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Clear Cell and Endometrioid Carcinomas : are their differences attributable to distinct cells of origin? Cochrane, Dawn Renee; Tessier-Cloutier, Basile; Lawrence, Katherine M.; Nazeran, Tayyebeh; Karnezis, Anthony N.; Salamanca, Clara; Cheng, Angela S.; McAlpine, Jessica N.; Hoang, Lien N.; Gilks, C. Blake; Huntsman, David G. Apr 23, 2017

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Clear	Cell	and	Endometrioid	Carcinomas:		are	their	differences	attributable	to	distinct	cells	of	origin?	Dawn	R	Cochrane1,	Basile	Tessier-Cloutier2,	Katherine	M	Lawrence1,	Tayyebeh	Nazeran2,	Anthony	N.	Karnezis2,,	Clara	Salamanca1,	Angela	S.	Cheng1,	Jessica	N	McAlpine3,	Lien	N	Hoang2,4,	C	Blake	Gilks2,4	and	David	G	Huntsman1,2,3,4.	1.	Department	of	Molecular	Oncology,	BC	Cancer	Agency,	Vancouver,	BC,	Canada	2.	Department	of	Pathology	and	Laboratory	Medicine,	University	of	British	Columbia,	Vancouver,	BC,	Canada	3.	Department	of	Gynecology	and	Obstetrics,	University	of	British	Columbia,	Vancouver,	BC,	Canada	4.	Department	of	Anatomical	Pathology,	Vancouver	General	Hospital,	Vancouver,	BC,	Canada			Running	Title:	Distinct	cellular	origins	for	endometrioid	and	clear	cell	cancers	Abstract	Endometrial	epithelium	is	the	presumed	tissue	of	origin	for	both	eutopic	and	endometriosis-derived	clear	cell	and	endometrioid	carcinomas.		We	had	previously	hypothesized	that	the	morphological,	biological	and	clinical	differences	between	these	cancers	are	due	to	subtype-restricted	underlying	mutations.	Although	some	mutations	and	genomic	landscape	features	are	more	likely	to	be	found	in	one	of	these	histotypes,	we	were	not	able	to	identify	a	single	class	of	mutations	that	was	exclusively	present	in	one	histotype	and	not	the	other.	This	lack	of	genomic	differences	led	us	to	an	alternative	hypothesis	that	these	cancers	arise	from	distinct	cells	of	origin	within	endometrial	tissue,	and	it	is	the	cellular	context	that	accounts	for	their	differences.		In	a	proteomics	screen,	we	have	identified	CTH	as	a	marker	for	clear	cell	carcinoma	differentiation,	as	it	is	expressed	at	high	levels	in	clear	cell	carcinomas	of	the	ovary	and	endometrium.		We	analyzed	normal	Müllerian	tissues	and	found	CTH	was	expressed	in	ciliated	cells	of	endometrium	(both	eutopic	endometrium	and	endometriosis)	and	fallopian	tube.		We	have	since	determined	that	other	ciliated	cell	markers	are	expressed	in	clear	cell	carcinomas	whereas	endometrial	secretory	cell	markers	are	expressed	in	endometrioid	carcinomas.		To	determine	whether	the	ciliated	endometrial	cells	are	uterine	derived	we	developed	a	3D	organoid	culture	system,	which	reliably	produced	both	ciliated	and	secretory	cells.		Clear	cell	carcinoma	is	an	IL-6	driven	tumour	and	lineage	experiments	on	bronchial	epithelium	have	shown	that	IL-6	is	an	essential	pathway	in	maintaining	the	population	of	ciliated	cells.		Taken	together	we	hypothesize	that	endometrioid	carcinomas	are	derived	from	cells	of	secretory	cell	lineage	whereas	clear	cell	carcinomas	are	derived	from	cells	of	endometrial	origin	that	share	features	with	a	ciliated	cell	lineage.			Keywords	(3-10)	Clear	cell	cancer;	endometrioid	cancer;	endometriosis;	ciliated	cells	Introduction		 Ovarian	cancers	are	subdivided	into	histotypes	that	display	distinct	histological	and	clinical	features	[1-3].	The	different	properties	of	the	histotypes	can	be	partially	explained	by	their	histogenesis.	The	majority	of	ovarian	cancers	are	proposed	to	have	extra-ovarian	origins	with	the	ovary	providing	a	rich	“soil”,	permissive	for	the	growth	of	cancerous	cells	(as	reviewed	by	[4]).	For	example	it	is	likely	that	the	majority	of	high-grade	serous	(HGS)	ovarian	cancers	originate	in	the	fallopian	tube	[5-7].	The	model	of	HGS	genesis	begins	with	the	appearance	of	p53	signature	in	fallopian	tube	secretory	cells,	progressing	to	a	serous	tubal	intraepithelial	carcinoma	(STIC)	lesion	prior	to	becoming	invasive	cancer	[8-10].	The	origins	of	the	other	subtypes	of	ovarian	cancer	are	not	as	well	characterized.	Clear	cell	ovarian	cancer	(CCOC)	and	endometrioid	ovarian	cancer	(ENOC)	are	both	associated	with	ovarian	endometriotic	cysts,	or	endometriomas,	which	are	thought	to	be	the	precursor	lesion	of	these	cancers	[11].	The	presence	of	endometrioma	confers	up	to	a	3	times	increased	risk	of	CCOC	and	ENOC	[12].	