n Puget Sound, Washington, sloughing  or r e j e c t i o n o f tumors under l a b o r a t o r y  a t a r a t e o f 70% over a two month p e r i o d . (Wellings et. a l . , 1964; were r e p o r t e d  his  conditions  In other  M i l l e r and W e l l i n g s ,  reported  studies  1971), tumors  to progress i n the l a b o r a t o r y ; no evidence o f  n e c r o s i s or tumor r e g r e s s i o n was evidence i s p o s s i b l y due tumor m a t u r i t y ,  found.  This  contradictory  t o s i z e d i f f e r e n c e s , and  o f the f i s h s e l e c t e d f o r study.  consequently Mature  tumors would seem more l i k e l y to show evidence o f  necrosis  or r e g r e s s i o n than would young, p r o g r e s s i v e  stages.  There are a t l e a s t two of tumor-like  instances  growths i n other  o f r e g r e s s i o n or r e j e c t i o n  families of f i s h .  a h y p e r p l a s t i c epidermal d i s e a s e , and  tumor  appears t o be  Fish  self-limiting  i s p r o b a b l y c o n t r o l l e d by temperature and/or the  F i s h pox  season.  l e s i o n s have been observed to undergo r e g r e s s i o n  (Lucke and t i o n ) has  pox,  Schlumberger, 1948).  N. Peters  (personal  observed t h a t European e e l s , a f f l i c t e d  c a u l i f l o w e r - l i k e papillomas,  communica-  with  o f t e n r e j e c t tumorous t i s s u e s .  Evidence t h a t the drop i n tumor frequency w i t h i n c r e a s i n g s i z e i s caused by the death o f tumor-bearing  fish  is inconclusive. per  Analyses o f the average number o f l e s i o n s  f i s h w i t h r e l a t i o n t o s i z e (Figure 8) shows an i r r e g u l a r ,  but d e f i n i t e , d e c l i n e as f i s h l e n g t h i n c r e a s e s . was shown i n the p r e s e n t e a r l i e r studies 1971) .  This phenomenon  study o f lemon s o l e as w e l l as i n  (Wellings e t a l . , 1965; M i l l e r and W e l l i n g s ,  Presumably, i f the tumors are not being  sloughed-off  or r e j e c t e d , then the absence o f l e s i o n s i n f i s h over 180 mm i n l e n g t h must i n d i c a t e t h a t the disease  is fatal.  I f this  i s the case, then h e a v i l y - i n f e c t e d f i s h would be expected to be removed from the p o p u l a t i o n others;  a t a f a s t e r r a t e than  t h i s would m a n i f e s t i t s e l f i n a decrease i n the  average number:of tumors p e r f i s h as f i s h s i z e  increases.  P a r t , i f not a l l , o f t h i s decrease can be e x p l a i n e d by o b s e r v a t i o n s  i n d i c a t i n g t h a t very o f t e n the spreading  edges o f many AENs merge t o form a s i n g l e l a r g e EP.  Cooper  and K e l l e r (1969), i n t h e i r study o f l e s i o n s on lemon s o l e i n San F r a n c i s c o Bay, presented evidence which i n d i c a t e d t h a t the d i s e a s e had no l e t h a l e f f e c t s on the p o p u l a t i o n . M i l l e r and W e l l i n g s shown a marked r e d u c t i o n  (1971), on the other hand, have  i n growth r a t e s o f tumor-bearing  f l a t h e a d s o l e i n the second year o f l i f e ,  as compared w i t h  normal f i s h ; t h i s i n d i c a t e s t h a t the l e s i o n s have a d e t r i mental e f f e c t on the h e a l t h o f the f i s h .  