Detecting^Cancer^ ^ ^ ^ ^ Cancer^ is^a^ leading^cause^of^disease^worldwide.^Certain^types^of^cancer^ have^a^high^ chance^of^cure^if^they^are^detected^at^an^early^stage^and^ad equately^treated.^^However,^ many^cancers^are^detected^at^a^ late^stage^and^delays^ in^cancer^diagnosis^can^occur^ throughout^ the^ diagnostic^ pathway.^ ^ This^ can^ include^ patients^ failing^ to^ recognise^^ symptoms^or^delaying^see^a^healthcare^provider.^^Doctors^may ^not^recognise^symptoms^ of^cancers^and^so^may^not^investigate^them^appropriately^or^ refer^on^time.^^Furthermore,^ some^cancers^are^difficult^to^detect.^^Late^diagnosis^is^a^m ajor^cause^of^cancer^mortality.^^^ ^ Renal^cell^carcinoma^(RCC)^is^the^cause^of^approximately^5,0 00^cancer^deaths^in^the^^ UK^ per^ year^ and^ non-small^ cell^ lung^ cancer^ (NSCLC)^ causes^ approximately^ 35,000^ cancer^deaths^in^the^UK^per^year,^with^a^combined^annual^NHS ^cost^of^over^£3bn.^This^ human^and^financial^cost^is^replicated^around^the^world.^^Mo re^than^40%^of^NSCLC^and^ 10-20%^of^RCC^cases^are^diagnosed^late,^representing^a^major ^cause^of^mortality.^More^ than^40%^of^patients^diagnosed^with^non-small^cell^lung^canc er^(NSCLC)^present^with^^ late-stage^disease^(Stage^3-4),^when^the^cancer^has^spread^a nd^where^5-year^survival^ rates^are^ dismal^ (90%^mortality).^As^a^ result,^NSCLC^ is^ the^ leading^ cause^of^ cancer^ death^both^worldwide^and^in^the^UK,^where^the^disease^causes ^over^20%^of^the^160’000^ annual^cancer^deaths.^In^addition,^NSCLC^incurs^significant^ resource^burden(s)^costing^ the^NHS^an^estimated^£2.4bn^each^year^and^a^global^cost^est imated^to^exceed^£150bn.^^ Patients^diagnosed^with^Stage^1^NSCLC^are^ten^times^more^lik ely^to^survive^5^years^than^ those^diagnosed^at^stage^4,^engendering^efforts^for^early^de tection^of^NSCLC.^^ ^ NSCLC^is^comprised^of^two^major^histological^subtypes;^^ ^ ^ i)^ lung^adenocarcinoma^(LUAD),^which^is^localised^to^the^lung^p arenchyma^in^ the^periphery^and^accounts^for^approximately^60-65%^of^disea se^and^^ ii)^ lung^squamous^cell^carcinoma^(LUSC)^is^found^in^the^central^ airways,^which^ constitutes^approximately^30%^of^disease.^ ^ Each^ subtype^has^ distinct^ forms^ of^ pre-malignant^ disease^ due^ to^ unique^ clinical^ and^ genomic^features^meaning^that^LUAD^and^LUSC^are^preferential ly^detected^via^specific^ screening^approaches.^ ^ ^ Low-dose^computerised^ tomography^ (LDCT)^ lung^ screening^ is^advocated^as^a^major^ pathway^for^early^lung^cancer^detection^and^predominantly^de tects^LUAD.^This^strategy^ increases^ early^ diagnosis^ and^ reduces^ mortality^ by^ 20-26%^ but^ even^ the^ perfectly^ executed^CT^screening^programme^will^only^prevent^20%^of^dea ths,^as^ it^ targets^ the^ highest-risk^ patients,^missing^ lighter^ and^ never^ smokers.^Additional^ disadvantages^ of^^ LDCT^ include^ resource^ intensiveness,^ radiation^ exposure^ and^ failure^ to^ detect^ most^ LUSCs.^LDCT^also^detects^pre-malignant^and^benign^peripheral ^lung^nodules,^but^these^ have^indeterminate^disease^penetrance^and^require^continual^ surveillance.^ ^ The^ pre-invasive^ stages^ of^ LUSC^ (‘pre-LUSC’)^ are^ characterised^ by^ asymptomatic^^ lesions^that^develop^in^a^stepwise^process^of^increasing^dys plasia^and^can^be^classified^ as^low-grade^(LG)^and^high-grade^(HG).^LG^lesions^are^associ ated^with^no^increased^ risk^of^NSCLC^but^40-90%^of^patients^with^HG^lesions^progres s.^Traditional^screening^of^ pre-LUSC^relies^on^sputum^cytology^which^has^poor^sensitivit y,^and^autofluorescence^ bronchoscopy^which^suffers^from^high^false-positive^rates,^ low^patient^throughput^and^^ the^ burden(s)^ associated^ with^ an^ invasive^ procedure.^ Similarly,^ whilst^ a^ 280-gene^ classifier^of^airway^bronchial^cells^has^been^successfully^u sed^to^predict^the^presence^of^ pre-LUSC^lesions^this^remains^an^expensive^and^invasive^appr oach.^^ ^ New^ non-invasive,^ tools^ to^ detect^ progressing^ lesions/nodules^ would^ address^ a^ vast^^ clinical^unmet^need.^Circulating^tumour^DNA^(ctDNA)^is^the^m ost^widely^proposed^future^ clinical^ strategy^ for^ liquid^multi-cancer^detection^but^detects^only^40-50%^of^ stage^ I-II^ NSCLC^cancers,^falling^to^as^low^as^10%^for^stage^I^LUAD^and ^is^expected^to^be^less^ effective^in^the^pre-invasive^setting.^^ ^ ^ Thus,^ current^ NSCLC^ detection^ protocols^ are^ often^ insensitive,^ non-specific,^ time- consuming,^resource-heavy,^invasive/painful^and^can^lead^to^ anxiety^and^over^diagnosis^ or^delayed^diagnosis.^^ ^ It^is^known^that^T^cell^differentiation^is^skewed^in^subject s^with^established^or^late^stage^^ cancer.^^The^balance^of^T^cell^populations^in^a^subject^shif ts^towards^the^majority^of^T^ cells^ exhibiting^ biomarkers^ that^ are^ associated^ with^ having^ identified^ their^ cognate^ antigen^on^a^tumour.^This^has^mostly^been^described^as^a^pro cess^within^the^tumour^or^ tissue,^with^sparse^evidence^that^this^could^also^be^seen^in ^the^blood.^ ^ However,^there^is^still^no^means^for^early^cancer^detection^ at^the^stage^of^detecting^a^^ progressing^ or^ high-grade^ pre-invasive^ lesion^ or^ nodule^ or^ solid^ tumours^ before^ the^ cancer^has^fully^developed^in^the^subject.^^^ ^ There^therefore^remains^an^unmet^and^urgent^need^for^early^d etection^of^NSCLC^and^ other^solid^cancers.^ In^particular,^a^new^blood^test^with^high^specificity^and^se nsitivity^^ could^fulfil^this^need^as^a^non-invasive^classifier^of^progr essing^preinvasive^disease^or^ early^established^disease.^ ^ The^ability^to^distinguish^between^subjects^with^early-stage ^solid^cancers^or^pre-invasive^ lesions^or^nodules^likely^to^progress^to^cancer,^versus^subj ects^with^no^malignancy^or^^ with^lesions^or^nodules^unlikely^to^progress^would^enable^ea rly,^curative^intervention^or^ targeted^surveillance,^saving^lives^and^significantly^reduci ng^resource^burden.^ ^ The^present^invention^ ^ ^ The^ present^ invention^ aims^ to^ solve^ these^ and^ other^ problems^ by^ providing^ a^ novel^ method^ for^determining^whether^a^subject^ is^at^ risk^ for^having^a^progressing^or^high- grade^pre-invasive^lesion^or^nodule^or^a^solid^malignant^tum our^of^any^stage.^The^present^ invention^ provides,^ for^ the^ first^ time,^ a^ non-invasive^ immune-based^ method^ for^ distinguishing^between^low-risk^pre-invasive^lesions^or^nodu les,^low^grade^pre-invasive^^ lesions^or^nodules^(or^lack^of^tumour),^and^progressing^pre- invasive^lesions^or^nodules,^ high^grade^pre-invasive^lesions^or^nodules^(or^having^a^soli d^tumour,^e.g.,^a^stage^I^solid^ tumour).^The^present^invention^also^detects^pre-invasive^les ions^or^nodules^(e.g.,^high^ grade^ pre-invasive^ lesions^ or^ nodules)^ that^ are^ at^ risk^ of^ progressing^ via^measuring^ immunological^markers^in^the^blood,^and^provides^a^readout^a s^compared^with^a^healthy^^ subject^or^a^plurality^of^healthy^subjects.^^ ^ The^ use^ of^ a^ combination^ multi-omic^ biomarkers^ to^ determine^ a^ subject’s^ T^ cell^ differentiation^state^in^a^blood^sample^obtained^from^a^subj ect,^that^can^be^used^in^the^ early^ detection^ setting^ to^ detect^ the^ presence^ of^ progressive^ pre-invasive^ lesions^ or^^ nodules,^or^early-stage^tumours^(e.g.,^stage^I^tumours)^is^d escribed^herein^for^the^first^ time.^ ^ When^T^cells^recognise^non-self^antigen^they^differentiate^f rom^a^naïve^or^resting^state^ into^ a^ range^ of^ activated,^ exhausted^ and^ memory^ T^ cells.^ ^ These^ changes^ can^ be^ measured^by^cytometry.^Cytometry^in^this^context,^is^used^to ^profile^the^frequency^and^^ intensity^of^biomarkers^on^T^cells.^^The^biomarkers^to^be^de tected^can^be^those^related^ to^ T^ cell^ activation,^ exhaustion^ and^ memory^ differentiation.^ Combinations^ of^ these^ biomarkers^ can^ be^ used^ to^ determine^ the^ strength^ or^ type^ of^ immune^ activation^ in^ disease^ states^ such^ as^ infection,^ autoimmunity,^ transplantation^ and^ cancer.^ These^ biomarkers^ can^be^measured^within^CD3+^ live^cells^ in^ the^blood^ (viable^T^ cells)^ and^^ amongst^the^major^T^cell^lineages^of^killer^cytotoxic^T^cell s^(‘CD8+^T^cells’)^or^helper^&^ regulatory^(‘CD4^T^cells’).^Other^methods^such^as^TCR-se q^are^also^disclosed^herein^ which^can^also^be^used^to^interrogate^the^immune^landscape^a nd^track^T^cell^traits^before^ neoplasia^ actually^ develops.^ The^ present^ invention^ therefore^ demonstrates^ multiple^ ways^to^track^T^cell^traits^and^predict^progression^into^can cer^by^measurements^made^in^^ the^periphery^using^only^blood.^The^combined^use^of^these^to ols^can^also^be^used^to^ more^ significantly^ differentiate^ between^ patient^ groups^ at^ risk^ of^ developing^ or^ with^ malignant^disease^as^an^early^cancer^detection^tool^to^detec t^pre-invasive^neoplasia.^^ ^ In^chronic^infections^like^HIV-1^and^HCV^there^is^well^estab lished^expansion^of^activated,^^ memory^and^exhausted^T^cells^at^the^expense^of^resting^and^n aïve^T^cells^in^the^blood.^ The^level^of^activated^and^exhausted^T^cells^reflects^the^pr esence^of^disease^compared^ to^healthy^individuals^and^the^amount^of^virus^detectable.^T his^shift^in^differentiation^is^ termed^T^cell^differentiation^skewing.^It^has^been^discovere d^that^a^similar^remodelling^ of^T^cell^differentiation^inside^the^tumours^of^patients^wit h^established^NSCLC^and^RCC;^^ is^ characterised^ by^ T^ cells^ co-expressing^ biomarkers^ of^ exhaustion^ activation^ and^ terminal^differentiation^and^loss^of^stem-like^populations^e xpressing^biomarkers^of^early-^ differentiation^and^progenitor^potential.^^ T^cell^differentiation^skewing^in^the^blood^of^cancer^patien ts^is^less^well^characterised.^^ The^present^inventors^have^shown^in^NSCLC,^that^(neo)antigen ^load^(inferred^by^tumour^^ mutational^burden)^correlated^with^T^cell^differentiation^sk ewing^inside^the^tumour^(loss^ in^resting,^progenitor^and^naïve^cells,^increase^in^termina lly^differentiated,^exhausted^and^ activated^T^cells).^^^ The^use^of^T^cell^differentiation^skewing,^defined^as^a^loss ^of^resting/naïve^T^cells^and^a^ gain^in^activated/exhausted^T^cells^as^a^method^of^early^can cer^detection,^such^as^early^ lung^cancer^detection,^has^not^previously^been^described.^Pr ior^to^the^present^invention^ there^ is^no^published^data^to^show^that^the^presence^of^pre-invasi ve^LUSC^lesions^or^ pre-LUAD^nodules^are^associated^with^blood^T^cell^differenti ation^skewing.^Equally^prior^ to^the^present^invention^there^was^no^data^to^support^an^equ ivalent^process^in^another^^ solid^tumour^type.^^ Prior^ to^ the^ present^ invention,^ existing^methods^and^ strategies,^ such^ as^ disclosed^ in^ US2018356420^A1^ have^ focused^ on^ using^ antibody^ panels^ to^ analyse^T^ cells^within^ patients^ with^ established^ cancer.^ However,^ these^ methods^ have^ limitations^ as^ they^ cannot^be^used^for^distinguishing^between^progressing^and^no n-progressing^lesions^or^^ nodules,^nor^for^determining^whether^a^subject^is^at^risk^fo r^having^a^progressing^or^high- grade^pre-invasive^lesion^or^nodule^or^having^a^very^early^s tage^(stage^1)^solid^tumour.^^ Elsewhere^ in^earlier^ studies,^ Li^ et^ al,^ Lung^Cancer^ 162^ (2021)^16–22^describes^ lung^ cancer-associated^T^ cell^ repertoire^ as^potential^biomarker^ for^ detection^of^stage^ lung^ cancer.^The^authors^do^not^propose^detection^of^lesions^or^n odules^before^a^cancer^is^^ established.^ ^ The^ authors^ do^ not^ propose^ analysis^ of^ the^ T^ cell^ repertoire^ for^ distinguishing^ between^ progressing^ and^ non-progressing^ lesions^ or^ nodules,^ nor^ for^ determining^whether^ a^ subject^ is^ at^ risk^ for^ having^ a^ progressing^ or^ high-grade^ pre- invasive^ lesion^or^nodule.^The^authors^ do^not^use^ the^ ratio^of^exhausted/activated^ to^ naïve/resting^T^cells^as^a^method^for^early^stage^cancer^de tection.^ ^ Mascaux^et^al,^570,^Nature,^Vol^571,^25^July^2019,^relates^t o^ immune^evasion^before^ tumour^invasion^in^early^lung^squamous^carcinogenesis^and^in dicates^that^the^adaptive^ immune^response^within^tumours^may^be^strongest^at^the^earli est^stage^of^carcinoma.^ However,^ there^ is^ no^ suggestion^ of^ distinguishing^ between^ progressing^ and^ non- progressing^lesions^or^nodules,^nor^determining^whether^a^su bject^is^at^risk^for^having^a^^ progressing^or^high-grade^pre-invasive^lesion^or^nodule^or^h aving^a^solid^tumour.^^ WO2021188941^A1^relates^to^methods^for^isolating^T^cells^and ^T-cells^receptors^from^ peripheral^blood^by^single-cell^analysis^for^immunotherapy,^ in^addition^to^preparing^and^ enriching^ a^ population^ of^ T^ cells^ having^ antigenic^ specificity^ for^ a^ target^ antigen.^ However,^ the^ authors^ focused^ on^ analysing^ and^ using^ the^ T^ cell^ repertoire^ as^ a^^ therapeutic^strategy^for^established^cancer^in^patients^and^ does^not^propose^a^method^ for^distinguishing^between^progressing^and^non-progressing^l esions^or^nodules,^nor^for^ determining^whether^ a^ subject^ is^ at^ risk^ for^ having^ a^ progressing^ or^ high-grade^ pre- invasive^lesion^or^nodule^or^having^a^solid^tumour.^^ Cancers,^vol.^14,^2022,^“Martinez-Gomez^et^al”^ identifies^certain^T^cell^biomarkers^as^ markers^for^tumor^specific^T^cells^or^T^cells^which^are^a^su rrogate^for^an^active^immune^ response.^However,^this^work^is^focused^on^samples^with^esta blished^disease,^with^no^ suggestion^or^teaching^of^what^biomarkers,^if^any,^could^be^ used^for^pre-invasive^or^early^^ stage^(e.g.,^stage^I)^disease.^^ ^ Immune^Network,^vol^20(6),^2020,^“Kim^et^al”^article^e48 ,^similarly^focuses^on^T^cells^in^ cancer^patients,^and^more^specifically^highlights^the^differ ence^between^those^that^do^or^ do^not^respond^to^checkpoint^inhibitors^with^clinical^benefi t.^However,^this^document^also^^ only^relates^to^later^stage^disease^and^response^to^a^therap y.^This^does^not^show^any^ patients^with^preinvasive^or^early-stage^disease^who^are^dis tinguished^by^ these^cells^ compared^with^no^disease.^^ ^ WO^ 2016/185182^ describes^ methods^ of^ assessing^ whether^ an^ individual^ has^ an^^ exhausted^CD8+^T^cell^or^lack^of^CD4+^T^cell^co-stimulation^ phenotype^to^determine^an^ individual’s^risk^of^autoimmune^disease^progression,^progr ession^of^a^chronic^infection^ or^ cancer^ progression.^ In^ contrast,^ the^ present^ invention^ relates^ to^measuring^ T^ cell^ differentiation^for^cancer^early^detection^(i.e.,^at^a^pre-i nvasive^stage,^or^early^stage^(e.g.,^ stage^ I),^ as^ opposed^ to^ forecasting^ progression^of^ patients^with^ established^ disease.^^^ There^is^no^suggestion^to^combine^different^T-cell^analyses, ^for^example,^using^cytometry^ and^TCR-seq,^let^alone^as^a^tool^for^the^early^detection^of^ cancer^(e.g.,^at^an^early^or^the^ pre-invasive^stage).^^ ^ Clinical^ Cancer^ Research,^ vol^ 27,^ 2021,^ Laumont^ et^ al^ focuses^ on^ the^ prognostic^^ potential^of^exhausted^T^cells^infiltrating^established^ovar ian^cancer.^However,^this^article^ does^ not^ allude^ to^ measuring^ phenotypic^ and/or^ TCR-seq^ features^ in^ the^ blood^ of^ individuals^ at^ risk^ of^ developing^ or^ having^ progressive^ pre-invasive^ or^ early-stage^ disease^for^the^purposes^of^early^cancer^detection.^This^art icle^also^does^not^describe^ combining^phenotypic^and^TCR-seq^features^in^the^blood.^^ ^ ^ Journal^of^Pathology,^vol.^251,^2020,^Guo^et^al,^pp^26-27^re ferences^using^TCR-seq^and^ transcriptional^measures^of^T^ cells^ in^ the^blood^of^patients^with^ renal^cell^ carcinoma.^ However,^this^document^does^not^teach^nor^suggest^what^trait s^can^be^used^to^forecast^ cancer^development^(e.g.,^at^a^pre-invasive^stage),^nor^does ^this^document^teach^how^^ to^ discriminate^ between^ progressive^ (e.g.,^ high-grade)^ vs^ low^ grade^ pre-invasive^ neoplasia.^ This^ document^ also^ fails^ to^ teach^ of^ a^method^ that^ can^be^ used^ for^ early^ detection^cancer^screening^via^a^non-invasive^blood^test.^Fi nally,^this^document^does^not^ disclose^ that^ multiple^ methods^ (e.g.,^ cytometry^ and^ TCR-seq)^ can^ be^ combined^ to^ forecast^cancer^development^using^a^multiomic^model.^^ ^ ^ Statements^of^the^Invention^ ^ The^present^invention^provides^a^method^for^determining^whet her^a^subject^is^at^risk^for^ having^a^progressing^or^high-grade^pre-invasive^lesion,^nodu le^or^small^mass,^or^having^ a^solid^malignant^tumour,^the^method^comprising^analysing^th e^proportion^of^T^cells^in^a^^ sample^of^blood^obtained^from^the^subject^which^are^activate d^and/or^exhausted^T^cells^ by^analysing^a^trait^of^the^T^cells.^In^some^embodiments,^th e^solid^malignant^tumour^is^a^ stage^I^malignant^tumour.^^ ^ The^present^invention^also^provides^a^method^for^determining ^whether^a^subject^is^at^risk^^ for^having^a^progressing^or^high-grade^pre-invasive^ lesion,^nodule^or^small^mass,^the^ method^comprising^analysing^the^proportion^of^T^cells^in^a^s ample^of^blood^obtained^from^ the^subject^which^are^activated^and/or^exhausted^T^cells^by^ analysing^a^trait^of^ the^T^ cells.^ ^ ^ Analysing^the^proportion^of^T^cells^in^the^sample^of^blood^o btained^from^the^subject^which^ are^activated^and/or^exhausted^may^comprise^analysing^a^plur ality^of^traits^of^the^T^cells.^ ^ The^method^may^further^comprise^combining^results^obtained^f rom^analysing^the^plurality^ of^ traits,^ preferably^ wherein^ the^ traits^ are^ (i)^ the^ diversity^ and/or^ clonality^ of^ T^ cell^^ receptors^ as^ determined^ by^ TCR^ Seq^ and^ (ii)^ biomarkers^ expressed^ by^ T^ cells^ as^ determined^by^flow^cytometry.^^^ ^ The^trait^may^be^a^phenotypic^trait.^ ^ ^ The^phenotypic^trait^may^be^the^diversity^(i.e.,^diversity^o r^clonality)^of^T^cell^receptors^on^ the^T^cells^in^the^sample^of^blood,^optionally^the^diversity ^^(i.e.,^diversity^or^clonality)^of^ CDR3B^on^TCRs^of^the^T^cells^in^the^sample^of^blood,^further ^optionally^using^TCR^Seq.^^ The^phenotypic^trait^may^be^the^diversity^or^clonality^of^T^ cell^receptors^on^the^T^cells^in^ the^sample^of^blood,^optionally^the^diversity^or^clonality^o f^CDR3B^on^TCRs^of^the^T^cells^^ in^the^sample^of^blood,^further^optionally^using^TCR^Seq.^^ ^ In^some^embodiments,^the^phenotypic^trait^is^biomarker^expre ssion^by^the^T^cells^in^the^ sample^of^blood^using^cytometry,^preferably^flow^cytometry.^ ^ ^ The^present^invention^also^provides^a^method^for^determining ^whether^a^subject^is^at^risk^^ for^ having^ a^progressing^ or^ high-grade^pre-invasive^ lesion,^ nodule^ or^ small^mass,^ or^ having^a^solid^malignant^tumour,^the^method^comprising^analy sing^the^proportion^of^T^ cells^in^a^sample^of^blood^obtained^from^the^subject^which^a re^activated^and/or^exhausted^ T^cells^by^analysing^a^trait^of^the^T^cells,^wherein^the^tra it^is^a^phenotypic^trait^which^is:^^ a)^the^diversity^or^clonality^of^T^cell^receptors^on^the^T^c ells^in^the^sample^of^blood,^and/or^^^ b)^biomarker^expression^by^the^T^cells^in^the^sample^of^bloo d,^optionally^using^cytometry.^ In^some^embodiments,^the^method^comprises^analysing^a^plural ity^of^traits^of^the^T^cells,^ and^ the^method^ comprises^ combining^ results^ obtained^ from^analysing^ the^plurality^of^ traits^of^the^T^cells,^which^involves^combining^results^obta ined^from^a)^and^b)^ ^ ^ The^present^invention^also^provides^a^method^for^determining ^whether^a^subject^is^at^risk^ for^having^a^progressing^or^high-grade^pre-invasive^ lesion,^nodule^or^small^mass,^ the^ method^comprising^analysing^the^proportion^of^T^cells^in^a^s ample^of^blood^obtained^from^ the^subject^which^are^activated^and/or^exhausted^T^cells^by^ analysing^a^trait^of^ the^T^ cells,^wherein^the^trait^is^a^phenotypic^trait^which^is^^ ^ a)^the^diversity^or^clonality^of^T^cell^receptors^on^the^T^c ells^in^the^sample^of^blood^and/or^^ b)^biomarker^expression^by^the^T^cells^in^the^sample^of^bloo d,^optionally^using^cytometry.^ In^some^embodiments,^the^method^comprises^analysing^a^plural ity^of^traits^of^the^T^cells,^ and^ the^method^ comprises^ combining^ results^ obtained^ from^analysing^ the^plurality^ of^ traits^of^the^T^cells,^which^involves^combining^results^obta ined^from^a)^and^b).^ ^ ^ he^present^invention^also^provides^a^method^for^determining^ whether^a^subject^is^at^risk^ for^ having^ a^progressing^ or^ high-grade^pre-invasive^ lesion,^ nodule^ or^ small^mass,^ or^ having^a^solid^malignant^tumour,^using^a^sample^of^blood^obt ained^from^the^subject,^the^ method^ combining^ two^or^more^T^ cell^ analyses^ comprising^a)^measuring^ the^ ratio^ of^^ activated^ and/or^ exhausted^ T^ cells:naïve^ and^ /or^ resting^ T^ cells^ using^ cytometry,^ or^ measuring^the^proportion^of^activated^and/or^exhausted^cells ^as^a^percentage^of^T^cells^ using^cytometry^and^^ b)^the^diversity^or^clonality^of^the^blood^TCR^repertoire.^ ^ ^ In^embodiments^of^the^above^methods,^the^present^invention^a lso^comprises^analysing^ a^ plurality^ of^ traits^ of^ T^ cells^ in^ a^ sample^ of^ blood^ obtained^ from^ the^ subject.^ ^ ^ The^ phenotypic^traits^(or^diversity^or^clonality)^can^be^diversi ty^of^T^cell^receptors,^which^may^ be^analysed,^for^example,^by^TCR^Seq,^and^biomarkers^express ed^by^T^cells,^which^may^ be^analysed,^for^example,^by^flow^cytometry.^^^ For^the^above^methods,^in^some^embodiments,^the^subject^is^a t^risk^for^having,^or^has,^^ a^ progressing^ or^ high-grade^ pre-invasive^ lesion,^ nodule^ or^ small^ mass;^ in^ some^ embodiments,^the^subject^is^at^risk^for^having,^or^has,^a^so lid^malignant^tumour,^e.g.,^a^ stage^I^solid^malignant^tumour.^^^ ^ For^all^the^above^methods,^the^subject^may^be^at^risk^if:^ ^ ^ a)^^the^diversity^of^the^repertoire^of^TCRs^is^lower^than^th e^diversity^of^the^repertoire^of^ TCRs^of^a^comparison^subject^or^a^plurality^of^comparison^su bjects,^^ ^ b)^D50^diversity^score^is^lower^than^a^D50^diversity^score^o btained^from^analysing^T^cells^^ in^a^sample^of^blood^obtained^from^a^comparison^subject^or^a ^plurality^of^comparison^ subjects,^^ ^ c)^a^clonality^score^is^higher^than^a^clonality^score^obtain ed^from^analysing^T^cells^in^a^ sample^ of^ blood^ obtained^ from^ a^ comparison^ subject^ or^ a^ plurality^ of^ comparison^^ subjects,^or^ ^ d)^Hill^number^is^lower^than^a^Hill^number^obtained^from^ana lysing^T^cells^in^a^sample^of^ blood^obtained^from^a^comparison^subject^or^a^plurality^of^c omparison^subjects.^ ^ ^ The^subject^may^be^at^risk^if:^ (e)^the^clonality^of^the^repertoire^of^TCRs^occupied^by^the^ top^n^clones^is^higher^than^the^ clonality^of^the^repertoire^of^TCRs^occupied^by^the^top^n^cl ones^in^a^comparison^subject^ or^ a^ plurality^ of^ comparison^ subjects,^ optionally^wherein^ n^ is^ between^ 50^ and^ 1000,^ preferably^wherein^n^=^100^ ^ (f)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^sma ll^clones^is^lower^than^the^ proportion^of^the^repertoire^of^TCRs^occupied^by^small^clone s^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects,^or^ (g)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^lar ge^clones^is^higher^than^the^ proportion^of^the^repertoire^of^TCRs^occupied^by^large^clone s^in^a^comparison^subject^or^^ plurality^of^comparison^subjects^or^ (h)^the^ratio^of^the^repertoire^of^TCRs^occupied^by^large^cl ones:^the^repertoire^of^TCRs^ occupied^by^small^clones^is^higher^than^the^ratio^of^the^rep ertoire^of^TCRs^occupied^by^ large^clones:^the^repertoire^of^TCRs^occupied^by^small^clone s^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects^ ^ ^ The^comparison^subject^or^the^plurality^of^comparison^subjec ts^is/are^healthy^subject(s)^ or^a^part^of^the^general^population.^^ ^ In^some^embodiments,^the^phenotypic^trait^is^biomarker^expre ssion^by^the^T^cells^in^the^^ sample^of^blood^using^cytometry,^preferably^spectral^or^flow ^cytometry.^^ ^ The^phenotypic^trait^may^be^biomarker^expression^by^the^T^ce lls^in^the^sample^of^blood,^ optionally^ using^ cytometry,^ further^ optionally^wherein^ cytometry^ is^ used^ to^detect^ the^ presence^or^absence^of^a^panel^of^biomarkers^comprising^Ki67 ^and^CD39.^ ^ ^ In^some^embodiments,^the^activated^and/or^exhausted^T^cells^ express^Ki67^and^CD39.^^ The^panel^of^biomarkers^may^further^comprise^one^or^more^bio markers^selected^from^ CD45RA,^CCR7,^PD-1,^CD57^or^CD38,^or^FoxP3.^^ ^ ^ The^panel^of^biomarkers^may^further^comprise^one^or^more^bio markers^selected^from^ CD3,^CD4^or^CD8.^In^some^embodiments,^the^T^cells^are^CD3+^T ^cells^(i.e.,^the^T^cells^ express^the^CD3^biomarker).^In^some^embodiments,^the^T^cells ^are^CD4+^T^cells^(i.e.,^ the^T^cells^express^the^CD4^biomarker)^or^CD8+^T^cells^(i.e. ,^the^T^cells^express^the^CD8^ biomarker).^In^some^embodiments,^the^T^cells^are^CD3+CD4+^T^ cells^(i.e.,^the^T^cells^^ express^the^CD3^and^CD4^biomarker)^or^CD3+CD8+^T^cells^(i.e. ,^the^T^cells^express^the^ CD3^and^CD8^biomarker).^ ^The^analysis^may^comprise^use^of^a^viability^dye^(e.g.,^ to^ determine^live^CD3+^T^cells).^ ^ The^ panel^ of^ biomarkers^may^ further^ comprise^CD45RA,^ or^ CCR7,^ or^ CD45RA^and^^ CCR7,^or^FoxP3.^ ^ The^panel^of^biomarkers^may^further^comprise^CD45RA,^CCR7,^a nd^PD-1.^or^CD45RA,^ CCR7,^PD-1^and^FoxP3^ ^ ^ The^panel^of^ biomarkers^may^ further^ comprise^CD45RA,^CCR7,^PD-1^and^CD57,^ or^ CD45RA,^CCR7,^PD-1^and^CD57^and^FoxP3.^^ The^panel^of^biomarkers^may^further^comprise^CD45RA,^CCR7,^P D-1,^CD57^and^CD38,^ or^CD45RA,^CCR7,^PD-1,^CD57,^CD38^and^FoxP3^ ^ The^panel^of^biomarkers^may^further^comprise^CD45RA,^CCR7,^C D57,^and^CD38,^or^^ CD45RA,^CCR7,^CD57,^CD38^and^FoxP3^ ^ The^panel^of^biomarkers^may^further^comprise^CD45RA,^PD-1^an d^CD57,^or^CD45RA,^ PD-1,^CD57^and^FoxP3.^ ^ ^ In^some^embodiments,^the^activated^and/or^exhausted^T^cells^ express^Ki67^and^CD39,^ wherein^the^T^cells^are^CD4^T^cells^or^CD4+CD3+^T^cells.^In^ some^embodiments,^the^ activated^and/or^exhausted^T^cells^express^Ki67^and^CD39,^wh erein^the^T^cells^are^CD8+^ T^cells^or^CD8+CD3+^T^cells.^In^some^embodiments,^the^activa ted^and/or^exhausted^T^ cells^express^Ki67^and^CD39,^and^do^not^express^CD45RA-,^CCR 7^and^PD1.^In^some^^ embodiments^the^T^cells^are^CD8+^T^cells^or^CD3+CD8+^T^cells .^^^^ ^ In^some^embodiments,^cytometry^is^used^to^detect^the^presenc e^of^a^panel^of^biomarkers^ comprising^FoxP3^and^CD4,^preferably^flow^cytometry.^^In^som e^embodiments,^the^panel^ of^biomarkers^may^further^comprise^one^or^more^biomarkers^se lected^ from^CD45RA,^^ CCR7,^PD-1,^CD57^Ki67,^or^CD39.^In^some^embodiments,^the^pan el^of^biomarkers^may^ further^ comprise^ CD3.^ In^ some^ embodiments,^ the^ analysis^ may^ comprise^ use^ of^ a^ viability^dye.^^ ^ In^some^embodiments,^the^activated^and/or^exhausted^T^cells^ express^FoxP3,^wherein^^ the^T^cells^are^CD4^T^cells.^In^some^embodiments^the^T^cells ^are^CD3+CD4+^T^cells.^In^ some^embodiments,^the^activated^and/or^exhausted^T^cells^exp ress^FoxP3^and^CD39,^ optionally^ wherein^ the^ activated^ and/or^ exhausted^ T^ cells^ further^ (i)^ express^ the^ biomarker^Ki67^or^(ii)^do^not^express^the^biomarker^CD45RA.^ ^ ^ ^ In^some^embodiments,^the^activated^and/or^exhausted^T^cells^ express^the^biomarkers^ Ki67^and^CD39.^^ ^ The^subject^may^be^at^risk^if:^ ^ ^ i)^the^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^ and/or^resting^T^cells^is^equal^to^ or^greater^than^^ a^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^and/o r^resting^T^cells^of^a^comparison^ subject,^^or^^ an^average^ratio^of^activated^and/or^exhausted^T^cells^:^na� �ve^and/or^resting^T^cells^of^a^ plurality^of^comparison^subjects,^or^ ^ ^ ii)^the^ratio^of^activated^and/or^exhausted^T^cells^:^T^cell s^which^are^not^activated^and/or^ exhausted^is^equal^to^or^greater^than^^ a^ ratio^of^activated^and/or^ exhausted^T^ cells^ :^ T^cells^which^are^not^activated^and/or^ exhausted^of^a^comparison^subject,^^or^^ ^ an^average^ratio^of^activated^and/or^exhausted^T^cells^:^T^c ells^which^are^not^activated^ and/or^exhausted^of^a^plurality^of^comparison^subjects,^or^^ ^ iii)^ the^proportion^of^ activated^and/or^exhausted^T^cells^as^a^percentage^of^T^ cells^ is^ higher^than^the^proportion^of^activated^and/or^exhausted^T^c ells^in^a^comparison^subject^^ or^a^plurality^of^comparison^subjects,^optionally^wherein^th e^activated^and/or^exhausted^ T^cells^are^CD4+^regulatory^T^cells^(Tregs)^(i.e.,^^the^acti vated^and/or^exhausted^T^cells^ are^CD4+^T^cells^which^express^FoxP3)^or^effector^regulatory ^T^cells.