Furthermore,	endometriosis,	both	adjacent	and	distal	to	cancer,	has	been	found	to	contain	mutations	common	to	the	associated	cancer,	indicating	a	clonal	relationship	[13,	14].	However,	as	these	mutations	can	be	found	in	endometriotic	lesions	without	associated	cancer	and	at	sites	with	minimal	transformational	potential,	the	role	they	play	in	oncogenesis	is	uncertain	[13,	15].	This	finding	also	suggests	that	clonal	relationships	between	adjacent	CCOC	and	ENOC	may	reflect	the	mutations	in	a	shared	endometriotic	cyst	and	not	a	common	process	of	transformation	or	cell	of	origin.			 It	remains	unclear,	however,	how	endometriosis	gives	rise	to	two	distinct	subtypes	of	ovarian	cancer.	ARID1A	mutations,	while	slightly	more	common	in	CCOC,	are	frequent	in	both	subtypes	[16].	Mutations	in	CTTNB1	are	more	frequent	in	ENOC,	but	can	also	be	found	in	CCOC	[17].	There	is	not	a	single	genetic	mutation	that	is	unique	to	either	subtype	and	genomic	landscape	features	are	not	mutually	exclusive	[18].	In	an	effort	to	further	understand	the	differences	between	the	three	most	common	subtypes	of	ovarian	cancer,	we	undertook	a	global	proteomic	analysis.	From	these	studies,	we	identified	cystathionine	gamma	lyase	(CTH)	to	be	highly	expressed	in	CCOC	compared	to	ENOC	and	HGS	[19].			 In	the	current	studies,	we	build	on	the	proteomic	results	to	provide	correlative	evidence	that	proteins	expressed	in	CCOC	tend	to	be	expressed	in	ciliated	cells	of	the	endometrium.	Conversely,	ENOC	and	secretory	epithelial	cells	tend	to	express	some	of	the	same	proteins.	We	propose	a	cell	context-based	model	of	endometriosis-associated	cancers	that	explains	how	endometriosis	gives	rise	to	two	different	ovarian	cancer	histotypes.	Methods	and	Materials	Immunohistochemistry	Tissue	microarrays	(TMAs)	were	constructed	using	duplicate	0.6mm	cores	of	formalin	fixed	paraffin	embedded	(FFPE)	materials	of	485	primary	ovarian	epithelial,	sex	cord	stromal	and	germ	cell	tumors.		4	μm	sections	of	the	TMAs	or	FFPE	embedded	normal	fallopian	tube	(5	cases),	endometrium	(5	cases),	typical	endometriosis	(4	cases),	atypical	endometriosis	adjacent	to	CCOC	(4	cases)	or	atypical	endometriosis	adjacent	to	ENOC	(3	cases)	were	sectioned	onto	Superfrost+	glass	slides.	The	slides	were	processed	using	the	automated	Ventana	Benchmark	and	Discovery	systems	(Ventana	Medical	Systems).	Description	of	antibodies	and	optimization	are	found	in	Supplemental	Methods.	This	include	a	modified	protocol	for	estogen	receptor	alpha	(ER)	staining	designed	to	provide	a	dynamic	range	of	expression	in	Müllerian	tissue,	as	opposed	to	the	protocol	used	for	staining	breast	carcinomas;	this	was	done	by	decreasing	the	primary	antibody	concentration	so	as	to	be	able	to	distinguish	between	weak	and	intermediate	or	strong	staining.	Slides	were	stained	with	antibodies	for	CTH	(1:1000,	LSBio);	Ezrin	(1:1000,	Sigma);	ER	(1:100,	ThermoFisher);	MTHFD1	(1:50,	Sigma)	and	Napsin		A	(1:100,	Cell	Marque).	Scoring	of	the	TMAs	was	assessed	by	an	anatomical	pathologist	(BT-C).	Positivity	was	evaluated	by	H-Score,	a	combination	of	staining	intensity	and	percentage	of	tumor	cell	staining.	Staining	intensity	was	scored	as	0	(none	or	staining	in	less	than	10%	tumor	cells),	1	(weak),	2	(moderate),	or	3	(strong)	and	each	score	multiplied	by	the	percentage	of	cells	(0-100%)	staining.	Therefore,	H	scores	ranged	from	0	to	300. Immunofluorescence	Heat	immobilized	sections	(4µm)	were	deparaffinised	and	subjected	to	steam	heat	for	antigen	retrieval.	Slides	were	blocked	with	normal	goat	serum	and	probed	with	primary	antibodies	as	follows:	CTH	(1:100;	mouse	monoclonal	clone	1E12,	LSBio);	CTH	(1:50;	rabbit	polyclonal,	LS-C312801,	Lifespan	Bioscience);	Ezrin	(1:100;	rabbit	polyclonal,	HPA021616,	Sigma);	ER	(1:200;	rabbit	monoclonal	SP1,	ThermoFisher);	FOXJ1	(1:100;	mouse	clone	3-19,	Abcam)	and	MTHFD1	(1:100;	rabbit	polyclonal,	HPA000704,	Sigma).	Secondary	antibodies	used	were	goat	anti-Mouse	IgG	conjugated	Alexa	594	(1:500;	A11005,	ThermoFisher)	and	goat	anti-Rabbit	IgG	conjugated	Alexa	488	(1:500;	A11008,	ThermoFisher).	Slides	were	mounted	in	Fluorshield	mounting	media	containing	DAPI	(Sigma).		Organoid	culture	Organoid	culture	of	normal	endometrium	was	performed	using	a	modification	of	a	previously	described	protocol	for	fallopian	tube	organoid	culture	[20].	