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Targeted chemoprevention of head and neck cancer

de Boer, D.V.

2021

document version

Publisher's PDF, also known as Version of record

Link to publication in VU Research Portal

citation for published version (APA)

de Boer, D. V. (2021). Targeted chemoprevention of head and neck cancer.

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Establishment and genetic landscape of

precancer cell model systems from the head

and neck mucosal lining

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INTRODUCTION

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Culture samples

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No genetic analysis WGs

WGs and Targeted sequencing

Figure 1: Population doublings of precancer cell cultures. )URP SDWLHQWV ELRSVLHV RI VXUJLFDO

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RESULTS

Collection of mucosal biopsies from surgical margins and tumor tissue

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Copy number changes

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Panel A Margin cultures

Patient CNA CDKN2A TP53-mutation Other somatic mutations PD reached Dysplasia margins

+10 1RUPDO na na No +10 1RUPDO 0LOG +10 1RUPDO Severe +10 1RUPDO Severe +10 1RUPDO 0LOG +10 1RUPDO 0LOG #27-HN539-M2 TORVV IRFDO 1RUPDO No VU-preSCC-M3a _SORVV TJDLQ SORVV TJDLQ JDLQ Loss /4: NOTCH1 ! <HV Panel B (Erythro)Leukoplakia

Sample CNA CDKN2A

TP53-mutation Other mutations

D4 ! Loss < CASP8, PIK3CA, FBXW7

D19 ! Loss 5/<& FAT1

D20 ! 1RUPDO 54 CASP8, PIK3CA, CDKN2A, MLL2 D34 ! 1RUPDO 70&6 D35 ! Loss na na %ROG&XOWXUHVFRQWDLQLQJHLWKHU&1$VPXWDWLRQVRUERWK a_{3UHYLRXVO\HVWDEOLVKHGYDQ=HHEXUJet al.} QDQRWDVVHVVHG 7KHREWDLQHGÀYHHU\WKUROHXNRSODNLDFXOWXUHVZHUHDQDO\]HGOLNHZLVHIRUJHQHWLF DEHUUDWLRQV 7KHVH DOO VKRZHG DEHUUDQW &1 SURÀOHV ZLWK PDQ\ +16&&DVVRFLDWHG FKDQJHV 7DEOH E $QDO\VLV RI WKH ÀYH HVWDEOLVKHG QHZ WXPRU FHOO FXOWXUHV DOVR VKRZHGDZLGHYDULHW\RI&1$VVHH)LJXUHFIRUDUHSUHVHQWDWLYHH[DPSOH7KHVH &1$VDUHFRPPRQIRU+16&&LQFOXGLQJORVVHVDWSSSSDQGS SDQGDPSOLÀFDWLRQVRIFKURPRVRPHVTTDQGTTT_. ,QDGGLWLRQDOOÀYHWXPRUFXOWXUHVFRQWDLQHGDWOHDVWRQHPXWDWLRQLQTP53DQGRXW RIDOVRGLVSOD\HGDORVVRIWKHCDKN2AUHJLRQRQFKURPRVRPHS7DEOH6

Table 1: Genetic characterization of selected margin cultures and the (erythro)leukoplakia cultures revealed numerous CNAs and HNSCC-associated mutations.

Panel A Margin cultures

Patient CNA CDKN2A TP53-mutation Other somatic mutations PD reached Dysplasia margins

#1-HN433-M1 TJDLQ SORVVS JDLQIRFDO Loss No +10 1RUPDO na na No #2-HN436-M1a SORVV IRFDO SORVV

Loss 5& NOTCH1 6HYHUH CIS

+10 1RUPDO No

#4-HN439-M1 1RUPDO NOTCH1 No

#4-HN439-M2 SORVVS

ORVVIRFDO Loss [)$7NOTCH1 No

#4-HN439-M3 TORVVS

ORVVS ORVVIRFDO

Loss 5+ NSD1 25 No

#4-HN439-M4 SORVVS

ORVVIRFDO Loss FAT1, NOTCH1 No

#6-HN441-M3 TJDLQ

IRFDO 1RUPDO na na 6HYHUHCIS +10 1RUPDO na na No +10 1RUPDO No +10 1RUPDO na na No +10 1RUPDO No +10 1RUPDO No +10 1RUPDO No #12-HN472-M1 SORVV IRFDOS ORVVS JDLQIRFDO 1RUPDO *6 5 Severe #12-HN472-M4 TORVVS ORVVIRFDO TORVV IRFDO Loss Severe #14-HN470-M2 TORVV