^ ^ v)^the^proportion^of^naïve^T^cells^as^a^percentage^of^T^cel ls^is^lower^than^the^proportion^^ of^ naïve^ T^ cells^ as^ a^ percentage^ of^ T^ cells^ in^ a^ comparison^ subject^ or^ a^ plurality^ of^ comparison^subjects,^optionally^wherein^the^naïve^cells^are ^naïve^CD4+^T^cells.^ ^ A^comparison^subject^may^be^selected^from:^ a)^a^subject^known^to^have^a^progressing^or^high-grade^pre-i nvasive^lesion,^nodule^or^^ small^ mass^ or^ an^ established^ solid^ malignant^ tumour,^^ b)^the^subject^at^a^different^time^point.^^ ^ A^plurality^of^comparison^subjects^may^be^selected^from:^ a)^a^plurality^of^subjects^known^to^have^a^progressing^or^hi gh-grade^pre-invasive^lesion,^^ nodule^or^small^mass^or^an^established^solid^malignant^tumou r,^^ b)^ a^ plurality^ of^ healthy^ subjects,^ or^ c)^a^plurality^of^subjects^of^the^general^population.^^^ ^ For^all^above^embodiments,^the^cytometry^may^comprise^one^or ^more^of^flow^cytometry,^^ spectral^ cytometry^ or^ mass^ cytometry,^ optionally^ the^ cytometry^ comprises^ flow^ cytometry.^^^ The^present^invention^also^provides^for^a^method^for^determi ning^whether^a^subject^is^at^ risk^for^having^a^progressing^or^high-grade^pre-invasive^les ion,^nodule^or^small^mass,^or^ having^a^solid^malignant^tumour,^using^a^sample^of^blood^obt ained^from^the^subject,^the^ method^combining^two^or^more^T^cell^analyses^comprising:^ ^ a)^measuring^the^ratio^of^activated^and/or^exhausted^T^cells :naïve^and^/or^resting^T^cells^ determined^by^flow^cytometry,^or^measuring^the^proportion^of ^activated^and/or^exhausted^ cells^as^a^percentage^of^T^cells^determined^by^flow^cytometr y^and^^ b)^ the^ diversity^ or^ clonality^ of^ the^ blood^ TCR^ repertoire.^ In^ some^ embodiments,^ the^ activated^and/or^exhausted^T^ cells^express^ the^biomarkers^Ki67^and^CD39.^ In^ some^^ embodiments,^ the^ activated^ and/or^ exhausted^T^ cells^ express^ the^ biomarker^ FoxP3,^ wherein^the^T^cells^are^CD4+^T^cells,^(optionally^in^combina tion^with^CD39,^and^further^ optionally^(i)^in^combination^with^CD39^and^Ki67,^or^(ii)^in ^combination^with^CD39^and^in^ the^absence^of^CD45RA).^The^diversity^and^clonality^or^the^b lood^can^be^measured^by^ any^suitable^technique.^In^some^embodiments^and^examples,^th e^diversity^or^clonality^of^^ the^blood^is^measured^using^the^D50^index.^In^some^embodimen ts^and^examples,^the^ diversity^or^clonality^of^the^blood^is^measured^using^the^ra tio^of^the^repertoire^of^TCRs^ occupied^by^large^clones:^the^repertoire^of^TCRs^occupied^by ^small^clones.^^ ^ The^present^invention^also^provides^a^method^of^treating^a^s ubject^determined^to^be^at^^ risk^for^having^a^progressing^or^high-grade^pre-invasive^les ion,^nodule^or^small^mass,^or^ having^ a^ solid^malignant^ tumour,^ comprising^ analysing^ the^ proportion^ of^ T^ cells^ in^ a^ sample^of^blood^obtained^from^the^subject^which^are^activate d^and/or^exhausted^T^cells^ by^ analysing^a^ trait^ of^ the^ T^ cells,^wherein^ the^ trait^ is^ a^ phenotypic^ trait^wherein^ the^ analysing^comprises^ ^ a)^analysing^ the^proportion^of^T^cells^ in^a^sample^of^blood^obtained^ from^ the^subject^ which^ are^ activated^ and/or^ exhausted^ T^ cells^ using^ biomarker^ expression,^ optionally^ using^cytometry,^wherein^the^subject^is^at^risk^if^ ^ (i)^the^ratio^of^activated^and/or^exhausted^T^cells^:^naïve ^and/or^resting^T^cells^is^equal^to^^ or^greater^than^a^ratio^of^activated^and/or^exhausted^T^cell s^:^naïve^and/or^resting^T^cells^ of^a^comparison^subject,^^or^in^a^plurality^of^comparison^su bjects,^or^ (ii)^the^ratio^of^activated^and/or^exhausted^T^cells^:^T^cel ls^which^are^not^activated^and/or^ exhausted^is^equal^to^or^greater^than^a^ratio^of^activated^a nd/or^exhausted^T^cells^:^T^ cells^which^are^not^activated^and/or^exhausted^of^a^comparis on^subject,^^or^^ ^ in^a^plurality^of^comparison^subjects.^ (iii)^the^proportion^of^activated^and/or^exhausted^T^cells^i s^higher^than^the^proportion^of^ activated^and/or^exhausted^T^cells^in^a^comparison^subject^o r^a^plurality^of^comparison^ subjects,^^ ^ ^ and/or^^ ^ b)^determining^the^diversity^or^clonality^of^T^cells^in^a^sa mple^of^blood^obtained^from^the^ subject,^optionally^using^TCR-seq,^wherein^the^subject^is^at ^risk^if^ ^^ ^ (i)^^the^diversity^of^the^repertoire^of^TCRs^is^lower^than^t he^diversity^of^the^repertoire^of^ TCRs^of^a^comparison^subject^or^a^plurality^of^comparison^su bjects,^^ ^ ii)^D50^diversity^score^is^lower^than^a^D50^diversity^score^ obtained^from^analysing^T^cells^ in^a^sample^of^blood^obtained^ from^a^comparison^subject^or^a^plurality^of^comparison^^ subjects,^^ ^ iii)^a^clonality^score^is^higher^than^a^clonality^score^obta ined^from^analysing^T^cells^in^a^ sample^ of^ blood^ obtained^ from^ a^ comparison^ subject^ or^ a^ plurality^ of^ comparison^ subjects,^or^ ^ ^ iv)^the^Hill^number^is^lower^than^a^Hill^number^obtained^fro m^analysing^T^cells^in^a^sample^ of^blood^obtained^from^a^comparison^subject^or^a^plurality^o f^comparison^subjects,^or^^ ^ v)^the^clonality^of^the^repertoire^of^TCRs^occupied^by^the^t op^n^clones^is^higher^than^the^^ clonality^of^the^repertoire^of^TCRs^occupied^by^the^top^n^cl ones^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects,^optionally^wherein^n^ is^from^50-1000,^and^preferably^ wherein^n^=^100,^or^^ ^ vi)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^sma ll^clones^is^lower^than^the^^ proportion^of^the^repertoire^of^TCRs^occupied^by^small^clone s^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects,^or^ ^ vii)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^la rge^clones^is^higher^than^the^ proportion^of^the^repertoire^of^TCRs^occupied^by^large^clone s^in^a^comparison^subject^or^^ a^plurality^of^comparison^subjects^or^ ^ viii)^the^ratio^of^the^repertoire^of^TCRs^occupied^by^large^ clones:small^clones^is^higher^ than^ the^ ratio^ of^ the^ repertoire^ of^ TCRs^ occupied^ by^ large^ clones:small^ clones^ in^ a^ comparison^ subject^ or^ a^ plurality^ of^ comparison^ subjects,^ optionally^ wherein^ the^ comparison^ subject^ is^ a^ healthy^ subject,^ or^ the^ plurality^ of^ comparison^ subjects^ are^^ healthy^subjects,^and^^ treating^the^subject^if^the^subject^is^at^risk.^^^ ^ In^ some^ embodiments,^ the^ treating^ may^ comprise^ administering^ an^ anti-cancer^ therapeutic.^ In^ some^ embodiments,^ the^ treating^ may^ comprise^ administering^ a^^ therapeutic^suitable^for^treating^pre-invasive^neoplasia^and /or^a^high^grade^pre-invasive^ lesion,^ nodule^ or^ small^ mass.^ ^ In^ some^ embodiments,^ the^ treating^ may^ comprise^ administering^a^therapeutic^suitable^for^treating^a^solid^ma lignant^tumour,^for^example,^a^ stage^ I^ solid^ malignant^ tumour.^ ^ In^ some^ embodiments,^ the^ treating^ may^ comprise^ electrocautery,^argon^plasma^coagulation^(APC),^cryotherapy^ and^photodynamic^therapy^^ (PDT).^The^latter^are^all^minimally^invasive^treatment^optio ns^that^may^be^used^for^the^ treatment^of^high-grade^pre-invasive^lesions,^as^described^i n^Daniels^et^al;^Ther.^Adv.^ Med^Oncol.^2013^Jul;^5(4);^235-248.^^^ ^ The^novelty^and^inventiveness^of^the^present^disclosure^does ^not^rely^on^the^techniques^^ to^ identify^ T-cell^ traits^ themselves^ (e.g.,^ using^ flow^ cytometry^ and/or^ TCR-seq,^ since^ these^ are^ well-known^ techniques),^ but^ the^ use^ of^ these^ techniques^ to^ analyse^ the^ peripheral^ blood^ from^ pre-invasive^ cancer^ patients,^ e.g.,^ pre-invasive^ lung^ cancer^ patients.^Most^of^the^research^is^carried^out^in^early-stage ^invasive^disease,^highlighting^ the^rarity^of^the^cohort^used.^These^samples^have^been^used^ to^interrogate^the^immune^^ landscape^ even^ before^ invasive^ neoplasia^ actually^ develops.^ The^ present^ invention^ demonstrates^multiple^ways^to^track^T^cell^traits^and^predic t^progression^into^cancer^in^ the^periphery^using^only^blood.^While^prior^research^has^als o^looked^at^the^presence^of^ activated/exhausted^T^ cells^ and^has^ used^TCR^clonality^ to^ identify^ tumour-reactive^T^ cells,^such^techniques^have^only^previously^been^demonstrate d^in^established^disease.^^ For^the^first^time,^the^present^inventors^have^uniquely^demo nstrated^that^these^tools^can^ be^used^for^the^early^detection^of^cancer^in^the^context^of^ pre-invasive^disease^(e.g.,^a^ high^grade^pre-invasive^lesion,^nodule^or^small^mass,^or^pre -invasive^neoplasia).^ ^ Further,^ the^ present^ inventors^ have^ demonstrated^ that^ the^ combined^ use^ of^ flow^^ cytometry^ and^ TCR-seq^ can^ be^ used^ as^ an^ early^ detection^ tool^ for^ cancer.^ These^ techniques^capture^different^parts^of^the^peripheral^biology ^and^differentiation^skewing^ occurring^in^pre-invasive^patients,^therefore^providing^a^sy nergistic^effect.^^Prior^methods^ have^not^used^a^combination^of^these^techniques,^let^alone^i n^the^context^of^pre-invasive^ disease.^^^ ^ ^ In^some^examples,^the^term^“comprising^analysing^the^propo rtion^of^T^cells^in^a^sample^ of^ blood^ obtained^ from^ the^ subject^ which^ are^ activated^ and/or^ exhausted^ T^ cells^ by^ analysing^a^trait^of^the^T^cells”^may^encompass^or^be^used ^interchangeably^with^the^term^ “determining^a^subject’s^T^cell^differentiation^state^in ^a^blood^sample”.^Therefore,^also^ disclosed^herein^ is^a^method^ for^determining^whether^a^subject^ is^at^ risk^ for^having^a^^ progressing^or^high-grade^pre-invasive^lesion,^nodule^or^sma ll^mass,^or^having^a^solid^ malignant^tumour,^the^method^comprising^determining^a^subjec t’s^T^cell^differentiation^ state^in^a^blood^sample^obtained^from^a^subject.^^In^some^em bodiments,^a^subject’s^T^ cell^differentiation^state^is^determined^(i)^using^cytometry ^(and^optionally^flow^cytometry)^ to^ detect^ the^ presence^ or^ absence^ of^ a^ panel^ of^ biomarkers^ and/or^ (ii)^ TCR-seq^ to^^ determine^the^diversity^or^clonality^of^the^blood^TCR^repert oire.^In^some^embodiments,^ the^solid^malignant^tumour^is^a^stage^I^malignant^tumour.^^ ^^ In^some^examples,^the^term^“high-grade^pre-invasive^lesion ,^nodule^or^small^mass”^may^ encompass^ or^ be^ used^ interchangeably^ with^ the^ term^ “pre-invasive^ neoplasia”,^ “pre-^ invasive^neoplastic^lesion”,^as^well^as^other^terms^descri bed^elsewhere^herein.^^ ^ For^any^method^defined^herein^describing^a^solid^malignant^t umour,^the^solid^malignant^ tumour^is^a^stage^I^solid^malignant^tumour^(i.e.,^an^early-s tage^solid^malignant^tumour).^ This^is^distinguished^from^more^established^tumours^in^later ^stages.^^ ^ ^ Brief^description^of^the^drawings^ ^ For^a^better^understanding^of^the^present^invention,^and^to^ show^more^clearly^how^it^may^ be^ carried^ into^ effect,^ reference^ will^ now^ be^ made,^ by^ way^ of^ example,^ to^ the^^ accompanying^drawings,^in^which:^ ^ Figure^1^shows^the^use^of^biomarkers^(CD39,^Ki67)^to^classif y^low^from^high^grade^pre- LUSC.^The^data^uses^a^combination^of^CD4^and^CD8^T^cells.^In ^this^example^the^data^ is^shown^as^a^generated^TEDI^score^ (short^hand^ for^ the^T^cell^early^detection^ index,^^ developed^by^the^inventors)^that^shows^the^capacity^to^class ify^low^from^high-grade^pre- LUSC.^A)^Workflow^ for^how^ to^generate^ the^TEDI^ score,^B)^ result^of^ non^parametric^ Mann-Whitney,^One-tailed^test^of^the^Minimal.^TEDI^score^bet ween^low-grade^and^high- grade^ pre-LUSC^ patients^ as^ described^ above.^ C)^ Gating^ strategy^ for^ identifying^ cell^ populations^required^to^generate^the^score^and^D)^the^associ ated^calculation.^ ^ ^ Figure^ 2^ shows^ a^ workflow^ showing^ the^ protocol^ of^ how^ the^ computational^ high^ dimensional^analysis^and^clustering^of^the^flow^cytometry^da ta^was^completed^following^ the^analysis^steps^outlined.^The^Figure^shows^each^major^ste p^in^how^a^skilled^operator^ should^ analyse^ files^ from^ a^ flow^ cytometer^ using^ the^ R^ programming^ environment^ (https://www.r-project.org/)^to^obtain^a^list^of^all^T^cell^ clusters^within^a^set^of^samples,^^ and^the^proportion^of^each^cluster^in^each^sample,^including ^the^exhausted/activated^and^ resting/naïve^ T^ cell^ clusters^ used^ in^ the^ invention.^All^ packages^ described^ are^ freely^ available^to^download^at^https://cran.r-project.org/^or^http s://www.bioconductor.org/.The^ person^skilled^in^the^art^will^know^that^the^workflow^provid ed^is^merely^an^example^in^the^ context^of^the^Examples^provided^herein^and^is^not^intended^ to^limit^the^invention^in^any^^ way.^ ^ Figure^3^shows^that^systemic^T^cell^differentiation^skewing^ distinguishes^patients^with^ high^ vs^ low-grade^ pre-invasive^ central^ airway^ lesions.^ A)^ Flow^ cytometry^ data^ from^ PBMCs^showing^Uniform^Manifold^Approximation^Projections^(UM APs)^of^CD4^(left)^and^^ CD8^(right)^viable^T^cells^from^66^samples^(31^high^vs^35^lo w^grade)^of^30^patients^(14^ high^vs^16^low^Grade)^with^pre-invasive^lesions.^This^shows^ which^clusters^of^T^cells^are^ present^across^all^samples^and^their^relative^abundance.^Ind ividual^clusters^and^major^ subsets^of^cells^are^indicated.^B)^Heatmaps^of^biomarker^exp ression^in^each^cluster^for^ CD4^and^CD8^T^cells.^This^shows^that^there^are^31^different^ clusters^T^cell^present^in^the^^ blood^of^patients^with^pre-LUSC,^and^details^which^biomarker s^describe^those^different^ clusters.^ Clusters^ are^ in^ rows^ and^ biomarkers^ are^ in^ columns.^ For^ each^ cluster^ the^ drawing^shows^whether^a^biomarker^is^highly,^ lowly,^or^intermediately^expressed.^The^ lines^on^the^left^are^a^dendogram,^which^shows^how^similar^c lusters^of^T^cells^are^related.^ All^clusters^within^CD8^T^cells^are^on^the^left^heatmap,^and ^those^within^CD4^T^cells^are^^ on^ the^ right.^ This^ shows^ that^ there^ are^ multiple^ types^ of^ activated/exhausted^ and^ resting/naïve^T^cell^present^in^the^pool^of^samples.^C)^Vol cano^plots^showing^significant^ differences^in^cluster^frequency^for^CD8^(left)^and^CD4^(rig ht)^T^cells.^Clusters^enriched^ in^high-grade^disease^are^shown^on^the^left^of^each^plot^and ^those^enriched^in^low^grade^ disease^on^the^right.^Samples^from^patients^that^progressed^ or^regressed^between^lesion^^ grades^were^excluded^for^analysis.^This^shows^which^clusters ^of^T^cell^are^increased^in^ high-grade^disease,^and^which^are^increased^in^low^grade^dis ease.^Activated/exhausted^ T^cells^are^increased^in^high-grade^and^resting/naïve^are^i ncreased^in^low^grade^disease.^ D)^The^ratio^of^clusters^enriched^in^high^vs^low^grade^(acti vated/^exhausted^vs^resting^ progenitor)^was^developed^as^a^potential^classifier^and^plot ted^per^patient^for^CD8^(left),^ CD4^(centre)^or^all^T^cell^subsets^(right).^This^shows^that^ the^ratio^of^activated/exhausted^^ :^naïve/resting^T^cells^is^significantly^higher^in^high^vs^ low^grade^disease.^E)^Receiver^ operator^characteristic^curves^(ROCs)^for^the^ratio^of^activ ated/exhausted^:^naïve/resting^ T^cells^within^CD4^and^CD8^T^cells,^labelled^as^CD4^TEDI^and ^CD8^TEDI,^respectively.^ This^ shows^ that^92-94%^of^patients^can^be^correctly^classified^as^High^ or^Low-grade^ using^the^invention.^P^values^from^one-tailed,^unpaired^Wilc oxon^test.^E)^ROC^curves^of^^ the^metrics^from^flow^cytometry^and^data^indicated,^AUC,^Are a^Under^Curve.^TEDI,^T^ cell^early^detection^index.^^ ^ Figure^4^ shows^CD4^T^cell^ differentiation^ skewing^ in^pre-invasive^ lung^neoplasia.^A)^ Summary^of^biomarker^expression^in^clusters^that^show^signif icantly^different^abundance^^ in^high^vs^low^grade^disease^according^to^sample^level^analy sis.^The^mean^frequency^of^ each^cluster^was^calculated^per^patient^and^shown^in^B)^volc ano^plot^and^C)^bar^plots.^ D)^Cluster^names,^median^biomarker^expression^and^annotation ^of^enrichment^in^high- grade^or^low-grade.^This^Figure^shows^which^clusters^of^T^ce ll^are^present^amongst^CD4^ T^cells^and^highlights^those^that^are^significantly^differen t^in^high^or^low-grade^disease^in^^ detail,^indicating^expression^of^key^biomarkers^CD39^and^Ki6 7^and^additional^biomarkers^ PD1,^CCR7,^CD45RA,^CD57^and^CD38.^^ ^ Figure^5^ shows^CD8^T^cell^ differentiation^ skewing^ in^pre-invasive^ lung^neoplasia.^A)^ Summary^of^biomarker^expression^in^clusters^that^show^signif icantly^different^abundance^^ in^high^vs^low^grade^disease^according^to^sample^level^analy sis.^The^mean^frequency^of^ each^cluster^was^calculated^per^patient^and^shown^in^B)^volc ano^plot^and^c)^bar^plots.^D)^ Cluster^ names,^median^ biomarker^ expression^ and^ annotation^ of^ enrichment^ in^ high- grade^or^low-grade.^This^Figure^shows^which^clusters^of^T^ce ll^are^present^amongst^CD8^ T^cells^and^highlights^those^that^are^significantly^differen t^in^high^or^low-grade^disease^in^^ detail,^indicating^expression^of^key^biomarkers^CD39^and^Ki6 7^and^additional^biomarkers^ PD1,^CCR7,^CD45RA,^CD57^and^CD38.^^ ^ Figure^6^shows^a^basic^workflow^showing^how^to^take^a^blood^ sample^and^measure^the^ change^in^CD4^or^CD8^clusters^as^a^single^score^referred^to^ as^the^T^cell^early^detection^^ index^(TEDI).^To^generate^TEDI,^a^ratio^of^[sum^freq.^of^all ^CD4^T^cell^clusters^enriched^ in^HG^disease]/^[sum^freq.^of^all^clusters^enriched^in^LG^di sease]^was^calculated^and^the^ process^was^repeated^ for^CD8^T^cells.^Activated/exhausted^T^cells^were^ increased^ in^ high-grade^and^resting/naïve^are^increased^in^low-grade^dis ease.^ This^is^a^key^step^in^using^the^invention.^^The^person^skill ed^in^the^art^will^know^that^the^ workflow^provided^is^merely^an^example^in^the^context^of^the ^Examples^provided^herein^^ and^is^not^intended^to^limit^the^invention^in^any^way.^ ^ Figure^ 7^ shows^ T^ cell^ early^ detection^ indices^ (TEDI)^ in^ high^ vs^ low-grade^ disease.^ Clusters^ associated^ with^ high-grade^ disease^were^ summed^ and^ divided^ by^ the^ sum^ frequency^ of^ clusters^ associated^ with^ low^ grade^ disease^ to^ generate^ a^ single^ score^^ referred^to^as^the^TEDI.^This^was^calculated^per^patient^acc ording^to^clusters^in^CD4^or^ CD8^T^cells^or^the^average^of^both^(combined).^Each^dot^is^a ^patient,^p^values^from^one^ way^Wilcoxon^tests,^dotted^lines^represent^the^sensitivity^a djusted^cut-off^values^in^the^ table^below.^This^drawing^summarises^the^key^results^from^th e^example,^showing^that^ the^ratio^of^exhausted/activated^:^naïve^resting^T^cells^is ^significantly^higher^in^high^vs^^ low-grade^disease.^The^person^skilled^in^the^art^will^know^t hat^the^use^of^a^TEDI^provided^ is^ an^ in-house^ term^ that^ is^ specific^ to^ the^ inventors^ and^ that^ various^ methods^ and^ techniques^discussed^herein^can^be^performed^using^a^variety ^of^different^apparatus,^ assay^conditions,^computational^hardware^and^software^compon ents^that^may^result^in^ different^numerical^values^obtained,^but^which^equate^to^the ^same^biological^results^to^^ the^Examples^provided^herein.^^Therefore,^the^use^of^a^TEDI^ is^merely^an^example^in^the^ context^of^the^Examples^provided^herein^and^is^not^intended^ to^limit^the^invention^in^any^ way.^ ^ Figure^ 8^ shows^ the^ gating^ strategy^ for^ manual^ identification^ of^ high^ and^ low^ grade^^ associated^T^cell^populations.^Gating^(from^left^to^right^up per^panel)^shows^identification^ of^live,^single,^CD3+^lymphocytes,^within^live^T^cells^CD4^a nd^CD8^T^cells^were^identified^ and^then^gating^of^key^CD8^and^CD4^T^cell^subsets^that^were^ enriched^in^high-grade^ samples^and^ low-grade^samples.^This^drawing^shows^how^a^skilled^ researcher^could^ bypass^computational^analysis^to^identify^several^activated/ exhausted^and^naïve/resting^^ T^ cell^ clusters^manually^ using^ compensated^FCS^ (Flow^Cytometry^ Standard)^ files^ in^ FlowJo^software.^ ^ Figure^ 9^ shows^ data^ from^ RCC^ patients^ showing^ an^ increase^ in^ systemic^ T^ cell^ differentiation^ in^malignant^ (n=16^samples^ from^10^patients)^ vs^benign^ (n=3^samples^^ from^3^patients)^disease.^A)^UMAP^of^FlowSOM^defined^T^cell^ clusters^from^PBMC^of^all^ samples^stained^with^31^biomarkers^and^analysed^by^spectral^ cytometry.^5000^live^CD3+^ events^ per^ samples^ were^ down-sampled^ for^ analysis.^ B)^ The^ ratio^ of^ progenitor^ vs^ exhausted^CD4^(left),^CD8^(centre)^or^combined^(right)^T^cel l^subsets.^P^values^ from^ one-tailed,^ unpaired^ Wilcoxon^ test.^ This^ drawing^ shows^ that^ the^ ratio^ of^ activated/exhausted:^naïve/resting^T^cells^is^increased^in^ the^blood^of^patients^with^renal^^ cancer^vs^patients^with^benign^disease.^ ^ Figure^10^shows^TCRseq^data^ from^pulmonary^pre-invasive^neoplasia.^A-B)^TCRseq^ analysis^on^PBMCs^from^22^patients.^Each^sample^is^a^differe nt^patient.^Proportion^of^ the^ repertoire^ occupied^ by^ clonotypes^ of^ the^ indicated^ size^ was^ analysed^ and^ B)^^ compared^between^high^vs^low^grade^disease.^C-E)^TCRseq^metr ics^in^high-^and^low- grade^patients.^Boxplots^display^C)^D50^diversity^ index,^D)^clonality^score,^and^E)^Hill^ number^ Significance^ determined^ by^ unpaired,^ one-tailed^ Wilcoxon^ test.^ The^ Figure^ indicates^that^there^is^a^significant^difference^in^some^of^ these^TCRseq^metrics^between^ high-^and^low-grade^patients,^possibly^reflective^of^persist ent^TCR^engagement^from^a^^ chronic^antigen^burden.^D50^(AUC^=^88.4%),^Hill^number^(AUC^ =^86%).^ ^ Figure^11^shows^an^ROC^curve^for^significant^TCRseq^metrics^ identified^from^Figure^10.^ ROC^ curves^ for^ the^ TCRseq^ metrics^ listed^ for^ all^ analysed^ TCRseq^ PBMCs^ in^ 22^ patients.^Hill^number^AUC^86%,^D50^AUC^88.4%.^^The^Figure^sh ows^that^as^the^D50^^ has^the^most^significant^difference^between^high-^and^low-gr ade^disease^(Figure^10C)^ and^has^the^greatest^diagnostic^power^(Figure^11),^this^metr ic^has^the^highest^potential.^ ^ Figure^12^shows^a^multi-omic^approach^and^the^combining^of^d ata^from^a^plurality^of^T^ cell^analyses.^ ^ Figure^ 12^ shows^ROC^curves^of^ the^metrics^ from^ flow^cytometry^and^^ TCRseq^data,^in^addition^to^results^using^a^combination^of^t hese^techniques.^(A)^AUC^ values^ and^ ROC^ analysis^ from^ important^ flow^ cytometry^ (CD4^ and^ CD8^ TEDI)^ and^ TCRseq^analysis^used^alone.^(B)^The^relative^importance^of^m ultiple^metrics^from^flow^ cytometry^ and^TCRseq^assays^ (‘feature^ importance’)^ as^determined^ by^ the^XGBoost^ machine^ learning^ library.^ This^ is^ a^ combined^ methodology^ used^ multiple^ metrics^ to^^ classify^patients^with^high^vs^ low^grade^disease^and^shows^ that^TEDI^CD4^from^ flow^ cytometry^in^addition^to^the^D50^index^from^TCRseq^are^ important^classifiers^within^a^ combined^multivariate^classifier^.^The^combined^methodology^ showed^improved^results^ (C)^ for^ classifying^ patients^ with^ high^ vs^ low^ grade^ disease^ compared^ to^ the^ use^ of^ cytometry^and^TCRseq^alone,^as^shown^by^ROC^analysis.^This^F igure^demonstrates^that^^ both^cytometry^and^TCRseq^approaches^can^be^used^to^effectiv ely^distinguish^subjects^ into^ high-grade^ and^ low-grade^ disease.^ These^ techniques^ can^ also^ be^ combined^ together^by^incorporating^multiple^metrics^from^these^techni ques^into^a^single^analysis,^ to^generate^data^that^further^improves^distinguishing^subjec ts^into^high-grade^and^low- grade^disease,^as^demonstrated^with^ROC^analysis.^ (AUC^=^Area^Under^curve)^ ^ ^ Figure^12A^shows^ROC^curves^of^ the^metrics^ from^ flow^cytometry^and^TCRseq^data^ indicated.^ ^Figure^12B^shows^feature^ importance^scores^of^ indicated^variables^ from^a^ machine^learning^model^using^70^percent^of^data^to^train^the ^model^(30^percent^of^the^ data^was^ then^used^ to^ test^ the^model).^The^model^was^developed^with^ the^XGBoost^^ algorithm^using^multiple^metrics^derived^from^flow^and^TCRse q^data.^^Figure^12C^shows^ the^performance^of^the^trained^multivariate^classifier^when^ used^to^classify^patients^with^ high^ vs^ low^ grade^ disease^ as^ shown^ by^ROC^analysis^ in^ test^ data^ (30%^ of^ all^ data^ available^from^the^TCRseq^and^flow^cytometry^analysis^of^the ^pre-LUSC^cohort).^ ^ ^ Figure^13^shows^a^workflow^showing^how^to^combine^data^obtai ned^from^a^plurality^of^ T^cell^analyses^^ ^ Figure^14^Top)^depicts^the^pre-invasive^data^and^shows^the^T ^cell^early^detection^ indices^(TEDI)^in^high^grade^pre-invasive^samples^versus^low ^grade^samples,^Bottom)^^ shows^the^T^cell^early^detection^indices^(TEDI)^in^healthy^p atients^versus^NSCLC^ (LUAD^+^LUSC)^patients^(the^majority^of^which^have^stage^I^d isease)^deriving^from^the^ ASCENT^analysis.^Each^datapoint^is^a^patient^and^the^p-value ^is^from^a^one-tailed^MW^ test.^^This^is^demonstrated^by^the^TEDI^CD39^Ki67^(CD3),^whe re^NSCLC^patients^have^ a^higher^proportion^of^CD3^CD39+Ki67+^cells^than^healthy.^^ ^ ^ Figure^15^shows^the^T^cell^early^detection^indices^(TEDI)^of ^the^pre-invasive^data^(i.e.,^ high^grade^and^low-grade^samples)^and^the^ASCENT^data^(i.e., ^healthy^and^NSCLC^ samples)^combined.^Each^datapoint^is^a^patient^with^the^medi an^value^shown^(top)^or^ the^mean^of^all^patients^in^each^group^(bottom)^and^p-values ^are^from^a^Kruskal-Wallis^^ test,^where^only^significant^values^are^shown.^Error^bars^re present^SEM.^An^increase^in^ the^TEDI^CD39^Ki67^ratio^from^healthy^samples^to^low-grade^s amples^to^high-grade^ samples^is^observed,^with^the^signal^peaking^at^high-grade^s amples^and^dropping^off^at^ the^lung^cancer^stage.^^ ^ ^ Figure^16^shows^the^lower^frequency^of^naïve^CD4+^T^cells^i n^NSCLC^patients^ compared^with^healthy^patients^(p^=^0.1,^one-tailed^MW^test) .^Naïve^CD4+^T^cells^are^ shown^as^a^percentage^of^effector^CD4+^T^cells^(eCD4),^meani ng^non-regulatory^CD4+^ T^cells,^or^FoxP3-^CD4+^T^cells.^ ^ Figure^17^shows^the^combined^data^for^(i)^healthy^and^low^gr ade^samples^(n=66)^^ compared^with^(ii)^high-grade^pre-invasive^samples^and^NSCLC ^patients^(N=88).^T^cell^ early^detection^indices^(TEDI);^%^Total^Treg,^%^Treg^CD39+,^ %^Tref^CD39+Ki67+^and^ %^naïve^CD4+^T^cells^as^a^proportion^of^total^CD4^cells^are ^all^provided.^p-values^are^ all^from^a^one-tailed^MW^test.^^ ^ ^ Figure^18^shows^the^%^Treg,^Treg^CD39+^and^Treg^CD39+Ki67+^a s^a^proportion^of^ total^CD4^cells^for^both^high^grade^pre-invasive^and^low^gra de^samples^(top)^and^for^ healthy^and^NSCLC^patients^(bottom).^Each^datapoint^is^a^sam ple^(top)^or^patient^ (bottom)^and^the^p-value^is^derived^from^a^one-tailed^MW.^Tr eg^stands^for^a^T^ regulatory^cell,^which^are^CD4+^T^cells^that^express^the^bio marker^FoxP3.^ ^ ^ Figure^19^shows^the^%^Treg,^Treg^CD39+^and^Treg^CD39+Ki67+^a s^a^proportion^of^ total^CD4^cells^for^healthy,^low^grade^pre-invasive^samples, ^high^grade^pre-invasive^ samples^and^NSCLC^patients.^Each^datapoint^is^a^patient^(hea lthy/NSCLC)^or^sample^ (low-/high-grade)^and^uncorrected^KW^p-values^are^shown^for^ all.^Treg^stands^for^a^T^^ regulatory^cell,^which^are^CD4+^T^cells^that^express^the^bio marker^FoxP3.^ ^ Figure^20^shows^a^volcano^plot^of^CD8+^T^cell^populations^de termined^by^ computational^clustering^of^flow^cytometry^data^of^pre-invas ive^samples^that^are^ significantly^enriched^in^high^grade^(HG;^upper^right^quadra nt)^pre-invasive^or^low^^ grade^(LG,^upper^left^quadrant)^samples.^ ^ Figure^21^shows^box^plots^for^naïve^and^Tem.Prolif.CF39hi^p opulations^at^the^sample^ level^for^both^patients^with^both^low^grade^and^high^grade^p re-invasive^species.^ Tem.Prolif.CF39hi^cells^are^significantly^enriched^in^high^g rade^pre-invasive^samples,^^ and^naïve^cells^are^significantly^enriched^in^low^grade^pre -invasive^samples.^Both^are^ shown^as^a^proportion^of^total^CD8+^T^cells.^^ ^ Figure^22^Top)^shows^a^volcano^plot^of^CD8+^T^cell^populatio ns^determined^by^ computational^clustering^of^flow^cytometry^data^that^are^sig nificantly^enriched^in^LUSC^^ (upper^right^quadrant).^Bottom)^shows^a^box^plot^for^the^Tem .Prolif.CF39hi^population^ which^is^significantly^enriched^in^LUSC^patients^compared^to ^a^healthy^group.^^ ^ Figure^23^shows^a^combined^box^plot^of^values^from^the^pre-i nvasive^flow^cytometry^ analysis^and^the^ASCENT^flow^cytometry^analysis^to^display^t he^change^in^Tem.^Prolif.^ CD39hi^population.^Each^dot^represents^a^patient^(healthy/LU SC)^or^sample^(low/high).^^ Shown^as^a^proportion^of^total^CD8+^T^cells.^ ^ Figure^24^shows^the^flow^cytometry^gating^strategy^of^CD8+^T ^cells^for^the^pre- invasive^data^samples^and^ASCENT^data^samples^used^in^Exampl e^9^and^Figures^20- 23.^Manual^gating^frequencies^are^used^to^validate^results^f rom^computational^ ^ clustering.