Detailed	protocol	can	be	found	in	Supplemental	Methods.			Results	Clear	cell	ovarian	cancer	markers	are	also	expressed	in	normal	Müllerian	tissue	To	better	understand	the	biology	underlying	the	different	histotypes	of	ovarian	cancer,	we	have	previously	performed	whole	proteome	profiling	of	HGS,	ENOC	and	CCOC	tumors	[19].	Of	particular	interest	were	the	differences	in	ENOC	and	CCOC,	as	these	tumors	usually	arise	from	the	same	precursor	lesion,	endometriosis.	We	found	cystathionine	gamma	lyase	(CTH)	was	higher	in	CCOC	than	in	ENOC.	These	findings	were	confirmed	by	immunohistochemistry	(IHC)	on	a	tissue	microarray	(TMA)	containing	different	subtypes	of	ovarian	cancer	(Supplemental	Figure	1).	We	also	find	that	CTH	stains	highly	in	endometrial	clear	cell	carcinoma	compared	to	endometrial	endometrioid	cancer	(Supplemental	Figure	1).		To	determine	the	expression	pattern	of	CTH	in	normal	Müllerian	tissues,	we	performed	IHC	on	normal	endometrium	(secretory	phase	shown)	and	fallopian	tube.	In	both	tissues,	CTH	was	very	highly	expressed	in	ciliated	cells,	with	much	lower	expression	in	secretory	cells	(Figure	1).	Since	endometriosis	is	thought	to	be	the	precursor	lesion	of	CCOC,	we	examined	expression	of	CTH	and	found	that	there	was	high	CTH	expression.	These	observations	led	us	to	examine	the	expression	of	a	ciliated	cell	protein,	Ezrin	[21].	Similar	to	CTH,	Ezrin	is	also	highly	expressed	in	ciliated	cells	of	the	endometrium,	fallopian	tube	and	in	endometrioma,	with	little	or	no	expression	in	secretory	cells	(Figure	1).	In	the	endometrioma,	the	cells	with	strong	Ezrin	staining	have	visible	cilia.	We	next	examined	expression	of	Napsin	A,	a	known	CCOC	marker	[22-24].	We	found	weakly	positive	expression	of	Napsin	in	typical	endometriosis,	and	no	Napsin	expression	observed	in	the	normal	endometrium	and	fallopian	tube	(Figure	1).	As	we	had	observed	previously,	CTH	was	highly	expressed	in	CCOC,	and	here	we	find	CTH	also	highly	expressed	in	atypical	endometriosis	adjacent	to	the	tumor	and	clear	cell	borderline	tumor	(Figure	2).	Ezrin	and	Napsin	are	also	expressed	in	CCOC	tumor,	borderline	tumor	and	adjacent	endometriosis	(Figure	2).	Staining	of	the	ovarian	tumor	TMA	for	Ezrin	and	Napsin	revealed	that	they	are	both	expressed	at	higher	levels	in	CCOC	than	in	ENOC	(Supplemental	Figure	1).	The	expression	of	ciliated	markers	in	CCOC	led	us	to	postulate	that	ciliated	cells	are	the	cell	of	origin	for	this	cancer.	Since	Napsin	is	not	expressed	in	the	normal	Müllerian	tissues,	we	hypothesize	it’s	expression	does	not	indicate	cell	of	origin,	but	results	from	metabolic	or	other	perturbations	that	occur	later	in	the	transformation	process.		The	observation	that	there	are	shared	proteins	in	CCOC	and	ciliated	cells	led	us	to	postulate	the	cells	of	origin	of	CCOC	are	similar	to	ciliated	cells	in	ectopic	endometrium.	By	extension,	we	questioned	whether	ENOC	arises	from	a	different	cell	population,	i.e.	the	secretory	cells.		Endometrioid	ovarian	cancer	markers	are	expressed	in	secretory	cells	of	normal	Müllerian	tissue	To	determine	whether	there	will	be	shared	expression	of	some	proteins	in	ENOC	and	secretory	cells	of	Müllerian	epithelium	we	studied	methylenetetrahydrofolate	dehydrogenase	1	(MTHFD1)	a	protein	expressed	at	a	higher	level	in	ENOC	compared	to	CCOC	in	our	proteomic	data	[19]	and	the	estrogen	receptor	alpha	(ER)	which	has	been	previously	reported	to	be	rarely	expressed	in	CCOC	[25-27].	We	note	that	ER	was	not	found	to	be	differentially	expressed	in	the	proteomics	screen	likely	because	ER	can	also	be	expressed	in	the	stroma.	We	examined	the	expression	of	MTHFD1	and	ER	by	IHC	staining	of	the	ovarian	tumor	TMA	and	found	that	both	are	more	highly	expressed	in	ENOC	compared	to	CCOC	(Figure	3A).	In	the	normal	Müllerian	tissue,	we	found	that	MTHFD1	displayed	diffuse	staining	of	the	glandular	epithelial	cells,	which	are	largely	secretory	cells	(Figure	3B).	In	the	fallopian	tube,	where	ciliated	cells	are	more	numerous,	it	is	more	apparent	that	MTHFD1	expression	is	primarily	in	the	secretory	cells,	with	little	or	no	expression	in	the	ciliated	cells.	Furthermore,	MTHFD1	is	expressed	in	the	secretory	cells	in	endometriosis.	Similarly,	ER	is	expressed	in	the	secretory	cells	of	the	endometrium,	fallopian	tube,	and	in	endometriosis,	but	expressed	at	much	lower	levels	in	ciliated	cells.	