Wellings  and h i s  co-workers (1964) found t h a t i t c o u l d n o t be shown t h a t the r a t e o f disappearance o f tumorous f i s h from the p o p u l a t i o n exceeded t h a t o f normal f i s h . can be made concerning bearing  Although no d e f i n i t e statement  the f a t e o f the l e s i o n s o r o f f i s h  the l e s i o n s , i t seems c e r t a i n t h a t some f l a t f i s h  must d i e from the a f f l i c t i o n o f EPs. or  Although no  metastases  t r u l y i n v a s i v e growths were observed, f a t a l i t i e s c o u l d be  caused by i n d i r e c t e f f e c t s o f the d i s e a s e , o f which t h e r e are  many p o s s i b i l i t i e s .  The p o s i t i o n o f l e s i o n s p r o b a b l y  has an obvious e f f e c t on the h e a l t h o f the f i s h , when they occur about the g i l l s , eyes, or mouth.  especially F i n tumors  c o u l d be expected t o cause s l u g g i s h n e s s and would decrease the  f i s h ' s a b i l i t y t o escape p r e d a t o r s .  Any  debilitating  e f f e c t on the h e a l t h o f the f l a t f i s h i s l i k e l y t o i n c r e a s e the  s u s c e p t i b i l i t y o f the f i s h to o t h e r agents (e.g.  b a c t e r i a l i n f e c t i o n s ) , p r e d a t o r s , or harsh environmental conditions. P o s s i b l y , both f a c t o r s  ( f i s h death and tumour  n e c r o s i s and l o s s ) are a t work i n the removal o f numbers of tumour-bearing  f l a t f i s h from the p o p u l a t i o n . C o n c e i v a b l y ,  s u r v i v a l o f tumour-bearing  f i s h long enough f o r the tumours  to mature, and necrose, i s r e q u i s i t e f o r the phenomenon o f t i s s u e n e c r o s i s and  sloughing-off.  The nature o f the u n i 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 o f f l a t f i s h s k i n l e s i o n s remains, t o some e x t e n t , u n c e r t a i n . have been proposed  (Brooks et^ al^., 1969):  Two  1) the c e l l s  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 formed f i s h c e l l s .  cells  i s similar,  unidentified cells of  are  are t r a n s -  The u n i d e n t i f i e d c e l l s were p o s t u l a t e d  to be u n i c e l l u l a r p a r a s i t e s because: the  hypotheses  1) the morphology o f  i n some ways, t o protozoans, 2) the  are o f t e n found, i n the stroma, i n cords  two to f o u r , 3) the c e l l s a r e u s u a l l y surrounded by a  91 50 nm wide c e l l c o a t , 4) the stroma shows evidence o f an inflammatory response, 5) c o l l a g e n and f i b r o b l a s t p r o c e s s e s o f t e n e n c i r c l e the c e l l s , and 6) macrophages can be seen i n the p r o c e s s of e n g u l f i n g the u n i d e n t i f i e d c e l l s . c o a t s were observed around the c e l l s  No  i n the p r e s e n t study.  The c e l l s s t u d i e d i n the lemon s o l e showed no r e a l to  any d e s c r i b e d u n i c e l l u l a r organism  cell  resemblance  (see P i t e l k a ,  although o t h e r workers r e p o r t e d a resemblance  1963),  to a h a p l o -  s p o r i d i a n p a r a s i t e o f f i s h as d e s c r i b e d by P e r k i n s 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 o f the  lemon s o l e possessed no e v i d e n t l y f u n c t i o n a l c y t o p l a s m i c o r g a n e l l e s ; those c e l l s which d i d , appeared t o be t i o n s between normal epidermal c e l l s and the new The i n i t i a l appearance  o f t h i s new  transicell  c e l l type e v i d e n t l y  o c c u r s , i n the stroma, a f t e r the a n g i o b l a s t i c and  fibro-  b l a s t i c p r o l i f e r a t i o n and, i n the e p i d e r m i s , a f t e r an typical hyperplasia.  