^^ ^ Figure^25^shows^the^flow^cytometry^panel^used^for^analysis^o f^the^ASCENT^data-set.^^ ^ Figure^26^shows^the^D50^diversity^index^in^Top)^low^and^high ^grade^samples,^and^^ Bottom)^in^healthy^subjects^and^patients^with^NSCLC.^ ^ Figure^27^shows^the^Top)^the^Hill^number^(aka^entropy^score) ^in^low-grade^pre- invasive^samples^and^high^grade^pre-invasive^samples^(left), ^and^healthy^patients^and^ patients^with^NSCLC^(right),^and^Bottom)^the^proportion^of^r epertoire^occupied^by^the^^ top^100^clones^in^low^and^high^grade^pre-invasive^samples^(l eft),^and^healthy^subjects^ and^patients^with^NSCLC^(right).^ ^ Figure^28^shows^the^proportion^of^repertoire^occupied^by^sma ll^(<0.01%^of^repertoire),^ medium^(0.01-0.1%^of^repertoire),^large^(0.1-1%^of^repertoir e)^and^hyperexpanded^(>^^ 1%^of^repertoire)^clones^in^high^grade^pre-invasive^samples, ^low-grade^pre-invasive^ samples^(top),^healthy^subjects^and^patients^with^NSCLC^(bot tom).^ ^ Figure^29^shows^a^ratio^of^large:^small^clones^occupying^the ^repertoire^for^healthy,^low- grade,^high^grade,^and^NSCLC^patients.^Each^dot^represents^t he^mean^value^for^each^^ group,^and^error^bars^represent^SEM.^ ^ Figure^30^shows^Top)^the^proportion^of^repertoire^occupied^b y^small^(<0.01%^of^ repertoire),^medium^(0.01-0.1%^of^repertoire),^large^(0.1-1% ^of^repertoire)^and^ hyperexpanded^(>^1%^of^repertoire)^clones,^Bottom^Left)^t he^large:^small^clones^ratio^^ occupying^the^repertoire,^and^Bottom^Right)^^the^entropy^sco re^(aka^Hill^number)^for^ healthy^subjects^and^patients^with^NSCLC^generated^from^a^di fferent,^publicly-available^ data-set.^ ^ Figure^31^shows^Top^Left)^the^proportion^of^repertoire^occup ied^by^the^top^100^clones,^^ Top^Right)^the^D50^Diversity^Index^and^Bottom)^the^clonality ^index^for^healthy^subjects^ and^patients^with^NSCLC^generated^from^a^different,^publicly -available^data-set.^^ ^ Figure^32^shows^the^D50^diversity^index,^the^Hill^number/ent ropy^score,^the^ratio^of^ large:^small^clones^occupying^the^repertoire^and^the^proport ion^of^repertoire^occupied^^ by^the^top^100^clones^for^a^combined^healthy^+^low^grade^dat a,^^and^a^combined^high^ grade^+^NSCLC^data.^LG=low^grade,^HG=high^grade.^^ ^ Figure^33^shows^Kaplan-Meier^analysis^of^combined^metric^fro m^flow^cytometry^and^ TCR-seq^showing^a^significant^difference^when^future^lung^ca ncer^diagnosis^is^used^as^^ outcome.^^ ^ Figure^34^Top)^shows^feature^importance^scores^of^indicated^ variables^from^a^machine^ learning^model^ developed^with^ the^XGBoost^ algorithm^using^multiple^metrics^derived^ from^ flow^ cytometry^ and^ TCRseq^ data.^ ^ Bottom)^ shows^ the^ ROC^ analysis^ of^ the^^ combination^of^these^techniques^alongside^standard^clinical^ metrics^into^a^single^analysis^ show^promise^ in^ distinguishing^ low-grade^ from^high-grade^pre-invasive^subjects.^The^ model^ was^ tested^ on^ ASCENT^ data^ (AUC=1)^ and^ validated^ on^ pre-invasive^ data^ (AUC=0.76).^ ^ ^ Figure^35^shows^another^example^flow^cytometry^manual^gating ^strategy^for^the^ manual^gating^analysis^of^the^pre-invasive^CD4+^T^cell^data^ (i.e.,^high^grade^and^low^ grade^pre-invasive^samples)^and^ASCENT^flow^cytometry^CD4+^T ^cell^data^(i.e.,^ healthy^subjects^and^NSCLC^patients),^used^in^Example^8,^inc luding^analysis^that^ selects^for^regulatory^T^cells^using^the^biomarker^FoxP3.^Al l^manual^gating^analysis^^ was^carried^out^on^FlowJo^v10.8.1.^Populations^gated^are^sho wn^on^a^concatenated^ file^of^all^samples^from^one^batch.^Frequencies^from^total^C D4^were^used^to^validate^ cluster^significance^from^the^high-dimensional^clustering^pi peline^analysis.^^ ^ Figure^36^Top)^shows^the^%^Treg^CD45RA-CD39+^as^a^proportion ^of^total^CD4^cells^^ for^both^high^and^low^grade^pre-invasive^samples^(left)^and^ for^healthy^and^NSCLC^ patients^(right).^Bottom^shows^a^combined^data^from^the^pre- invasive^flow^cytometry^ analysis^and^the^ASCENT^flow^cytometry^to^display^the^change ^in^Treg^CD45RA- CD39+^population.^ ^ Figure^37^shows^a^Forest^plot^displaying^odds^ratios^calcula ted^from^multivariate^^ analysis^logistic^regression^models^accounting^for^listed^cl inical^variables^in^the^pre- invasive^data.^This^analysis^shows^that^all^three^metrics^fr om^flow^cytometry^(Treg^ CD45RA-CD39+)^and^TCR-seq^(large:small^ratio,^proportion^of^ top^100^clones)^remain^ significant^even^after^accounting^for^the^listed^clinical^va riables.^ ^ ^ ^ The^present^ invention^provides^a^blood^test^which^examines^ the^state^of^blood^T^cell^^ differentiation^to^determine^whether^a^subject^has^or^is^at^ risk^for^having^a^progressive^ or^high-grade^pre-invasive^lesion^or^nodule^or^progressive^s mall^mass.^^It^is^understood^ that^ progressive^ or^ high-grade^ pre-invasive^ lesions^ or^ nodules^ or^ progressive^ small^ masses^are^of^concern^because^they^can^develop^into^a^solid^ malignant^tumour.^^ ^ ^ The^methods^of^the^present^invention^can^also^provide^a^bloo d^test^which^examines^the^ state^of^blood^T^cell^differentiation^to^determine^whether^a ^subject^has^or^ is^at^risk^of^ having^a^solid^malignant^tumour.^^This^is^because^similar^or ^the^same^patterns^of^T^cell^ differentiation^ skewing^ may^ be^ identified^ if^ a^ subject^ has^ a^ solid^ malignant^ tumour^^^ Therefore^the^present^invention^provides^a^method^for^determ ining^whether^a^subject^is^^ at^risk^for^having^a^progressing^or^high-grade^pre-invasive^ lesion,^nodule^or^small^mass,^ and/or^having^a^solid^malignant^tumour,^by^analysing^T^cell^ differentiation^skewing^in^a^ sample^of^blood^obtained^from^the^subject.^Prior^to^the^pres ent^invention^there^was^no^ effective^non-invasive,^screening^method^ for^early^detection^of^a^progressive^or^high- grade^pre-invasive^lesion^or^nodule^or^small^mass^that^uses^ T^cell^differentiation^for^the^^ test.^^ ^ T^cells^in^humans^with^a^challenged^immune^system^are^known^ to^look^more^activated,^ exhausted^ or^ differentiated,^ compared^ to^ those^ who^ are^ not^ challenged.^ Prior^ to^ the^ present^ invention^ it^was^not^known^ that^ such^a^challenged^ immune^ system^could^ be^^ detected^in^the^blood^of^individuals^who^do^not^yet^have^an^ established^solid^malignant^ tumour^but^harbour^a^lesion^or^nodule^or^small^mass^of^the^t ype^that^can^be^expected^to^ develop^into^disease.^The^present^invention^leverages^this^c oncept^by^generating^a^test^ that^measures^T^cell^differentiation,^exhaustion^and^activat ion.^^^ ^ The^present^ invention^ is^concerned^with^early^detection^of^cancer.^ ^Prior^art^methods^^ have^been^concerned^with^detection^of^cancer^at^stage^1^or^2 ,^rather^than^detection^at^ stage^3^or^4^when^the^prognosis^is^very^poor,^and^this^has^s ometimes^been^referred^to^ as^“early^detection”.^^In^contrast^early^detection^in^th e^context^of^the^present^invention^ includes^detection^of^pre-invasive^or^high-grade^lesions^or^ nodules^that^can^develop^into^ a^solid^ tumour.^ ^Detection^prior^ to^ the^establishment^of^ a^ tumour^ can^ lead^ to^greater^^ treatment^options^and^improved^prognosis.^Detection^of^such^ lesions^or^nodules^currently^ occurs^ via^ low^ dose^ CT^ screening^ or^ ineffective^ and/or^ costly^ and^ /^ or^ invasive^ and^ burdensome^ methods.^ A^ high^ sensitivity^ and^ specificity^ blood^ test^ using^ T^ cell^ differentiation^has^not^been^described.^ ^ ^ The^methods^of^the^present^invention^may^be^used^for^detecti on^of^a^progressing^or^high- grade^pre-invasive^lesion^or^nodule^(or^pre-invasive^neoplas ia).^^The^present^invention^ may^also^be^valuable^to^detect^solid^malignant^tumours^at^st age^I^or^later,^and^in^some^ embodiments,^preferably^stage^I^solid^malignant^tumours.^^ ^ ^ Pre-invasive^generally^refers^to^a^cluster^of^malignant^cell s^or^lesions^that^have^not^left^ their^original^focal^or^spread^to^other^parts^of^the^body^an d^are^not^yet^considered^to^be^ invasive.^Nodule^generally^refers^to^a^growth^or^lump^that^m ay^be^malignant^or^benign.^ The^early^cancer^detection^in^the^present^disclosure^is^conc erned^with^changes^prior^to^ stages^1-4^of^established^cancer.^^Therefore,^pre-invasive^a s^described^herein^may^refer^^ to^a^stage^of^neoplasia^development^before^stages^1-4^cancer .^Optionally,^this^disclosure^ may^be^concerned^with^very^early^(stage^1)^solid^tumours,^i. e.,^the^detection^of^very^early^ (stage^1)^solid^tumours.^^ ^ In^the^context^of^this^invention,^pre-invasive^lesions^or^no dules^or^small^masses^are^or^^ can^ be^ classified^ into^ low-grade^ and^ high-grade.^ High-grade^ pre-invasive^ lesions,^ nodules^ or^ small^ masses^ have^ a^ well-established^ meaning^ in^ the^ art.^ This^ is^ demonstrated^in^at^ least^by^Pennycuick^et^al;^Cancer^Discov.^2020^Oct;^10(10):^ 1489- 1499;^Banerjee^et^al;^Journal^of^Thoracic^Oncology,^Volume^4 ,^Issue^4,^April^2009,^Pages^ 545-551;^ D.^Moro‐Sibilot^ et^ al;^ European^Respiratory^ Journal^2004^24:^24-29,^ among^^ others.^^ ^ The^term^high-grade^pre-invasive^lesion,^nodule^or^small^mas s^may^also^encompass^or^ may^be^used^ interchangeably^with^pre-invasive^neoplasia^and^pre-invasive ^neoplastic^ lesion.^In^some^embodiments,^the^term^high-grade^pre-invasiv e^lesion,^nodule^or^small^ mass^may^also^encompass^or^be^used^interchangeably^with^the^ following^terms^in^the^^ Table^below:^ ^ ^ High-grade^lesion^is^an^umbrella^term^encompassing^carcinoma ^in^situ^(CIS)^and^severe^ dysplasia^ lesions^ of^ the^ airway.^ Low-grade^ lesions^ include^ hyperplasia,^ squamous^^ metaplasia,^mild^dysplasia,^and^moderate^dysplasia.^These^ar e^all^different^histological^ states^of^different^levels^of^neoplasia^of^the^airway^that^c an^be^identified^from^a^lesion^ biopsy^by^a^histopathologist.^These^different^histological^s tates^are^classified^into^low-^or^ high-grade^groups^to^facilitate^downstream^analysis,^and^sep arated^in^this^way^based^on^ the^clinical^risk^each^group^carries.^Low-grade^lesions^carr y^no^significant^clinical^risk^of^^ developing^into^invasive^disease/lung^cancer,^whereas^50%^of ^high-grade^lesions^have^ been^ reported^ to^ progress^ into^ invasive^ disease/lung^ cancer^ (P^ J^ George^ et^ al.^ Surveillance^for^the^detection^of^early^lung^cancer^in^patie nts^with^bronchial^dysplasia.^ Thorax^2007;62:43-50.^doi^:^10.1136/thx.2005.052191).^ ^ In^some^embodiments,^the^high^grade^pre-invasive^lesion,^nod ule^or^solid^mass^may^be^ a^high-grade^bronchial^ lesion,^a^pre-invasive^ lesion^of^ the^bronchus,^a^bronchial^pre- invasive^lesion,^a^bronchial^dysplasia,^bronchial^lesion^or^ an^endobronchial^lesion.^^^ ^ ^ In^the^present^ invention,^classification^of^ lesions^or^nodules^or^small^masses^ into^ low- grade^ or^ high-grade^ can^ be^ performed^ using^ a^ ratio^ of^ differing^ T^ cell^ phenotypes,^ identified^using^^detection^biomarkers^on/within^the^T^cells .^Classification^of^ lesions^or^ nodules^or^small^masses^into^low-grade^or^high-grade^can^be^ performed^by^determining^ the^proportion^of^T^cells^comprising^one^or^more^detection^b iomarkers^on/within^the^T^^ cells^as^a^percentage^of^T^cells.^ ^ Skewing^of^T^cell^differentiation^can^be^detected^by^analysi ng^a^trait^of^the^T^cells.^ ^ Alternatively,^skewing^of^T^cell^differentiation^can^be^dete cted^by^analysing^a^plurality^of^^ traits^of^the^T^cells.^^The^information^obtained^from^analys ing^a^plurality^of^traits^can^be^ combined^(e.g.,^using^TCR-seq^and^cytometry).^^Combining^inf ormation^from^analysing^ a^plurality^of^ traits^may^provide^a^ further^ improved^method^ for^determining^whether^a^ subject^is^at^risk^for^having^a^progressing^or^high-grade^pr e-invasive^lesion,^nodule^or^ small^mass,^or^having^a^solid^malignant^tumour.^^^ ^ ^ A^trait^of^T^cells^to^be^analysed^can^be^a^phenotypic^trait. ^^^ ^ Examples^of^methods^for^analysing^skewing^of^T^cell^differen tiation^are^described^herein.^ ^^ ^ Skewing^ of^ T^ cell^ differentiation^ can^ be^ detected^ by^ determining^ a^ ratio^ of^ activated^ and/or^exhausted^T^cells^:^naïve^and/or^resting^T^cells^in^ a^sample^of^blood^obtained^from^ the^subject,^or^by^determining^a^proportion^of^activated^and /or^exhausted^T^cells^as^a^ percentage^ of^ T^ cells^ (i.e.,^ total^ T^ cells).^ ^ Or^ a^ subpopulation^ of^ T^ cells^ (i.e.,^ total^ subpopulation^of^T^cells).^Optionally^skewing^of^T^cell^diff erentiation^can^be^detected^by^^ determining^a^ratio^of^activated^and/or^exhausted^T^cells^:^ T^cells^which^are^not^activated^ and/or^exhausted^T^cells^in^a^sample^of^blood^obtained^from^ the^subject.^Where^a^subject^ is^shown^to^have^more^activated^and/or^exhausted^T^cells^(e. g.,^based^on^detection^of^a^ combination^of^T^cell^biomarker^expression),^this^would^be^i ndicative^of^the^subject^being^ at^risk^of^having^a^progressing^or^high-grade^pre-invasive^l esion^or^nodule^or^small^mass,^^ or^having^a^solid^malignant^tumour^(e.g.,^a^stage^1^solid^ma lignant^tumour).^Optionally^ skewing^of^T^cell^differentiation^can^be^detected^by^determi ning^a^proportion^of^activated^ and/or^exhausted^T^cells^as^a^proportion^of^T^cells,^or^a^su bpopulation^of^T^cells,^ in^a^ sample^of^blood^obtained^from^the^subject.^Where^a^subject^i s^shown^to^have^a^greater^ proportion^of^activated^and/or^exhausted^T^cells^(e.g.,^base d^on^detection^of^a^specific^ biomarker^expression),^this^would^be^indicative^of^the^subje ct^being^at^risk^of^having^a^^ progressing^or^high-grade^pre-invasive^lesion^or^nodule^or^s mall^mass,^or^having^a^solid^ malignant^tumour^(e.g.,^a^stage^1^solid^malignant^tumour).^^ ^ ^ Skewing^of^T^cell^differentiation^can^be^detected^by^analysi ng^the^diversity^(i.e.,^diversity^ and/or^clonality)^of^T^cell^receptors^on^the^T^cells^in^the^ sample^of^blood^obtained^from^^ the^subject.^^This^may^include^analysing^the^diversity^of^CD R3B^on^TCRs^of^the^T^cells^ in^the^sample^of^blood^obtained^from^the^subject.^^Such^anal ysis^may^be^performed^using^ TCR^Seq.^ ^Where^ a^ subject^ is^ shown^ to^ have^ a^ lower^ or^ a^ reduced^ diversity^ of^ the^ repertoire^ of^ TCRs^ this^ would^ be^ indicative^ of^ the^ subject^ being^ at^ risk^ of^ having^ a^ progressing^or^high-grade^pre-invasive^lesion^or^nodule^or^s mall^mass,^or^having^a^solid^^ malignant^ tumour.^ Since^ clonality^ is^ inversely^ related^ to^ diversity,^ diversity^ may^ be^ measured^directly^by^one^or^more^diversity^metrics^or^indire ctly^by^one^or^more^clonality^ metrics.^^Since^clonality^is^inversely^related^to^diversity, ^when^a^subject^is^shown^to^have^ a^ higher^ clonality,^ this^ would^ be^ indicative^ of^ the^ subject^ being^ at^ risk^ of^ having^ a^ progressing^or^high-grade^pre-invasive^lesion^or^nodule^or^s mall^mass,^or^having^a^solid^^ malignant^ tumour.^ Similarly,^ skewing^ of^ T^ cell^ differentiation^ can^ be^ detected^ by^ analysing^the^clonality^of^T^cell^receptors^on^the^T^cells^i n^the^sample^of^blood^obtained^ from^the^subject,^or^a^proxy^of^clonality.^^ ^ Skewing^of^T^cell^differentiation^can^be^detected^by^analysi ng^T^cells^in^a^sample^of^^ blood^obtained^from^a^subject^using^more^than^one^technique. ^^For^example,^ Therefore,^skewing^of^T^cell^differentiation^can^be^detected ^by^both:^ a)^determining^a^ratio^of^activated^and/or^exhausted^T^cells ^:^naïve^and/or^resting^T^ cells^in^a^sample^of^blood^obtained^from^the^subject,^or^by^ determining^a^ratio^of^ activated^and/or^exhausted^T^cells^:^T^cells^which^are^not^a ctivated^and/or^exhausted^T^^ cells^in^a^sample^of^blood^obtained^from^the^subject^(for^ex ample^cytometry),^^ and^ b)^analysing^the^diversity^and/or^clonality^of^T^cell^recept ors^on^the^T^cells^in^the^sample^ of^blood^obtained^from^the^subject^(TCR^Seq).^ ^ ^ Skewing^of^T^cell^differentiation^can^also^be^detected^by^bo th:^ a)^determining^a^proportion^of^activated^and/or^exhausted^T^ cells^(or^a^subpopulation^of^ T^cells)^as^a^percentage^of^T^cells^(or^subpopulation^of^T^c ells)^in^a^sample^of^blood^ obtained^from^the^subject^(for^example^cytometry),^^ and^ b)^analysing^the^diversity^and/or^clonality^of^T^cell^recept ors^on^the^T^cells^in^the^sample^^ of^blood^obtained^from^the^subject^(TCR^Seq).^ ^ In^the^context^of^the^invention,^low-grade^or^“LG”,^as^u sed^herein,^generally^refers^to^the^ pre-invasive^stages^of^cancer^development^characterised^by^s quamous^metaplasia,^mild^ dysplasia^and^moderate^dysplasia.^Generally,^a^low-grade^pre -invasive^lesion^or^nodule^^ or^small^mass^may^not^need^further^clinical^intervention^sin ce^it^is^not^expected^to^develop^ into^cancer.^^^ ^ Whereas^high-grade^or^“HG”,^as^used^herein,^generally^re fers^to^the^pre-invasive^disease^ stages^of^cancer^development^characterised^by^severe^dysplas ia^and^carcinoma^in^situ.^^ Generally,^a^high-grade^pre-invasive^lesion^or^nodule^or^sma ll^mass^should^be^the^focus^ of^further^clinical^ follow^up.^This^has^a^well-established^term^in^ the^art.^This^ term^may^ encompass^or^may^be^used^ interchangeably^with^pre-invasive^neoplasia^and/or^other^ terms^as^described^elsewhere^herein.^^ ^ ^ The^ utility^ of^ the^ present^ invention^ is^ shown^ herein^ in^ two^ types^ of^ lung^ cancer^with^ differing^ clinical^ and^ genomic^ features^ and^ in^ renal^ cancer.^ ^ These^are^ cancers^with^ differing^body^locations^and^differing^mutational^background s.^^Therefore,^the^underlying^ principle^of^T^cell^activation^or^T^cell^differentiation^ske wing^ in^the^presence^of^a^high- grade^or^progressing^pre-invasive^nodule,^ or^ lesion,^which^ could^ lead^ to^a^malignant^^ tumour^is^broad^and^encompasses^solid^tumours^generally.^^Th erefore,^the^utility^of^this^ test^for^early^detection^is^pan-cancer.^^^ ^ Advantages^ ^ ^ The^present^invention^provides^at^least^the^following^advant ages^over^previously^known^ methods.^ ·^ A^non-invasive^method^for^detecting^whether^a^subject^is^at^ risk^for^having^a^ progressing^or^high-grade^pre-invasive^lesion^or^nodule^or^h aving^a^solid^ tumour^using^T^cells^as^a^read^out.^ ^ ·^ A^method^for^distinguishing^between^a^progressing^or^high-gr ade^pre-invasive^ lesion^or^nodule^and^a^non-progressing^or^low-grade^lesion^o r^nodule.^^ ·^ An^in^vitro^method.^ ·^ A^method^that^only^requires^a^blood^sample^to^determine^whet her^a^subject^is^at^ risk.^Whole^blood^or^peripheral^blood^mononuclear^cell^(PBMC )^may^be^used.^ ·^ A^non-invasive^procedure^that^encourages^high^compliance^wit h^a^population^^ screening^program^requiring.^ ·^ A^method^that^has^high^sensitivity^and^specificity.^ ·^ A^method^that^is^valuable^for^early^detection^of^a^wide^rang e^of^solid^tumours,^ such^as^RCC,^LUAD^and^LUSC^which^therefore^provides^a^valuab le^tool^as^the^ test^works^on^a^variety^of^cancers,^even^when^they^come^from ^different^causes.^^^ ·^ A^method^that^can^be^used^as^a^screening^tool^on^healthy^peo ple^that^can^be^ used^to^see^if^the^subject^is^healthy^and^can^be^incorporate d^in^standard^blood^ tests^to^see^the^likelihood^that^someone^has^a^risk^of^progr essing^or^high-grade^ pre-invasive^lesion^or^nodule^or^having^a^solid^tumour.^ ·^ A^method^that^can^be^personalised^to^be^used^as^subject’s^ baseline,^that^can^^ be^referred^to^in^later^tests^to^see^if^the^subject’s^risk ^of^developing^progressing^ or^high-grade^pre-invasive^lesion^or^nodule^or^having^a^soli d^tumour^ ·^ A^method^that^is^or^can^be^validated^by^one^or^more^differen t^techniques^(e.g.,^ using^flow^cytometry^and^TCR-seq)^ ·^ A^method^that^can^utilise^more^than^one^different^technique^ in^combination^^ (e.g.,^using^flow^cytometry^and^TCR-seq)^to^more^significant ly^differentiate^ between^sample^types,^as^a^more^effective^tool^for^early^det ection^of^cancer^ (e.g.,^at^the^pre-invasive^stage,^or^in^early^stage^(e.g.,^s tage^1)^cancer.^^^^ ^ Unless^ defined^ otherwise,^ all^ technical^ and^ scientific^ terms^ used^ herein^ have^ the^^ meaning^ commonly^understood^by^ a^person^ skilled^ in^ the^ art^ to^which^ this^ invention^ belongs.^Certain^terms^are^defined^below^for^the^sake^of^cla rity^and^ease^of^reference.^ ^ The^present^invention^provides^a^method^or^methods^for^deter mining^whether^a^subject^ is^at^risk^for^having^a^progressing^or^high-grade^pre-invasi ve^lesion^or^nodule^or^having^^ a^solid^tumour.^^This^or^these^methods^can^also^be^understoo d^as^distinguishing^a^subject^ having^ a^ progressing^ or^ high-grade^ pre-invasive^ lesion^ or^ nodule^ or^ having^ a^ solid^ tumour,^ from^a^ subject^not^ having^a^progressing^or^ high-grade^pre-invasive^ lesion^or^ nodule^ or^ not^ having^ a^ solid^ tumour^ or^ having^ a^ non-progressing^ or^ low-grade^ pre- invasive^lesion^or^nodule.^The^method^or^methods^disclosed^h erein^also^can^distinguish^^ a^subject^having^a^progressing^or^high^grade^pre-invasive^le sion^or^nodule,^or^having^a^ solid^tumour^(e.g.,^a^stage^I^solid^tumour)^from^a^healthy^s ubject.^^ ^ The^subject^is^a^mammal.^The^subject^may^be^a^human.^^Altern atively,^the^subject^may^ be^in^the^Primates,^Rodentia,^Canidae,^Felidae,^Equidae^orde r^or^other^mammal^orders.^ The^subject^may^be^a^horse,^cat,^dog^or^other^companion^anim al.^^The^subject^can^be^^ healthy^or^asymptomatic.^^The^subject^may^be^a^patient.^The^ subject^can^be^suspected^ of^having^a^cancer.^^The^subject^can^have^a^genetic^pre-disp osition^to^cancer^or^have^ lifestyle^factors^increasing^the^likelihood^for^developing^c ancer.^^The^subject^may^have^ previously^received^treatment^for^a^cancer.^^The^methods^of^ the^present^invention^may^ also^be^used^for^population^screening^and/or^be^used^as^a^st andard^clinical^tool^in^routine^^ blood^work,^also^referred^to^as^mass^testing.^^^ ^ A^subject^is^at^risk,^ if^the^method^of^the^present^invention^indicates^that^the^su bject^is^ likely^to^have,^or^has,^a^high-grade^pre-invasive^lesion^or^ nodule.^^A^subject^is^also^at^ risk^if^the^method^of^the^present^invention^indicates^that^t he^subject^is^likely^to^have,^or^^ has,^a^solid^tumour,^e.g.,^a^stage^1^solid^malignant^tumour. ^^ ^ The^term^solid^tumour^generally^refers^to^an^abnormal^mass^o f^tissue^that^usually^does^ not^ contain^ cysts^ or^ liquid^ areas.^ Solid^ tumours^ may^ be^ benign,^ or^ malignant.^ ^ The^ methods^of^ the^present^allow^to^determine^if^a^subject^ is^at^risk^of^developing^a^solid^^ malignant^ tumour^ or^ at^ risk^ of^ having^ a^ solid^malignant^ tumour^ (e.g.,^ a^ stage^ I^ solid^ tumour).^The^term^is^meant^to^exclude^liquid^tumours^or^haem atological^malignancies.^A^ lesion^ generally^ refers^ to^ an^ area^ of^ abnormal^ tissue.^ A^ lesion^ may^ be^ benign^ or^ malignant,^or^premalignant^and^if^premalignant^can^represent ^moderate^or^mild^or^severe^ dysplasia,^metaplasia^or^carcinoma^in^situ.^A^nodule^general ly^refers^to^a^growth^or^lump^^ that^may^be^malignant,^benign^or^ indeterminate.^ ^Low-grade^or^ “LG”,^as^used^herein,^ generally^refers^to^the^pre-invasive^disease^stages^of^cance r^development^characterised^ by^squamous^metaplasia,^mild^dysplasia^and^moderate^dysplasi a.^^Whereas^high-grade^ or^ “HG”,^ as^ used^ herein,^ generally^ refers^ the^ pre-invasive^ disease^ stages^ of^ cancer^ development^characterised^by^to^severe^dysplasia^and^carcino ma^in^situ.^This^has^a^well-^ established^term^in^the^art.^This^term^may^encompass^or^may^ be^used^interchangeably^ with^pre-invasive^neoplasia^and/or^other^terms^as^described^ elsewhere^herein.^^ ^ The^methods^of^the^present^invention^are^performed^on^a^bloo d^sample^obtained^from^a^ subject^and^hence^occur^in^vitro.^^The^methods^can^use^whole ^blood.^^The^methods^can^^ use^PBMCs.^ ^ Analysis^of^T^cells^ ^ The^present^invention^provides^a^method^for^determining^whet her^a^subject^is^at^risk^for^ having^a^progressing^or^high-grade^pre-invasive^lesion,^nodu le^or^small^mass,^or^having^^ a^solid^malignant^tumour^(e.g.,^a^stage^I^solid^malignant^tu mour),^the^method^comprising^ analysing^the^proportion^of^T^cells^in^a^sample^of^blood^obt ained^from^the^subject^which^ are^activated^and/or^exhausted^T^cells^by^analysing^a^trait^ of^the^T^cells.^^ ^ The^method^may^ include^ analysing^ the^ proportion^ of^ T^ cells^ in^ the^ sample^ of^ blood^^ obtained^from^the^subject^which^are^activated^and/or^exhaust ed^comprises^analysing^a^ plurality^of^traits^of^the^T^cells^(e.g.,^by^TCR^Seq,^and^bi omarkers^expressed^by^T^cells,^ which^may^be^analysed,^for^example,^by^flow^cytometry).^^ ^ A^trait^of^the^T^cells^relates^to^a^property^of^the^T^cells^ which^can^be^analysed.^^The^trait^^ may^be^a^phenotypic^trait.^^The^phenotypic^trait^can^be^dive rsity^(or^diversity^or^clonality)^ of^T^cell^receptors^,^which^may^be^analysed,^for^example,^by ^TCR^Seq.^^The^phenotypic^ trait^can^be^biomarkers^expressed^by^T^cells,^which^may^be^a nalysed,^for^example,^by^ flow^cytometry.^^^ ^ ^ The^present^invention^also^embraces^analysing^a^plurality^of ^traits^of^T^cells^in^a^sample^ of^blood^obtained^from^the^subject.^^^The^phenotypic^traits^ can^be^diversity^(or^diversity^ or^clonality)^of^T^cell^receptors,^which^may^be^analysed,^fo r^example,^by^TCR^Seq,^and^ biomarkers^ expressed^ by^ T^ cells,^ which^ may^ be^ analysed,^ for^ example,^ by^ flow^ cytometry.^^The^diversity^or^clonality^of^T^cell^of^T^cell^r eceptors^(e.g.,^analysed^by^TCR^^ Seq)^and^the^biomarkers^expressed^by^T^cells^(e.g.,^analysed ^by^flow^cytometry)^may^be^ determined^by^any^suitable^method^as^described^herein.^ ^ Diversity^of^T^cell^receptors^ ^ ^ The^present^invention^provides^a^method^for^determining^whet her^a^subject^is^at^risk^for^ having^a^progressing^or^high-grade^pre-invasive^lesion,^nodu le^or^small^mass,^or^having^ a^solid^malignant^tumour,^the^method^comprising^analysing^th e^proportion^of^T^cells^in^a^ sample^of^blood^obtained^from^the^subject^which^are^activate d^and/or^exhausted^T^cells^ by^analysing^a^trait^of^the^T^cells.^^ ^ ^ The^trait^may^be^a^phenotypic^trait.^^The^phenotypic^trait^m ay^be^the^diversity^of^T^cell^ receptors^on^ the^T^cells^ in^ the^sample^of^blood,^optionally^ the^diversity^of^CDR3B^on^ TCRs^of^ the^T^ cells^ in^ the^sample^of^ blood,^ further^ optionally^using^TCR^Seq.^Since^ clonality^is^inversely^related^to^diversity,^diversity^may^i nclude^one^or^more^diversity^or^^ clonality^metrics.^Therefore,^the^phenotypic^trait^may^be^th e^diversity^and/or^clonality^of^ T^cell^receptors^on^the^T^cells^in^the^sample^of^blood,^opti onally^the^diversity^of^CDR3B^ on^TCRs^of^the^T^cells^in^the^sample^of^blood,^further^optio nally^using^TCR^Seq.^ ^ In^some^embodiments,^the^diversity^and/or^clonality^of^T^cel l^receptors^on^the^T^cells^in^^ the^sample^of^blood^may^be^determined^using^any^suitable^div ersity^metric^or^clonality^ metric.^The^term^clonality^and/or^clonality^metric^may^encom pass^metrics^which^analyse^ clonal^expansion,^therefore,^ in^some^embodiments,^the^clonality^of^T^cell^receptors^on^ the^T^cells^in^the^sample^of^blood^may^be^determined^by^anal ysing^clonal^expansion^of^ the^T^cell^receptors^on^the^T^cells^in^the^sample^of^blood.^ Suitable^methods^of^determining^^ the^diversity^and/or^clonality^are^described^in^a^Table^belo w.^^ ^ ^ In^some^embodiments,^the^subject^may^be^at^risk^if^the^diver sity^of^the^repertoire^of^TCRs^ is^less^than^the^diversity^of^the^repertoire^of^TCRs^of^a^co mparison^subject^or^a^plurality^ of^comparison^subjects.^In^this^context^a^comparison^subject ^can^be^a^healthy^subject,^^ or^a^plurality^of^comparison^subjects^can^be^plurality^of^he althy^subjects^or^a^member^of^ the^ general^ population.^ In^ some^ embodiments^ and^ examples^ described^ herein,^ the^ diversity^metric^is^analysed^using^a^D50^diversity^score^and /or^a^Hill^number.^^ ^ In^some^embodiments,^the^subject^may^be^at^risk^if^the^clona lity^of^the^repertoire^of^TCRs^^ is^higher^than^the^clonality^of^the^repertoire^of^TCRs^of^a^ comparison^subject^or^a^plurality^ of^comparison^subjects.^In^this^context^a^comparison^subject ^can^be^a^healthy^subject,^ or^a^plurality^of^comparison^subjects^can^be^plurality^of^he althy^subjects^or^a^member^of^ the^ general^ population.^ In^ some^ embodiments^ and^ examples^ described^ herein,^ the^ clonality^metric^may^be^analysed^using^a^clonality^index,^th e^clonality^of^the^repertoire^of^^ the^TCRs^occupied^by^the^top^n^clones^(e.g.,^wherein^n^is^50 -1000^clones,^or^n^=^100),^ and/or^the^the^ratio^of^the^repertoire^of^TCRs^occupied^by^l arge^clones:the^repertoire^of^ TCRs^occupied^by^small^clones.