4.1 FV#gene#expression#in#breast#cancer#
Data!from!the!breast!cancer!materials!of!the!OsloII!study!and!the!GOBO!dataset!
(described!in!section!3.1.1)!was!studied!to!gain!insight!in!similarities!and!differences!of!
FV!gene!expression!levels!between!breast!cancer!molecular!subtypes!and!tumor!
characteristics,!and!associations!to!survival.!!
!
4.1.1 FV#expression#levels#in#breast#cancer#molecular#subtypes#and#tumor#
characteristics#in#OsloII#and#GOBO#
FV#tumor#mRNA#expression#and#FV#plasma#protein#levels#across#breast#cancer#
tumor#characteristics#in#the#material#of#the#OsloII#study##
FV!mRNA!expression!levels!in!tumors!and!FV!protein!levels!in!plasma!were!studied!in!
the!OsloII!material.!This!was!mainly!done!using!SPSS,!where!the!first!step!was!to!check!
whether!the!data!followed!normal!distribution.!Data!of!FV!mRNA!expression!did!not!
follow!normal!distribution,!hence!a!log10!transformation!was!done!to!fit!this!trend.!The!
following!steps!were!done!individually!for!both!FV!mRNA!and!FV!protein!levels.!Data!
was!distributed!across!breast!cancer!tumor!characteristics,!into!the!subgroups!estrogen!
receptor!(ER)Tstatus,!progesterone!receptor!(PR)Tstatus,!HER2Tstatus,!histological!
tumor!grade,!tumor!status!(TTstatus),!hormone!receptor!(HR)!negative!status,!triple!
negative!status,!lymph!node!spread!and!TP53!mutation!status.!Independent!sample!tT tests!and!Levenes!tests!were!performed!to!see!whether!there!were!significant!
differences!in!FV!expression!levels!and!FV!protein!levels!between!the!subgroups!within!
each!tumor!characteristic.!Boxplots!for!FV!mRNA!expression!and!FV!protein!levels!
according!to!each!group!were!made!for!a!visual!effect!of!variances!within!each!group.!
Moreover,!a!distribution!across!PAM50!subgroups!using!ANOVA!was!made,!for!both!FV!
mRNA!tumor!expression!and!FV!protein!plasma!levels.!!
!
FV#tumor#mRNA#expression#levels#across#breast#cancer#molecular#subtypes#and#
tumor#characteristics#in#pooled#GOBO#dataset#
The!online!tool!GOBO!provides!several!options!for!expression!analyses!of!breast!cancer!
genes!and!was!used!to!study!the!pooled!GOBO!dataset.!The!Gene!Set!Analysis!(GSA)!
application!of!GOBO,!including!analyses!of!tumors!and!cell!lines,!was!used!for!multiple!
analyses!of!FV!mRNA!expression!in!tumors!regarding!the!two!FV!probe!sets,!
204713_s_at!and!204714_a_st,!both!comprising!11!probes.!Analyses!of!each!probe!set,!as!
well!as!of!the!two!probe!sets!merged!(equal!to!FV)!was!done.!From!the!GSATtumor!
application!FV!tumor!mRNA!expression!levels!distributed!across!breast!cancer!subtypes!
were!derived,!visualized!in!boxplot!format.!To!correlate!FV!tumor!mRNA!expression!
levels!according!to!outcome,!as!well!as!breast!cancer!subtypes,!KaplanTMeier!plots!were!
derived.!10!years!censoring!endpoint!for!all!tumors!were!used!as!parameters.!In!
addition,!queries!were!run!with!overall!survival!(OS),!distant!metastasisTfree!survival!
(DMFS)!and!relapseTfree!survival!(RFS)!as!endTpoint,!all!with!data!divided!into!both!
medianTcut!and!tertiles.!The!data!was!divided!according!to!the!FV!mRNA!expression!
level!of!each!sample,!where!in!the!medianTcut!query!each!sample!was!classified!above!
(high!expression)!or!below!(low!expression)!the!median!of!all!tumors!within!the!chosen!
subgroup.!When!divided!into!tertiles,!data!in!each!group!comprised!a!third!of!the!
observed!data.!Being!the!center!observation!the!median!is!found!in!the!middle!of!the!
middle!group.!The!lower!cutTpoint!represents!the!third!of!the!data!with!lower!levels!of!
FV!mRNA!expression,!while!the!upper!cutTpoint!represents!the!third!of!the!data!with!
higher!levels!of!FV!mRNA!expression.!Multivariate!analysis!was!used!to!adjust!for!
covariates!that!may!have!an!effect!on!outcome.!The!covariates!included!were!ERTstatus,!
node!status,!histological!tumor!grade,!age!and!tumor!size.!!
!
4.1.2 Biological#function#of#FV#in#breast#cancer#
FV#coFexpressed#genes#and#relation#to#biological#function#
In!an!attempt!to!gain!insight!in!what!biological!functions!the!expression!of!F5!as!well!as!
genes!coTexpressed!with!F5!are!related!to,!the!online!tools!GOBO,!DAVID!and!Gene!
Ontology!(GO)!Term!Finder!of!LewisTSigler!Institute!of!Princeton!University!were!used.!!
!
The!expression!levels!of!FV!mRNA!and!the!correlation!to!genes!depending!on!their!
biological!functions!where!studied.!GOBO!stratify!genes!into!the!following!modules!
according!to!biological!functions:!stroma,!lipid!metabolism,!immune!response,!
checkpoint,!MTphase,!basal,!early!response!and!steroid!response.!The!correlation!was!
visualized!in!a!Spearman!correlation!plot,!stating!positive!or!negative!correlation.!!
!
A!list!of!genes!coTexpressed!with!F5!was!derived!using!the!CoTexpressed!Genes!feature!
of!the!GOBO!database.!Pearson!Correlation!method!for!ERTnegative!tumors!with!
correlation!cutToff!and!a!standard!deviation!cutToff!at!0,4!were!selected.!Minimum!
number!of!connections!was!set!to!2,!but!also!tested!for!3,!4!and!5.!Both!positive!and!
negative!correlations!were!chosen.!!The!output!gene!list!was!used!as!input!in!the!online!
functional!annotation!tool!DAVID!to!identify!biological!terms!associated!with!these!coT expressed!genes.!The!preTset!gene!list!for!Homo%sapiens!was!selected.!The!annotation!
clustering,!chart!and!table,!as!well!as!gene!ontology!(GOTERM_BP_FAT,!
GOTERM_CC_FAT!and!GOTERM_FF_FAT)!and!pathways!(KEGG)!were!studied.!For!
studies!of!gene!ontology!the!Genetic!Ontology!(GO)!Term!Finder!of!LewisTSigler!Institute!
of!Princeton!University!was!used,!in!addition!to!DAVID,!using!GOA!–!H.%sapiens!
(Humans)!as!annotation!and!function!ontology!and!process!ontology!as!aspects.!
!
4.1.3 FV#mRNA#expression#levels#in#cell#lines#
The!GSATcell!line!application!was!used!to!study!FV!mRNA!expression!levels!in!51!
individual!breast!cancer!cell!lines,!where!the!cell!lines!were!grouped!according!to!
luminal!and!basal!breast!cancer!subtypes.!FV!mRNA!expression!levels!were!also!studied!
in!breast!cancer!cell!lines!and!nonTbreast!cancer!cell!lines!from!inThouse!experiments.!!
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