type.  initial  These o b s e r v a t i o n s suggest t h a t these  u n i d e n t i f i e d c e l l s occur as a r e s u l t o f , and not as a cause of,  the t i s s u e r e a c t i o n s c h a r a c t e r i s t i c o f the d i s e a s e .  The inflammatory nature o f the AEN  stroma p o s s i b l y c o u l d  be e l i c i t e d by these c e l l s i f they underwent a n t i g e n i c changes due t o t r a n s f o r m a t i o n or i n f e c t i o n , s i m i l a r t o the phenomenon of  v i r u s - i n d u c e d tumor a n t i g e n s . C e l l u l a r hypertrophic reactions i n f i s h  of  cells  v a r i o u s s p e c i e s have been shown t o be e l i c i t e d by  agents  several  (Weissenberg, 1949); these agents i n c l u d e v i r u s e s  ( N i g r e l l i and R u g g i e r i , 1965; Weissenberg,  1965),  Rickettsia  (Wolke, 1970), b a c t e r i a protozoans  (Davis, 1953),  f u n g i , and p a r a s i t i c  ( N i g r e l l i and Smith, 1940; N i g r e l l i , 1948b;  1968; Weissenberg,  1968; Lom, 1970; Trager, 1974).  Sprague,  Ultra-  s t r u c t u r a l s t u d i e s o f the u n i d e n t i f i e d 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 t o any o f the above agents, o r t o any c y t o p a t h o l o g i c a l changes (except hypertrophy) e l i c i t e d by these agents. H i s t o l o g i c evidence presented i n t h i s  thesis  i n d i c a t e s t h a t the u n i d e n t i f i e d stromal and epidermal  cells  found i n f l a t f i s h l e s i o n s p r o b a b l y a r e transformed f i s h r a t h e r than protozoan p a r a s i t e s ; the transformed c e l l s from the normal  differ  i n t h a t they a r e extremely h y p e r t r o p h i e d and  show c h a r a c t e r i s t i c s o f extreme and i r r e v e r s i b l e dmage.  cells,  T h i s c o n c l u s i o n i s based on:  cellular  1) the absence  evidence i n d i c a t i v e o f protozoan o r other p a r a s i t i c  o f any 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) o b s e r v a t i o n s o f a number o f m o r p h o l o g i c a l i n t e r m e d i a t e s between normal  epidermal  c e l l s and the g i a n t c e l l types found i n the l e s i o n s .  The  f o l l o w i n g d i s c u s s i o n i l l u s t r a t e s what I have concluded t o be a l i k e l y p r o g r e s s i o n o f c y t o p a t h i c events i n v o l v e d i n the t r a n s f o r m a t i o n o f normal c e l l s t o g i a n t c e l l t y p e s . Probably the f i r s t r e a c t i o n o f the epidermal i s a l o s s o f c e l l - t o - c e l l c o n t a c t , which i n some cases  cells appears  to r e s u l t i n an i n c r e a s e i n i n t r a c e l l u l a r space and i n the formation o f s t e l l a t e or " p r i c k l e " c e l l s , a c o n d i t i o n o f t e n noted i n h i s t o l o g i c a l s t u d i e s o f h y p e r p l a s t i c t i s s u e s 44).  Presumably  (Figure  the c e l l s then lose desmosomal c o n t a c t and  93 " b a l l up" (Figure 4 1 ) . V a c u o l a t i o n o f the c e l l a p p a r e n t l y f o l l o w s , as does l o s s o f c e l l volume r e g u l a t i o n .  The  i n c r e a s i n g l y severe v a c u o l a t i o n i s c o r r e l a t e d w i t h the d i s appearance o f a l l c y t o p l a s m i c o r g a n e l l e s ( F i g u r e s 41 and 4 2 ) . Very o f t e n the vacuoles tend t o be e l o n g a t e d , s t r u c t u r e s which resemble  horseshoe-shaped  d i l a t e d endoplasmic r e t i c u l u m  (Figure 4 8 ) ; other l a r g e r , more rounded v a c u o l e s o f t e n c o n t a i n membrane remnants and resemble chondria.  