Next,	we	examined	ENOC	tumors	and	adjacent	endometriosis.	We	found	that	CTH,	ER	and	MTHFD1	expression	in	the	tumors	phenocopied	what	is	observed	in	the	adjacent	endometriosis	(Figure	3C).	Namely,	that	CTH	has	a	weak	diffuse	pattern	of	staining	(much	weaker	than	what	is	observed	in	CCOC	tumors),	while	ER	and	MTHFD1	positively	stain	both	the	tumor	and	adjacent	endometriosis.		CCOC	and	ENOC	markers	in	normal	endometrial	tissue	To	verify	that	CTH	was	staining	the	ciliated	cells	and	not	the	secretory	cells,	dual	immunofluorescence	in	normal	endometrium	was	performed.	Dual	staining	of	CTH	and	FOXJ1,	a	transcription	factor	specific	for	ciliated	cells	[28],	reveals	that	CTH	and	FOXJ1	stain	the	same	cells	(Figure	4A).	Similarly,	Ezrin	and	CTH	also	both	stain	the	same	cells	in	the	normal	endometrium	(Figure	4B).	To	verify	that	CTH	was	not	staining	the	secretory	cells,	dual	IF	was	performed	for	CTH/ER	and	CTH/MTHFD1.	In	both	cases,	the	staining	of	CTH	was	mutually	exclusive	to	the	secretory	cell	markers	(Figures	4C,	4D).	Similar	results	were	observed	in	normal	fallopian	tube	(Supplemental	Figure	2).		Ciliated	cells	in	the	endometrium	are	often	thought	to	be	metaplastic	in	nature	[29].	To	ensure	that	we	are	observing	endometrium	derived	ciliated	cells,	we	turned	to	an	in	vitro	model	system.	We	grew	organoid	cultures	from	normal	endometrium	samples	(Figure	5A).	The	organoids	have	a	pseudostratified	columnar	appearance,	comparable	to	the	pattern	that	is	often	observed	in	normal	endometrial	glands.	The	organoids	are	composed	mostly	of	secretory	cells,	with	occasional	ciliated	cells,	similar	to	the	composition	of	normal	endometrial	glands.	The	dual	immunofluorescent	staining	pattern	recapitulates	the	pattern	observed	in	the	normal	endometrial	glands	(Figure	5B).	The	organoid	culture	demonstrates	that	the	stem	cell	population	of	the	normal	endometrium	is	able	to	differentiate	into	both	secretory	and	ciliated	cells,	a	finding	that	has	been	recently	observed	by	others	using	a	similar,	but	not	identical,	protocol	[30].		Discussion	The	importance	of	cell	context	in	shaping	the	oncogenic	potential	of	mutations	as	well	as	the	phenotype	of	resulting	neoplasms	is	well	known	[31-33].	Clinical	examples	include	the	many	unusual	tumours	that	share	the	ETV6:NTRK3	gene	fusion	events	[33,	34]	or	the	presence	of	KRAS	mutations	as	driver	events	in	many	cancers	but	also	in	endometriosis,	a	diseases	generally	thought	to	be	non-neoplastic.	This	concept	has	been	recapitulated	in	mouse	models,	including	the	demonstration	that	the	same	mutations	lead	to	distinct	cancer	phenotypes	depending	on	which	follicular	epithelial	cells	express	canonical	cancer	mutations	[35].	In	a	model	of	gynecologic	cancer,	the	combined	loss	of	PTEN	and	APC	in	ovarian	surface	epithelium	lead	to	high	grade	endometrioid-like	carcinomas,	whereas	the	same	events	in	secretory	cells	in	the	oviduct	lead	to	conventional	low	grade	endometrioid	carcinomas	that	more	accurately	resemble	the	clinicopathological	and	immunohistochemical	features	of	human	endometrioid	carcinoma,	indicating	the	Müllerian	cell	of	origin	[36,	37].	For	CCOC	and	ENOC,	the	finding	of	identical	mutations;	ARID1a,	PIK3CA	and	CTNNB1,	albeit	at	different	frequencies	in	these	histotypes,	suggests	that	their	clinical	and	phenotypic	distinctions	are	not	determined	by	mutation	alone.	Given	these	mutations	can	be	found	in	non	atypical	deep	infiltrating	endometriosis,	as	well	as	cancer	associated	ovarian	endometriomas,	the	usual	precursor	for	CCOC	and	ENOC,	it	is	uncertain	where	in	the	development	of	endometriosis	the	mutations	occur	and	what	role	they	play	in	that	process	[15].	We	have	recently	shown	that	although	genomic	landscape	features	correlate	with	ovarian	cancer	histotype	the	relationship	is	not	absolute	[18].	The	APOBEC	signature	is	most	commonly	found	in	CCOC	but	can	occur	in	ENOC,	and	whilst	we	found	microsatellite	instability	only	in	ENOC	in	our	cohort,	MSI	has	been	described	in	CCOC	in	other	studies	[38,	39].		It	is	not	known	when	in	the	transition	from	endometriosis	to	carcinoma	these	landscape	features	become	apparent.	 In	our	proposed	model	of	CCOC	and	ENOC	genesis,	CCOC	tumors	arise	from	cells	sharing	features	with	a	ciliated	cell	lineage,	whereas	ENOC	tumors	come	from	secretory	cells	or	their	precursors	(Figure	5).	