^^ ^ The^subject^may^be^at^risk^if:^ a)^the^diversity^of^ the^repertoire^of^TCRs^ is^ less^than^the^diversity^of^ the^repertoire^of^ TCRs^of^a^comparison^subject^or^a^plurality^of^comparison^su bjects,^^ b)^D50^diversity^score^is^lower^than^a^D50^diversity^score^o btained^from^analysing^T^cells^ in^a^sample^of^blood^obtained^from^a^comparison^subject^or^a ^plurality^of^comparison^^ subjects,^^ c)^a^clonality^score^is^higher^than^a^clonality^score^obtain ed^from^analysing^T^cells^in^a^ sample^ of^ blood^ obtained^ from^ a^ comparison^ subject^ or^ a^ plurality^ of^ comparison^ subjects,^or^ d)^Hill^number^is^lower^than^a^Hill^number^obtained^from^ana lysing^T^cells^in^a^sample^of^^ blood^obtained^from^a^comparison^subject^or^a^plurality^of^c omparison^subjects,^or^^ e)^the^clonality^of^the^repertoire^of^the^TCRs^occupied^by^t he^top^n^clones^is^^ higher^than^the^clonality^of^the^repertoire^of^TCRs^occupied ^by^the^top^n^clones^in^a^ comparison^subject^or^a^plurality^of^comparison^subjects,^wh erein^n^is^optionally^ between^50^and^1000,^e.g.,^wherein^n^=^100^ ^ (f)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^sma ll^clones^is^lower^than^the^ proportion^of^the^repertoire^of^TCRs^occupied^by^small^clone s^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects,^or^ (g)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^lar ge^clones^is^higher^than^the^ proportion^of^the^repertoire^of^TCRs^occupied^by^large^clone s^in^a^comparison^subject^^ or^plurality^of^comparison^subjects^or^ (h)^the^ratio^of^the^repertoire^of^TCRs^occupied^by^large^cl ones:^the^repertoire^of^TCRs^ occupied^by^small^clones^is^higher^than^the^ratio^of^the^rep ertoire^of^TCRs^occupied^by^ large^clones:^the^repertoire^of^TCRs^occupied^by^small^clone s^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects^ ^ ^ In^this^context^a^comparison^subject^can^be^a^healthy^subjec t,^or^a^plurality^of^comparison^ subjects^can^be^plurality^of^healthy^subjects^or^a^member^of ^the^general^population,^ ^ The^subject^may^be^at^risk^if:^ ^ a)^the^diversity^of^the^repertoire^of^TCRs^is^similar^to^or^ greater^than^the^diversity^of^the^ repertoire^of^TCRs^of^a^comparison^subject^or^a^plurality^of ^comparison^subjects,^^ b)^D50^diversity^score^is^similar^to^or^greater^than^a^D50^d iversity^score^obtained^from^ analysing^T^cells^in^a^sample^of^blood^obtained^from^a^compa rison^subject^or^a^plurality^ of^comparison^subjects,^^ c)^a^clonality^score^is^similar^to^or^greater^than^a^clonali ty^score^obtained^from^analysing^ T^ cells^ in^ a^ sample^ of^ blood^ obtained^ from^ a^ comparison^ subject^ or^ a^ plurality^ of^ comparison^subjects,^or^ d)^Hill^number^is^similar^to^or^greater^than^a^Hill^number^o btained^from^analysing^T^cells^^ in^a^sample^of^blood^obtained^ from^a^comparison^subject^or^a^plurality^of^comparison^ subjects.^ e)^the^clonality^of^the^repertoire^of^TCRs^occupied^by^the^t op^n^clones^is^similar^to^or^ lower^than^the^clonality^of^the^repertoire^of^TCRs^occupied^ by^the^top^n^clones^in^a^ comparison^subject^or^a^plurality^of^comparison^subjects,^op tionally^wherein^n^is^50^to^^ 1000,^e.g.,^^wherein^n^=^100^ (f)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^sma ll^clones^is^similar^to^or^ greater^than^the^proportion^of^the^repertoire^of^TCRs^occupi ed^by^small^clones^in^a^ comparison^subject^or^ a^plurality^of^comparison^subjects,^or^ ^ (g)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^lar ge^clones^is^similar^to^or^ lower^than^the^proportion^of^the^repertoire^of^TCRs^occupied ^by^large^clones^in^a^ comparison^subject^or^plurality^of^comparison^subjects^or^ (h)^the^ratio^of^the^repertoire^of^TCRs^occupied^by^large^cl ones:^the^repertoire^of^TCRs^ occupied^by^small^clones^is^similar^to^or^lower^than^the^rat io^of^the^repertoire^of^TCRs^^ occupied^by^large^clones:^the^repertoire^of^TCRs^occupied^by ^small^clones^in^a^ comparison^subject^or^a^plurality^of^comparison^subjects.^^ ^ In^ this^ context^ a^ comparison^ subject^ or^ a^ plurality^ of^ comparison^ subjects^ are^ those^ known^ to^ can^ be^ a^ subject^ known^ to^ have^ a^ progressing^ or^ high-grade^ pre-invasive^^ lesion,^nodule^or^small^mass,^or^known^to^have^a^solid^malig nant^tumour.^ ^ Based^on^the^above,^the^person^skilled^in^the^art^of^TCR^Seq ^knows^whether^identifying^ greater,^similar^or^lower^diversity^of^TCRs^is^indicative^of ^the^subject^being^at^risk^based^ on^the^disease^status^(having^cancer^or^being^healthy)^of^th e^^comparison^subject(s).^^ ^ The^ term^ “T^ cell^ receptor”^ (TCR),^ as^ used^ herein,^ generally^ refers^ to^ the^molecular^ structure^on^T^cells^that^is^composed^of^two^different^prote in^chains,^with^the^vast^majority^ of^ human^ T^ cells^ having^TCR^ containing^ α^ (alpha)^ and^ β^ (beta)^ chains^ and^minority^ comes^from^TCR^containing^γ^(gamma)^chain^and^one^δ^(delta )^chains.^In^the^majority^^ of^T^cells,^diversity^of^TCR^comes^from^genes^encoding^alpha ^(Tcra)^and^beta^chains^ (Tcrb)^having^multiple^non-contiguous^gene^segments^which^ include^variable^ (V)^and^ joining^(J)^for^the^Tcra^gene^and^variable^(V),^diversity^(D ),^and^joining^(J)^segments^for^ the^Tcrb^gene.^The^process^of^genetic^recombination^of^the^D NA^encoded^segments^in^ individual^T^cells^occurs,^that^results^in^random^combinatio ns^of^the^V,^D,^and^J^segments^ for^ TCR^ beta^ chains^ and^ V^ and^ J^ segments^ for^ TCR^ alpha^ chains.^ This^ results^ in^^ formation^of^CDR3,^which^ is^ essential^ for^ the^T^cell’s^ ability^ to^ recognise^ its^ cognate^ antigen^and^is^therefore^highly^variable.^ ^ The^ term^ “TCRseq”,^ as^ used^ herein,^ generally^ refers^ to^ the^ well-established,^ high^ throughput^sequencing^method^for^studying^TCR^repertoires,^i ncluding^allowing^greater^^ insight^into^CDR3^diversity^within^a^T^cell^population.^Here ^DNA^or^RNA^starting^material^ is^combined^with^PCR-based^amplification^methods.^One^such^m ethod^is^multiplex^PCR,^ where^primers^for^the^J^alleles^and^all^known^V^alleles^are^ combined^with^the^sample^to^ amplify^the^V^and^J^alleles.^These^amplified^products^are^th en^combined^with^appropriate^ sequencing^adaptors^ and^ analysed^ via^ next^ generation^sequencing^ (NGS).^ The^ data^^ obtained^from^the^NGS^provides^information^into^variables^su ch^as^the^abundance^of^a^ particular^T^cell^clone.^^ ^ The^term^“repertoire”^as^used^herein,^generally^refers^t o^the^unique^T-cell^receptor^(TCR)^ genetic^ rearrangements^ within^ the^ adaptive^ immune^ system.^ In^ the^ context^ of^ the^^ invention,^ this^ includes^ determining^ the^ abundance^ of^ a^ given^ TCR^ (and^ therefore^ determining^the^diversity^of^TCR)^within^a^subject’s^blood ^sample,^which^when^compared^ to^a^comparison^subject,^it^may^indicate^the^subject^is^at^r isk^for^having^a^progressing^or^ high-grade^pre-invasive^lesion,^nodule^or^small^mass,^or^hav ing^a^solid^malignant^tumour.^^ ^ ^ The^terms^“D50”,^“Hill^number”^and^“clonality^scor e^or^“clonality^index”,^“proportion^of^the^ repertoire^ occupied^ by^ the^ top^ n^ clones,^ e.g.,^ top^ 100^ clones”^ or^ grouping^ of^ TCR^ clonotypes^ as^ small,^ medium,^ large^ or^ hyperexpanded,^ as^ used^ herein,^ will^ be^ understood^by^ the^person^skilled^ in^ the^art^ to^be^values^ that^are^ relevant^ to^TCRseq^ analysis^when^determining^ the^diversity^and/or^clonality^of^ the^ repertoire^of^TCR^ in^a^^ sample.^They^may^be^determined^according^to^the^methods^desc ribed^herein^(i.e.,^in^the^ Examples^section).^For^example,^the^Hill^score^and^the^D50^s core^may^be^determined^ using^the^“repDiversity”^function^from^the^immunarch^pac kage^(v0.6.9).^To^determine^the^ grouping^of^TCR^clonotypes^as^small^clones^(defined^as^<0 .01%^of^repertoire),^medium^ clones^ (defined^ as^ 0.01-0.1%^ of^ repertoire),^ large^ clones^ (defined^ as^ 0.1-1%^ of^^ repertoire)^or^hyperexpanded^clones^(defined^as^>1%^of^re pertoire),^the^“repClonality”^ function^may^be^used^from^the^immunarch^package^(v0.6.9),^e. g.,^as^described^herein.^ To^calculate^the^large:small^ratio,^the^proportion^of^clones ^deemed^as^large^(0.1-1%^of^t^ repertoire)^can^be^divided^by^the^proportion^of^clones^deeme d^as^small^(<^0.001%^of^the^ repertoire),^e.g.,^as^described^herein.^The^proportion^of^th e^repertoire^occupied^by^the^ top^ n^ clones^may^ be^ determined^ by^ taking^ the^ top^ n^ clones^ from^ each^ patient^ and^^ clustered^ using^ the^ “gliph2”^ function^ from^ the^ turbogliph^ package^ (v0.99.2),^ e.g.,^ as^ described^herein,^wherein^n^may^be^from^50^to^1000,^and^in^a n^example,^n^is^100.^The^ clonality^index^may^be^determined^using^the^entropy^package^ (v1.3.1),^e.g.,^as^described^ herein,^using^the^(1-[‘entropy’^function]).^The^Hill^num ber^described^herein^may^also^be^ known^as,^and^be^referred^to^as,^the^entropy^score,^Shannon^ index^or^Shannon^entropy^^ score^herein.^^ ^ The^person^skilled^in^the^art^will^know^that^based^on^the^co mparison^of^a^given^subject,^ a^subject’s^D50,^Hill^number^and/or^clonality^scores^(incl uding^the^clonality^index,^The^ proportion^of^ the^repertoire^occupied^by^ the^ top^n^clones,^and^ the^ratio^of^ large:small^^ clonotypes^ in^ the^ repertoire)^ may^ be^ higher^ or^ lower^ than^ a^ comparison^ subject,^ depending^ on^ whether^ they^ are^ a^ healthy^ subject^ or^ a^ plurality^ of^ healthy^ subjects,^ whether^the^subject^is^assessed^at^a^different^time^point,^a nd/or^relative^to^a^plurality^of^ subjects^ from^ the^ general^ population.^ ^ A^ skilled^ person^ would^ also^ be^ aware^ that^ measures^of^TCR^diversity^or^clonality^can^also^be^assessed^ through^alternative^metrics^^ or^techniques^and^that^metrics^that^measure^with^TCR^sequenc e^similarity^in^a^sample^ could^provide^similar^results.^^ ^ Cytometry^Techniques^ ^ ^ Provided^ in^ the^ present^ disclosure^ is^ an^ example^ of^ generating^ a^ ratio^ of^ T^ cell^ differentiation^that^can^be^used^to^classify^a^subject^as^ha ving^non-progressing^or^low- grade^ pre-invasive^ lung^ lesions^ (posing^ no^ risk^ of^ cancer)^ or^ a^ subject^ having^ a^ progressing^or^high-grade^pre-invasive^lung^lesions^(high^ri sk^of^developing^cancer),^a^ healthy^subject^or^cancer^(e.g.,^stage^I^cancer).^Provided^i n^the^present^disclosure^is^also^^ an^example^of^determining^the^proportion^of^T^cells^(or^subp opulation^of^T^cells)^that^are^ activated^and/or^exhausted^as^a^percentage^of^T^cells^(or^a^ subpopulation^of^T^cells)^that^ can^be^used^to^classify^a^subject^has^having^a^non-progressi ng^or^low-grade^pre-invasive^ lung^lesions^(posing^no^risk^of^cancer)^or^a^subject^having^ a^progressing^or^high-grade^ pre-invasive^ lung^lesions^(high^risk^of^developing^cancer),^a^healthy^sub ject^or^cancer^^ (e.g.,^stage^I^cancer)^ ^ In^one^embodiment,^I^method^of^the^present^invention^compris es^determining^a^ratio^of^ activated^and/or^exhausted^T^cells^:^naïve^and/or^resting^T ^cells^in^a^sample^of^blood^ obtained^from^the^subject,^wherein^the^determining^comprises ^analysing^T^cells^using^ cytometry^to^detect^the^presence^or^absence^of^a^panel^of^bi omarkers^comprising^Ki67^^ and^CD39.^^ ^ Optionally^the^method^of^the^present^invention^comprises^det ermining^a^ratio^of^ activated^and/or^exhausted^T^cells^:^T^cells^which^are^not^a ctivated^and/or^exhausted^T^ cells^in^a^sample^of^blood^obtained^from^the^subject,^wherei n^the^determining^ ^ comprises^analysing^T^cells^using^cytometry^to^detect^the^pr esence^or^absence^of^a^ panel^of^biomarkers^comprising^Ki67^and^CD39.^ ^ A^subject^having^more^T^cells^showing^Ki67^and^CD39^expressi on,^vs^T^cells^in^which^ there^is^no^detection^of^expression^of^one^or^both^of^Ki67^a nd^CD39,^in^comparison^^ with^a^comparison^subject^or^a^plurality^of^comparison^subje cts^is^considered^to^be^at^ risk^of^having^a^progressing^or^high-grade^pre-invasive^lesi on^or^nodule.^^It^is^also^ noted^that^a^subject^having^more^T^cells^showing^Ki67^and^CD 39^expression,^vs^T^cells^ in^which^there^is^no^detection^of^expression^of^one^or^both^ of^Ki67^and^CD39,^in^ comparison^with^a^comparison^subject^or^subjects^is^consider ed^to^be^at^risk^of^having^^ a^solid^tumour^(e.g.,^a^stage^1^solid^tumour).^^^^In^this^co ntext^the^comparison^subject^or^ subjects^can^include:^ ^-^a^healthy^subject^or^a^plurality^of^healthy^subjects,^^ ^-^the^subject^at^a^different^time^point,^^ ^-^a^plurality^of^subjects^from^the^general^population.^^^ ^ ^ A^subject^having^the^same^amount^or^more^T^cells^showing^Ki6 7^and^CD39^expression,^ vs^T^cells^in^which^there^is^no^detection^of^expression^of^o ne^or^both^of^Ki67^and^CD39,^ in^comparison^with^a^comparison^subject^or^a^plurality^of^co mparison^subjects^is^ considered^to^be^at^risk^of^having^a^progressing^or^high-gra de^pre-invasive^lesion^or^^ nodule.^^It^is^also^noted^that^a^subject^having^the^same^amo unt^or^more^T^cells^showing^ Ki67^and^CD39^expression,^vs^T^cells^in^which^there^is^no^de tection^of^expression^of^ one^or^both^of^Ki67^and^CD39,^in^comparison^with^a^compariso n^subject^or^subjects^is^ considered^to^be^at^risk^of^having^a^solid^tumour^(e.g.,^a^s tage^1^solid^tumour).^^In^this^ context,^the^comparison^subject^or^subjects^can^include:^ ^ ^-^a^subject^or^a^plurality^of^subjects^known^or^have^a^soli d^tumour^or^known^to^have^a^ progression^or^high-grade^pre-invasive^lesion^or^nodule,^^ ^-^the^subject^at^a^different^time^point,^^ ^-^a^plurality^of^subjects^from^the^general^population.^^^ ^ ^ The^validity^of^comparing^results^in^this^way^is^supported^t hrough^the^data^provided^ herein^where^subjects^with^known^progressing^or^high-grade^p re-invasive^lesion^or^ nodule^or^having^a^solid^tumour^have^more^T^cells^showing^Ki 67^and^CD39^expression,^ and^conversely,^subjects^with^low-grade^disease^have^less^T^ cells^with^Ki67^and^CD39^ expression^(Figure^1),^as^well^as^healthy^subjects^(Figure^1 5).^ ^ ^ In^another^embodiment,^the^method^of^the^present^invention^c omprises^determining^the^ proportion^of^activated^and/or^exhausted^T^cells^as^a^percen tage^of^T^cells^in^the^blood^ obtained^from^the^subject,^wherein^the^determining^comprises ^analysing^T^cells^using^ cytometry^to^detect^the^presence^or^absence^of^a^panel^of^bi omarkers^comprising^^ FoxP3,^wherein^the^T^cells^are^CD4^T^cells.^In^some^embodime nts,^the^panel^of^ biomarkers^comprises^CD4^to^identify^the^CD4^T^cells^also^us ing^cytometry.^In^some^ embodiments,^the^CD4^T^cells^are^pre-selected^for^before^the ^cytometry^step,^e.g.,^ using^a^T^cell^enrichment^or^purification^kit^and^magnetic^s election^(as^described^ elsewhere^herein).^In^some^embodiments,^the^panel^of^biomark ers^further^comprises^^ CD39.^In^some^embodiments,^the^panel^of^biomarkers^further^c omprises^CD39^and^ Ki67,^or^CD39^and^CD45RA.^In^some^embodiments,^the^activated ^and/or^exhausted^T^ cells^express^FoxP3^and^CD39,^wherein^the^T^cells^are^CD4^T^ cells.^^In^some^ embodiments,^the^activated^and/or^exhausted^T^cells^express^ FoxP3^and^CD39^in^ combination^with^Ki67,^wherein^the^T^cells^are^CD4^T^cells.^ In^some^embodiments,^the^^ activated^and/or^exhausted^T^cells^express^FoxP3^and^CD39^an d^do^not^express^ CD45RA,^wherein^the^T^cells^are^CD4^T^cells.^^^ ^ In^another^embodiment,^the^method^of^the^present^invention^c omprises^determining^the^ proportion^of^CD4^regulatory^T^cells^as^a^percentage^of^CD4^ T^cells^in^the^blood^^ obtained^from^the^subject,^wherein^the^determining^comprises ^analysing^CD4^T^cells^ using^cytometry^to^detect^the^presence^or^absence^of^a^panel ^of^biomarkers^comprising^ FoxP3.^In^some^embodiments,^the^panel^of^biomarkers^further^ comprises^CD39.^In^ some^embodiments,^the^panel^of^biomarkers^further^comprises^ CD39^and^Ki67,^or^ CD39^and^CD45RA.^In^some^embodiments,^the^CD4^regulatory^T^c ells^are^proliferating^^ CD4^regulatory^cells^and^further^express^CD39^and^Ki67.^ ^ In^another^embodiment,^the^method^of^the^present^invention^c omprises^determining^the^ proportion^of^regulatory^T^cells^as^a^percentage^of^T^cells^ in^the^blood^obtained^from^ the^subject,^wherein^the^determining^comprises^analysing^T^c ells^using^cytometry^to^ detect^the^presence^or^absence^of^a^panel^of^biomarkers^comp rising^FoxP3,^wherein^^ the^T^cells^are^CD4^T^cells.^In^some^embodiments,^the^activa ted^and/or^exhausted^T^ cells^express^FoxP3^and^CD39,^wherein^the^T^cells^are^CD4^T^ cells.^In^some^ embodiments,^the^activated^and/or^exhausted^T^cells^express^ FoxP3^and^CD39^in^ combination^with^Ki67^(and^optionally^CD4),^wherein^the^T^ce lls^are^CD4^T^cells.^In^ some^embodiments,^the^activated^and/or^exhausted^T^cells^exp ress^FoxP3^and^CD39^^ and^do^not^express^CD45RA,^wherein^the^T^cells^are^CD4^T^cel ls.^^^ ^ A^subject^having^more^CD4+^T^cells^showing^FoxP3^expression^ (preferably^in^ combination^with^CD39^expression^and^further^preferably^in^c ombination^with^(i)^Ki67^ expression,^or^(ii)^no^detection^and/or^no^expression^of^CD4 5RA),^vs^CD4^T^cells^in^^ which^there^is^no^detection^of^expression^of^FoxP3^(preferab ly^in^combination^with^no^ detection^or^expression^of^CD39,^and^further^preferably^in^c ombination^with^(i)^no^ detection^or^expression^of^Ki67,^or^(ii)^expression^and/or^d etection^of^CD45RA,^in^ comparison^with^a^comparison^subject^or^a^plurality^of^compa rison^subjects^is^ considered^to^be^at^risk^of^having^a^progressing^or^high-gra de^pre-invasive^lesion^or^^ nodule.^^It^is^also^noted^that^a^subject^having^more^CD4+^T^ cells^showing^FoxP3^ expression^(preferably^in^combination^with^CD39^expression^a nd^further^preferably^in^ combination^with^(i)^Ki67^expression,^or^(ii)^no^detection^a nd/or^expression^of^ CD45RA),^vs^CD4+^T^cells^in^which^there^is^no^detection^of^e xpression^of^FoxP3^ (preferably^in^combination^with^no^detection^or^expression^o f^CD39,^and^further^^ preferably^in^combination^with^(i)^no^detection^or^expressio n^of^Ki67,^or^(ii)^expression^ of^CD45RA),^in^comparison^with^a^comparison^subject^or^subje cts^is^considered^to^be^ at^risk^of^having^a^solid^tumour^(e.g.,^a^stage^1^solid^tumo ur).^^In^this^context^the^ comparison^subject^or^subjects^can^include:^ ^-^a^healthy^subject^or^a^plurality^of^healthy^subjects,^^ ^ ^-^the^subject^at^a^different^time^point,^^ ^-^a^plurality^of^subjects^from^the^general^population.^^^ ^ A^subject^having^the^same^amount^or^more^T^cells^showing^Ki6 7^and^CD39^expression,^ vs^T^cells^in^which^there^is^no^detection^of^expression^of^o ne^or^both^of^Ki67^and^CD39,^^ in^comparison^with^a^comparison^subject^or^a^plurality^of^co mparison^subjects^is^ considered^to^be^at^risk^of^having^a^progressing^or^high-gra de^pre-invasive^lesion^or^ nodule.^^It^is^also^noted^that^a^subject^having^the^same^amo unt^or^more^T^cells^showing^ Ki67^and^CD39^expression,^vs^T^cells^in^which^there^is^no^de tection^of^expression^of^ one^or^both^of^Ki67^and^CD39,^in^comparison^with^a^compariso n^subject^or^subjects^is^ considered^to^be^at^risk^of^having^a^solid^tumour.^^In^this^ context,^the^comparison^^ subject^or^subjects^can^include:^ ^-^a^subject^or^a^plurality^of^subjects^known^or^have^a^soli d^tumour^or^known^to^have^a^ progression^or^high-grade^pre-invasive^lesion^or^nodule,^^ ^-^the^subject^at^a^different^time^point,^^ ^-^a^plurality^of^subjects^from^the^general^population.^^^ ^ ^ The^methods^of^the^present^invention^are^valuable^for^early^ detection^of^a^variety^of^ solid^tumours,^including^stage^1^solid^tumours/stage^1^solid ^malignant^tumours.^^Early^ detection^may^be^for^NSCLC^including^LUAD^or^LUSC,^mesotheli oma,^renal^cancer,^ melanoma,^pancreatic^cancer,^head^and^neck^cancer,^prostate^ cancer,^brain^cancer^^ including^glioblastoma,^breast^cancer,^bowel^cancer,^liver^c ancer.^^ ^ A^ratio^as^used^herein,^generally^refers^to^the^relationship ^of^the^frequency^of^activated^ and/or^exhausted^T^cells^compared^with^the^frequency^of^naï ve^and/or^resting^T^cells^in^ a^subject.^^Alternatively,^a^ratio^can^refer^to^the^relation ship^of^the^frequency^of^ ^ activated^and/or^exhausted^T^cells^amongst^all^T^cells^or^am ongst^a^subset^of^T^cells^in^ a^subject^(i.e.,^the^proportion^of^activated/exhausted^cells ^as^a^percentage^of^T^cells,^or^ amongst^a^subset^of^T^cells^in^a^subject,^e.g.,^in^a^sample^ of^blood^in^a^subject).^^^^ ^ A^ratio^indicative^of^a^whether^a^subject^is^at^risk^can^be^ inferred^from^having^a^high^^ percentage^of^activated/exhausted^T^cells^in^a^sample^obtain ed^from^the^subject,^or^a^ percentage^of^activated/exhausted^T^cells^in^a^sample^obtain ed^from^the^subject^which^ is^greater^than^the^average^percentage^of^activated^and/or^e xhausted^T^cells^in^a^ comparison^subject^or^plurality^of^subjects^is^indicative^of ^a^subject^at^risk.^^ ^ ^ An^activated^T^cell^as^used^herein,^generally^refers^to^a^T^ cell^that^has^recognised^its^ cognate^antigen,^on^a^target^cell^expressing^the^antigen^inc luding^an^epithelial^cell^or^ other^cell^that^may^progress^to^cancer^or^be^part^of^a^lesio n^or^nodule^or^tumour^ triggering^activation^and/or^proliferation^of^that^T^cell.^^ Repetitive^encounter^of^an^ antigen^or^a^high^affinity^interaction^with^that^antigen^can ^cause^terminal^differentiation.^^ Chronic^antigen^stimulation^with^or^without^inhibitory^signa lling^or^defective^co- stimulation^with^or^without^inflammation^may^result^in^T^cel l^exhaustion^or^T^cell^ dysfunction.^^An^activated^T^cell^may^or^may^not^be^prolifer ating.^ Activated^T^cells^may^include:^^ -Any^subset^of^CD4^or^CD8^T^cell^or^CD3^positive^cell^expres sing^CD39^and^/^or^Ki67^in^^ the^presence^or^absence^of^other^commonly^used^activation^ma rkers^obvious^to^a^ skilled^researcher.^^ ^-^CD8^Terminally^differentiated^effector^memory^cells^(RA)^ that^are^activated^ (TEMRA.Act).^Such^cells^may^have^one^or^more^of^the^biomarke rs^CD39,^Ki67^ CD45RA^or^CD57.^^ ^ ^-^CD4^Regulatory^T^cells^that^are^proliferating^and^may^exp ress^PD-1^(Teg.prolif.PD-1)^ and^may^express^one^or^more^of^the^biomarkers^CD39,^Ki67^or^ PD-1^(i.e.,^along^with^ FoxP3).^^^ ^-^CD4^Effector^memory^CD4^T^cells^(TEM)^which^may^express^o ne^or^more^of^the^ biomarkers^CD39^and^Ki67.^Such^cells^may^lack^the^biomarkers ^CCR7^and^CD45RA.^^^ ^-^CD4^Regulatory^T^cells^(Treg)^which^may^express^one^or^mo re^of^the^biomarkers^ CD39^and^Ki67^(i.e.,^along^with^FoxP3).^^ ^-^CD4^Proliferating^regulatory^T^cells^(Treg.prolif)^may^ex press^one^or^more^of^the^ biomarkers^CD39,^Ki67hi^or^PD-1^(i.e.,^along^with^FoxP3).^^ -^^CD4^Proliferating^effector^memory^CD4^T^cells^(Tem.Prolif )^which^may^express^one^^ or^more^of^the^biomarkers^CD39,^Ki67hi^and^such^cells^may^la ck^one^or^more^of^the^ biomarkers^CCR7^and^CD45RA.^^ -^cytolytic^CD4^T^cells^(Cytolytic)^which^may^be^terminally^ differentiated^and^cytotoxic^ CD4^T^cells^and^may^have^the^biomarker^CD57.^ ^ ^ A^proliferating^T^cell,^as^used^herein,^generally^refers^to^ a^T^cell^that^has^interacted^with^ their^cognate^antigen^or^other^activating^stimuli^(thereby^b ecoming^activated)^and^has^ increased,^are^increasing^or^in^the^process^of^increasing^th e^numbers^of^that^T^cell^ through^cell^growth^and^division.^A^proliferating^T^cell^is^ usually^activated^but^may^not^ always^be.^ ^ ^ An^exhausted^T^cell^as^used^herein,^generally^refers^to^a^st ate^of^T^cell^dysfunction^ which^can^be^defined^by^biological^changes^in^the^T^cell,^in cluding^poor^effector^ function^and/or^expression^of^inhibitory^receptors^and^a^tra nscriptional^state^and/or^ phenotype^and^/or^epigenetic^state^which^is^distinct^from^th at^of^effector^or^memory^or^^ naïve^T^cells.^Exhausted^T^cells^may^be^referred^to^as^Tex^ cells,^dysfunctional^T^cells^ or^Tdys^cells.^ Exhausted^T^cells^may^include:^ exhausted,^proliferating^CD8^T^cells^(Tex.Prolif)^which^may^ express^one^or^more^of^the^ biomarkers^PD-1,^Ki67^or^CD39.^^ Exhausted,^proliferating^CD4^T^cells^(Tex.Prolif)^which^may^ express^one^or^more^of^the^^ biomarkers^PD-1,^Ki67hi^or^CD39.^^ Progenitor,^exhausted^CD4^T^cells^(TPEX),^an^early^version^o f^exhausted^CD4^T^cells^ and^may^express^one^or^more^of^the^biomarkers^CD39,^Ki67^or^ PD-1.^^ ^ A^naïve^T^cell,^as^used^herein,^generally^refers^to^a^T^cel l,^which^may^be^a^CD4+^^ helper^T^cell^or^a^CD8+^cytotoxic^T^cell,^that^has^not^yet^e ncountered^their^cognate^ antigen.^^ Naïve^T^cells^may^include:^^ naïve^CD8^T^cells^(Naïve)^which^may^express^one^or^more^of ^the^biomarkers^CD45RA,^ CCR7.^^ ^ Naïve^CD4^T^cells^that^may^have^lost^TCF7^expression^(Naïv e.TCF7neg),^and/or^which^ may^express^one^or^more^of^the^biomarkers^CD45RA,^CCR7^or^CD 27.^^ CD4^native^T^cells^(Naïve)^which^may^express^one^or^more^of ^the^biomarkers^CD45RA^ and^CCR7.^ ^ ^ A^resting^T^cell,^as^used^herein,^generally^refers^to^a^quie scent^T^cell^that^has^stopped^ or^is^not^proliferating^or^expressing^biomarkers^of^activati on.^ Resting^T^cells^includes:^ CD8^terminally^differentiated^effector^memory^cells^(RA)^(TE MRA.Rest)^which^may^ express^the^biomarker^CD45RA.^^Such^cells^may^lack^one^or^mo re^of^the^biomarkers^^ CCR7^and^CD57.^^ Resting^central^memory^CD4^T^cells^which^are^an^early^differ entiated^resting^memory^ cells^that^are^unstimulated,^which^may^express^the^biomarker ^CCR7.^^Such^cells^may^ lack^one^or^more^of^CD45RA^and^CD38.^^ ^ ^ In^some^methods,^the^analysis^may^be^performed^on^a^T^cell^s ubtype^or^subpopulation.^ In^some^embodiments,^the^analysis^may^be^performed^on^CD3^T^ cells,^CD4^T^cells,^CD8^ T^cells,^CD3+CD8+^T^cells,^or^CD4+CD3+^T^cells.^^ ^ In^some^embodiments,^the^T^cell^subtype^or^subpopulation^may ^be^regulatory^T^cells.^^ Regulatory^ T^ cells^ express^ the^ FoxP3^ biomarker.^ In^ some^ embodiments,^ prior^ to^ analysis,^a^preliminary^step^of^identifying^regulatory^T^cel ls^may^be^performed,^e.g.,^using^ an^antibody^specific^to^the^biomarkers^FoxP3.^This^analysis^ is^performed^on^CD4^T^cells.^ In^some^embodiments,^prior^to^analysis,^a^preliminary^step^o f^identify^CD4^T^cells^may^ be^performed,^e.g.,^using^an^antibody^specific^to^CD4.^^ ^ ^ In^some^embodiments,^the^T^cell^subtype^or^subpopulation^may ^be^an^effector^memory^ cell^ (Teffm).^ effector^memory^ T^ cells^may^ be^ absent^ of^CCD7,^CD45RA^and/or^ PD1^ biomarkers.^In^some^embodiments,^prior^to^analysis,^a^prelim inary^step^of^identifying^an^ effector^memory^cell^may^be^performed,^e.g.,^using^one^or^mo re^antibodies^specific^to^ CCD7,^ CD45RA,^ and/or^ PD1.^ This^ analysis^ may^ be^ performed^ in^ CD8^ T^ cells,^ or^^ CD3+CD8+^T^cells.^ ^ The^methods^of^the^present^invention^can^detect^biomarkers^u sing^cytometry,^which^as^ used^herein,^generally^refers^to^techniques^for^measurement^ of^properties^of^the^cells.^ Cytometry^embraces^flow^cytometry,^spectral^cytometry^and^ma ss^cytometry^(including^^ time^of^flight^cytometry^or^cytometry^by^time^of^flight^(CyT OF).^^^ ^ The^ cytometry^ can^ be^ flow^ cytometry.^ ^ Flow^ cytometry^ refers^ to^ a^ well-established^ technique^ that^ can^ be^ used^ to^ detect^ and^ analyse^ populations^ of^ cells^ and^ provide^ information^ regarding^ the^ physical^ or^ chemical^ characteristics^ of^ those^ cells.^ This^^ detection^and/or^analysis^includes^identifying^subsets^of^ce lls^within^a^population^of^cells^ to^be^analysed^that^express^a^specific^biomarker^or^specific ^biomarker^combinations.^^A^ biomarker^may^be^a^cell^surface^antigen^or^may^be^an^intrace llular^molecule.^To^identify^ subsets^of^cells,^one^application^of^flow^cytometry^may^invo lve^performing^multi-coloured^ analysis^using^antibodies^each^of^which^targets^biomarker^of ^interest^(e.g.,^according^to^^ the^ examples^ disclosed^ herein).^ ^ Single^ cells^ are^ transferred^ in^ the^ flow^ cytometer^ through^a^stream^of^fluid^and^passed^by^a^light^source^which ^may^activate^a^fluorescent^ antibody^bound^to^a^cell^flowing^past^the^light^source.^^Flu orescent^light^emitted^by^an^ antibody^may^be^detected^by^an^electronic^detection^apparatu s^within^the^flow^cytometer^ that^in^turn^relays^this^information^computationally^through ^creation^of^gates/gating^that^^ classifies^ cells^ that^ are^ expressing^ a^ biomarker^ and^ those^ that^ are^ not.^ In^ some^ examples,^ gating^ is^ performed^ using^ FlowJo^ software,^ for^ example,^ FlowJo^ v10.8.1^ software,^although^other^gating^software^may^be^used.