d i l a t e d , degenerated  mito-  The m a t r i x o f such vacuoles i s c l e a r ; membranes  resembling c r i s t a e tend t o be extremely s m a l l , i r r e g u l a r , and l o c a t e d a t the vacuole p e r i p h e r y (Figure 4 9 ) . These o b s e r v a t i o n s tend t o suggest t h a t these "mitochondria" (Wellings e t a l . , 1967; Brown e t a l . , 1969) a r e n o n - f u n c t i o n a l , d e g e n e r a t i v e structures.  The c y t o p l a s m i c vacuoles can be found bounded by  e i t h e r a s i n g l e or a double presumably depending  (the more common) membrane,  on t h e i r o r i g i n  ( F i g u r e s 48 and 4 9 ) .  On the b a s i s o f the above o b s e r v a t i o n s , I b e l i e v e these vacuoles t o be the product o f damaged and d i l a t e d mitochondria and endoplasmic  reticulum.  the cytoplasm, another 38 and 4 9 ) .  Often membrane  "whorls" occur i n  i n d i c a t i o n o f c e l l u l a r damage ( F i g u r e s  The c y t o p l a s m i c ground substance tends t o become  i r r e g u l a r l y amorphic and may become extremely l i g h t The n u c l e i o f g i a n t c e l l e n l a r g e d ; chromatin  (Figure 46) .  types i n mature l e s i o n s tend t o be  i s always clumped i n t o d i s c r e t e , random  clumps, n u c l e o l i are e n l a r g e d , prominent, s i v e l y of ribosome-like p a r t i c l e s  and c o n s i s t e x c l u -  ( F i g u r e 4 6 ) . These  o b s e r v a t i o n s tend t o i n d i c a t e degenerative p r o c e s s e s ; the  94 c y t o p a t h i c changes d e s c r i b e d and u l t i m a t e n e c r o s i s 1971).  are changes t o n o n - f u n c t i o n a l i t y  (Trump and Ginn, 1969;  Trump and  Arstila,  I t i s important t o note here t h a t the s u b c e l l u l a r  p a t t e r n o f n e c r o s i s of f l a t f i s h  " X - c e l l s " d i f f e r s from the  p a t t e r n o f n e c r o s i s found i n o t h e r c e l l s i n j u r i e s to the c e l l .  i n r e a c t i o n to l e t h a l  In p a r t i c u l a r , n e c l e o l a r d i s i n t e g r a t i o n  and k a r y o l y s i s , common changes i n n e c r o t i c c e l l s 1968), do n o t occur i n f l a t f i s h tumour c e l l s . suggest t h a t the n e c r o t i c changes d e s c r i b e d  (Ginn e t a l . ,  T h i s would  in flatfish  cells  are not the r e s u l t o f a n o n - s p e c i f i c l e t h a l i n j u r y t o the cell  (e.g. a n o x i a ) , but may  i n s t e a d be the 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 ( i . e . , changes i n the genome) or o f a s p e c i f i c s u b c e l l u l a r i n j u r y 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 o f both the hypert r o p h i c epidermal c e l l types as w e l l as the analogous c e l l type.  C e l l s c l a s s i f i e d as type A g i a n t 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 f u r t h e r trophy and v a c u o l a t i o n cells.  stromal  hyper-  than have the s m a l l e r , type B, g i a n t  They are c h a r a c t e r i z e d by the appearance  of large  numbers o f extremely e l e c t r o n - d e n s e c y t o p l a s m i c i n c l u s i o n s o f unknown o r i g i n and composition (Figure 46 and 51). and h i s co-workers  Brooks  (1969) i n d i c a t e d t h a t 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 r e q u i r e d , s i n c e the c e l l s are found i n both stromal and epidermal components o f the l e s i o n .  This  hypothesis need not be the c a s e .  