Central	to	this	model	is	an	endometrial	progenitor	cell,	which	can	differentiate	towards	a	secretory	or	ciliated	cell	lineage,	depending	on	cues	provided	from	the	microenvironment.	In	normal	endometrium	and	in	organoid	culture,	the	secretory	cell	population	is	much	more	prevalent,	however,	a	progenitor	cell	in	an	endometrioma	will	be	exposed	to	different	factors	which	may	promote	ciliated	cell	differentiation	in	addition	to	mutation,	en	route	to	CCOC.		One	aspect	of	this	model	that	needs	to	be	addressed	is	the	differences	in	prevalence	of	clear	cell	and	endometrioid	cancers	of	the	ovary	and	the	endometrium.	There	is	roughly	equal	prevalence	of	clear	cell	and	endometrioid	subtypes	in	the	ovary	[40].	Conversely,	there	is	a	vast	difference	in	the	incidence	of	endometrioid	and	clear	cell	cancers	of	the	endometrium,	with	endometrioid	being	common	and	clear	cell	tumors	being	very	rare	[41].	One	explanation	can	be	the	relative	numbers	of	ciliated	and	secretory	cells	in	the	endometrium,	where	ciliated	cells	are	by	far	the	minor	population.	Throughout	the	menstrual	cycle,	ciliated	cells	range	from	5-20%	of	the	epithelial	population	of	the	endometrium	[42-44].	Tubal	metaplasia	of	the	endometrial	glandular	epithelium	is	more	prevalent	in	post-menopausal	endometrium,	which	is	when	the	majority	of	endometrial	clear	cell	cancers	occur,	although	the	two	have	not	been	linked	before	[29].		It	is	likely	that	the	environment	in	the	endometriotic	cyst	contributes	to	the	transformation	process.	Inside	the	endometriotic	cyst,	trapped	blood	leads	to	high	levels	of	reactive	oxygen	species,	proteolytic	enzymes	and	inflammation	[45-47].	We	believe	that	this	environment	may	act	on	the	progenitor	cells	to	increase	the	number	of	cells	on	the	ciliated	cell	lineage.	Metaplasia	is	often	linked	to	chronic	inflammation	and	is	thought	to	be	a	reprogramming	of	stem	cells	[48].	In	the	trachea,	IL-6	has	been	demonstrated	to	promote	ciliated	cell	differentiation	[49].	The	inflammatory	cytokines	such	as	IL-6	could	be	originating	from	the	inflammatory	cells	within	the	endometriotic	cyst,	or	from	the	ovarian	stroma	[50-52].	A	large	portion	of	CCOCs	express	IL-6	[53-55],	and	this	autocrine	IL-6	(or	another	bypass	mechanism)	may	be	an	integral	step	in	achieving	stromal	independence	during	oncogenesis.	If	inflammation	drives	cells	towards	a	ciliated	lineage	and	the	potential	for	transformation	to	CCOC,	then	other	microenvironmental	factors,	perhaps	in	combination	with	specific	mutations	or	epigenomic	changes	could	lead	to	oncognesis	along	a	sectreotry	lineage	towards	ENOC.	The	organoid	culture	system	described	could	be	used	to	model	the	interplay	between	mutations,	microenvironmental	factors,	and	other	modifiable	risk	factors,	such	as	hormonal	manipulation,	to	determine	their	role	in	differentiation	and	transformation.			It	is	interesting	to	note	that	in	the	CTH-high	cells	observed	in	the	endometriosis	adjacent	to	CCOC	do	not	have	any	apparent	cilia.	These	could	either	be	cells	earlier	on	the	ciliated	cell	lineage	that	have	not	differentiated	to	the	point	of	expressing	cilia,	or	could	be	a	de-differentiation	of	ciliated	cells.	Napsin	is	a	marker	for	CCOC	[22-24],	however	it	is	not	expressed	to	any	appreciable	levels	in	normal	fallopian	tube,	endometrium	and	has	weak	expression	in	endometriosis.	It	appears	that	some	CCOC	markers	give	us	insight	into	the	cell	of	origins	(CTH,	Ezrin),	while	others,	like	Napsin,	may	be	expressed	as	secondary	events	occurring	later	in	the	transformation	process.	Napsin	and	HNF1B,	another	well	known	marker	for	CCOC,	as	well	as	CTH	are	also	expressed	in	hypersecretory	glands	within	decidualized	endometrium,	the	Arias	–Stella	reaction	[56]	(data	not	shown).	Although	Arias-Stella	reaction	can	be	a	phenocopy	of	CCOC,	it	is	not	a	precursor	lesion,	and	may	speak	to	the	plasticity	of	Müllerian	secretory	cells	in	specific	stromal	microenvironments.								We	note	that	a	simple,	yet	intriguing	question	must	be	addressed	before	our	proposed	hypothesis	that	ciliated	cells	of	the	endometrium	(or	cells	which	share	a	lineage	with	terminally	differentiated	ciliated	cells)	are	the	cells	of	origin	for	clear	cell	carcinomas	of	the	uterus	and	ovary	is	accepted.		