^Typica lly,^a^negative^control,^such^ as^an^antibody^for^a^biomarker^ that^ is^not^expressed^on^ the^population^of^cells^being^ analysed^ is^used^ to^set^ the^gating^ for^cells^ that^will^be^classified^as^not^expressing^a^^ biomarker.^ The^ set^ gating^ is^ then^ used^ for^ the^ rest^ of^ the^ antibody^ panel^ for^ the^ biomarkers^ of^ interest.^ The^ information^ is^ then^ visualised^ to^ correlate^ the^ amount^ of^ detected^ fluorescence^ with^ the^ number^ of^ cells^ that^ are^ expressing^ the^biomarker^ of^ interest^and^the^amount^of^the^biomarker^being^expressed^by^ those^cells.^This^technique^ provides^ a^ fast,^ sensitive^ and^high-throughput^ analysis^ of^ a^ population^of^ cells.^ Flow^ cytometry^may^be^used^to^profile^the^frequency^and^intensity ^of^biomarkers^expressed^^ by^T^cells.^^^ ^ The^ biomarkers^ to^ be^ detected^ are^ biomarkers^ associated^ with^ T^ cell^ activation,^ exhaustion,^regulatory^or^memory^differentiation.^^Detection ^of^a^biomarker^or^a^specific^ combination^of^biomarkers^can^be^used^to^determine^the^stren gth,^frequency^and^type^of^^ immune^activation.^^^Therefore,^in^embodiments^where^flow^cy tometry,^or^spectral^or^time^ of^ flight^or^another^form^of^cytometry^ is^used^ to^detect^ the^presence^or^absence^of^a^ presence^of^biomarkers,^ the^detecting^comprises^detecting^ the^signal^of^one^or^more^ fluorescently^ labelled^ antibodies^ that^ bind^ to^ the^ panel^ of^ biomarkers^ of^ interest.^ For^ example,^when^the^panel^or^biomarkers^includes^(i)^Ki67^–^ the^detecting^may^comprise^^ detecting^the^signal^of^a^fluorescently^labelled^Anti-Ki67^a ntibody,^and/or^(ii)^CD39,^the^ detecting^ may^ comprise^ detecting^ the^ signal^ of^ a^ fluorescently^ labelled^ Anti-CD39^ antibody,^and/or^ (iii)^ for^FoxP3,^ the^detecting^may^comprise^detecting^ the^signal^of^a^ fluorescently^labelled^Anti-FoxP3^antibody.^The^presence^and /or^absence^of^the^other^ named^biomarkers^herein,^when^used^in^the^panel^of^biomarker s,^can^also^be^determined^^ by^similarly^detecting^the^signal^of^a^fluorescent^labelled^ antibody^targeted^against^the^ specific^biomarker^of^interest.^^ ^ Cytometry^also^ includes^ spectral^ cytometry^ or^ spectral^ flow^cytometry^ a^ technique^ in^ which^an^emission^spectrum^of^multiple^fluorescing^molecules ^may^be^captured^by^a^set^^ of^ detectors^ across^ a^ defined^wavelength^ range.^ The^ fluorescence^ spectrum^ can^ be^ recognized,^ recorded^as^a^spectral^signature,^and^used^as^a^ reference^ in^multicolour^ applications.^^ ^ Cytometry^also^includes^mass^cytometry^which^is^a^variation^ of^flow^cytometry^in^which^^ antibodies^ are^ labelled^ with^ heavy^ metal^ isotopes^ rather^ than^ fluorochromes.^ The^ isotopes^are^analysed^through^the^cells^being^ionised^and^th e^ions^are^separated^by^their^ mass-to-charge^ ratio^ and^ detected^ as^ an^ electrical^ signal^ at^ the^ terminal^ gate^ of^ the^ spectrometer.^The^readout^is^typically^by^time-of-flight^mas s^spectrometry.^ ^ ^ In^the^methods^of^the^present^invention^the^analysis^may^com prise^using^at^least^one^of^ flow^cytometry,^spectral^cytometry^and/or^mass^cytometry,^op tionally^flow^cytometry.^^ In^ some^ embodiments,^ the^ cytometry^ analysis^ (e.g.,^ the^ flow^ cytometry^ analysis)^ comprises^using^computational^clustering^for^a^particular^su bpopulation^of^T^cells^and/or^ T^ cells^which^express^or^which^ do^ not^ express^one^or^more^ particular^biomarkers^ of^ interest.^Example^computational^clustering^strategies^are^de scribed^in^further^detail^ in^^ the^Examples^section^herein.^The^computational^clustering^st rategies^described^herein^ are^merely^an^example^and^the^person^skilled^in^the^art^woul d^be^aware^of^the^various^ software^packages^and^protocols^that^could^be^used^to^analys e^the^cytometry^data^and^ obtain^the^same^biological^result.^ ^ ^ In^some^embodiments,^the^cytometry^analysis^(e.g.,^flow^cyto metry^analysis)^comprises^ gating^or^gating^for^a^particular^subpopulation^of^T^cells^a nd/or^T^cells^which^express^or^ which^do^not^express^a^particular^biomarker^of^interest.^The ^terms^“gating”^or^“gates”,^as^ used^herein,^generally^refers^to^the^use^of^software^that^is ^associated^with^flow^cytometry,^ such^as^manually^drawing^two-dimensional^gates^with^a^mouse^ on^a^computer^screen,^^ based^on^the^density^contour^lines^that^are^provided^by^soft ware^tools.^Gates^are^typically^ used^to^distinguish^between^cells^that^are^expressing^a^biom arker^of^interest^and^those^ that^are^not.^The^cells^falling^in^a^gate^may^be^selected^(g ated^in)^or^excluded^(gated^out)^ and^the^process^may^be^repeated^for^different^two-dimensiona l^(or^higher)^projections^of^ the^gated^cells^(e.g.,^two-dimensional^dot^plots),^thus^resu lting^in^a^sequence^of^gates^^ that^ describe^ subpopulations^ of^ the^multivariate^ flow^ cytometry^ data.^ Specific^ gating^ strategies^are^described^ in^ further^detail^ in^ the^Examples^section^herein.^The^specific^ gating^strategies^described^herein^are^merely^an^example^and ^the^person^skilled^in^the^ art^would^be^aware^of^the^various^software^packages^and^prot ocols^that^could^be^used^ to^ analyse^ the^ flow^ cytometry^ data^ and^ obtain^ the^ same^ biological^ result.^ In^ some^^ examples,^gating^(i.e.,^manual^gating)^is^performed^using^Fl owJo^software,^for^example,^ FlowJo^v10.8.1^software,^although^other^gating^software^may^ be^used.^ ^ A^biomarker^as^used^herein,^is^as^a^phenotypic^biomarker^tha t^reflects^the^activation^or^ differentiation^state^of^a^T^cell^in^human^blood.^Biomarkers ^that^may^be^detected^in^the^^ methods^ of^ the^ present^ invention^ can^ include^ Ki67^ and^ CD39^ and/or^ FoxP3.^ ^ The^ methods^detect^the^presence^or^absence^of^a^panel^of^biomark ers^comprising^Ki67^and^ CD39,^ and/or^ FoxP3,^ optionally^ in^ combination^ with^ other^ biomarkers^ listed^ herein.^^ Hence^the^panel^of^biomarkers^of^the^present^invention^compr ises^Ki67^and^CD39^and/or^ FoxP3,^optionally^in^combination^with^other^biomarkers^liste d^herein.^ ^ ^ An^activated^and/or^exhausted^T^cell^may^express^CD39^and^Ki 67^(CD39+^and^Ki67+^T^ cells).^^Detection^of^expression^of^CD39^and^Ki67^is^conside red^to^indicate^a^T^cell^that^ is^activated^and/or^exhausted.^An^activated^and/or^exhausted ^T^cell^may^also^be^a^CD4^ Regulatory^T^cell.^^A^CD4^Regulatory^T^cell^expresses^the^bi omarker^FoxP3.^In^some^^ embodiments,^the^CD4^Regulatory^T^cell^also^expresses^CD39^( i.e.,^in^combination^ with^FoxP3).^Detection^of^FoxP3^expression^(and^preferably^i n^combination^with^CD39)^ can^indicate^a^CD4+T^cell^that^is^activated^and/or^exhausted .^A^CD4+^T^cell^that^ expresses^FoxP3^is^otherwise^referred^to^as^a^T^regulatory^c ell^herein.^In^some^ embodiments,^the^CD4^Regulatory^T^cell^may^express^FoxP3,^Ki 67^and^CD39.^In^some^^ embodiments,^the^CD4^Regulatory^T^cell^may^express^FoxP3,^CD 39,^and^may^lack^ CD45RA.^^ ^ A^naïve^and/or^resting^T^cell^does^not^express^CD39^or^Ki67 ^(CD39-^and^Ki67-^cells).^ Lack^of^detection^of^expression^is^CD39^and^Ki67^is^or^can^b e^considered^to^indicate^a^^ T^cell^is^naïve^and/or^resting.^^^ ^ T^cells^which^are^not^activated^and/or^exhausted^do^not^expr ess^one^or^both^of^CD39^ or^Ki67^(CD39-^and^Ki67+^T^cells,^CD39+^and^Ki67-^T^cells,^o r^CD39-^and^Ki67-^T^ cells).^Lack^of^detection^of^expression^of^one^or^both^of^CD 39^or^Ki67^is^or^can^be^^ considered^to^indicate^a^T^cell^that^is^not^an^activated^and /or^exhausted^T^cell.^^^ ^ In^this^disclosure^reference^to^a^T^cell^not^expressing^a^bi omarker^marker^can^refer^a^ lack^of^detection^of^expression^of^that^biomarker.^ ^ ^ The^panel^of^biomarkers^may^comprise^further^biomarkers^(i.e .,^in^addition^to^Ki67,^CD39^ and/or^FoxP3).^^The^panel^of^biomarkers^may^include^one^or^m ore^further^biomarkers^ selected^from^CD45RA,^CCR7,^PD-1,^CD57^or^CD38.^^These^bioma rkers^are^considered^ to^further^assist^in^distinguishing^between^activated^and/or ^exhausted^T^cells^and^naïve^ and/or^resting^T^cells.^ ^ ^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39^and^CD45RA.^In ^some^embodiments,^ the^activated^and/or^exhausted^cells^express^Ki67^and^CD39^b ut^may^or^may^not^express^ CD45RA.^^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39^and^CCR7.^In^s ome^embodiments,^the^^ activated^and/or^exhausted^cells^express^Ki67^and^CD39^but^m ay^or^may^not^express^ CCR7.^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39^and^PD-1.^In^s ome^embodiments,^the^ activated^and/or^exhausted^cells^express^Ki67^and^CD39^but^m ay^or^may^not^express^ PD-1^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39^and^CD57.^In^s ome^embodiments,^.the^^ activated^and/or^exhausted^cells^express^Ki67^and^CD39^but^m ay^or^may^not^express^ CD57.^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39^and^CD38.^In^s ome^embodiments,^the^ activated^and/or^exhausted^cells^express^Ki67^and^CD39^but^m ay^or^may^not^express^ CD38.^ ^ A^ panel^ of^ biomarkers^ may^ comprise^ Ki67,^ CD39,^ CD45RA^ and^ CCR7.^ In^ some^ embodiments,^the^activated^and/or^exhausted^cells^express^Ki 67^and^CD39^but^may^or^ may^not^express^CD45RA^and^may^or^may^not^express^CCR7.^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39,^CD45RA,^CCR7, ^and^PD-1.^In^some^ embodiments,^the^activated^and/or^exhausted^cells^express^Ki 67^and^CD39^but^may^or^^ may^not^express^CD45RA,^may^or^may^not^express^CCR7,^and^may ^or^may^not^express^ PD-1.^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39,^CD45RA,^CCR7, ^PD-1^and^CD57.^In^ some^embodiments,^the^activated^and/or^exhausted^cells^expre ss^Ki67^and^CD39^but^ may^or^may^not^ express^CD45RA,^may^or^may^not^ express^CCR7,^may^or^may^not^^ express^PD-1^and^may^or^may^not^express^CD57.^^^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39,^CD45RA,^CCR7, ^PD-1,^CD57^and^ CD38.^ In^some^embodiments,^ the^activated^and/or^exhausted^cells^express^Ki67^and^ CD39^but^may^or^may^not^express^CD45RA,^may^or^may^not^expre ss^CCR7,^may^or^may^ not^express^PD-1,^may^or^may^not^express^CD57^and^may^or^may ^not^express^CD38.^^^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39,^CD45RA,^CCR7, ^CD57,^and^CD38.^In^ some^embodiments,^the^activated^and/or^exhausted^cells^expre ss^Ki67^and^CD39^but^ may^or^may^ not^ express^CD45RA,^may^or^may^not^ express^CCR7,^may^or^may^not^ express^CD57^and^may^or^may^not^express^CD38.^ A^panel^of^biomarkers^may^comprise^Ki67,^CD39,^CD45RA,^PD-1^ and^CD57.^In^some^^ embodiments,^the^activated^and/or^exhausted^cells^express^Ki 67^and^CD39^but^may^or^ may^not^express^CD45RA,^may^or^may^not^express^PD-1^and^may^ or^may^not^express^ CD-57.^^ ^ In^some^embodiments,^the^activated^and/or^exhausted^cells^ex press^Ki67^and^CD39^but^^ do^not^express^CD45RA.^^In^some^embodiments,^the^activated^a nd/or^exhausted^cells^ express^Ki67^and^CD39^but^do^not^express^PD-1.^In^some^embod iments,^the^activated^ and/or^ exhausted^ cells^ express^ Ki67^ and^CD39^ but^ do^ not^ express^CCR7.^ In^ some^ embodiments,^the^activated^and/or^exhausted^cells^express^Ki 67^and^CD39^but^do^not^ express^CD45RA,^CCR7^and^PD-1.^The^activated^and/or^exhauste d^T^cells^may^CD8+^ T^cells,^or^CD8+CD3+^T^cells.^^ ^ ^ The^above^panel^of^biomarkers^may^be^combined^with^(i)^CD3,^ CD4^or^CD8,^or^(ii)^CD3^ and^CD4^or^(iii)^CD3^and^CD8.^The^presence^of^CD3^identifies ^live^T-cells.^^The^presence^ of^CD4^identifies^CD4+^T^cells,^i.e.,^T^helper^cells.^^The^p resence^of^CD8^identifies^CD8+^ T^cells,^i.e.,^cytotoxic^T^cells.^ ^ A^panel^of^biomarkers^may^comprise^FoxP3^(and^optionally^CD4 ).^In^some^embodiments,^ the^activated^and/or^exhausted^cells^are^CD4+^T^cells^that^e xpress^FoxP3.^ ^ A^panel^of^biomarkers^may^comprise^FoxP3,^and^CD39^(and^opti onally^CD4).^In^some^ embodiments,^the^activated^and/or^exhausted^cells^are^CD4+^T ^cells^that^express^FoxP3^^ and^CD39.^ ^ A^panel^of^biomarkers^may^comprise^FoxP3,^CD39^and^Ki67^and^ optionally^CD4).^ In^ some^embodiments,^the^activated^and/or^exhausted^cells^are^C D4+^T^cells^that^express^ FoxP3,^CD39^and^Ki67.^ ^ ^ A^panel^of^biomarkers^may^comprise^FoxP3,^CD39^and^CD45RA^(a nd^optionally^CD4).^ In^ some^ embodiments,^ the^ activated^ and/or^ exhausted^ cells^ are^ CD4+^ T^ cells^ that^ express^FoxP3,^CD39^and^do^not^express^CD45RA.^ ^ ^ The^above^panel^of^biomarkers^may^be^combined^with^CD3.^^ ^ Ki67^generally^refers^to^the^proliferation^biomarker^protein ^Ki-67,^encoded^by^the^MKI67^ gene^and^is^widely^used^as^a^biomarker^to^assess^cell^prolif eration.^ ^ ^ CD39^generally^refers^to^Cluster^of^Differentiation^39,^enco ded^by^the^ENTPD1^gene^and^ is^a^ectonucleoside^triphosphate^diphosphohydrolase.^ ^ CD45RA^generally^ refers^ to^the^ long^isoform^of^the^cell-surface^tyrosine^phosphatase^ CD45^(lymphocyte^common^antigen)^encoded^by^the^PTPRC^gene.^ ^ ^ CCR7^generally^refers^to^C-C^motif^chemokine^receptor^7^enco ded^by^the^CCR7^gene.^ It^is^a^member^of^the^G^protein-coupled^receptor^family.^ PD-1^(also^known^as^PD1)^generally^refers^to^Programmed^cell ^death^protein1,^encoded^ by^the^PDCD1^gene^and^is^a^inhibitory^receptor^on^antigen^ac tivated^T-cells^that^plays^a^^ critical^role^in^induction^and^maintenance^of^immune^toleran ce^to^self.^ ^ CD38,^generally^refers^to^Cluster^of^Differentiation^38^enco ded^by^the^CD38^gene^and^is^ a^cyclic^ADP^ ribose^hydrolase^ found^on^ the^ surface^of^many^ immune^cells,^ including^ lymphocytes.^ ^ ^ FoxP3^is^a^member^of^the^forkhead^transcription^factor^famil y^and^is^a^master^regulatory^ of^the^regulatory^pathway^in^the^development^and^function^of ^regulatory^T^cells.^^ ^ The^ term^ “hi”^ as^ used^ herein,^ generally^ refers^ to^ a^ higher/greater^ abundance^ of^ a^^ particular^biomarker^relative^to^other^T^cell^types.^The^ter m^“lo”^as^used^herein,^generally^ refers^to^a^lower/less^abundance^of^a^particular^biomarker^r elative^to^other^T^cell^types.^ The^term^“prolif”^as^used^herein^generally^refers^to^a^p roliferating^T^cell.^^ ^ Biomarkers^may^be^measured^by^directly^conjugated^antibodies ^which^are^available^from^^ multiple^ commercial^ sources.^ Alternatively,^ the^ skilled^ person^ may^ use^ a^ primary^ antibody^specific^ to^ the^biomarker^and^a^ secondary^antibody^ to^detect^ the^biomarker^ indirectly.^Antibodies^may^be^used^to^stain^peripheral^blood ^mononuclear^cells^(PBMCs)^ isolated^from^whole^blood^by^centrifugation.^Staining^may^al so^be^performed^on^whole^ blood.^The^biomarker^profiles^(the^frequency^of^cells^expres sing^the^biomarker)^can^be^^ determined^by^cytometry^using^instrumentation^and^software^k nown^to^the^person^skilled^ in^the^art.^^Specific^antibodies^ to^detect^biomarkers^of^T^cell^activation^or^proliferation^ state^may^be^used.^^These^antibodies^may^be:^^ ^ 1.^ Anti-CD39^(to^identify^activated^and^exhausted^T^cells^[Tex] ,^tumour^reactive^T^^ cells)^ 2.^ Anti-Ki67^(to^identify^proliferating^T^cells)^and/or^ 3.^ Anti-FoxP3^(to^identify^the^biomarker^FoxP3,^which^can^be^us ed^to^identify^CD4+^ T^regulatory^cells,^e.g.,^with^the^antibody^Anti-CD4)^ ^ ^ As^described^above^additional^biomarkers^may^be^detected^and ^methods^may^use^one^ or^more^the^following^antibodies:^ ^ 4.^ Anti-CD4,^to^identify^the^major^CD4+^T^cell^lineage^ 5.^ Anti-CD8,^to^identify^the^major^CD8+^T^cell^lineage^ 6.^ Anti-CD57^(to^identify^terminally^differentiated^T^cells)^ ^ 7.^ Anti-CD38^(to^identify^activated^and^exhausted^T^cells)^ 8.^ Anti-CD45RA^(to^identify^Naïve^and^terminally^differentiate d^T^cells)^ 9.^ Anti-PD-1^(to^identify^activated^and^exhausted^T^cells)^ 10.^Anti-CCR7^(to^identify^naïve^and^early^differentiated^m emory^cells).^ ^ ^ In^ some^ methods,^ a^ preliminary^ step^ of^ identifying^ live^ T^ cells^ for^ analysis^ may^ be^ performed.^^This^step^may^involve^antibodies/^dyes^for^2^bio markers^as^follows:^^ ^ 11.^A^viability^dye^(e.g.^fixable^live/dead,^which^can^be^ob tained^from^Thermofisher)^ to^exclude^dead^cells^ ^ 12.^Anti-CD3,^to^identify^T^cells.^^ ^ These^ are^ general^ purpose^ biomarkers^ of^ which^ the^ skilled^ person^ is^ aware.^ An^ alternative^to^using^CD3^is^available^to^the^skilled^person^ which^could^be^to^use^a^T^cell^ enrichment^or^purification^kit^and^magnetic^selection^ including^a^CD3^or^CD4^or^CD8^^^ negative^ or^ positive^ selection^ kit^ containing^ a^ cocktail^ of^ biotinylated^ or^ otherwise^ conjugated^antibodies^specific^to^CD3^or^CD4^or^CD8^or^multi ple^biomarkers^on^blood^ cells^excluding^CD3,CD4^or^CD8.^^Such^kits^may^also^be^used^ in^flow^cytometry,^when^ co-stained^with^streptavidin^or^secondary^antibodies^contain ing^fluorophores.^^ ^ ^ It^will^be^understood^that^the^term^“T^cell^differentiatio n^index”/”TEDI”,^as^used^herein,^^ provides^a^means^to^represent^ the^data^obtained^herein^ to^aid^ in^ interpretation^of^ the^ scope^of^the^invention^and^is^not^intended^to^limit^the^scop e^of^the^invention.^^The^person^ skilled^ in^ the^art^will^ understand^ that^ such^a^methodology^ is^an^ in-house^ term^ that^ is^ specific^to^the^inventors^and^that^various^methods^and^techn iques^discussed^herein^can^^ be^ performed^ using^ a^ variety^ of^ different^ apparatus,^ assay^ conditions,^ hardware^ and^ software^components^that^may^result^in^different^numerical^v alues^obtained,^but^which^ equate^to^the^same^biological^results^to^the^examples^provid ed^herein;^that^the^ratio^of^ exhausted/activated^T^cells^to^naïve/^resting^T^cells^is^hi gher^in^subjects^with^progressive,^ pre-cancerous^lesion^or^nodule^or^ in^the^presence^of^a^malignant^tumour^relative^to^a^^ subject^with^an^ indolent^or^ regressive^or^non-progressing^or^ low^grade^disease^or^no^ disease^or^a^benign^solid^tumour.^^ ^ The^term^“T^cell^differentiation^index”/”TEDI”,^as^u sed^herein,^generally^refers^to^the^ratio^ of^activated^and/or^exhausted^T^cells^:^naïve^and/or^restin g^T^cells^within^a^subject^which^ can^be^ represented^numerically^ in^ the^ form^of^an^ index^when^a^ subject’s^ results^ are^^ compared^against^the^average^ratio^of^activated^and/or^exhau sted^T^cells^:^naïve^and/or^ resting^T^cells^obtained^from^a^plurality^of^patients^known^ to^have^progressing^or^high- grade^pre-invasive^lesions^or^known^to^have^a^cancer^to^infe r^the^risk^that^the^subject^ has^ a^ progressing^ or^ high-grade^ pre-invasive^ lesion^ or^ nodules^ or^ having^ a^ solid^ malignant^tumour.^A^subject’s^results^may^also^be^compared ^against^their^own^previously^^ determined^ ratio^ to^ indicate^whether^ that^subject^ is^at^ risk^of^having^a^progressing^or^ high-grade^pre-invasive^lesion^or^nodules^or^having^a^solid^ malignant^tumour.^ ^ The^methods^of^the^present^invention^are^performed^on^a^bloo d^sample^obtained^from^a^ subject^and^hence^occur^in^vitro.^^The^methods^can^use^whole ^blood.^^The^methods^can^^ use^PBMCs.^ ^ The^present^invention^also^provides^a^kit^comprising^a^set^o f^lyophilised^antibodies^or^a^ fragment^thereof,^comprising^antibodies^binding^to^(i)^CD39^ and^Ki67,^and/or^(ii)^FoxP3^ (optionally^ in^ combination^ with^ CD4).^ ^ Such^ a^ kit^ may^ be^ used^ in^ or^ to^ perform^ the^^ methods^of^the^present^invention.^^In^embodiments,^the^kit^m ay^comprise^one^or^more^ further^lyophilised^antibodies^selected^from^antibodies^bind ing^to^one^or^more^biomarkers^ selected^from^CD45RA,^CCR7,^PD-1,^CD57^or^CD38.^^The^kit^may ^be^used^in^a^method^ for^determining^whether^a^subject^is^at^risk^for^having^a^pr ogressing^or^high-grade^pre- invasive^lesion^or^nodule^or^having^a^solid^tumour.^ ^ ^ The^present^invention^also^provides^a^device^comprising^mean s^for^receiving^a^sample^ of^blood^and^contacting^the^sample^with^a^set^of^ lyophilised^antibodies^or^a^ fragment^ thereof,^comprising^antibodies^binding^to^(i)^CD39^and^Ki67^ and/or^(ii)^FoxP3^(optionally^ in^combination^with^CD4).^Such^a^device^may^be^used^in^or^to ^perform^the^methods^of^^ the^present^invention.^^The^device^may^be^used^in^a^method^f or^determining^whether^a^ subject^is^at^risk^for^having^a^progressing^or^high-grade^pr e-invasive^lesion^or^nodule^or^ having^a^solid^tumour.^ ^ It^will^be^understood^that^the^present^invention^may^be^of^u se^in^providing^information^on^^ whether^a^subject^is^at^risk^for^having^a^progressing^or^hig h-grade^pre-invasive^lesion^or^ nodule^or^having^a^solid^malignant^tumour.^^The^methods^of^t he^present^invention^can^ also^be^implemented^for^valuable^healthcare^outcomes,^exampl es^by^way^of^illustration,^ are^set^out^below.^^ ^ A)^^^^^^^^Multi-cancer^screening^ ^ At^ risk^ populations^ for^ any^ cancer^ to^ receive^ blood^ test^ (e.g.^ those^ with^ hereditary^ predispositions^due^to^germline^mutations).^A^positive^resul t^would^trigger^a^confirmatory^ second^ test.^A^positive^second^test^ then^ triggers^clinical^ follow^up^ for^ relevant^cancer^ type.^ The^ invention^ therefore^ provides^ a^ valuable^ tool^ which^ works^ on^ a^ variety^ of^ cancers,^even^when^they^come^from^different^causes.^ ^ ^ B)^ Screening^for^lung^cancer^or^pre-invasive^lung^neoplasia.^ ^At-risk^populations^such^as^ smokers^or^ ex-smokers^over^ 50^ years^ of^ age^ to^ receive^ blood^tests.^A^positive^result^would^trigger^a^confirmatory^ second^test.^A^positive^second^ test^then^triggers^sputum^cytology,^bronchoscopy^and^LDCT^sc reen.^^ ^ ^ C)^ Screening^for^renal^cell^carcinoma.^^ At^risk^populations^to^receive^blood^test.^A^positive^result ^would^trigger^a^confirmatory^ second^test.^A^positive^second^test^then^triggers^clinical^f ollow^up^including^ultrasound,^ MRI^X-ray^or^CT^scan.^^ ^ ^ D)^Personalised^Dynamic^Immune^monitoring^^ ^ An^initial^negative^test^is^used^to^calibrate^a^personalised ^baseline^value.^A^statistically^ significant^increase^in^ratio^of^activated^and/or^exhausted^ T^cells^:^naïve^and/or^resting^T^^ cells^in^any^future^test^triggers^a^follow^up^test,^where^a^ positive^result^triggers^further^ clinical^evaluation^for^cancer^types^relevant^to^the^demogra phic/^subject.^ ^ E)^Healthy^adult^population^level^screening^(mass^testing)^ ^ ^ Adults^above^the^age^of^40^can^be^screened,^a^positive^resul t^would^trigger^a^confirmatory^ second^test.^A^positive^second^test^then^triggers^broad^clin ical^follow^up^to^screen^for^ cancers^of^major^incidence^in^the^demographic.^A^negative^te st^is^used^to^establish^an^ individual’s^personalised^baseline.^^ ^ ^ F)^Detection^of^cancer^recurrence^^ ^ Subjects^with^a^history^of^cancer^may^be^screened.^Scores^ob tained^with^methods^of^the^ present^invention^decrease^post^resection,^suggesting^that^t imepoints^early^after^tumour^ removal/^ eradication^may^ be^ suitable^ to^ establish^ a^ baseline.^ Statistically^ significant^ increases^trigger^a^second^test^and^/or^clinical^follow^up.^ This^would^be^to^detect^either^^ minimal^residual^disease^and^or^recurrence.^ ^ G)^Inclusion^in^standard^clinical^blood^work^^ ^ The^invention^disclosed^herein^could^be^incorporated^into^st andard^clinical^blood^work^^ alongside^tests^such^as^ESR/CRP/white^blood^cell^count^or^fu ll^blood^count.^ ^ Multi-omic^analysis^ ^ Recent^ advances^ in^ technology^ have^ permitted^ integration^ of^ different^ data^ types^ in^^ analytical^ strategies^ referred^ to^ as^multi-modal^ or^ multi-omics.^ In^ cancer^ diagnostics^ combinations^of^multiple^metrics^from^one^or^more^platform^h ave^been^shown^to^improve^ both^specificity^ and^ sensitivity.^For^ example,^ the^ integration^of^ imaging^data^ from^CT^ scans^with^pathology^assessed^histological^subtype^and^stage ^definition^yields^improved^ diagnostic^and^prognostic^evaluation.^^^ ^ ^ The^method^of^the^present^invention^may^include^analysing^th e^proportion^of^T^cells^in^ the^sample^of^ blood^obtained^ from^ the^subject^which^are^activated^and/or^exhausted^ comprises^analysing^a^plurality^of^traits^of^the^T^cells^and ^or^using^a^plurality^of^T^cell^ analysis^techniques.^ ^ ^ Combining^data^from^more^than^one^technique^for^the^analysis ^of^T^cells^in^a^sample^of^ blood^obtained^from^a^subject^may^provide^a^further^ improved^method^for^determining^ whether^the^subject^is^at^risk^for^having^a^progressing^or^h igh-grade^pre-invasive^lesion,^ nodule^or^small^mass,^or^having^a^solid^malignant^tumour^(e. g.,^a^stage^I^solid^malignant^^ tumour).^ ^ Combining,^ data^ from^ a^ plurality^ of^ T^ cell^ analyses^ can^ provide^ improved^ specificity^and^sensitivity^in^early^cancer^detection.^^Comb ining,^data^from^a^plurality^of^ T^cell^analyses^can^provide^improved^diagnostic^and/or^progn ostic^results.^ ^ Therefore^the^present^invention^provides^a^method^for^determ ining^whether^a^subject^is^^ at^risk^for^having^a^progressing^or^high-grade^pre-invasive^ lesion,^nodule^or^small^mass,^ or^having^a^solid^malignant^tumour^(e.g.,^a^stage^I^solid^ma lignant^tumour),^the^method^ comprising^analysing^the^proportion^of^T^cells^in^a^sample^o f^blood^obtained^from^the^ subject^which^are^activated^and/or^exhausted^T^cells^by^perf orming^a^plurality^of^T^cell^ analyses.^^ A^plurality^means^more^than^one.^^A^plurality^may^also^mean^ two^or^more.^^ ^ ^ In^preferred^embodiments,^ the^plurality^ of^ T^ cell^ analyses^ comprises^using^ cytometry^ (preferably^ flow^cytometry,^e.g.,^using^a^method^as^described^elsewhere^ herein),^and^ TCR-seq.^In^some^embodiments,^The^T^cell^analyses^may^compri se^(i)^determining^the^ diversity^and/or^clonality^of^T^cell^receptors^by^TCR^Seq^an d^(ii)^determining^biomarkers^^ expressed^by^T^cells^by^cytometry,^preferably^flow^cytometry .^^^ ^ Therefore,^ the^ present^ invention^ also^ provides^ a^ method^ of^ determining^ whether^ a^ subject^is^at^risk^for^having^a^progressing^or^high-grade^pr e-invasive^lesion,^nodule^or^ small^mass,^or^having^a^solid^malignant^tumour,^using^a^samp le^of^blood^obtained^from^^ the^ subject,^ the^ method^ combining^ two^ or^ more^ T^ cell^ analyses^ comprising^ a)^ (i)^ measuring^the^ratio^of^activated^and/or^exhausted^T^cells:na ïve^and^/or^resting^T^cells^ determined^ by^ flow^ cytometry,^ or^ (ii)^ measuring^ the^ proportion^ of^ activated^ and/or^ exhausted^cells^as^a^percentage^of^T^cells^determined^by^flo w^cytometry^and^^ b)^the^diversity^or^clonality^of^the^blood^TCR^repertoire.^ ^ ^ The^present^invention^also^provides^a^method^of^determining^ whether^a^subject^is^at^ risk^for^having^a^progressing^or^high-grade^pre-invasive^les ion,^nodule^or^small^mass,^ or^having^a^solid^malignant^tumour,^the^method^comprising^an alysing^the^proportion^of^ T^cells^in^a^sample^of^blood^obtained^from^the^subject^which ^are^activated^and/or^^ exhausted^T^cells^by^analysing^a^trait^of^the^T^cells,^where in^the^trait^is^a^phenotypic^ trait^which^is^^ a)^the^diversity^or^clonality^of^T^cell^receptors^on^the^T^c ells^in^the^sample^of^blood,^and^^ b)^biomarker^expression^by^the^T^cells^in^the^sample^of^bloo d,^optionally^using^ cytometry.^ ^ ^ The^present^invention^is^the^first^T^cell^focused^multi-omic s^machine^learning^method^for^ early^cancer^detection.^^ ^ The^inventors^have^shown^that^individual^measures^of^blood^T ^cells^can^help^to^detect^^ malignant^tumours^or^high^grade^preinvasive^disease.^Namely^ ^ ^ i)^ the^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^and ^/or^resting^T^cells^ determined^by^ flow^ cytometry,^ using^ key^ biomarkers^Ki67^ and^CD39,^and^ optionally^ one^ or^more^ biomarkers^ selected^ from^ CD45RA,^ CCR7,^CD38,^ PD1,^or^CD57,^which^the^inventors^refer^to^as^the^T^cell^ear ly^detection^index^^ (TEDI).^Specifically,^ this^ ratio^within^CD4^T^cells^ is^a^powerful^ classifier^of^ patients^with^low^vs^high^grade^disease,^or^the^proportion^o f^activated^and/or^ exhausted^T^cells^as^a^percentage^of^T^cells,^using^key^biom arkers^such^as^ (i)^FoxP3^and^CD4,^and/or^(ii)^CD39^and^Ki67,^and^optionally ^one^or^more^ one^or^more^biomarkers^selected^from^CD45RA,^CCR7,^CD38,^PD1 ,^or^CD57^^^ ^ ii)^ The^diversity^of^the^T^cell^repertoire,^measured^by^TCR^seq^ data,^optionally^ the^D50^ index,^which^corresponds^ to^ the^percentage^of^unique^sequences^ that^account^for^50%^of^the^total^number^of^sequences,^or^th e^clonality^of^the^ T^cell^repertoire,^measured^by^TCR^seq^data,^optionally^the^ a)^repertoire^of^^ TCRs^occupied^by^the^top^n^clones^(optionally^wherein^n^is^f rom^50-1000,^for^ example,^n^=^100)^or^b)^the^ratio^of^the^repertoire^of^TCRs^ occupied^by^large^ clones:^the^repertoire^of^TCRs^occupied^by^small^clones^ ^ The^ inventors^ have^ further^ shown^ that^ coupling^ these^ metrics^ together^ may^ yield^^ improved^ classifier^ performance^ to^ generate^ a^ multi-omics^ approach^ for^ enhanced^ cancer^early^detection.