IRFDO 1RUPDO NOTCH1 5 Severe

#14-HN470-M4 SORVV SJDLQ Loss 5/DQGframeshift H[RQ Severe +10 1RUPDO No +10 1RUPDO na na 0LOG (continued)

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Figure 3: Waterfall plot of the copy-number segments.&RS\QXPEHUGDWDRIDOOFXOWXUHVZHUHVHJ

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Table 2: Take rate of cancer and precancer cell cultures in nude mice. Tumor cell lines

Cell line Take rate (%) Lag time (days)

806&&$ 986&&2( 986&& No 806&& 25 986&& )D'X 986&& 806&& No 986&& 806&&% 986&&

Precancer cell lines

Cell line Take rate (%) Lag time (days)

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Figure 4: Overview of the mutational landscape of the precancer cell cultures.2IWKHVHTXHQFHG

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Normal and precancer cell cultures

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Figure S3: &1 SURÀOHV RI WKH FXOWXUHV RI SDWLHQW +1 UHYHDOHG PXOWLSOH ÀHOGV QRW UHODWHG WR the tumor. a.&1SURÀOHRIFXOWXUH+10VKRZHGWZRIRFDODEHUUDWLRQVDORVVDWFKURPRVRPH

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Figure S2: Frequency plots of CNAs detected in tumor and precancer samples. a.$IUHTXHQF\SORW

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T

able S1: Overview of our patient cohort of which we successfully brought cells in culture. Patient Study number Mucosa samples Site Age Gender TNM PDs

Culture genetically characterised Any genetic changes

Relapse Relapse type Dysplasia Smoking status +1 2 &DYXP2ULV 0DOH T2N2 No #2 +1 3LULIRUPVLQXV 0DOH 71 6HYHU H&,6 +1 3 UR FDOYLQI )HPDOH 71 No + 1 6XSUDJORWWLFODU\Q[ 0 DO H 7 1 No #5 +1 2 )ORRURIPRXWK )HPDOH 71 Sever e +1 0 RELOHWRQJXH 0 DOH 71 6HYHU H&,6 +1 2 3U RFDOYLQI )HPDOH 71 No +1 ) ORRURIPRXWK 0DOH 71 No +1 0 RE LOHWR Q JXH ) HP DOH 7 1

6PDOOVHFRQG SULPDU\ WXPRU

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7DEOH62YHUYLHZRIWKHRULJLQRIWKHÀYHYLVLEOHHU\WKUROHXNRSODNLDFXOWXUHVZHREWDLQHGIURP others.

Samplea _Source _Age _Gender _{Stage of dysplasia}

' /HXNRSODNLD 0DOH CIS ' (U\WKUROHXNRSODNLD 0DOH 6HYHUH&,6 ' /HXNRSODNLD 0DOH Moderate ' /HXNRSODNLD )HPDOH Moderate ' (U\WKUROHXNRSODNLD 0DOH 6HYHUH&,6 a_{3UHYLRXVO\HVWDEOLVKHG0F*UHJRUet al.DQG}

7DEOH6*HQHWLFFKDUDFWHUL]DWLRQRIWKHÀYHHVWDEOLVKHGWXPRUFXOWXUHV

Patient CNA CDKN2A TP53-mutation Other mutations

+1 ! Loss S*5 na +1 ! Loss *!$VSOLFHVLWHH[S" na +1 ! 1RUPDO S4GHOS*6 na +1 ! 1RUPDO S&< na +1 ! Loss S<& na QDQRWDVVHVHG Patient Study number Mucosa samples Site Age Gender TNM PDs