and  Staining properties  u l t r a s t r u c t u r a l s t u d i e s o f the two c e l l types i n d i c a t e c e r t a i n  d i f f e r e n c e s between the epidermal and s t r o m a l unknown c e l l s . These d i f f e r e n c e s might a r i s e i f the o r i g i n o f the c e l l types were d i f f e r e n t :  a degenerative, n e c r o t i c disease  p r o c e s s presumably would be capable o f c a u s i n g s i m i l a r c y t o p a t h i c e f f e c t s i n stromal f i b r o b l a s t s , f o r i n s t a n c e , and i n epidermal c e l l s . observed  The stromal u n i d e n t i f i e d c e l l type was  never  t o undergo extreme c e l l u l a r hypertrophy t o the e x t e n t  t h a t i t s analogous  epidermal c e l l type can.  the stromal c e l l type i s unknown; an obvious  The o r i g i n o f 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 t o whether the f l a t f i s h  epidermal  l e s i o n s should be c o n s i d e r e d as h y p e r p l a s t i c or n e o p l a s t i c processes  (Wellings e t a l . , 1968;  Mawdesley-Thomas, 1972).  There might a l s o be doubt as t o whether the l e s i o n s r e p r e s e n t a h y p e r t r o p h i c p r o c e s s w i t h compensatory h y p e r p l a s i a o f s u p e r f i c i a l and e n v e l o p i n g c e l l s , or whether the l e s i o n s are t r u e h y p e r p l a s t i c or n e o p l a s t i c r e a c t i o n s p r e c e d i n g a secondary, or  and i n c i d e n t a l , c e l l u l a r hypertrophy.  Discussion  s p e c u l a t i o n without f u r t h e r s t u d i e s would be i n c o n c l u s i v e  at best.  Some workers (Wellings e t a l . , 1965;  W e l l i n g s , 1971) Nigrelli  Miller  and  b e l i e v e d the l e s i o n s to be t r u e neoplasms.  and h i s co-workers (1965) p o s t u l a t e d t h a t the l e s i o n s  were h y p e r p l a s t i c i n n a t u r e .  The d i f f e r e n c e between  p l a s i a and n e o p l a s i a u s u a l l y cannot be determined h i s t o l o g i c a l s t u d i e s , as noted i n the case o f f i s h (Schlumberger  and Lucke, 1948).  hyper-  by pox  According t o the d e f i n i t i o n  of neoplastic hyperplasia  (Prehn, 1972)  as:  "that form o f  h y p e r p l a s i a which i s caused, a t l e a s t i n p a r t , by an  intrinsic  i n h e r i t a b l e abnormality i n the i n v o l v e d c e l l s " , animal neoplasms must be t r a n s f e r r a b l e by the i n o c u l a t i o n o f  living  neoplastic c e l l s .  flatfish  Transplantation  tumors have a l l proved negative Wellings,  1964; The  McArn, 1968;  be  Wellings,  Chuinard  above; the r e l a t i o n s h i p o f these p a r t i c l e s t o the has  i n the given disease  not been determined.  V i r u s e s have been proven to be the cause o f c e l l u l a r hypertrophic c y s t i s disease  d i s e a s e o f f i s h epidermis,  (Weissenberg, 1965;  epidermal h y p e r p l a s t i c d i s e a s e s  Wolf e t a l . , 1966),  (Nigrelli,  1952;  in fish.  1970), and  and  other  These l a t t e r  Mawdesley-Thomas  Bucke, 1967), the c a u l i f l o w e r d i s e a s e  one  lympho-  have been i m p l i c a t e d as a probable cause o f s e v e r a l  i n c l u d e f i s h pox  process;  f a c t o r approach a p p l i e s t o f l a t f i s h  Evidence of v i r u s - l i k e p a r t i c l e s p r e s e n t  i f any,  Many  o f any d i s e a s e  c e l l s o f mature epidermal l e s i o n s o f lemon s o l e was  process,  and  1969).  f l a t f i s h remains an enigma.  i n v o l v e d i n the genesis  probably t h i s multiple lesions.  (Good, 1940;  a e t i o l o g y o f epidermal p a p i l l o m a - l i k e l e s i o n s  a f f l i c t i n g P a c i f i c coast f a c t o r s may  s t u d i e s done on  of eels  and  (Koops e t a l . ,  other h y p e r p l a s t i c r e a c t i o n s o f f i s h e p i d e r m i s  (Walker, 1966  and  1968).  E l e c t r o n microscopic  v i r u s - l i k e p a r t i c l e s has been g i v e n  evidence o f  in this report  52-54) as w e l l as i n other r e p o r t s o f s t u d i e s o f epidermal l e s i o n s (Wellings e t al_., 1964,  1965  (Figures  flatfish  and  1967;  McArn, 1968;  K e l l y , 1971).  The use o f v i r a l techniques i n  i s o l a t i o n or t r a n s m i s s i o n o f the v i r u s has proved n e g a t i v e (Wellings §_t a l . , 1965;  McArn, 1968;  W e l l i n g s , 1969).  From the evidence presented i n t h i s t h e s i s , i t seems reasonable to conclude t h a t :  1) e p i t h e l i a l tumours i n P a c i f i c  c o a s t f l a t f i s h p r o g r e s s from i n i t i a l a n g i o e p i t h e l i a l  nodules  t o mature epidermal p a p i l l o m a - l i k e growths, and 2) t h i s p r o g r e s s i o n i s m a n i f e s t i n h i s t o l o g i c a l and changes, as documented above.  cytological  Other evidence presented i n  t h i s t h e s i s suggests t h a t the u n i d e n t i f i e d  " X - c e l l s " which  compose the mature t i s s u e are transformed, n e c r o t i c  fish  epidermal c e l l s , r a t h e r than i n t r a - o r e x t r a c e l l u l a r p a r a s i t i c protozoans  (as has been h y p o t h e s i z e d ) .  Evidence f o r  the p r o g r e s s i o n o f normal epidermal c e l l s i n t o  "X-cells"  i s p r i m a r i l y the presence o f morphologic i n t e r m e d i a t e s , the t i m i n g o f appearance o f the new and the l a c k o f resemblance  c e l l type i n the t i s s u e ,  o f the " X - c e l l s " to any known  protozoan.  F u r t h e r work must be done t o d e f i n i t e l y  this fact.  The  establish  f u l l b i o l o g i c a l nature o f the l e s i o n s  (i.e.,  whether the growths are the r e s u l t o f h y p e r p l a s t i c , neop l a s t i c , o r h y p e r t r o p h i c p r o c e s s e s ) i s unknown, and work i s c e r t a i n l y needed and d e s i r a b l e .  further  Autoradiographic  s t u d i e s o f the v a r i o u s tumour stages would show where (by which c e l l s ) h y p e r p l a s t i c or n e o p l a s t i c growth was o c c u r r i n g . F u r t h e r , and more e x t e n s i v e , o b s e r v a t i o n s o f f i s h under l a b o r a t o r y c o n d i t i o n s may  tumour-bearing  be u s e f u l i n determining  the h y p e r p l a s t i c or n e o p l a s t i c nature o f the l e s i o n s .  The  nature o f f l a t f i s h tumours i s p r e s e n t l y u n c l e a r and s i m i l i t u d e o f these growths and seems q u e s t i o n a b l e .  t r u e epidermal  p a p i l l o m a s , carcinomas, t o warrant f u r t h e r  papillomas  C e r t a i n l y the histomorphology  f l a t f i s h lesions differs s u f f i c i e n t l y  o f the  from other known  or other d i s e a s e s o f f i s h  study.  the  epidermis  99  LITERATURE CITED A n g e l l , C.L.  and M i l l e r , B.S.  E n g l i s h Sole  (Parophrys  J . F i s h Res.  Bd. Can.  (1974), Ecology o f Tumor-bearing v e t u l u s ) i n Puget Sound, Washington.  (submitted).  ( C i t e d by Mearns and  Sherwood, 1974). Andrew, W.  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