^^ ^ The^ present^ invention^ therefore^ also^ provides^ a^ method^ for^ determining^ whether^ a^ subject^is^at^risk^for^having^a^progressing^or^high-grade^pr e-invasive^lesion,^nodule^or^^ small^mass,^or^having^a^solid^malignant^tumour,^using^a^samp le^of^blood^obtained^from^ the^subject,^the^method^combining^two^or^more^T^cell^analyse s^comprising^a)^measuring^ the^ratio^of^activated^and/or^exhausted^T^cells:naïve^and^/ or^resting^T^cells^determined^ by^flow^cytometry,^or^measuring^the^proportion^of^activated^ and/or^exhausted^cells^as^a^ percentage^of^T^cells^determined^by^flow^cytometry^and^^ ^ b)^the^diversity^or^clonality^of^the^blood^TCR^repertoire.^ ^ The^present^invention^provides^a^method^for^determining^whet her^a^subject^has^a^high^ grade^ or^ progressing^ preinvasive^ lesion^ or^ nodule^ or^ small^ mass^ or^ solid^ malignant^ tumour^vs^has^no^malignant^tumour,^or^a^benign^tumour^or^a^b enign^or^non^progressing^^ or^low^grade^lesion^or^nodule^or^small^mass,^using^a^sample^ of^blood^obtained^from^the^ subject,^the^method^comprising^combining^two^or^more^T^cell^ analyses^comprising:^ -^ measuring^the^ratio^of^activated^and/or^exhausted^T^cells^:^ naïve^and^/or^resting^ T^cells^determined^by^flow^cytometry,^and^^ -^ the^diversity^or^clonality^of^the^blood^TCR^repertoire.^Meas ures^of^the^diversity^or^ clonality^of^the^blood^TCR^repertoire^may^include^the^D50^in dex.^^ ^ ^ The^present^invention^also^provides^a^method^for^determining ^whether^a^subject^is^at^risk^ for^ having^ a^progressing^ or^ high-grade^pre-invasive^ lesion,^ nodule^ or^ small^mass,^ or^ having^a^solid^malignant^tumour,^using^a^sample^of^blood^obt ained^from^the^subject,^the^ method^combining^two^or^more^T^cell^analyses^comprising:^^ ^ -^ measuring^the^ratio^of^activated^and/or^exhausted^T^cells^:^ naïve^and^/or^resting^ T^cells^determined^by^flow^cytometry,^and^^ -^ the^diversity^or^clonality^of^the^blood^TCR^repertoire.^Meas ures^of^the^diversity^or^ clonality^of^the^blood^TCR^repertoire^may^include^the^D50^in dex.^^ ^ ^ Combining^ data^ obtained^ from^ two^ different^ methods^ and/or^ the^ two^ or^ more^ T^ cell^ analyses^ may^ involve^ one^ or^ more^ of^ machine^ learning,^ artificial^ intelligence^ or^ mathematical^modelling.^ In^ some^ embodiments^ and^ examples,^ the^machine^ learning^ uses^the^XGBoost^algorithm,^e.g.,^using^the^methods^describe d^herein.^However,^other^ suitable^algorithms^may^be^used.^^ ^ ^ In^preferred^embodiments,^the^diversity^or^clonality^of^the^ blood^TCR^repertoire^is^ determined^by^TCR-seq.^The^diversity^or^clonality^of^the^blo od^TCR^repertoire^may^be^ determined^using^one^or^more^diversity^or^clonality^metrics^ as^described^herein.^ ^ ^ The^measuring^the^ratio^of^activated^and/or^exhausted^T^cell s:naïve^and^/or^resting^T^ cells^determined^by^flow^cytometry,^or^measuring^the^proport ion^of^activated^and/or^ exhausted^cells^as^a^percentage^of^T^cells^determined^by^flo w^cytometry^may^be^as^ determined^using^any^of^the^methods^or^embodiments^of^the^me thods^described^ herein.^^ ^ In^some^preferred^embodiments,^the^activated^and/or^exhauste d^T^cells^express^the^ biomarkers^Ki67^and^CD39.^^ ^ In^some^preferred^embodiments,^the^activated^and/or^exhauste d^T^cells^express^the^ biomarker^FoxP3,^wherein^the^T^cells^are^CD4+^T^cells,^optio nally^in^combination^with^^ CD39.^In^some^embodiments,^the^CD4+^T^cells^express^FoxP3,^C D39^and^Ki67.^In^ some^embodiments,^the^CD4^T^cells^express^FoxP3,^CD39^and^do ^not^express^ CD45RA.^ ^ In^some^embodiments^and^examples,^the^diversity^of^the^blood ^is^measured^using^the^ D50^index.^In^other^embodiments^and^examples,^the^diversity^ of^the^blood^is^measured^ using^the^Hill^number.^However,^other^methods^of^measuring^t he^diversity^of^the^TCR^^ blood^repertoire^may^be^used.^^ ^ In^some^embodiments^and^examples,^the^clonality^of^the^blood ^is^measured^using^the^ ratio^of^the^repertoire^of^TCRs^occupied^by^large^clones:^th e^repertoire^of^TCRs^ occupied^by^small^clones.^This^may^be^determined^using^the^m ethod^disclosed^herein.^^^ ^ Methods^of^Treatment^^ ^ Also^disclosed^herein^is^a^method^of^treating^a^subject^dete rmined^to^be^at^risk^for^ having^a^progressing^or^high-grade^pre-invasive^lesion,^nodu le^or^small^mass,^or^^ having^a^solid^malignant^tumour,^comprising^analysing^the^pr oportion^of^T^cells^in^a^ sample^of^blood^obtained^from^the^subject^which^are^activate d^and/or^exhausted^T^cells^ by^analysing^a^trait^of^the^T^cells,^and^treating^the^subjec t^determined^to^be^at^risk.^The^ analysing^may^be^as^described^elsewhere^herein.^The^subject^ may^be^determined^to^ be^at^risk^using^any^method^described^elsewhere^herein.^^ ^ ^ Also^disclosed^herein^is^a^method^of^treating^a^subject^dete rmined^to^be^at^risk^for^ having^a^progressing^or^high-grade^pre-invasive^lesion,^nodu le^or^small^mass,^or^ having^a^solid^malignant^tumour,^comprising^analysing^the^pr oportion^of^T^cells^in^a^ sample^of^blood^obtained^from^the^subject^which^are^activate d^and/or^exhausted^T^cells^^ by^analysing^a^trait^of^the^T^cells,^wherein^the^trait^is^a^ phenotypic^trait^and^wherein^the^ analysing^comprises^ a)^analysing^the^proportion^of^T^cells^in^a^sample^of^blood^ obtained^from^the^subject^ which^are^activated^and/or^exhausted^T^cells^using^biomarker ^expression,^optionally^ using^cytometry,^wherein^the^subject^is^at^risk^if^ ^ (i)^the^ratio^of^activated^and/or^exhausted^T^cells^:^naïve ^and/or^resting^T^cells^is^equal^ to^or^greater^than^a^ratio^of^activated^and/or^exhausted^T^c ells^:^naïve^and/or^resting^T^ cells^of^a^comparison^subject,^^or^in^a^plurality^of^compari son^subjects,^or^ (ii)^the^ratio^of^activated^and/or^exhausted^T^cells^:^T^cel ls^which^are^not^activated^ and/or^exhausted^is^equal^to^or^greater^than^a^ratio^of^acti vated^and/or^exhausted^T^^ cells^:^T^cells^which^are^not^activated^and/or^exhausted^of^ a^comparison^subject,^^or^^ in^a^plurality^of^comparison^subjects.^ (iii)^the^proportion^of^activated^and/or^exhausted^T^cells^i s^higher^than^the^proportion^of^ activated^and/or^exhausted^T^cells^in^a^comparison^subject^o r^a^plurality^of^comparison^ subjects,^^ ^ ^ and/or^^ ^ b)^determining^the^diversity^or^clonality^of^T^cells^in^a^sa mple^of^blood^obtained^from^the^ subject,^optionally^using^TCR-seq,^wherein^the^subject^is^at ^risk^if^ ^^ ^ (i)^^the^diversity^of^the^repertoire^of^TCRs^is^lower^than^t he^diversity^of^the^repertoire^of^ TCRs^of^a^comparison^subject^or^a^plurality^of^comparison^su bjects,^^ ^ ii)^D50^diversity^score^is^lower^than^a^D50^diversity^score^ obtained^from^analysing^T^ cells^in^a^sample^of^blood^obtained^from^a^comparison^subjec t^or^a^plurality^of^ ^ comparison^subjects,^^ ^ iii)^a^clonality^score^is^higher^than^a^clonality^score^obta ined^from^analysing^T^cells^in^a^ sample^of^blood^obtained^from^a^comparison^subject^or^a^plur ality^of^comparison^ subjects,^or^ ^ ^ iv)^the^Hill^number^is^lower^than^a^Hill^number^obtained^fro m^analysing^T^cells^in^a^ sample^of^blood^obtained^from^a^comparison^subject^or^a^plur ality^of^comparison^ subjects,^or^^ ^ ^ v)^the^clonality^of^the^repertoire^of^TCRs^occupied^by^the^t op^n^clones^is^higher^than^the^ clonality^of^the^repertoire^of^TCRs^occupied^by^the^top^n^cl ones^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects,^optionally^wherein^n^ =^100,^or^^ ^ vi)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^sma ll^clones^is^lower^than^the^^ proportion^of^the^repertoire^of^TCRs^occupied^by^small^clone s^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects,^or^ ^ vii)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^la rge^clones^is^higher^than^the^ proportion^of^the^repertoire^of^TCRs^occupied^by^large^clone s^in^a^comparison^subject^^ or^a^plurality^of^comparison^subjects^or^ ^ viii)^the^ratio^of^the^repertoire^of^TCRs^occupied^by^large^ clones:small^clones^is^higher^ than^the^ratio^of^the^repertoire^of^TCRs^occupied^by^large^c lones:small^clones^in^a^ comparison^subject^or^a^plurality^of^comparison^subjects,^op tionally^wherein^the^ comparison^subject^is^a^healthy^subject,^or^the^plurality^of ^comparison^subjects^are^^ healthy^subjects,^and^^ ^ treating^the^subject^if^the^subject^is^at^risk.^^^ ^ In^ some^ embodiments,^ the^ treating^ may^ comprise^ administering^ an^ anti-cancer^^ therapeutic.^ In^ some^ embodiments,^ the^ treating^ may^ comprise^ administering^ a^ therapeutic^suitable^for^treating^pre-invasive^neoplasia^and /or^a^high^grade^pre-invasive^ lesion,^ nodule^ or^ small^ mass.^ ^ In^ some^ embodiments,^ the^ treating^ may^ comprise^ administering^a^therapeutic^suitable^for^treating^a^solid^ma lignant^tumour,^for^example,^a^ stage^I^solid^malignant^tumour.^^ ^ ^ Also^disclosed^herein^is^an^anti-cancer^therapeutic^for^use^ in^a^method^of^treatment^of^ a^subject^determined^to^be^at^risk^for^having^a^progressing^ or^high-grade^pre-invasive^ lesion,^nodule^or^small^mass,^or^having^a^solid^malignant^tu mour,^comprising^analysing^ the^proportion^of^T^cells^in^a^sample^of^blood^obtained^from ^the^subject^which^are^^ activated^and/or^exhausted^T^cells^by^analysing^a^trait^of^t he^T^cells,^wherein^the^anti- cancer^therapeutic^is^administered^to^the^subject^determined ^to^be^at^risk.^The^ analysing^may^be^as^described^elsewhere^herein.^The^subject^ may^be^determined^to^ be^at^risk^using^any^method^described^elsewhere^herein.^^ ^ ^ In^some^embodiments,^the^analysing^comprises^analysing^a^tra it^of^the^T^cells,^wherein^ the^trait^is^a^phenotypic^trait^and^wherein^the^analysing^co mprises^ a)^analysing^the^proportion^of^T^cells^in^a^sample^of^blood^ obtained^from^the^subject^ which^are^activated^and/or^exhausted^T^cells^using^biomarker ^expression,^optionally^ using^cytometry,^wherein^the^subject^is^at^risk^if^ ^ (i)^the^ratio^of^activated^and/or^exhausted^T^cells^:^naïve ^and/or^resting^T^cells^is^equal^ to^or^greater^than^a^ratio^of^activated^and/or^exhausted^T^c ells^:^naïve^and/or^resting^T^ cells^of^a^comparison^subject,^^or^in^a^plurality^of^compari son^subjects,^or^ (ii)^the^ratio^of^activated^and/or^exhausted^T^cells^:^T^cel ls^which^are^not^activated^ and/or^exhausted^is^equal^to^or^greater^than^a^ratio^of^acti vated^and/or^exhausted^T^^ cells^:^T^cells^which^are^not^activated^and/or^exhausted^of^ a^comparison^subject,^^or^^ in^a^plurality^of^comparison^subjects.^ (iii)^the^proportion^of^activated^and/or^exhausted^T^cells^i s^higher^than^the^proportion^of^ activated^and/or^exhausted^T^cells^in^a^comparison^subject^o r^a^plurality^of^comparison^ subjects,^^ ^ ^ and/or^^ ^ b)^determining^the^diversity^or^clonality^of^T^cells^in^a^sa mple^of^blood^obtained^from^the^ subject,^optionally^using^TCR-seq,^^ ^ ^ In^some^embodiments,^the^subject^is^at^risk^if^ ^^ (i)^^the^diversity^of^the^repertoire^of^TCRs^is^lower^than^t he^diversity^of^the^repertoire^of^ TCRs^of^a^comparison^subject^or^a^plurality^of^comparison^su bjects,^^ ^ ^ ii)^D50^diversity^score^is^lower^than^a^D50^diversity^score^ obtained^from^analysing^T^ cells^in^a^sample^of^blood^obtained^from^a^comparison^subjec t^or^a^plurality^of^ comparison^subjects,^^ ^ iii)^a^clonality^score^is^higher^than^a^clonality^score^obta ined^from^analysing^T^cells^in^a^^ sample^of^blood^obtained^from^a^comparison^subject^or^a^plur ality^of^comparison^ subjects,^or^ ^ iv)^the^Hill^number^is^lower^than^a^Hill^number^obtained^fro m^analysing^T^cells^in^a^ sample^of^blood^obtained^from^a^comparison^subject^or^a^plur ality^of^comparison^^ subjects,^or^^ ^ v)^the^clonality^of^the^repertoire^of^TCRs^occupied^by^the^t op^n^clones^is^higher^than^the^ clonality^of^the^repertoire^of^TCRs^occupied^by^the^top^n^cl ones^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects,^optionally^wherein^n^ =^100,^or^^ ^ ^ vi)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^sma ll^clones^is^lower^than^the^ proportion^of^the^repertoire^of^TCRs^occupied^by^small^clone s^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects,^or^ ^ vii)^the^proportion^of^the^repertoire^of^TCRs^occupied^by^la rge^clones^is^higher^than^the^ proportion^of^the^repertoire^of^TCRs^occupied^by^large^clone s^in^a^comparison^subject^ or^a^plurality^of^comparison^subjects^or^ ^ ^ viii)^the^ratio^of^the^repertoire^of^TCRs^occupied^by^large^ clones:small^clones^is^higher^ than^the^ratio^of^the^repertoire^of^TCRs^occupied^by^large^c lones:small^clones^in^a^ comparison^subject^or^a^plurality^of^comparison^subjects,^op tionally^wherein^the^ comparison^subject^is^a^healthy^subject,^or^the^plurality^of ^comparison^subjects^are^ healthy^subjects.^ ^ ^ ^ ^

Examples^Section^ ^ In^this^section,^Examples^are^described^for^analysing^T^cell s^isolated^from^a^subject.^The^ methods^in^the^Examples^provided^herein^for^analysing^T^cell s^isolated^from^a^subject^^ can^ be^ implemented^ using^ a^ variety^ of^ methodologies,^ including^ using^ a^ variety^ of^ apparatus,^ hardware^ and^ software^ components^ that^ include^ (but^ are^ not^ limited^ to)^ different^ cytometry^ devices,^ assay^ conditions,^ computational^ hardware^ and^ analysis^ software.^It^will^be^understood^to^the^person^skilled^in^the ^art^that^in^general,^the^methods^ provided^herein^in^the^Examples^are^merely^an^example.^The^p erson^skilled^in^the^art^^ will^know^that^that^the^various^steps^and^techniques^discuss ed^herein^can^be^performed^ using^a^variety^of^ different^apparatus,^ assay^conditions,^ computational^ hardware^and^ software^components^that^may^result^in^different^numerical^v alues^obtained,^but^which^ equate^ to^ the^ same^ biological^ results^ to^ the^ Examples^ provided^ herein.^ It^ will^ be^ understood^to^the^person^skilled^in^the^art^that^in^general, ^the^methodologies^and^results^^ provided^ herein^ in^ the^ below^ Examples^ are^ not^ intended^ to^ limit^ the^ scope^ of^ the^ invention.^^^ ^ ^ Table^1:^List^of^antibodies^used^in^Examples^1-3^ ^ Samples^were^acquired^on^FACSymphony^A5^High-Parameter^Cell^ Analyser^from^BID^ Biosciences.^^ ^ ^ An^example^protocol^for^processing^of^blood^samples^for^flow ^cytometry^analysis^^ Blood^samples^were^collected^in^Vacutainer^EDTA^blood^collec tion^tubes^(BD),^PBMCs^^ isolated^ by^ gradient^ centrifugation^ (750g^ for^ 10minutes)^ on^ Ficoll^ Paque^ Plus^ (GE^ Healthcare).^The^interface^was^washed^twice^with^complete^RP MI-1640,^resuspended^in^ 90%^FBS^with^10%^DMSO^(Sigma)^and^cryopreserved^ in^ liquid^nitrogen^or^ in^a^ -180^ degrees^Celsius^freezer^system^prior^ to^staining.^To^characterise^T^cell^differentiation^ profiles^of^HG^vs^LG^samples^cryopreserved^PBMCs^from^were^t hawed^with^warm^R20^ media^ (RPMI^ with^ 20%^ FBS,^ L-glutamine,^ HEPES^ and^ Penicillin/Streptomycin)^ and^^ washed^with^R10^media^containing^DNase^I^grade^II^37.5μg/mL ^(Roche,^10104159001).^ The^samples^were^subjected^to^20^mins^incubation^of^Live/Dea d^Fixable^Aqua^at^RT^in^ the^dark.^Samples^were^then^washed^in^PBS^and^resuspended^wi th^FACS^buffer^(PBS^ +^2%^FBS^+^2mM^EDTA)^and^plated^on^a^96-well^U^bottom^plate^ containing^Fc^Receptor^ Binding^ Inhibitor^ Polyclonal^ Antibody^ (Thermo^ Fisher,^ 14-9161-73)^ and^ surface^^ antibodies^for^chemokine^receptors^(CXCR4,^CCR7)^at^room^tem perature^(RT)^for^15^ mins,^followed^by^another^30^mins^incubation^with^all^additi onal^surface^antibodies,^on^ ice.^ Plates^ were^washed^ twice^with^ FACS^ buffer^ and^ fixed^with^ Foxp3^ Transcription^ Factor^Fixation/Permeabilization^Concentrate^and^Diluent^sol utions^(Thermo^Fisher,^00- 5521-00)^for^30^mins^at^RT.^Cells^were^washed^twice^with^X1^ Permeabilization^Buffer^^ (Thermo^Fisher,^00-8333)^followed^by^1^hour^ incubation^with^a^cocktail^of^ intracellular^ antibodies^ at^ RT.^ The^ plates^ were^ washed^ 3^ times^ and^ resuspended^ with^ X1^ Permeabilization^ Buffer^ before^ sample^ acquisition^ using^ FACSymphony^ (BD^ Biosciences.^ ^ ^ An^example^protocol^for^acquisition^and^processing^^ ^ Samples^ were^ acquired^ using^ a^ BD^ Symphony^ cytometer^ according^ to^ the^ manufacturer’s^ instructions^ using^ the^BD^ FACS^DIVA^ software.^ Raw^ FCS^ files^ were^ exported^ and^ files^ imported^ into^ FlowJo.^ Compensation^matrices^ were^ calculated^ in^^ FACS^DIVA^from^single-stain^compensation^bead^controls^and^o ptimised^in^FlowJo.^To^ do^so,^ files^ from^each^batch^ (two^ in^ total^ from^consecutive^days)^were^ concatenated^ separately^as^a^reference^file^and^optimised^matrices^applie d^to^each^batch.^Dead^cells^ and^compensation^artefacts^were^excluded^as^described^previo usly^(NC)^and^5000^live^ CD3+CD4+^or^2000^CD3+CD8+^events^were^down-sampled^and^expor ted^per^file.^^^ ^ An^example^protocol^for^clustering^ ^ Figure^ 2^ shows^ a^ workflow^ showing^ the^ protocol^ of^ how^ the^ clustering^ of^ the^ flow^ cytometry^data^was^completed^following^the^analysis^steps^ou tlined.^ Only^samples^with^minimum^1000^live^CD3+^cells^were^analysed .^FCS^files^underwent^ quality^control^of^signal^acquisition,^assessed^by^the^FlowA I^package^(v1.24).^An^Arcsinh^ transformation^was^applied^to^the^data,^using^the^prepData^f unction^from^the^CATALYST^ package^(v1.18.1),^with^the^cofactor^set^to^150.^Any^biomark ers^with^a^poor^contribution^^ to^phenotypic^variance^were^excluded^pre-clustering.^These^w ere^determined^using^the^ PCA-based^non-redundancy^score^ (NRS)^as^described^ in^ the^Nowicka^et^al.^pipeline^ (Nowicka^M,^Krieg^C,^Crowell^HL^et^al.^CyTOF^workflow:^diffe rential^discovery^in^high- throughput^high-dimensional^cytometry^datasets^ [version^4;^peer^ review:^2^approved].^ F1000Research^ 2019,^ 6:748^ (https://doi.org/10.12688/f1000research.11622.4).^ In^ the^^ initial^analysis^(e.g.,^of^the^pre-invasive^data)^FoxP3,^CD1 03,^Tim3,^CXCR4,^TIGIT,^were^ all^excluded^from^clustering^(live/dead,^CD3,^CD8^and^CD4^we re^excluded^as^FCS^files^ only^included^cells^pre-gated^on^live^CD4^or^CD8^T^cells).^I n^subsequent^analysis,^FoxP3^ was^included^in^the^clustering^panel.^^ ^ ^ Data^was^clustered^using^the^FlowSOM^package^(v2.2.0)^onto^a ^15x15^node^square^self- organising^map^ (SOM).^Nodes^were^clustered^by^ the^ConsensusCenterPlus^package^ (v1.58.0),^ as^ described^ by^ Nowicka^ et^ al.^ ^ The^ UMAP^ algorithm^ was^ applied^ as^ a^ dimension^ reduction^analysis^ to^better^understand^ the^phenotypic^ relevance^between^ individual^ clusters.^The^ resulting^clusters^were^manually^merged^based^on^biomarker^^ expression^similarity,^occupation^of^space^on^the^UMAP,^and^ previously^defined^T^cell^ states.^16^ clusters^ of^CD4^T^ cells^ and^15^ clusters^ of^CD8^T^cells^were^ resolved.^ All^ computational^ packages^ were^ sourced^ from^ https://cran.r-project.org/^ and^ https://www.bioconductor.org/.^^ ^ ^ ^ Table^2:^A^list^of^T^cell^clusters^that^were^differentially^ abundant^between^high^grade^or^ low^grade^pre-invasive^lesion^or^nodule.^^ ^ Clusters^ found^ to^ be^ enriched^ in^ high-grade^ were^ found^ to^ express^ biomarkers^ of^^ activation^and^late^differentiation,^proliferation^or^exhaus tion,^in^particular^the^biomarkers^ CD39,^known^to^be^expressed^on^tumour^specific^T^cells,^and^ the^marker^Ki67^which^ denotes^that^cells^are^actively^proliferating.^TEDI^scores^w ere^generated^by^dividing^high- grade-associated^clusters^by^the^low^grade^associated^cluste rs^within^each^lineage^(e.g.^ sum^of^high^grade^clusters^/^sum^of^low^grade^clusters*^for^ CD4^=^CD4.TEDI)^to^develop^^ a^metric^ that^would^have^ the^highest^discriminatory^power,^which^ reflects^the^state^of^ differentiation^within^ that^ lineage.^The^same^was^completed^for^CD8^T^cells^and^all^T^ cells^combining^CD4^and^CD8.^^ ^ Unsupervised^clustering^refers^to^using^computational^analys is^to^determine^which^type^^ of^T^cell^is^present^in^a^given^sample.^This^is^a^preferred^ alternative^for^initial^analysis^of^ flow^cytometry^data^to^‘supervised’^analysis^such^as^man ually^gating^cells^in^a^sample^ using^prior^knowledge^and^selecting^specific^marker^combinat ions.^^ ^ ^ Example^1^ ^ In^Example^1,^the^inventors^used^the^example^protocols^set^o ut^above.^^ ^ ^ Table^3:^^Clinical^variables^of^patients^used^to^generate^da ta^in^Example^1.^^ ^ ^ ^ Analysis^of^cluster^distribution^in^high^versus^low^grade^sa mples^ ^ ^ The^frequency^of^each^cluster^was^expressed^as^a^%^of^parent ^(CD4^or^CD8)^for^each^ of^the^66^samples^and^Mann-Whitney^tests^were^performed^in^R ^to^compare^average^ cluster^frequency^between^high-grade^(HG)^vs^low-grade^(LG)^ samples^for^each^cluster.^ Separate^rounds^of^analysis^focused^on^CD4^and^CD8^T^cell^su bsets^were^performed,^^ with^multiple^correction^adjustment^via^benjamini-hochberg,^ applying^a^false^discovery^ rate^ of^ 0.05.^ To^ account^ for^ multiple^ samples^ being^ drawn^ from^ individual^ patients,^ analysis^was^repeated^using^the^mean^frequency^of^a^given^cl uster^in^each^patient,^using^ all^available^samples,^yielding^similar^results.^^ ^ ^ 12^ (of^ 16)^ clusters^ of^ CD4^ T^ cell^ clusters^ and^ 4^ (of^ 15)^ CD8^ T^ cell^ clusters^ were^ significantly^differentially^abundant^in^HG^vs^LG^(FDR<0. 05^sample^level^analysis^and^ p<0.05^in^patient^level^analysis).^The^clusters^enriched^ in^HG^patients^represented^late^ T^cell^differentiation,^activation^and^exhaustion^within^bot h^CD4^and^CD8^T^cells.^This^ shows^ which^ clusters^ of^ T^ cells^ are^ present^ across^ all^ samples^ and^ their^ relative^ abundance.^Thereby^showing^ that^T^cells^can^be^classified^based^on^their^phenotype^ into^low-grade^and^high-grade^disease.^^Figure^3C.^The^prese nce^of^HG^(vs^LG)^lesions^ is^ associated^ with^ a^ significantly^ increased^ frequency^ of^ activated,^ memory^ and^^ exhausted^CD8^and^CD4^T^cells^and^a^loss^of^resting^Tcm^and^ naïve^cells^in^the^blood^ (Figure^ 3A),^ This^ shows^ that^ there^ are^ multiple^ types^ of^ activated/exhausted^ and^ resting/naïve^T^cell^present^in^the^pool^of^samples.^furthe r^demonstrated^with^significantly^ different^expression^of^CD45RA,^CCR7,^Ki67,^CD39,^CD57^and^C D38^biomarkers^ in^ activated^and/or^exhausted^T^cell^types^compared^to^naïve^a nd/or^resting^T^cell^types^^ (Figure^3B-C).^The^ratio^of^[T^cell^clusters^significantly^e nriched^in^high-grade^disease^ samples^ :^ T^ cell^ clusters^ significantly^ enriched^ in^ low^grade^ samples]^ for^ CD4^ (AUC^ 94.2%)^or^CD8^(AUC^92.6%)^T^cells^were^able^ to^help^discriminate^patients^with^HG^ lesions^in^ROC^analysis,^meaning^that^92-94%^of^patients^can ^be^correctly^classified^as^ having^High^or^Low-grade^disease^using^the^invention.^Figure ^3D-E.^^ ^ ^ In^CD4^ T^ cells^ 8^ of^ the^ 9^ subsets^ significantly^ over-represented^ in^ patients^ with^HG^ disease^expressed^at^least^one^of^the^following^biomarkers:^ PD-1^(a^biomarker^of^antigen^ driven^ activation^ and^ exhaustion;^ 6/9^HG-clusters)^ CD39^ (a^ biomarker^ of^ exhausted,^ regulatory^ and^ tumour^ reactive^ T^ cells;^ 8/9^ HG-clusters)^ and^ Ki67^ (an^ activation^^ biomarker^expressed^on^proliferating^T^cells;^8/9^HG-cluster s).^Moreover,^CD39^and^Ki67^ co-expression^defined^7^of^the^9^T^cell^subsets^enriched^in^ HG^disease.^HG^associated^ clusters^of^CD4^T^cells^also^lacked^CD45RA^and^7^of^9^also^l acked^high^levels^of^ the^ early^differentiation^biomarker^CCR7.^In^contrast,^all^3^CD4 ^T^cell^clusters^enriched^in^ LG^were^devoid^of^the^activation^and^exhaustion^biomarkers^P D-1,^Ki67,^and^CD39^(i.e.^^ PD-1-KI67-CD39-),^ and^ all^ 3/3^ expressed^ CCR7^ and^ 2/3^ expressed^ CD45RA.^ This^ imbalance^in^CD4^T^cell^differentiation^is^indicative^of^inc reased^antigen^exposure^with^ HG^ disease^ and^ suggests^ that^ HG^ vs^ LG^ patients^ can^ be^ distinguished^ using^ a^ combination^of^ the^biomarkers^PD-1,^Ki67,^CD39,^CCR7^and^CD45RA.^ In^addition,^a^ single^cluster^of^Cytolytic^phenotype^CD4^T^cells^enriched^i n^HG^disease^that^could^be^^ identified^by^unique^expression^of^the^terminal^differentiat ion^biomarker^CD57.^Figure^4.^ This^shows^that^expression^of^CD45RA,^CCR7,^Ki67,^CD39,^CD57 ^and^CD38^biomarkers^ were^significantly^different^in^activated^and/or^exhausted^T ^cell^types^compared^to^naïve^ and/or^resting^T^cell^types^and^that^these^biomarkers^can^be ^used^to^determine^the^ratio^ of^ activated^ and/or^ exhausted^ T^ cells^ compared^ to^ naïve^ and/or^ resting^ T^ cells^ in^ a^^ subject,^thereby^distinguishing^between^high-grade^and^low-g rade^disease.^^^^ ^ Skewing^in^T^cell^differentiation^was^also^observed^in^CD8^T ^cells,^where^two^clusters^ were^enriched^in^HG^disease^that^represent^end^points^of^lat e-stage^differentiation^and^ activation^in^the^blood.^These^2^clusters^once^again^both^co -expressed^CD39^and^Ki67,^ but^ could^ be^ distinguished^ as^ exhausted^ (PD-1^ high)^ or^ terminally^ differentiated^^ (CD57+CD45RA+)^ based^ on^ additional^ biomarkers.^Figure^ 5^As^ observed^ in^CD4^T^ cells,^CD8^T^cell^clusters^enriched^ in^LG^disease^were^PD-1-CD39-Ki67-^ indicating^a^ resting^ state.^ These^ clusters^ were^ naïve^ cells^ known^ to^ be^ enriched^ in^ less^ antigen^ experienced^individuals,^and^resting^memory^cells.^Identific ation^of^these^4^populations^ from^all^remaining^CD8^T^cells^via^clustering^required^the^s ame^6^biomarkers^as^CD4^T^^ cells;^ namely^ PD-1,^ Ki67,^ CD39,^ CCR7,^ CD45RA,^ CD57.^ In^ addition,^ the^ biomarker^ CD38^was^required^to^identify^the^activated,^terminally^diff erentiated^population.^Figure^ 5.^ This^ shows^ that^ expression^ of^ CD45RA,^ CCR7,^ Ki67,^ CD39,^ CD57^ and^ CD38^ biomarkers^ were^ significantly^ different^ in^ activated^ and/or^ exhausted^ T^ cell^ types^ compared^to^naïve^and/or^resting^T^cell^types^and^that^thes e^biomarkers^can^be^used^to^^ determine^ the^ ratio^ of^ activated^ and/or^ exhausted^ T^ cells^ compared^ to^ naïve^ and/or^ resting^T^cells^ in^a^subject,^ thereby^distinguishing^between^high-grade^and^ low-grade^ disease.^^^This^data^therefore^supports^measuring^systemic^T ^cell^differentiation^as^an^ innovative^strategy^for^early^detection.^Specifically,^ the^data^show^that^measuring^ the^ ratio^of^activated,^exhausted^and^late^memory^T^cell^subsets ^:^Naïve,^resting^and^earlier^^ differentiated^T^cells^is^a^novel^metric^that^distinguishes^ individuals^with^less^pathogenic^ lesions^(LG)^from^patients^with^lesions^more^likely^to^progr ess^to^NSCLC^(HG).^ ^ Generation^of^an^example^of^a^T^cell^Early^Detection^Index^( TEDI)^ ^ ^ To^simplify^these^results,^the^change^in^CD4^or^CD8^clusters ^was^converted^to^a^single^ score^referred^to^as^the^T^cell^early^detection^index^(TEDI) .^To^generate^TEDI^a^ratio^of^ [sum^freq.^of^all^CD4^T^cell^clusters^enriched^in^HG^disease ]/^[sum^freq.^of^all^clusters^ enriched^ in^LG^disease]^was^calculated^and^ the^process^ repeated^ for^CD8^T^cells,^a^ simple^workflow^is^shown^in^Figure^6^^ ^ ^ In^addition,^ the^mean^average^of^CD4^and^CD8^T^cell^TEDI^scores^was^calcu lated^to^ generate^a^Combined^TEDI^score.^Receiver^operator^characteri stic^(ROC)^curves^and^ calculated^the^areas^under^the^curve^(AUC)^were^generated^fo r^each^TEDI^score,^shown^ in^Figure^7.^^This^Figure^summarises^the^key^results^from^Ex ample^1,^showing^that^the^^ ratio^of^exhausted/activated^:^naïve^resting^T^cells^is^sig nificantly^higher^in^high^vs^low^ grade^disease.^The^same^data^is^also^shown^in^Figure^3C^^ ^ Table^4:^Sensitivity^and^specificity^values^with^their^corre sponding^threshold^probabilities^ for^ROC^analysis^ ^ ^ To^ determine^ the^ optimum^ TEDI^ cut-off^ values^ from^ the^ ROC^ analysis,^ all^ possible^ sensitivity^ and^specificity^ values^with^ their^ corresponding^ threshold^ probabilities^were^ calculated,^ as^ROC^curves^ represent^ a^ trade-off^ between^ the^ true^and^ false^positive^ rates.^ The^ threshold^ probability^ that^ yielded^ the^ highest^ combined^ sensitivity^ and^ specificity^ was^ selected,^ known^ as^ the^ Youden^ Index,^ a^ commonly^ used^ method^ to^^ estimate^ the^ optimal^ cut-off^ point^ for^ a^ diagnostic^ Fluss^ R,^ Faraggi^ D,^ Reiser^ B.