That	is	the	question	of	why	given	the	vast	numbers	of	ciliated	cells	in	the	fallopian	tube	that	we	never	see	clear	cell	carcinomas	of	that	organ.	It	is	possible	that	the	explanation	goes	beyond	the	importance	of	cell	context	and	mutation	underpinning	the	development	of	cancers,	and	demands	consideration	of	the	influence	of	particular	stromal	microenvironments.		The	fallopian	tube	is	a	highly	evolved	structure	designed	for	two	purposes:	1)	The	transit	and	nutrient	support	for	egg	and	sperm,	and	2)	The	prevention	of	implantation	and	resulting	ectopic	pregnancy,	which	would	have	been	frequently	lethal	in	the	pre-surgical	era.	The	stroma	of	the	endometrium	is	absolutely	distinct	[57],	in	that	it	has	a	key	role	in	the	maintenance	and	replenishment	of	uterus	as	an	ideal	site	for	implantation	and	invasion,	and	ultimately	the	growth	of	an	embryo.		We	therefore	hypothesize	that	the	paracrine	interactions	that	underpin	the	tumour	suppressive	microenvironment	of	the	fallopian	tube	and	the	invasion	enhancing	microenvironment	of	the	uterus	may	ultimately	explain	the	differences	in	the	oncogenic	pathways	that	are	opened	in	these	organ	systems.		This	consideration	may	also	explain	why	the	most	common	subtype	of	ovarian	cancer,	high-grade	serous,	which	are	derived	from	secretory	epithelial	cells	of	the	fallopian	tube	present	as	mass	lesions	outside	of	the	proposed	organ	of	origin.		Herein	we	have	provided	correlative	evidence	to	formulate	a	new	model	of	how	endometriosis-associated	ovarian	cancers	develop.	We	recognize	that	ciliated	cells	are	assumed	to	be	terminally	differentiated	[58]	and	the	cell	of	origin	for	CCOC	may	share	features	with	this	lineage	as	opposed	to	being	directly	related	to	it.	We	also	recognize	that	the	rare	cases	of	a	true	mixed	CCOC	and	ENOC	may	reflect	tumour	cell	plasticity.	The	model	described	is	aligned	with	the	burgeoning	recognition	of	the	importance	of	cell	context	in	shaping	oncogenic	opportunity	[31,	32,	35].	Although	functional	evidence	from	in	vitro	and	likely	in	vivo	modeling	of	the	events	described	here	will	be	required	to	test	its	validity;	the	model	described	offers	a	potential	explanation	for	one	of	the	most	vexing	questions	in	gynecologic	pathology.	Namely,	how	can	two	very	different	cancers	emerge	from	the	same	precursor	lesion,	and	contain	the	same	mutations?			Acknowledgements	This	work	was	funded	by	A	Canadian	Cancer	Society	Research	Institute	Impact	grant	to	DGH.	We	wish	to	thank	the	Gray	Family	Ovarian	Clear	Cell	Carcinoma	Research	Resource,	which	has	provided	funding	for	this	research.	We	also	wish	to	acknowledge	further	support	from	the	BC	Cancer	Foundation	and	the	VGH	and	UBC	Hospital	Foundations.	DGH	holds	a	Canada	Research	Chair.			Statement	of	Author	Contributions	DRC	and	DGH	were	responsible	for	conceptualization	of	the	experimental	design	and	development	of	the	model	of	clear	cell	development	from	ciliated	cells.	DRC	performed	the	organoid	culture	experiments	and	optimization	of	dual	IF.	BT-C	scored	all	TMAs.	BT-C,	TN,	ANK,	LNH	and	CBG	provided	pathology	support,	review	of	cases	and	contributed	to	IHC	antibody	optimization.	KML	performed	all	IF	and	fluorescent	microscopy.	CS	assisted	with	organoid	culture	experiments.	ASC	performed	IHC	experiments.	JNM	directs	the	tumor	bank	where	the	endometrial	tissue	for	organoid	culture	was	obtained.	Manuscript	was	prepared	by	DRC	and	DGH,	with	input	from	CBG.		Conflicts	of	Interest	The	authors	have	no	conflicts	of	interest	in	relation	to	this	manuscript.			References	1. Kobel	M,	Kalloger	SE,	Boyd	N,	et	al.	Ovarian	carcinoma	subtypes	are	different	diseases:	implications	for	biomarker	studies.	PLoS	Med	2008;	5:	e232.	2. Kobel	M,	Kalloger	SE,	Lee	S,	et	al.	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Images	of	CTH,	Ezrin	and	Napsin	A	immunohistochemical	staining	in	normal	endometrium,	fallopian	tube	and	typical	endometriosis,	40X	magnification.	Insets	show	high	magnification	(100X)	of	ciliated	cells	when	visible.	Figure	2.	Expression	of	clear	cell	ovarian	cancer	markers	in	clear	cell	ovarian	cancer,	clear	cell	borderline	tumor	and	adjacent	endometriosis.	Top	image	shows	clear	cell	ovarian	tumor	with	adjacent	clear	cell	borderline	tumor	and	atypical	endometriosis.	Below	are	images	of	CTH,	Ezrin	and	Napsin	A	immunohistochemical	staining	in	representative	areas,	40X	magnification.		Figure	3.	