^ Estimation^ of^ the^ Youden^ Index^ and^ its^ associated^ cutoff^ point.^ Biom^ J.^ 2005^ Aug;47(4):458-72.^doi:^10.1002/bimj.200410135.^PMID:^1616180 4.^ The^Youden-determined^ cut-offs^were^ adjusted^ to^prioritise^sensitivity^ (reducing^ false^ negatives)^over^specificity^(increasing^false^positive),^yie lding^threshold^probabilities^of^^ the^TEDI^score^required^ for^correct^high-grade^patient^classification,^with^ the^greatest^ accuracy.^These^adjusted^cut^off^values^represent^the^values ^at^which^a^test^using^the^ TEDI^indices^would^be^classified^as^positive^or^negative.^ ^ ^ ^ ^ ^ Manual^Gating^ ^ The^FlowJo^software^(BD)^was^used^to^manually^gate^the^compu tationally^resolved^T^^ cell^clusters^of^interest^(those^significantly^enriched^in^H G^or^LG^samples^after^using^the^ mean^frequency^of^each^patient’s^samples).^Figure^8^This^s hows^the^person^skilled^in^ the^art^could^bypass^computational^analysis^to^identify^seve ral^activated/exhausted^and^ naïve/resting^ T^ cell^ clusters^ manually^ using^ compensated^ FCS^ (Flow^ Cytometry^ Standard)^files^in^FlowJo^software.^^In^this^Example,^this^s tep^is^not^only^necessary^to^ validate^output^of^the^unsupervised^clustering^analysis^but^ also^to^ensure^that^equivalent^ results^can^be^generated^with^ a^minimal^panel^of^ antibodies.^ This^demonstrates^ that^ standard^ methodologies^ can^ be^ used^ that^ do^ not^ require^ computational^ expertise,^ development^of^a^TEDI^or^other^similar^forms^of^statistical^ indexing/classification.^This^^ means^that^a^subject’s^population^of^T^cells^can^be^analys ed^and^where^the^ratio^of^that^ subject’s^ T^ cells^ show^ more^ activated^ and/or^ exhausted^ T^ cell’s^ compared^ to^ naïve/resting^T^cells^based^on^detection^of^a^combination^o f^biomarker^expression,^a^ subject^can^be^deemed^at^risk^of^having^a^progressing^or^hig h-grade^pre-invasive^lesion^ or^nodule^or^having^a^solid^tumour.^A^subject^can^then^be^ad ministered^to^further^testing,^^ thereby^simplifying^clinical^implementation.^Such^standard^m ethodology^can^be^applied^ to^a^point^of^care^device^ (not^ shown)^where^a^subject^applies^a^blood^sample^ to^ the^ device^containing^a^panel^of^antibody^biomarkers^for^analysi s^with^the^subject’s^T^cells^ and^where^the^combination^of^biomarkers^present^on^the^subje ct’s^T^cells^will^provide^an^ indication^ whether^ the^ subject^ is^ at^ risk^ of^ having^ a^ progressing^ or^ high-grade^ pre-^ invasive^ lesion^or^nodule^or^having^a^solid^ tumour.^ In^preliminary^analysis^of^ the^pre- invasive^data,^the^results^showed^that^a^panel^of^CD45RA,^CC R7,^Ki67,^CD39,^CD57^ and^CD38^biomarkers^can^be^applied^to^manually^gate^the^clus ters^of^CD8^and^CD4^T^ cells^that^were^significantly^different^in^high-grade^or^low -grade^disease.^Figure^8.^ ^ ^ Alternative^manual^gating^methods^using^a^different^panel^of ^biomarkers^are^shown^in^ Figure^24^and^Figure^35,^where^CD8^T^cells^and^CD4^T^cells^( including^T^regulatory^ subsets)^are^analysed^separately.^These^are^used^for^both^th e^pre-invasive^and^the^ ASCENT^data^(see^below).^In^both^cases,^manual^gating^freque ncies^can^be^used^to^ validate^computational^clustering^frequencies^and^may^be^use d^interchangeably.^^^^ ^ Example^2^ ^ The^inventors^performed^further^analysis^of^the^data^obtaine d^in^Example^1^to^determine^ key^ biomarkers^ which^ are^ to^ form^ the^ inventive^ panel^ of^ biomarkers.^ ^ A^ part^ of^ this^^ analysis^used^ the^biomarkers^Ki67^and^CD39^ to^manually^gate^ the^clusters^of^ total^T^ cells^isolated^from^a^blood^sample^using^CD3.^Surprisingly,^ the^expression^of^these^two^ biomarkers^were^ showed^ significantly^ differences^ between^ high-grade^ and^ low-grade^ disease.^ ^The^ inventors^were^able^ to^show^ that^subjects^can^be^separated^ into^high- grade^or^low-grade^disease^based^on^detection^of^Ki67^and^CD 39^expression^on^T^cells.^^^ Figure^1.^This^provides^a^simple^and^powerful^tool^requiring ^detection^of^expression^of^ Ki67^and^CD39.^^Based^on^the^expression^of^Ki67^and^CD39^bio markers,^a^subject^can^ be^identified^as^being^at^risk^for^having^a^progressing^or^h igh-grade^pre-invasive^lesion^ or^nodule^or^having^a^solid^tumour.^The^analysis^is^based^on ^a^subject^having^more^Ki67^ and^CD39^expressing^T^cells^compared^with^the^T^cells^in^whi ch^expression^of^one^or^^ both^of^Ki67^CD39^is^not^detected.^^^ ^ Example^3^ ^ To^assess^ if^ the^methodology^demonstrated^in^Examples^1^and^2^could^be^us ed^ in^a^^ cancer^type^with^a^different^mutational^landscape,^blinded^P BMC^samples^from^patients^ with^benign^and^malignant^renal^masses^were^assessed.^T^cell ^subsets^were^clustered^ and^derived^TEDI^scores^by^deriving^the^ratio^of^exhausted:^ resting^T^cell^clusters.^Figure^ 9.^ The^ results^ show^ an^ increase^ in^ systemic^ T^ cell^ differentiation^ in^malignant^ (n=16^ samples^from^10^patients)^vs^benign^(n=3^samples^from^3^pati ents)^disease.^A)^UMAP^^ of^FlowSOM^defined^T^cell^clusters^from^PBMC^of^all^samples^ stained^with^31^biomarkers^ and^analysed^by^spectral^cytometry.^5000^ live^CD3+^events^per^samples^were^down- sampled^for^analysis.^B)^The^ratio^of^progenitor^vs^exhauste d^CD4^(left),^CD8^(centre)^or^ combined^(right)^T^cell^subsets.^P^values^from^one-tailed,^u npaired^Wilcoxin^test.^Figure^ 9^shows^that^the^ratio^of^activated/exhausted:^naïve/restin g^T^cells^ is^increased^in^the^^ blood^ of^ patients^ with^ renal^ cancer^ vs^ patients^ with^ benign^ disease.^ ^ These^ results^ replicated^ the^ flow^cytometry^ findings^ from^pre-LUSC^data^suggesting^broad^utility^ of^ systemic^T^cell^differentiation^in^multi-cancer^early^detect ion^that^can^be^used^in^all^solid^ tumours^from^a^variety^of^cancers.^ ^ ^ It^is^to^be^understood^that^while^the^invention^has^been^des cribed^in^conjunction^with^ the^detailed^description^thereof,^the^foregoing^description^ is^intended^to^illustrate^and^ not^limit^the^scope^of^the^invention,^which^is^defined^by^th e^scope^of^the^appended^ claims.^Other^aspects,^advantages,^and^modifications^are^wit hin^the^scope^of^the^ following^claims.^ ^ ^ Example^4^ ^ Example^ 4^ was^ performed^ using^ TCRseq.^ The^ methodology^ provided^ below^ is^ an^ example^of^a^method^of^performing^TCRseq.^The^person^skilled ^in^the^art^will^know^that^^ other^methodologies^for^performing^TCRseq^are^available,^suc h^as^is^provided^in^(Rosati^ E,^ Dowds^ CM,^ Liaskou^ E,^ Henriksen^ EKK,^ Karlsen^ TH,^ Franke^ A.^ Overview^ of^ methodologies^ for^ T-cell^ receptor^ repertoire^ analysis.^ BMC^ Biotechnol.^ 2017^ Jul^ 10;17(1):61.^ doi:^ 10.1186/s12896-017-0379-9.^ PMID:^ 28693542;^ PMCID:^ PMC5504616.)^that^may^result^in^different^numerical^values^o btained,^but^which^equate^ to^the^same^biological^results^to^the^Example^provided^herei n.^It^will^be^understood^to^^ the^ person^ skilled^ in^ the^ art^ that^ in^ general,^ the^methodologies^ and^ results^ provided^ herein^in^the^below^Example^are^not^intended^to^limit^the^sc ope^of^the^invention.^^^ ^ Pre-processing^&^sequencing^of^the^TCR^ ^ ^ Blood^samples^were^collected^in^Vacutainer^EDTA^blood^collec tion^tubes^(BD),^PBMCs^ isolated^ by^ gradient^ centrifugation^ (750g^ for^ 10minutes)^ on^ Ficoll^ Paque^ Plus^ (GE^ Healthcare).^The^interface^was^washed^twice^with^complete^RP MI-1640,^resuspended^in^ 90%^FBS^with^10%^DMSO^(Sigma)^and^cryopreserved^ in^ liquid^nitrogen^or^ in^a^ -180^ degrees^Celsius^freezer^system^prior^ to^staining.^To^characterise^T^cell^differentiation^^ profiles^of^HG^vs^LG^samples^cryopreserved^PBMCs^from^were^t hawed^with^warm^R20^ media^ (RPMI^ with^ 20%^ FBS,^ L-glutamine,^ HEPES^ and^ Penicillin/Streptomycin)^ and^ washed^with^R10^media^containing^DNase^I^grade^II^37.5μg/mL ^(Roche,^10104159001).^ TCR^α-chain^and^β-chain^sequencing^was^performed^by^utiliz ing^whole^DNA^extracted^ from^cryopreserved^PBMC^samples^as^above,^using^the^MN^Nucle oSpin^Tissue^kit.^The^^ samples^ were^ sequenced^ using^ immunoSEQ^ from^ Adaptive^ Biotechnologies^ (https://www.immunoseq.com/)^ (deep^ sequencing^ depth).^ The^ assay^ in^ this^ Example^ utilises^ a^ multiplex^ PCR-based^ methodology^ that^ amplifies^ rearranged^ TCR^ CDR3^ sequences^ and^ exploits^ the^ capacity^ of^ high^ throughput^ sequencing^ technology,^ characterises^tens^of^thousands^of^TCRB^CDR3^chains^simultan eously.^ ^ ^ ^ Data^processing^and^analysis^ ^ After^sequencing,^the^raw^files^were^passed^through^our^cust om^analysis^pipeline^in^R,^^ to^generate^the^following^metrics:^TCR^clone^expansion,^D50^ diversity,^clonality^score,^ and^Hill^number^(Figure.^10).^ ^ TCR^clone^expansion,^D50^diversity,^Hill^number^ ^ ^ The^raw^files^were^loaded^into^R^and^analysed^using^the^Immu narch^(v0.6.9)^package.^ TCR^ clone^ expansion^ was^ calculated^ using^ the^ ‘repClonality’^ function^ with^ “homeo”^ specified^ in^ the^command^ line.^This^will^provide^the^proportion^of^TCRs^ in^ the^overall^ repertoire^that^are^defined^as^hyperexpanded^(0.01^<^X^&l t;=^1),^large^(0.001^<^X^<=^0.01),^ medium^(1e-04^<^X^<=^0.001),^small^ (1e-05^<^X^<=^1e-04),^or^ rare^ (0^<^X^<=^1e-05),^ where^X^is^a^clonotype^(Figure^10A-B).^The^‘repDiversity� �^function^was^used^to^calculate^ the^D50^diversity^metric^(with^“d50”^specified^ in^the^command^line)^(Figure^10C),^and^ the^Hill^number^(Figure^10E)^(again,^“hill”^specified^in ^the^command^line).^Hill^numbers^^ are^a^mathematically^unified^family^of^diversity^indices^tha t^differ^among^themselves^only^ by^an^exponent,^q).^q=1^was^set^because^ it^ reaches^maximal^diversity^to^ identify^ the^ number^of^unique^TCR^CDR3B^amino^acid^sequences.^The^D50^is^ a^recently^developed^ immune^diversity^estimate^ that^calculates^ the^minimum^number^of^distinct^ clonotypes^ amounting^ to^ greater^ than^ or^ equal^ to^ 50%^ of^ a^ total^ of^ sequencing^ reads^ obtained^^ following^amplification^and^sequencing.^^ ^ ^ Clonality^score^ ^ ^ All^TCRseq^raw^files^were^imported^into^R^and^filtered^to^on ly^include^productive^TCR^ sequences^(i.e.,^no^failed^sequence^reads).^Next,^the^propor tion^each^unique^clonotype^ holds^within^the^repertoire^was^calculated.^After^this^initi al^QC^step,^1000^TCR^ sequences^were^randomly^subsampled^1000^times,^with^the^clon ality^score^calculated^ for^each^round^of^sampling,^using^the^entropy^(v1.3.1)^packa ge.^The^average^clonality^^ score^across^the^1000^subsamples^was^taken^and^transformed^i nto^a^Z-score,^which^ indicates^how^much^a^given^value^deviates^from^the^mean,^wit hin^a^dataset^(Figure^ 10D).^ ^ ^ ^ TCRseq^metrics^to^predict^HG^vs^LG^disease^ ^ Figure^10^ indicates^there^is^a^significant^difference^in^some^of^these ^TCRseq^metrics^ between^high-^and^low-grade^patients,^possibly^reflective^of ^persistent^TCR^engagement^ from^ a^ chronic^ antigen^burden.^ Receiver^ operator^ characteristic^ (ROC)^ analysis^was^^ completed^to^test^the^diagnostic^potential^of^the^different^ metrics^using^the^‘roc’^function^ from^ the^pROC^(v1.18.0)^ package^(Figure^11).^All^ possible^sensitivity^ and^specificity^ values^with^their^corresponding^threshold^probabilities^were ^calculated,^as^ROC^curves^ represent^a^trade-off^between^the^true^and^false^positive^ra tes.^The^threshold^probability^ that^yielded^the^highest^combined^sensitivity^and^specificit y^was^selected,^known^as^the^^ Youden^ Index,^ a^ commonly^ used^method^ to^ estimate^ the^ optimal^ cut-off^ point^ for^ a^ diagnostic^ test.^The^Youden-determined^cut-offs^were^adjusted^ to^prioritise^sensitivity^ (reducing^false^negatives)^over^specificity^(increasing^fals e^positive),^yielding^threshold^ probabilities^of^ the^TCR^metrics^ required^ for^correct^High^grade^patient^classification.^ These^adjusted^cut^off^values^represent^the^values^at^which^ a^test^using^the^TCR^metrics^ would^be^classified^as^positive^or^negative.^As^the^D50^had^ the^most^significant^difference^ between^ high-^ and^ low-grade^ disease^ (Figure^ 10C)^ and^ has^ the^ greatest^ diagnostic^ power^(Figure^11),^this^metric^has^the^highest^potential.^^ ^ ^ Example^4^demonstrates^that^the^state^of^the^T^cell^repertoi re^in^the^blood^can^be^used^ as^a^means^to^determine^if^an^individual^is^harbouring^a^pro gressing^or^high-grade^pre- invasive^ lesion,^ nodule^ or^ small^ mass,^ or^ having^ a^ solid^ malignant^ tumour.^ In^ this^ Example,^ a^ score^ beyond^ a^ given^ number^ indicates^ a^ chronic^ and^ ongoing^ immune^ challenge.^It^has^shown^that^multiple^TCRseq^metrics^have^th e^potential^to^be^used^as^a^^ diagnostic^predictor.^These^include:^^ ^ i)^ D50^(AUC^=^88.4%)^ ii)^ Hill^ number^ (AUC^ =^ 86%)^ (also^ known^ as^ “entropy^ score”,^ Shannon^ index^ or^ Shannon^entropy^score)^ ^ ^ In^ this^ Example,^ patients^ with^ advanced^ preinvasive^ lesions^ of^ the^ lung^ (likely^ to^ progress)^vs^those^with^low^grade^pre-invasive^lesions^of^th e^lung^(unlikely^to^progress),^ are^more^likely^to^fall^below^the^cut-off^values^list^above^ for^these^metrics^listed:^D50^and^ Hill^number.^Whilst^utility^of^ these^TCRseq^metrics^ is^here^shown^ in^ lung^cancer,^ the^^ underlying^ principle^ of^ blood^ immune^ activation^ linked^ to^ an^ underlying^ preinvasive^ nodule/lesion^is^broad^and^encompasses^all^solid^cancers,^an d^therefore^the^utility^of^this^ test^for^early^detection^is^pan-cancer.^ ^ Example^5^ ^ ^ The^inventors^combined^T^cell^flow^cytometry^data^and^TCRseq ^data^from^the^cohort^of^ 30^ patients^ with^ high^ or^ low^ grade^ pre-LUSC^ as^ described^ above.^ Analysis^ was^ performed^in^each^platform^as^described^above^ then^combined^multiple^metrics^ listed^ below^from^each^method,^using^the^XGBoost^model,^via^an^open -source^package^in^the^^ programming^language^R.^The^XGBoost^package^can^be^downloade d^at^^ ^ https://cran.r-project.org/web/packages/xgboost/index.html^ ^ ^ ^ XGBoost^ is^shorthand^ for^Extreme^Gradient^Boosting,^which^ is^a^scalable,^distributed^ gradient-boosted^ decision^ tree^ (GBDT)^ machine^ learning^ library.^ XGBoost^ provides^ parallel^ tree^ boosting^ and^ is^ the^ leading^ machine^ learning^ library^ for^ regression,^ classification,^and^ranking^problems.^ ^ ML/AI^analysis^workflow^^ ^ ^ Combining^data^involves^adding^values^from^each^set^of^analy sis^into^a^machine^learning^ library.^70%^of^the^data^was^used^to^train^the^model,^wherei n^the^library^computes^which^ combination^of^metrics^ from^each^analysis^ best^ classifies^ group^ 1^ vs^ group^2^ via^an^ iterative^process^using^decision^ trees,^ in^which^ the^best^decision^ is^boosted^ until^ the^^ model^is^optimised.^These^2^groups^could^be^patients^with^vs ^without^cancer^or^in^the^ present^examples^patients^with^ low^vs^high^grade^preinvasive^neoplasia.^^The^trained^ model^is^then^applied^to^the^remaining^(‘unseen’)^30%^of ^the^data^to^test^that^it^works^on^ a^different^set^of^samples.^The^steps^are^outlined^in^Figure ^13.^^^ ^ ^ Input^data^ All^metrics^were^added^from^the^flow^cytometry^(e.g.,^as^des cribed^in^Examples^1^and^2)^ and^ TCRseq^ analysis^ (e.g.,^ from^ Example^ 4)^ to^ train^ the^ machine^ learning^ model.^ TCRseq^variables^included:^D50^diversity^metric,^Hill,^and^T CR^expansion^metrics^(i.e.,^ small^(1e-05^<^X^<=^1e-04),^medium^(1e-04^<^X^<= ^0.001),^large^(0.001^<^X^<=^0.01),^^ hyperexpanded^(0.01^<^X^<=^1)^expansion^proportions).^ CD4^and^CD8^TEDI^scores^were^ derived^from^the^flow^cytometry^metrics^as^described^above.^ ^^ ^ Data^processing^ All^analysis^was^completed^in^R^(v1.4.1106).^The^data^was^pa rtitioned^into^a^training^and^^ a^test^data^set^with^a^70/30^split,^using^the^‘createDataP artition’^function^from^the^caret^ package^(v6.0-92).^The^inventors^ensured^the^outcome^ratio^( i.e.,^the^high^to^low^grade^ ratio)^was^maintained^in^both^data^sets.^Outcome^was^defined ^as^a^high/low^grade^(1/0)^ lesion^for^each^patient.^^ ^ ^ Hyperparameter^tuning^ To^select^the^optimum^values^for^the^hyperparameters^to^yiel d^best^model^accuracy,^a^ random^ search^ method^ was^ completed,^ where^ 10000^ iterations^ of^ the^ model^ are^ generated^with^different^values^for^each^of^the^defined^hype rparameters^(maximum^tree^ depth:^2-6;^eta:^0.01-0.3;^subsample:^0.7-1;^subsample^ratio ^of^columns:^0.6-1;^cover:^0-^ 10).^We^selected^the^hyperparameter^values^which^yielded^the ^greatest^model^accuracy^ for^the^final,^tuned^model.^The^final^model^was^trained^usin g^the^‘xgb.train’^function^from^ the^xgboost^(v1.6.0.1)^package.^ ^ Feature^importance^ ^ The^ ‘xgb.importance’^ function^was^used^ to^determine^ the^ relative^ importance^of^each^ variable^within^the^overall^model.^Any^variables^with^a^feat ure^importance^score^<0.05^ were^excluded.^^ ^ Results^ ^ ^ Figure^3^ shows^ that^ the^ flow^cytometry^defined^CD4^TEDI^ (AUC=94.2%),^CD8^TEDI^ (AUC=92.6%)^are^strong^classifiers^of^patients^with^high^vs^ low^grade^pre-LUSC.^Figure^ 10^ also^ shows^ that^ the^ TCRseq^ defined^measures,^ including^ the^ D50^ index^ (AUC=^ 88.4%)^have^discriminatory^ power^ to^specifically^detect^ patients^with^high^grade^pre-^ LUSC.^The^drawing^then^shows^that^combining^flow^cytometry^a nd^TCRseq^metrics^in^ the^XGBoost^package^yields^improved^classification^of^high-g rade^vs^low-grade^disease^ (AUC=100%).^This^demonstrates^that^combining^multiple^differ ent^metrics^from^T^cell^ analyses^in^the^blood^can^generate^a^powerful^novel^tool^tha t^detects^preinvasive^lesions^ which^are^likely^to^progress^to^lung^cancer.^In^principal^th is^could^be^applied^to^other^solid^^ cancer^types.^^ ^ In^more^detail^the^figures^show^that^there^are^multiple^CD4^ and^CD8^T^cell^phenotypes^ in^ the^ blood^of^ patients^with^preinvasive^ lung^disease,^Figure^3A.^Patients^with^high^ grade^disease^have^more^activated^and^exhausted^CD8^and^CD4^ T^cells,^and^ fewer^^ naïve^and^resting^CD4^and^CD8^T^cells,^Figure^3C.^This^can^ be^converted^into^ratio(s)^ which^divides^these^patient^populations,^Figure^3D.^In^addit ion,^TCRseq^analysis^shows^ that^patients^with^high^grade^disease^have^ larger^ clonal^T^cell^expansions^and^ fewer^ small^T^cell^expansions^and^lower^TCR^diversity,^Figures^10A ,^10B^and^10C.^Finally,^ that^these^can^be^combined^in^the^XGBoost^model^with^superio r^performance^to^metrics^^ from^either^assay^alone,^where^the^top^ranking^features^of^e ach^assay^are^shown^to^be^ the^CD4^TEDI^(Flow^cytometry)^and^D50^index^(TCRseq),^Figure s^12A,^12B^and^12C.^^^ ^ ^ Figure^12A^demonstrates^that^both^cytometry^and^TCRseq^appro aches^can^be^used^to^^ effectively^distinguish^subjects^into^high-grade^and^low-gra de^disease.^These^techniques^ can^also^be^combined^together,^incorporating^multiple^metric s^from^these^techniques^into^ a^single^analysis,^to^generate^data^that^further^improves^di stinguishing^subjects^into^high- grade^and^low-grade^disease,^as^demonstrated^with^ROC^analys is^Figure^12B-C.^^This^ is^demonstrated^by^the^AUC^increasing^from^88%^with^TCRseq^a nalysis^alone^to^100%^ with^TCRseq^analysis^+^flow^cytometry^analysis,^see^Figure^1 2.^^AUC^of^flow^cytometry^ analysis^alone^is^93-94%^but^adding^TCRseq^in^a^combined^mod el^shows^100%.^This^is^^ the^comparison^between^Figure^12a^and^Figure^12c.^Therefore^ Figure^12^shows^for^ the^first^time^that^combining^different^measures^of^blood^T^ cell^differentiation^results^in^a^ stronger^blood^test^than^using^each^alone.^This^would^ lead^to^an^improved^method^of^ detecting^cancer^or^nodules^or^lesions^or^masses^that^lead^t o^cancer^compared^to^a^test^ that^used^either^technology^alone.^ ^ ^ Further^Exemplification^–^Detecting^Biomarkers^Expression^ of^T-cells^in^the^blood^using^ flow^cytometry^ ^ ASCENT^ ^ ^ ASCENT^is^an^observational^study^of^patients^with^screen-det ected^lung^cancer^through^ the^SUMMIT^longitudinal^surveillance^study^via^LDCT^scans.^B lood^(and^tissue)^samples^ are^collected^from^surgery^at^the^point^of^resection.^For^th e^ASCENT^flow^analysis,^86^ patients^ were^ included,^ of^ which^ there^ were^ 57^ NSCLC^ (35^ LUAD^ (Lung^^ adenocarcinoma)^+^22^LUSC^(Lung^squamous^cell^carcinoma)^and ^29^Healthy.^Healthy^ age-matched^ blood^ samples^ were^ collected^ from^ patients^ going^ in^ for^ orthopaedic^ surgery,^with^no^active^infections^or^auto-immune^conditions .^~90%^of^NSCLC^samples^ used^ in^ASCENT^analysis^are^Stage^ I^ (51^Stage^ I,^ 4^Stage^ II,^ 2^Stage^ III).^ The^ flow^ cytometry^panel^used^for^this^data-set^is^shown^in^Figure^25 .^Samples^were^acquired^on^^ a^ID7000^Spectral^Cell^Analyser^from^Sony^Biotechnology.^ ^ Example^7^ The^findings^relating^to^skewing^of^T^cell^differentiation^ in^the^blood^from^pre-invasive^ patients^were^validated^ in^a^ larger^cohort^of^patients^with^early-stage^(mainly^stage^I), ^^ established^non-small^cell^lung^cancer^(NSCLC).^ ^ As^shown^by^the^bottom^of^Figure^14,^in^early^stage^establis hed^disease,^a^higher^ratio^ of^activated:^resting^T^cells^is^also^observed^as^we^did^in^ pre-invasive^disease.^This^is^ demonstrated^ by^ the^ TEDI^CD39^Ki67^ (CD3),^where^ NSCLC^patients^ have^ a^ higher^^ proportion^of^CD3^CD39+Ki67+^cells^ than^healthy.^A^similar^plot^with^healthy^patients^ versus^LUSC^gave^a^p-value^of^0.14.^ ^ As^shown^in^Figures^15A^and^15B,^when^combining^pre-invasive ^and^NSCLC^data,^we^ also^see^an^ increase^ in^the^TEDI^CD39^Ki67^ ratio^ from^healthy^ to^ low-grade^ to^high- grade.^The^signal^peaks^at^high-grade^and^dropping^off^at^th e^lung^cancer^stage.^This^^ suggests^ the^ high-grade^ pre-invasive^ stage^ has^ the^ potential^ to^ be^ the^ most^ immunogenic^and^is^therefore^the^optimum^time^to^detect^mali gnancy^in^the^blood.^^ ^ As^well^as^lower^frequency^of^the^biomarkers^CD39^and^Ki67^i n^T^cells^in^lung^cancer^ patients,^we^also^observe^a^trend^of^lower^frequencies^of^na ïve^CD4+^T^cells^in^NSCLC^^ patients^compared^to^healthy^(p=0.1),^similar^to^what^was^pr eviously^observed^in^the^pre- invasive^setting^(see^Figure^16).^^When^combining^healthy^an d^low^grade^(n^=^66)^and^ combining^high^grade^and^HSCLC^(n=88),^there^is^still^a^sign ificantly^higher^CD3^TEDI^ score^ in^ the^ disease/high-risk^ group^ compared^ with^ the^ healthy/low-risk^ group^ (see^ Figure^17)^coupled^with^a^decrease^in^naïve^cells^in^the^CD 4^T^cell^compartment.^^^^ ^ Example^8^ ^ ^ In^further^analysis^of^the^pre-invasive^flow^cytometry^data, ^the^present^inventors^noted^^ the^ importance^of^ CD4+^ regulatory^ T^ cells^ (Tregs)^ expressing^ the^ biomarker^ FoxP3.^ Proliferating^Tregs^may^also^express^the^activation/prolifer ation^markers^CD39^and^Ki67.^ In^both^pre-invasive^and^ early-stage^ lung^ cancer,^ there^was^ a^significant^ increase^ in^ CD39+Ki67+^ Tregs^ in^ high-grade/early-stage^ lung^ cancer^ when^ compared^ with^ low- grade/healthy^ respectively.^ This^ is^ in^ accordance^ with^ more^ activated/memory^ cells^^ supporting^ the^ finding^ that^ T^ cell^ differentiation^ is^ skewed^ during^ pre-invasive^ and^ invasive^lung^cancer,^wherein^patients^having^a^higher^risk^ of^having^a^progressing/high- grade^ lesion/nodule/tumour^ to^ have^ higher^ frequencies^ of^ more^ activated/effort/regulatory^T^cells.^This^data^is^shown^in^F igure^18.^When^combining^this^ data^together,^there^is^an^increase^from^healthy^to^low^grad e^and^to^high^grade,^with^the^^ signal^from^activated/proliferating^Tregs^peaking^in^high^gr ade^pre-invasive^samples^(see^ Figure^19).^When^combining^healthy^and^low^grade^(n^=^66)^an d^combining^high^grade^ and^HSCLC^(n=88),^there^is^still^a^significantly^higher^freq uency^of^total^Tregs,^CD39+^ Tregs^and^CD39+Ki67+^T^regs^(see^Figure^17).^ ^ ^ Tregs^ (i.e.,^ CD4+^ T^ cells^ expressing^ the^ biomarker^ FoxP3),^ further^ expressing^ the^ biomarker^CD39^(CD39+)^ in^the^absence^of^ the^biomarker^CD45RA^(CD45RA-)^were^ also^ significantly^ enriched^ in^CD4+^ cells^ in^ high-grade/early-stage^ lung^ cancer^ when^ compared^with^low-grade/healthy^respectively^(see^Figure^36) ,^also^peaking^in^high^pre- invasive^samples.^^ ^ Example^9^^ ^ Another^ subpopulation^ of^ T^ cell,^ i.e.,^ Tem.Prolif.CD39hi^ cells,^ where^ Prolif^ =^ Ki67+,^ expressing^ both^ CD39+^ and^ Ki67+^ were^ also^ significantly^ enriched^ in^ high-grade^ samples^in^live^CD8^Cells^(CD3+CD8+^cells).^These^cells^cont ain^the^presence^of^CD39^ and^Ki67^biomarkers,^as^well^as^having^negative^expression^o f^the^biomarkers^CD45RA,^ CCR7^and^PD1.^^ ^ ^ For^the^pre-invasive^(PID)^data,^flow^cytometry^analysis^is^ of^68^samples^(31^high^grade,^ 37^low^grade)^which^passed^the^QC^for^having^>^3000^Live^ CD3+^and^CD8+^cells^used^ for^analysis.^Figure^20^shows^a^volcano^plot^of^populations^ determined^by^manual^gating^ on^ flow^cytometry^that^are^significantly^enriched^ in^high^grade^pre-invasive^ lesions^or^^ nodules^ or^ low^ grade^ pre-invasive^ lesions^ or^ nodules.^ ^ Significant^ enrichment^ of^ the^ Tem.Prolif.CD39hi^cells^(prolif^=^Ki67+)^was^observed^in^hig h^grade^pre-invasive^lesions^ or^nodules.^Figure^21^shows^box^plots^for^Top)^naïve^T^cell s^and^Bottom)^the^above- mentioned^Tem.Prolif.CD39hi^populations^(right)^at^sample^le vel.^^ ^ ^ For^the^ASCENT^data,^the^flow^cytometry^analysis^is^of^49^sa mples^(27^Healthy^and^22^ LUSC)^ which^ passed^ the^QC^ for^ having^ >1500^ Live^ CD3+CD8+^ cells^were^ used^ for^ analysis.^^Figure^22^(Top)^shows^a^volcano^plot^of^populatio ns^determined^by^manual^ gating^that^are^significantly^enriched^in^LUSC.^Figure^22^(B ottom)^shows^a^box^plot^for^ the^ Tem.Prolif.CD39hi^ population^ which^ is^ significantly^ enriched^ in^ LUSC^ (Lung^^ squamous^cell^carcinoma).^^ Figure^23^shows^the^combined^box^plot^values^from^both^the^P ID^(pre-invasive^data)^and^ ASCENT^flow^cytometry^analysis^to^display^the^change^in^Tem. Prolif.CD39hi^population.^^ Figure^24^shows^the^flow^cytometry^gating^strategy^for^both^ PID^(pre-invasive^data)^and^ ASCENT^manual^gating^used^in^this^particular^Example.^^ ^ ^ Further^ Exemplification^ –Determining^ the^ diversity^ (i.e.,^ using^ diversity^ and^ clonality^ metrics)^of^T-cells^in^the^blood^using^TCR-seq^ ^ Example^10^ The^findings^relating^to^the^remodelling^of^the^T^cell^recep tor^repertoire^in^the^blood^from^ pre-invasive^ patients^ were^ validated^ in^ a^ larger^ cohort^ of^ patients^ with^ early-stage,^ established^non-small^cell^lung^cancer^(NSCLC)^to^discern^pa tients^with^progressive^pre- invasive^disease^or^early-stage^invasive^neoplasia^(Stage^1^ NSCLC).^ ^ As^described^above,^ASCENT^is^an^observational^study^of^pati ents^with^screen-detected^ lung^cancer^through^the^SUMMIT^longitudinal^surveillance^stu dy^via^LDCT^scans.^Blood^ (and^tissue)^samples^are^collected^from^surgery^at^the^point ^of^resection.^^ ^ For^ASCENT^TCR-seq^analysis,^75^patients^were^included:^57^N SCLC^(35^LUAD^+^22^^ LUSC),^and^18^healthy^subjects.^Healthy^age-matched^blood^sa mples^were^collected^ from^ patients^ undergoing^ orthopaedic^ surgery,^ with^ exclusions^ to^ ensure^ no^ active^ infections^ or^ auto-immune^ conditions.^ ~90%^ of^ NSCLC^ samples^ used^ in^ ASCENT^ analysis^are^rarely^sampled^very^early^Stage^I^tumours^(51^S tage^I,^4^Stage^II,^2^Stage^ III).^ ^ ^ This^validation^cohort^was^analysed^as^before.^For^TCRseq^DN A^was^extracted^ from^ peripheral^ blood^ samples^ and^ sequenced^ using^ the^ same^ platform^ (Adaptive^ ImmunoSeq),^and^pre-processed^and^analysed^using^Immunarch^p ackage^on^R.^ ^ ^ In^further^analysis^of^pre-invasive^data,^a^trend^in^D50^div ersity^index^(higher^score^in^ low-grade^samples)^and^clonality^(higher^score^in^high-grade ^samples)^was^observed.^ Looking^at^the^same^metrics^in^ASCENT^(established^very^earl y-stage^lung^cancer),^the^ same^trends^were^observed^when^comparing^healthy^to^NSCLC^(s ee^Figure^26)^ ^ ^ Other^metrics^measuring^diversity^and^clonality^of^the^reper toires^also^showed^the^same^ trends^as^ the^pre-invasive^analysis^ including^D50^and^ the^proportion^of^ the^repertoire^ occupied^by^the^top^100^clones^as^a^proxy^for^clonality,^wit h^even^stronger^results.^The^ Hill^score^(aka^entropy^score)^and^the^proportion^of^the^rep ertoire^occupied^by^the^top^ 100^clones^further^show^the^same^differences^between^low-^vs ^high-grade,^and^healthy^^ vs^NSCLC^(see^Figure^27).