Endometrioid	ovarian	cancer	markers	are	expressed	in	secretory	cells	of	the	endometrium	and	fallopian	tube.	A.	Immunohistochemical	staining	of	ovarian	cancer	TMA	showing	expression	MTHFD1	(left)	and	ER	(right)	in	clear	cell	and	endometrioid	ovarian	cancers.	Error	bars	represent	standard	error	of	the	mean.	P-values	were	calculated	using	a	Mann-Whitney	non-parametric	test.	Representative	images	shown	below.	B.	Images	of	MTHFD1	and	ER	immunohistochemical	staining	of	normal	endometrium,	fallopian	tube	and	typical	endometriosis,	40X	magnification.	High	magnification	(100X)	of	ciliated	cells	when	apparent.	C.	Immunohistochemical	staining	of	CTH,	ER	and	MTHFD1	in	endometrioid	ovarian	tumor	and	endometriosis	adjacent	to	the	tumor.		Figure	4.	CTH	expression	in	normal	endometrium	is	restricted	to	ciliated	cells.	Dual	immunofluorescent	staining	of	normal	endometrial	glands	for	CTH	and	FOXJ1	(A),	CTH	and	Ezrin	(B),	CTH	and	ER	(C)	and,	CTH	and	MTHFD1	(D).	Nuclei	are	stained	with	DAPI	(blue).	63X	magnification.		Figure	5.	Organoid	cultures	of	normal	endometrium	express	both	secretory	and	ciliated	cells.	A.	Phase	contrast	image	of	organoid	culture	were	grown	from	normal	endometrium.	H&E	of	organoids	at	20X	and	50X	magnification	with	ciliated	cell	indicated	by	arrow.	B.	Dual	immunofluorescence	staining	of	CTH	and	FOXJ1	(top),	and	CTH	and	ER	(bottom),	with	nuclei	stained	with	DAPI,	63X	magnification.	Figure	6.	Model	of	endometriosis	associated	cancers.	Endometrial	progenitor	can	differentiate	into	ciliated	cells	or	secretory	cells	in	normal	endometrium	or	atypical	endometriosis.	Conditions	in	atypical	endometriosis	can	lead	to	the	development	of	clear	cell	ovarian	cancer	arising	from	a	ciliated	cell	lineage	or	endometrioid	ovarian	cancer	from	a	secretory	cell	lineage.	Supplemental	Figure	1.	CTH,	Ezrin	and	Napsin	expression	is	enriched	in	clear	cell	ovarian	cancer.	A.	CTH	staining	of	endometrial	clear	cell	and	endometrioid	cancers.	Staining	of	ovarian	cancer	TMA	showing	expression	CTH	(B),	Ezrin	(C)	and	Napsin	(D)	in	clear	cell	and	endometrioid	ovarian	cancers.	Error	bars	represent	standard	error	of	the	mean.	P-values	were	calculated	using	a	Mann-Whitney	non-parametric	test.	Representative	images	shown	below.	Supplemental	Figure	2.	CTH	expression	in	normal	fallopian	tube	in	ciliated	cells.	Dual	immunohistochemical	staining	of	normal	fallopian	tube	for	CTH	and	FOXJ1	(A),	CTH	and	Ezrin	(B),	CTH	and	ER	(C)	and,	CTH	and	MTHFD1	(D).	63X	magnification.	Figure	1	Endometrium	Fallopian	tube	Endometrioma	CTH	 Napsin	EZR	CTH	Napsin	Figure	2	Ezrin	Endometriosis	 Clear	Cell	Borderline	 CCOC	CCOC ENOC050100150200250 MTHFD1H ScoreFigure	3	Endometrium	 Fallopian	tube	 Endometriosis	Endometriosis		Adj	ENOC	ENOC	MTHFD1	ER	CTH	 ER	 MTHFD1	CCOC ENOC050100150200 ERH ScoreA	B	C	P<0.0001	P	=	0.0374	Figure	4	ER	 CTH	 Merge	DAPI	 ER	+	CTH	MTHFD1	 CTH		 Merge	DAPI	 MTHFD1	+	CTH	CTH	 FOXJ1		 Merge	DAPI	 CTH	+	FOXJ1	Ezrin	 CTH		 Merge	DAPI	 Ezrin	+	CTH	A	B	C	D	Figure	5	ER	 CTH	 Merge	DAPI	 ER	+	CTH	A	B	 CTH	 FOXJ1		 Merge	DAPI	 CTH	+	FOXJ1	Figure	6	CCOC	 ENOC	Progenitor	cell	Normal	Endometrium	CTH+	EZR+	Napsin-	ER-	MTHFD1-	CTH-	EZR-	Napsin-	ER+	MTHFD1+	CTH+	EZR+	Napsin+	ER-	MTHFD1-	CTH-	EZR-	Napsin-	ER+	MTHFD1+	Atypical	Endometriosis	Ovarian	Stroma	Muta=ons:	ARID1A	PIK3CA	others	Typical	Endometriosis	Inflamma/on	 ?	Muta=ons:	ARID1A	KRAS	others	?	?	CCOC ENOC0100200300CTHH ScoreSupplementary	Figure	S1	P<0.0001	CCOC ENOC050100150200EZRH ScoreP	=	0.0012	Endometrial	clear	cell	carcinoma	Endometrial	endometrioid	carcinoma	A	 B	C	 D	CCOC ENOC020406080100NapsinH ScoreP<0.0001	Supplementary	Figure	S2	ER	 CTH	 Merge	DAPI	 ER	+	CTH	MTHFD1	 CTH		 Merge	DAPI	 MTHFD1	+	CTH	CTH	 FOXJ1		 Merge	DAPI	 CTH	+	FOXJ1	Ezrin	 CTH		 Merge	DAPI	 Ezrin	+	CTH	A	B	C	D	Supplemental	Methods		Optimization	of	Immunohistochemistry		For	Napsin	A,	the	clinical	concentration	(1:100)	and	conditions	were	used.	All	other	antibodies	were	optimized	to	maximize	sensitivity	and	minimize	background	staining.	The	ER	antibody	used	clinically	was	originally	optimized	in	breast	tissue.	The	current	conditions	performed	on	the	gynecological	tissues	were	modified	from	the	original	protocol,	at	1:100	using	the	HQ	and	ChromoMap	Detection	Kit	instead.			For	each	antibody,	optimizations	were	performed	with	normal	fallopian	tube	sections	on	charged	glass	slides	baked	at	60°C	for	an	hour.	Initial	dilutions	as	determined	from	previous	publications	or	from	manufacturer’s	suggestions,	were	trialed	on	two	different	chromogenic	detection	systems	for	initial	comparisons.	Additional	antibody	dilutions,	antibody	diluent,	incubation	time,	antigen	retrieval	methods	and/or	chromogenic	detection	kits	were	assayed	and	altered	as	needed,	until	the	optimal	conditions	were	determined.		All	IHC	were	performed	on	the	automated	Ventana	Benchmark	and	Discovery	systems	(Ventana	Medical	Systems).		Protein	 CTH	 Ezrin	 ER	 MTHFD1	Manufacturer	 LSBio	 Sigma	 Thermo	Fisher	 Sigma	Antibody	details	 Mouse		Clone:	1E12	Rabbit	Polyclonal	Rabbit		Clone:	SP1	Rabbit		Polyclonal	Catalogue	Number	 LS-C337259	 HPA021616	 RM-9101	 HPA000704	Optimum	Dilution	 1:1000	 1:1000	 1:100	 1:50	Antigen	retrieval	 Cell	Conditioning	1	solution		(Ventana)	64	minutes,	95°C	Cell	Conditioning	1	solution		(Ventana)	64	minutes,	95°C	Cell	Conditioning	1	solution	(Ventana)	32	minutes,	95°C	Cell	Conditioning	1	solution		(Ventana)	64	minutes,	95°C	Antibody	incubation	time	60	minutes,	37°C	 60	minutes,	37°C	 32	minutes,	37°C	 60	minutes,	37°C	Chromogenic	detection	DABMap	kit	(Ventana)	DABMap	kit	(Ventana)	Anti	Rb	HQ	+		Anti	HQ-HRP		ChromoMap	DAB	Detection	Kit	(Ventana)	DABMap	kit	(Ventana)			Organoid	Culture		This	study	was	approved	by	the	Institutional	Review	Board	(IRB)	of	the	University	of	British	Columbia	and	British	Columbia	Cancer	Agency	(H05-60119),	and	all	patients	provided	written	informed	consent.	Endometrial	tissue	samples,	collected	as	part	of	the	OVCARE	gynecologic	tissue	bank,	were	obtained	from	women	undergoing	hysterectomy	for	benign	conditions	at	the	Vancouver	General	Hospital.	Tissue	was	processed	within	3h	of	surgery.	From	the	small	piece	of	uterus	provided	(approximately	4	cm	X	4	cm),	the	endometrium	was	macrodissected	with	a	scalpel	from	the	rest	of	the	tissue,	washed	in	DPBS	to	remove	excess	blood	and	cut	into	small	pieces	using	scissors.	To	dissociate	the	epithelial	cells	from	connective	tissue	and	extracellular	matrix,	the	minced	tissue	was	incubated	in	0.5	mg/ml	collagenase	(Sigma)	for	1h,	with	stirring.	After	centrifugation	(4	minutes,	300	x	g),	the	cell	pellet	was	resuspended	in	Advanced	DMEM/F12	(ThermoFisher)	containing	12	mM	HEPES	(ThermoFisher),	1%	GlutaMAX	(ThermoFisher),	5%	fetal	bovine	serum	(Hyclone),	10	ng/ml	hEGF	(ThermoFisher),	9	µM	ROCK	inhibitor	(Y-27632,	Tocris)	and	1%	penicillin/streptomycin	(ThermoFisher).	The	cells	were	plated	and	maintained	in	a	37°C,	humidified	incubator	with	5%	CO2.			After	4-10	days	in	culture,	when	the	cells	had	reached	80%	confluence,	the	cells	were	trypsinized	using	TrypLE	Express	(ThermoFisher).	Cells	were	centrifuged,	counted	and	resuspended	in	growth	factor-reduced	Matrigel	(ThermoFisher).	Cells	were	plated	into	24-well	plates	with	50	µl	of	Matrigel	containing	25,000	cells	per	well.	Matrigel	was	allowed	to	solidify	at	37°C	for	30	minutes.	The	Matrigel	was	overlaid	with	Expansion	Media:	Advanced	DMEM/F12	containing	12	mM	HEPES,	1%	GlutaMAX,	10	ng/ml	hEGF,	9	µM	ROCK	inhibitor,	2%	B27	supplement	(ThermoFisher),	1%	N2	supplement	(ThermoFisher),	100	ng/ml	human	noggin	(Peprotech),	100	ng/ml	human	FGF10	(Peprotech),	1	mM	nicotinamide	(Sigma)	and	0.5	µM	TGFβ type	I	receptor	inhibitor	(SB431542,	Tocris).	Cultures	were	maintained	in	37°C,	humidified	incubator	with	5%	CO2.	Media	was	changed	every	3-4	days	and	organoids	were	passaged	every	14-21	days.			To	passage	the	organoids,	cold	DMEM/F12	was	used	to	dissociate	the	Matrigel.	Organoids	were	disrupted	using	mechanical	force	by	vigorous	pipetting	with	a	glass	capillary	pipet.	Cells	were	pelleted	by	centrifugation	for	5	minutes	at	300	x	g,	4°C.	The	cell	pellet	was	resuspended	in	Matrigel	and	plated	in	24-well	plates.	The	matrigel	was	overlaid	with	Expansion	Media.			For	immunohistochemical	analysis,	organoid	cultures	were	formalin	fixed	and	paraffin	embedded.	The	Matrigel	was	dissolved	with	ice	cold	PBS,	and	the	organoids	fixed	in	4%	formaldehyde	for	1	hour	at	room	temperature.	After	dehydration	in	a	series	of	alcohols,	the	organoids	were	embedded	in	paraffin.	Sections	(4	mm)	were	cut	and	put	onto	Superfrost+	glass	slides	and	heat-immobilized	prior	to	immunostaining.		


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