^ ^ Looking^at^ the^ proportion^of^ the^ repertoire^occupied^by^ small^ (<0.01%^of^ repertoire),^ medium^(0.01-0.1%^of^repertoire),^large^(0.1-1%^of^repertoir e)^or^hyperexpanded^(>1%^ of^repertoire)^clones,^an^increase^in^large^clones^was^obser ved^present^in^the^blood^of^^ high-grade/progressive^ pre-invasive^ patients^ and^ established^ early-stage^ invasive^ neoplasia,^coupled^with^a^decrease^in^small^clones^ in^ low-grade/non-progressive^pre- invasive^patients^and^healthy^volunteer^samples.^This^demons trated^that^an^increased^ proportion^of^large^clones^occupying^the^subject’s^periphe ral^TCR^repertoire^can^also^be^ used^as^an^indicator^of^higher^risk^of^progressing^into^lung ^cancer,^and/or^the^presence^ of^a^progressive^lesion/nodule^or^early-stage^tumour^(see^Fi gure^28)^ ^ Similar^to^the^phenotypic^“TEDI^”^ratio^used^in^the^ flow^cytometry^data^representing^a^ shift^in^differentiation^skewing^in^the^blood,^a^ratio^of^la rge:^small^clones^occupying^the^ repertoire^ can^ be^ used^ to^ distinguish^ healthy^ vs^ pre-invasive^ disease^ vs^ early-stage^ NSCLC,^ with^ a^ new^ metric^ of^ clonal^ expansion.^ This^ signal^ can^ be^ tracked^ as^ carcinogenesis^develops,^with^the^ratio^signal^peaking^at^hi gh-grade/progressive^lesions.^^ This^demonstrates^the^technique’s^potential^to^be^used^in^ early^detection^of^cancer^(i.e.,^ before^cancer^formally^develops)^(see^Figure^29).^ ^ The^ same^ trends^were^also^ observed^when^ this^ novel^ analysis^was^ performed^ on^ a^ publicly^available^large^scale^data^set^acquired^via^the^sam e^wet^lab^and^bioinformatic^^ platform^comparing^healthy^subjects^and^NSCLC^patients^(see^ Figures^30^and^31).^^ ^ Combining^healthy^+^ low-grade^(no^tumour^and^low^risk^of^developing^a^ tumour),^and^ high-grade^ +^ NSCLC^ (high^ risk^ or^ with^ early^ stage^ cancer)^ therefore^ allows^ for^ a^ comparison^of^no/low^risk^of^developing^or^currently^having^ lung^cancer^to^high^risk^of^^ developing^or^currently^having^lung^cancer^in^situ.^This^com parison^aids^the^stratification^ of^ the^general^population^using^peripheral^blood^ for^bulk^TCR-seq.^The^ trends^ in^ the^ output^ metrics^ described^ above^ in^ each^ cohort^ remain^ the^ same^ when^ combined.^ Generally,^ the^ repertoires^ of^ higher^ risk^ (HG+NSCLC)^ patients^ are^more^ clonal^ and^ expanded^(higher^clonality^index,^higher^proportion^of^the^r epertoire^occupied^by^top^100^^ clones,^ higher^ large:small^expansion^ ratio),^ and^ less^diverse^ (lower^D50^ index,^ lower^ entropy^ score/hill^ number)^ than^ the^ repertoires^ of^ lower^ risk^ (Healthy+LG)^ patients.^ Notably,^ the^ large:small^ expansion^ ratio^ (which^ calculates^ a^ ratio^ similar^ to^ the^ phenotypic^ratio^of^exhausted/activated^T^cells^to^naïve/re sting^cells)^was^ found^to^be^ the^most^significant^metric^to^distinguish^these^two^groups^ (see^Figure^32).^ ^ ^ Further^ Exemplification^ –^ Combining^ Flow^ Cytometry^ and^ TCR-seq^ in^ a^ multi-omic^ approach.^ ^ The^ following^ findings^provide^further^evidence^of^using^a^multi-omic^mode l^using^the^^ combination^of^the^updated^T^cell^phenotype^and^TCR-seq^sign als^in^the^blood^for^early^ detection^of^cancer.^^ Example^11^ ^ Using^follow-up^time^available^from^the^pre-invasive^cohort, ^we^used^a^combination^of^ the^ flow^ cytometry^ phenotypic^ signal^ which^ uses^ some^ of^ our^ key^ biomarkers^ to^^ differentiate^between^sample^types^(e.g.,^FoxP3,^CD39+^to^id entify^effector^Treg^cells)^ and^the^ratio^of^large:small^clones^from^the^TCR-seq^analysi s^to^predict^the^probability^ of^a^patient^receiving^a^lung^cancer^diagnosis.^A^patient’ s^effector^Treg^CD39+^frequency^ is^multiplied^by^their^large:small^TCR^ratio^and^all^patient s^(low-^and^high-grade)^are^split^ by^their^median^multiplied^score.^^ ^ ^ Kaplan-Meier^analysis^of^this^combined^metric^from^flow^cyto metry^and^TCR-seq^shows^ a^ significant^ difference^ when^ future^ lung^ cancer^ diagnosis^ is^ used^ as^ outcome,^ demonstrating^the^predictive/diagnostic^potential^when^these ^two^techniques^(i.e.,^TCR- seq^and^flow^cytometry)^are^used^in^combination.^It^is^also^ important^to^note^that^using^^ both^metrics^in^combination^give^stronger^predictive^potenti al^than^using^either^the^flow^ cytometry^data^or^TCR^metrics^alone.^After^accounting^for^cl inical^data,^this^combined^ flow/TCR-seq^metric^is^able^to^predict^a^future^lung^cancer^ diagnosis,^further^highlighting^ the^diagnostic^potential^of^the^mentioned^peripheral^immune^ metrics^(see^Figure^33).^^ ^ ^ We^ updated^ the^ prior-mentioned^ machine^ learning^ model^ XGBoost,^ including^ the^ different^ TCR-seq^ metrics^ (proportion^ of^ repertoire^ occupied^ by^ top^ 100^ clones,^ hill^ number,^small^clones,^hyperexpanded^clones),^flow^cytometry^ metrics^(FoxP3^vs^CD39^ manually^gated^quadrant^frequencies^on^total^CD4,^i.e.,^FoxP 3-CD39-,^FoxP3-CD39+,^ FoxP3+CD39-,^ FoxP3+CD39+),^ and^ standard^ clinical^ metrics^ (age,^ gender,^ smoking^^ status).^ The^ model^ was^ now^ trained^ on^ 70%^ of^ the^ ASCENT^ cohort,^ tested^ in^ the^ remaining^30%^of^ASCENT,^and^then^validated^on^31^patients^f rom^PID.^(Previously^the^ model^was^trained^and^tested^on^22^PID^patients,^but^with^no ^validation^cohort).^Test^ AUC^in^ASCENT^was^100%,^and^validation^AUC^in^PID^was^76%.^A s^demonstrated^by^ ROC^analysis,^the^combination^of^these^techniques^alongside^ standard^clinical^metrics^^ into^a^single^analysis^show^great^promise^in^distinguishing^ low-^from^high-grade^subjects.^ The^relative^ importance^of^each^metric^used^(as^calculated^by^the^feature ^ importance^ score)^shows^that^peripheral^ immune^metrics^that^contribute^more^towards^ the^model^ than^clinical^variables^such^as^age,^which^is^already^common ly^and^strongly^associated^ with^lung^cancer^development^(see^Figure^34).^ ^ ^ Materials^and^Methods^ ^ High-dimensional^clustering^ Clustering^ of^ flow^ cytometry^ data^ was^ completed^ following^ a^ modified^ pipeline^ from^ Nowicka^ et^ al^ [https://f1000research.com/articles/6-748].^ FCS^ files^ underwent^ quality^^ control^ of^ signal^ acquisition,^ assessed^ by^ the^ FlowAI^ package^ (v1.24)^ [https://academic.oup.com/bioinformatics/article/32/16/2473/ 2240408?login=false].^ The^ arcsinh^ transformation^was^applied^ to^ the^data,^using^ the^prepData^ function^ from^ the^ CATALYST^package^(v1.18.1)^^ [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981006/],^wit h^ the^cofactor^set^ to^150^^ for^ standard^ fluorescence^ flow^ cytometry^ (PID^ cohort)^ and^ to^ 550^ for^ spectral^ flow^ cytometry^ (ASCENT^ cohort).^ Any^ markers^ with^ a^ poor^ contribution^ to^ phenotypic^ variance^were^excluded^pre-clustering.^These^were^determined ^using^the^PCA-based^ non-redundancy^ score^ (NRS)^ as^ described^ in^ the^ Nowicka^ et^ al^ [https://f1000research.com/articles/6-748].^ pipeline.^ CD103,^ Tim-3,^ TCF-7,^ CXCR4,^^ TIGIT,^and^CD4^were^excluded^from^clustering^due^to^poor^mar ker^separation^in^the^PID^ cohort,^as^well^as^the^ASCENT^cohort^for^consistency^between ^panels^(CD4^excluded^ as^FCS^files^only^included^CD4 ⁺ ^T^cells).^^ ^ Data^ was^ clustered^ using^ the^ FlowSOM^ package^ (v2.2.0)^^ [https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.226 25]^onto^a^10x10^node^square^ self-organising^ map^ (SOM).^ Nodes^ were^ clustered^ by^ the^ ConsensusCenterPlus^ package^ (v1.58.0)^ [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881355/],^ as^ described^ by^ Nowicka^ et^ al^ [https://f1000research.com/articles/6-748].^ The^ UMAP^ algorithm^was^applied^as^a^dimension^reduction^analysis^to^c haracterise^the^phenotypic^^ relevance^ between^ individual^ clusters.^ The^ resulting^ clusters^ were^manually^ merged^ based^on^marker^expression^similarity,^occupation^of^space^o n^the^UMAP,^and^previously^ defined^T^cell^states.^ ^ Manual^gating^ ^ Subsets^ resolved^ from^ high-dimensional^ clustering^ were^ manually^ identified^ by^ conventional^biaxial^gating^to^ensure^validity^of^clusters.^ PBMCs^with^³^x^viable^T^cells^ were^analysed^(PID^cohort^³^5000^CD4;^ASCENT^cohort^³^4000 ^CD4).^Manual^gating^ analysis^ was^ carried^ out^ on^ FlowJo^ v10.8.1.^ Populations^ gated^ are^ shown^ on^ a^ concatenated^file^of^all^samples^from^one^batch.^Frequencies ^from^total^CD4^can^be^used^^ to^validate^cluster^significance^from^the^high-dimensional^c lustering^pipeline^analysis^ ^ TCR^sequencing^ Sequencing^ of^ the^ CDR3^ regions^ of^ PBMC^ TCR-β^ chains^ was^ performed^ using^ the^ immunoSEQ®^Assay^(Adaptive^Biotechnologies,^Seattle,^WA)^as ^previously^described^ [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418738/,^ ^ https://www.nature.com/articles/s41467-019-14273-0#Sec12].^ In^ brief,^ extracted^ genomic^ DNA^ was^ amplified^ in^ a^ multiplex^ PCR,^ followed^ by^ high-throughput^ sequencing.^ Raw^ files^ were^ processed^ using^ the^ MixCR^ pipeline^ [https://www.nature.com/articles/nmeth.3364]^and^exported^as ^tsv^files.^^ ^ ^ Curation^of^publicly^available^TCRseq^dataset^ Publicly^ available^ data^ from^ the^ immuneACCESS^ portal^ (Adaptive^ Biotechnologies,^ Seattle,^WA)^was^ leveraged^ to^ curate^ an^additional^ validation^ dataset^ that^ had^been^ sequenced^on^the^sample^platform^as^the^PID^and^ASCENT^sampl es.^Healthy^controls^ were^ obtained^ from^ the^ immunoSEQ^ hsTCRB-V4b^ Control^ Data^^ [https://doi.org/10.21417/ADPT2020V4CD].^The^non-small^cell^ lung^cancer^cohort^was^ taken^from^Reuben^et^al.^[https://www.nature.com/articles/s4 1467-019-14273-0#Sec2].^^ ^ Analysis^of^TCR^sequencing^data^ Raw^ tsv^ files^ were^ processed^ to^ include^ only^ productive^ sequences.^ To^ pass^ QC,^^ repertoires^ required^ a^ minimum^ of^ 1000^ unique^ clones^ and^ <3^ counts^ per^ CDR3^ sequence^to^limit^the^effect^of^naïve^cells^and^sequencing^ artefacts.^^ ^ The^ ‘repClonality’^ function^ was^ used^ to^ calculate^ clonal^ expansion^ metrics^ from^ the^ immunarch^ package^ (v0.6.9)^ [https://zenodo.org/record/3367201].^ To^ calculate^ the^^ Large:Small^ratio,^the^proportion^of^clones^deemed^as^“lar ge”^(1%<^repertoire^>0.1%)^ was^divided^by^ the^proportion^of^ clones^ deemed^as^ “small”^ (<0.001%).^ The^diversity^ metrics^D50^and^Entropy/Hill^score^were^calculated^using^the ^‘repDiversity’^function^from^ the^same^package,^with^the^method^set^to^“d50”^and^“hi ll”,^respectively.^For^the^Entropy^ score,^Q=1^was^selected.^^ ^ ^ The^Renyi^entropy^is^a^generalised^measure^of^diversity^as^p reviously^described^[Joshi^ paper].^ Briefly,^ as^ the^ Renyi^ diversity^ approaches^ 0,^ greater^ importance^ is^ given^ to^ smaller/rarer^clones^but,^as^Renyi^diversity^tends^to^infini ty,^greater^importance^is^given^ to^the^more^common^clones.^The^Renyi^diversity^was^calculate d^as^a^range^of^values^^ using^ the^ ‘renyi’^ function^ from^ the^ vegan^ (v2.6-4)^ [https://CRAN.R- project.org/package=vegan]^package.^All^ repertoires^were^ randomly^sampled^ to^1000^ unique^ sequences,^ 100^ times^ before^ the^mean^Renyi^ score^was^ calculated^ for^ each^ sequence^for^each^value^of^Renyi^diversity.^^ ^ To^calculate^the^clonality^index,^all^productive^sequences^f or^each^repertoire^were^down-^ sampled^to^1000^clones.^The^clonality^was^estimated^for^this ^sample^using^(1^–^[‘entropy’^ function])^ from^ the^ entropy^ package^ (v1.3.1)^ [https://CRAN.R- project.org/package=entropy].^This^was^bootstrapped^1000^tim es^and^the^mean^clonality^ score^was^taken^for^each^sample.^^ ^ ^ For^ TCR^ clustering,^ the^ top^ 1000^ clones^ from^ each^ patient^ from^ all^ 3^ cohorts^ were^ downsampled^and^subsequently^clustered^together^using^the^� �gliph2’^function^from^the^ turbogliph^ package^ (v0.99.2)^ [https://www.nature.com/articles/s41587-020-0505-4].^ Clusters^were^assigned^to^a^histology^group^based^on^a^count ^proportion^threshold^of^ >50%.^Using^publicly^available^databases^like^VDJdb^(URL) ,^viral^cluster^specificity^was^^ assigned.^Only^one^sequence^within^a^cluster^ required^a^specificity^annotation^ for^the^ whole^cluster^to^be^deemed^as^viral.^^ ^ Statistical^analysis^ All^statistical^tests^were^performed^in^R^(version^1.4.1106) .^Tests^involving^differences^^ between^groups^were^done^using^a^‘wilcox.test’^using^unp aired^filters,^or^a^linear^mixed^ effects^ model^ from^ the^ lme4^ (v1.1-30)^ [https://www.jstatsoft.org/article/view/v067i01]^ package.^Details^of^the^statistical^ test^used^are^typically^outlined^ in^the^corresponding^ figure^legends.^When^required,^p-values^were^adjusted^using^ Benjamini-Hochberg^(BH)^ p-value^correction^[doi:10.1214/193940307000000158],^using^t he^‘p.adjust’^function.^^^ Correlation^ analyses^ completed^ using^ the^ ‘cor.test’^ function,^ with^ the^method^ set^ to^ ‘Spearman’^ and^ the^ alternative^ ‘two.sided’.^ Logistic^ regression^models^ to^ account^ for^ potentially^confounding^variables^were^completed^using^the^� ��glm’^function.^Coefficients^ were^exponentiated^to^get^odds^ratios^with^95%^confidence^in tervals.^Significance^was^ determined^with^a^Wald’s^test,^calculated^by^taking^the^cu mulative^probability^associated^^ with^the^absolute^value^of^the^Wald^test^statistic.^All^Wald ’s^test^p-values^are^two-tailed.^^ All^graphical^presentation^completed^using^the^ggplot2^packa ge^(v3.3.6)^[H.^Wickham.^ ggplot2:^Elegant^Graphics^for^Data^Analysis.^Springer-Verlag ^New^York,^2016.].^^ ^ Future^lung^cancer^diagnosis^analysis^ ^ Patients^were^split^ into^groups^(low/high)^based^on^ the^median^metric^value.^Kaplan- Meier^(KM)^analysis^was^completed^using^the^‘survfit’^fu nction^from^the^survival^package^ (v3.5-0)^ [https://CRAN.R-project.org/package=survival].^ Significance^ was^ determined^ using^a^log-rank^test.^For^the^Cox^proportional^hazards^mode ls,^the^‘coxph’^function^from^ the^survival^package^(v3.5-0)^ [https://CRAN.R-project.org/package=survival]^was^used^ to^estimate^ the^ coefficients^of^ each^ of^ the^ variables.^ To^ calculate^ the^ combined^ flow^^ cytometry^ and^ TCRseq^ immune^metric,^ the^ frequency^ of^ CD45RA-CD39 ⁺ ^ Tregs^was^ multiplied^by^the^Large:Small^ratio.^In^the^KM^and^Cox^model ^analyses,^the^outcome^was^ a^future^lung^cancer^diagnosis^(yes/no).^The^final^timepoint ^and^corresponding^follow-up^ time^ for^ each^ patient^ was^ selected.^ This^ reflected^ a^ real-life^ scenario^ better^ than^ averaging^immune^metrics^and^follow-up^times.^Lesion^grade^w as^omitted^from^the^Cox^^ models^because^no^low-grade^patients^had^a^future^lung^cance r^diagnosis^and^so^the^ model^was^unable^to^converge,^therefore^becoming^inaccurate. ^^ ^

Paragraphs^relating^to^certain^aspects^of^this^disclosure :^ ^ 1.^^A^method^for^determining^whether^a^subject^is^at^risk^fo r^having^a^progressing^or^ high-grade^pre-invasive^lesion,^nodule^or^small^mass,^or^hav ing^a^solid^malignant^^ tumour,^the^method^comprising:^ determining^a^ratio^of^activated^and/or^exhausted^T^cells^:^ naïve^and/or^resting^T^cells^ in^a^sample^of^blood^obtained^from^the^subject,^^ wherein^the^determining^comprises^analysing^T^cells^using^cy tometry^to^detect^the^ presence^or^absence^of^a^panel^of^biomarkers^comprising^Ki67 ^and^CD39.^ ^ ^ 2.^The^method^of^aspect^1,^wherein^the^subject^is^at^risk^if ^^ the^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^and /or^resting^T^cells^is^equal^to^ or^greater^than^^ a^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^and/o r^resting^T^cells^of^a^ ^ comparison^subject,^^or^^ an^average^ratio^of^activated^and/or^exhausted^T^cells^:^na� �ve^and/or^resting^T^cells^of^a^ plurality^of^comparison^subjects.^ ^ 3.^^A^method^for^determining^whether^a^subject^is^at^risk^fo r^having^a^progressing^or^^ high-grade^pre-invasive^lesion,^nodule^or^small^mass,^or^hav ing^a^solid^malignant^ tumour,^the^method^comprising:^ determining^a^ratio^of^activated^and/or^exhausted^T^cells^:^ T^cells^which^are^not^ activated^and/or^exhausted^T^cells^in^a^sample^of^blood^obta ined^from^the^subject,^^ wherein^the^determining^comprises^analysing^T^cells^using^cy tometry^to^detect^the^^ presence^or^absence^of^a^panel^of^biomarkers^comprising^Ki67 ^and^CD39.^ ^ 4.^^The^method^of^aspect^3,^wherein^the^subject^is^at^risk^i f^^ the^ratio^of^activated^and/or^exhausted^T^cells^:^T^cells^wh ich^are^not^activated^and/or^ exhausted^is^equal^to^or^greater^than^^ ^ a^ratio^of^activated^and/or^exhausted^T^cells^:^T^cells^whic h^are^not^activated^and/or^ exhausted^of^a^comparison^subject,^^or^^ an^average^ratio^of^activated^and/or^exhausted^T^cells^:^T^c ells^which^are^not^activated^ and/or^exhausted^of^a^plurality^of^comparison^subjects.^ ^ ^ ^ ^ ^ 5.^The^method^of^aspect^2^or^4^wherein,^a^comparison^subject ^is^selected^from:^ a)^a^subject^known^to^have^a^progressing^or^high-grade^pre-i nvasive^lesion,^nodule^or^ small^mass^or^an^established^solid^malignant^tumour,^^ b)^the^subject^at^a^different^time^point.^^ ^ ^ 6.^The^method^of^aspect^2^or^4^wherein,^a^plurality^of^compa rison^subjects^is^selected^ from:^ a)^a^plurality^of^subjects^known^to^have^a^progressing^or^hi gh-grade^pre-invasive^lesion,^ nodule^or^small^mass^or^an^established^solid^malignant^tumou r,^^ ^ b)^a^plurality^of^healthy^subjects,^or^ c)^a^plurality^of^subjects^of^the^general^population.^^^ ^ 7.^^The^method^of^any^one^of^the^preceding^aspects,^wherein^ an^activated^and/or^ exhausted^T^cell^expresses^CD39^and^Ki67^(CD39+^and^Ki67+^T^ cells).^ ^ ^ 8.^^The^method^of^any^one^of^the^preceding^aspects,^wherein^ naïve^and/or^resting^T^ cells^do^not^express^CD39^or^Ki67^(CD39-^and^Ki67-^T^cells). ^ ^ 9.^^The^method^of^any^one^of^the^preceding^aspects,^wherein^ T^cells^which^are^not^^ activated^and/or^exhausted^do^not^express^one^or^both^of^CD3 9^or^Ki67^(CD39-^and^ Ki67+^T^cells,^CD39+^and^Ki67-^T^cells,^or^CD39-^and^Ki67-^T ^cells).^ ^ 10.^The^method^of^any^one^of^the^preceding^aspects^wherein^t he^panel^of^biomarkers^ further^comprises^one^or^more^biomarkers^selected^from^CD45R A,^CCR7,^PD-1,^CD57^^ or^CD38.^^ ^ 11.^The^method^of^any^one^of^the^preceding^aspects^wherein^t he^panel^of^biomarkers^ further^comprises^one^or^more^biomarkers^selected^from^CD3,^ CD4^or^CD8.^ ^ ^ 12.^^The^method^of^any^one^of^the^preceding^aspects^wherein^ the^analysis^comprises^ use^of^a^viability^dye.^ ^ 13.^^The^method^of^any^one^of^aspects^1^to^10,^wherein^the^p anel^of^biomarkers^further^ comprises^CD45RA,^or^CCR7,^or^CD45RA^and^CCR7.^ ^ ^ 14.^The^method^of^any^one^of^aspects^1^to^10^wherein,^the^pa nel^of^biomarkers^further^ comprises^CD45RA,^CCR7,^and^PD-1.^ ^ 15.^^The^method^of^any^one^of^aspects^1^to^10,^wherein^the^p anel^of^biomarkers^further^^ comprises^CD45RA,^CCR7,^PD-1^and^CD57.^^ ^ 16.^The^method^of^any^one^of^aspects^1^to^10,^wherein^the^pa nel^of^biomarkers^further^ comprises^CD45RA,^CCR7,^PD-1,^CD57^and^CD38.^ ^ ^ 17.^^The^method^of^any^one^of^aspects^1^to^10,^wherein^the^p anel^of^biomarkers^further^ comprises^CD45RA,^CCR7,^CD57,^and^CD38.^ ^ 18.^The^method^of^any^one^of^aspects^1^to^10,^wherein^the^pa nel^of^biomarkers^further^ comprises^CD45RA,^PD-1^and^CD57.^^ ^ ^ 19.^The^method^of^any^one^of^the^preceding^aspects,^wherein^ the^cytometry^comprises^ one^or^more^of^flow^cytometry,^spectral^cytometry^or^mass^cy tometry,^optionally^the^ cytometry^comprises^flow^cytometry.^^^ ^ ^ 20.^^A^kit^comprising^a^set^of^lyophilised^antibodies^or^a^f ragment^thereof,^comprising^ antibodies^binding^to^CD39^and^Ki67.^ ^ 21.^The^kit^according^to^aspect^20,^wherein^the^set^of^lyoph ilised^antibodies^or^fragment^ thereof^comprises^antibodies^binding^to^the^further^biomarke rs^as^defined^in^any^one^of^^ aspects^10^to^18.^ ^ 22.^^Use^of^a^kit^according^to^aspect^20^or^21^in^a^method^f or^determining^whether^a^ subject^is^at^risk^for^having^a^progressing^or^high-grade^pr e-invasive^lesion,^nodule^or^ small^mass,^or^having^a^solid^malignant^tumour.^ ^ ^ 23.^^A^device^comprising:^^ (i)^means^for^receiving^a^sample^of^blood,^and^^ (ii)^a^set^of^lyophilised^antibodies^or^a^fragment^thereof,^ comprising^antibodies^binding^ to^CD39^and^Ki67.^ ^ ^ 24.^^A^method^of^treating^a^subject^determined^to^be^at^risk ^for^having^a^progressing^or^ high-grade^pre-invasive^lesion,^nodule^or^small^mass,^or^hav ing^a^solid^malignant^ tumour,^wherein^the^method^comprises:^ ^^^^^^^^^^^^determining^whether^a^subject^is^at^risk^for^hav ing^a^progressing^or^high-grade^^ pre-invasive^lesion,^nodule^or^small^mass,^or^having^a^solid ^malignant^tumour,^the^ method^comprising:^ determining^a^ratio^of^activated^and/or^exhausted^T^cells^:^ naïve^and/or^resting^T^cells^ in^a^sample^of^blood^obtained^from^the^subject,^^ wherein^the^determining^comprises^analysing^T^cells^using^cy tometry^to^detect^the^^ presence^or^absence^of^a^panel^of^biomarkers^comprising^Ki67 ^and^CD39,^and^^ wherein^the^subject^is^at^risk^if^^ the^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^and /or^resting^T^cells^is^equal^to^ or^greater^than^^ a^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^and/o r^resting^T^cells^of^a^ ^ comparison^subject,^^or^^ an^average^ratio^of^activated^and/or^exhausted^T^cells^:^na� �ve^and/or^resting^T^cells^of^a^ plurality^of^comparison^subjects,^ and^ ^^^^^^^^^^^^^^^providing^treatment^to^said^subject,^if^said^ subject^is^determined^to^be^at^risk.^^^ ^ 25.^^A^method^of^treating^a^subject^determined^to^be^at^risk ^for^having^a^progressing^or^ high-grade^pre-invasive^lesion,^nodule^or^small^mass,^or^hav ing^a^solid^malignant^ tumour,^wherein^the^method^comprises:^ ^^^^^^^^^^^^^^^determining^whether^a^subject^is^at^risk^for^ having^a^progressing^or^high-^ grade^pre-invasive^lesion,^nodule^or^small^mass,^or^having^a ^solid^malignant^tumour,^ the^method^comprising:^ determining^a^ratio^of^activated^and/or^exhausted^T^cells^:^ T^cells^which^are^not^ activated^and/or^exhausted^T^cells^in^a^sample^of^blood^obta ined^from^the^subject,^^ wherein^the^determining^comprises^analysing^T^cells^using^cy tometry^to^detect^the^^ presence^or^absence^of^a^panel^of^biomarkers^comprising^Ki67 ^and^CD39,^and^^ wherein^the^subject^is^at^risk^if^^ the^ratio^of^activated^and/or^exhausted^T^cells^:^T^cells^wh ich^are^not^activated^and/or^ exhausted^is^equal^to^or^greater^than^^ a^ratio^of^activated^and/or^exhausted^T^cells^:^T^cells^whic h^are^not^activated^and/or^^ exhausted^of^a^comparison^subject,^^or^^ an^average^ratio^of^activated^and/or^exhausted^T^cells^:^T^c ells^which^are^not^activated^ and/or^exhausted^of^a^plurality^of^comparison^subjects,^ and^ ^^^^^^^^^^^^^^providing^treatment^to^said^subject,^if^said^s ubject^is^determined^to^be^at^risk.^^ ^ 26.^^A^method^for^determining^whether^a^subject^is^at^risk^f or^having^a^progressing^or^ high-grade^pre-invasive^lesion,^nodule^or^small^mass,^or^hav ing^a^solid^malignant^ tumour,^the^method^comprising^analysing^the^proportion^of^T^ cells^in^a^sample^of^blood^ obtained^from^the^subject^which^are^activated^and/or^exhaust ed^T^cells^by^analysing^a^^ trait^of^the^T^cells.^ ^ 27.^A^method^for^determining^whether^a^subject^is^at^risk^fo r^having^a^progressing^or^ high-grade^pre-invasive^lesion,^nodule^or^small^mass,^the^me thod^comprising^analysing^ the^proportion^of^T^cells^in^a^sample^of^blood^obtained^from ^the^subject^which^are^^ activated^and/or^exhausted^T^cells^by^analysing^a^trait^of^t he^T^cells.^ ^ 28.^The^method^of^paragraph^27^or^28,^wherein^analysing^the^ proportion^of^T^cells^in^ the^sample^of^blood^obtained^from^the^subject^which^are^acti vated^and/or^exhausted^ comprises^analysing^a^plurality^of^traits^of^the^T^cells.^ ^ ^ 29.^The^method^of^paragraph^28,^wherein^the^method^further^c omprises^combining^ results^obtained^from^analysing^the^plurality^of^traits.^ ^ 30.^The^method^of^any^one^of^paragraphs^26-29,^wherein^the^t rait^is^a^phenotypic^trait.^^ ^ 31.^^The^method^of^paragraph^30,^wherein^the^phenotypic^trai t^is^the^diversity^of^T^cell^ receptors^on^the^T^cells^in^the^sample^of^blood,^optionally^ the^diversity^of^CDR3B^on^ TCRs^of^the^T^cells^in^the^sample^of^blood,^further^optional ly^the^phenotypic^trait^is^ analysed^using^TCR^Seq.^^ ^ ^ 32.^The^method^of^paragraph^31,^wherein^the^subject^is^at^ri sk^if:^ ^ a)^^the^diversity^of^the^repertoire^of^TCRs^is^less^than^the ^diversity^of^the^repertoire^of^ TCRs^of^a^comparison^subject^or^a^plurality^of^comparison^su bjects,^^ ^ ^ b)^D50^diversity^score^is^lower^than^a^D50^diversity^score^o btained^from^analysing^T^ cells^in^a^sample^of^blood^obtained^from^a^comparison^subjec t^or^a^plurality^of^ comparison^subjects,^^ ^ c)^a^clonality^score^is^lower^than^a^clonality^score^obtaine d^from^analysing^T^cells^in^a^^ sample^of^blood^obtained^from^a^comparison^subject^or^a^plur ality^of^comparison^ subjects,^or^ ^ d)^Hill^number^is^lower^than^a^Hill^number^obtained^from^ana lysing^T^cells^in^a^sample^ of^blood^obtained^from^a^comparison^subject^or^a^plurality^o f^comparison^subjects,^^ ^ optionally^wherein^the^comparison^subject^is^a^healthy^subje ct,^or^the^plurality^of^ comparison^subjects^are^healthy^subjects.^^ ^ 33.^The^method^of^paragraph^30,^wherein^the^phenotypic^trait ^is^biomarker^expression^^ by^the^T^cells^in^the^sample^of^blood,^optionally^using^cyto metry,^further^optionally^ wherein^cytometry^is^used^to^detect^the^presence^or^absence^ of^a^panel^of^biomarkers^ comprising^Ki67^and^CD39.^ ^ 34.^The^method^of^paragraph^33,^wherein^the^subject^is^at^ri sk^if:^ ^ ^ a)^the^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^ and/or^resting^T^cells^is^equal^ to^or^greater^than^^ a^ratio^of^activated^and/or^exhausted^T^cells^:^naïve^and/o r^resting^T^cells^of^a^ comparison^subject,^^or^^ ^ an^average^ratio^of^activated^and/or^exhausted^T^cells^:^na� �ve^and/or^resting^T^cells^of^a^ plurality^of^comparison^subjects,^or^ ^ b)^the^ratio^of^activated^and/or^exhausted^T^cells^:^T^cells ^which^are^not^activated^ and/or^exhausted^is^equal^to^or^greater^than^^ ^ a^ratio^of^activated^and/or^exhausted^T^cells^:^T^cells^whic h^are^not^activated^and/or^ exhausted^of^a^comparison^subject,^^or^^ an^average^ratio^of^activated^and/or^exhausted^T^cells^:^T^c ells^which^are^not^activated^ and/or^exhausted^of^a^plurality^of^comparison^subjects.^ ^ ^ 35.^The^method^of^paragraph^33^wherein,^a^comparison^subject ^is^selected^from:^ a)^a^subject^known^to^have^a^progressing^or^high-grade^pre-i nvasive^lesion,^nodule^or^ small^mass^or^an^established^solid^malignant^tumour,^^ b)^the^subject^at^a^different^time^point.^^ ^ 36.^The^method^of^paragraph^33^wherein,^a^plurality^of^compa rison^subjects^is^selected^^ from:^ a)^a^plurality^of^subjects^known^to^have^a^progressing^or^hi gh-grade^pre-invasive^lesion,^ nodule^or^small^mass^or^an^established^solid^malignant^tumou r,^^ b)^a^plurality^of^healthy^subjects,^or^ c)^a^plurality^of^subjects^of^the^general^population.^^^ ^ ^ 37.^The^method^of^any^one^of^paragraphs^33-36^wherein^the^pa nel^of^biomarkers^ further^comprises^one^or^more^biomarkers^selected^from^CD45R A,^CCR7,^PD-1,^CD57^ or^CD38.^^ ^ ^ 38.^The^method^of^any^one^of^paragraphs^33-37^,^wherein^the^ panel^of^biomarkers^ further^comprises^one^or^more^biomarkers^selected^from^CD3,^ CD4^or^CD8.^ ^ 39.^^The^method^of^the^preceding^paragraphs^33-37,^wherein^t he^analysis^comprises^ use^of^a^viability^dye.^ ^ ^ 40.^^The^method^of^paragraphs^33-36,^wherein^the^panel^of^bi omarkers^further^ comprises^CD45RA,^or^CCR7,^or^CD45RA^and^CCR7.^ ^ 42.^The^method^of^paragraphs^33-36,^wherein,^the^panel^of^bi omarkers^further^ ^ comprises^CD45RA,^CCR7,^and^PD-1.^ ^ 43.^^The^method^of^any^one^of^paragraphs^33-36,^wherein^the^ panel^of^biomarkers^ further^comprises^CD45RA,^CCR7,^PD-1^and^CD57.^^ ^ ^ 44.^The^method^of^paragraphs^33-36,^wherein^the^panel^of^bio markers^further^ comprises^CD45RA,^CCR7,^PD-1,^CD57^and^CD38.^ ^ 45.^^The^method^of^paragraphs^33-36,^wherein^the^panel^of^bi omarkers^further^ comprises^CD45RA,^CCR7,^CD57,^and^CD38.^ ^ ^ 46.^The^method^of^paragraphs^33-36,^wherein^the^panel^of^bio markers^further^ comprises^CD45RA,^PD-1^and^CD57.^^ ^ 47.^The^method^of^any^one^of^paragraphs^33-46,^wherein^the^c ytometry^comprises^one^^ or^more^of^flow^cytometry,^spectral^cytometry^or^mass^cytome try,^optionally^the^ cytometry^comprises^flow^cytometry.^^^ ^