Culture genetically characterised Any genetic changes

Relapse Relapse type Dysplasia Smoking status +1 2 0RELOHWRQJXH ) HPDOH 71 No #22 + 1 6XSUDJORWWLFODU\Q[ ) HP DO H 7 1 No +1 * ORWWLFODU\Q[ 0DOH 71 No +1 2 *ORWWLFODU\Q[ 0DOH 71 No #25 +1 0 RELOHWRQJXH ) HPDOH 71E 0 LOG +1 Cheek )HPDOH 71 No +1 * ORWWLFODU\Q[ 0DOH 71 No +1 + \SRSKDU\Q[ 0DOH 71 5 0RGHUDWH Sever e HN552 ) ORRURIPRXWK 0DOH 71 0LOG +1 0 RELOHWRQJXH ) HPDOH 71 No +1 ) ORRURIPRXWK )HPDOH 71 0LOG + 1 7UDQVJORWWLF/DU\Q[ 0 DO H 7 1 0 LO G +1 0 RELOHWRQJXH 0 DOH 71 na na na No 98SU H6&& 0 a * ORWWLFODU\Q[ 0DOH 71 < HV a3U HYLRXVO\HVWDEOLVKHGYDQ=HHEXU J et al. QDQRWDSSOLFDEOH 3'V DOOFXOWXU HV DQ\FXOWXU H *HQHWLFFKDQJHV QRQHRIWKHFXOWXU HVFRQWDLQHGDJHQHWLFDEH UUDWLRQ DQ\RIWKHFXOWXU HVGLGFRQWDLQDRUPXOWLSOHJHQHW LFDEHUUDWLRQV 6PRNLQJ QR FXUU HQWIRUPHU XQNQRZQ

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2

Panel A CNAs Sample HN441-M3 Region T Loss/Gain *DLQ Chromosomal location

Known reccurent region HNSCC?

No Sample HN470-M2 Region T Loss/Gain Loss Chromosomal location

No Sample HN470-M4 Region SS S S Loss/Gain Loss *DLQ *DLQ Chromosomal location

/DZU HQFH et al. 7R ZOH et al. 2014 /DZU HQFH et al. Sample HN472-M1 Region S S S Loss/Gain Loss Loss *DLQ Chromosomal location

No No 0DU HVFDOFR et al. T

able S4: Overview of all detected genetic alterations. Panel A

CNAs Sample HN433-M1 Region T SS SS S Loss/Gain *DLQ Loss *DLQ Loss Chromosomal location

No /DZU HQFH et al. 7R ZOH et al. No Sample HN436-M1a Region S SS Loss/Gain Loss Loss Chromosomal location

/DZU HQFH et al. Leemans et al. Sample HN439-M2 and M4 Region SS S Loss/Gain Loss Loss Chromosomal location

/DZU HQFH et al. No Sample HN439-M3 Region T T SS SS Loss/Gain Loss Loss Loss Loss Chromosomal location

/DZU HQFH et al. /DZU HQFH et al. /DZU HQFH et al. No (continued)

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Panel B Mutations Culture

Gene dbSNP ID Chr . Exon Function class T ype A A Ref. allele Alt. allele V A F Read depth Germline/ somatic &RQÀUPHG Sanger +10 MLL2 UV FKU H[ MISSENSE SNP $7 CT *HUPOLQH nn +10 NOTCH1 FKU H[ MISSENSE SNP &) CA Somatic <H V +1B0 FA T 1 FKU H[ MISSENSE SNP &: AC Somatic <H V +1B0 FA T 1 FKU H[ MISSENSE SNP <& TC Somatic <H V +1B0 NOTCH1 FKU H[ )5$0(B SHIFT 'HO *$ * Somatic <H V +1B0 NSD1 UV chr5 H[ NONSENSE SNP 5 CT Somatic <H V +1B0 TP53 UV FKU H[ MISSENSE SNP 5+ CT Somatic <H V +10 FA T 1 FKU H[ MISSENSE SNP <& TC Somatic <H V +10 NOTCH1 FKU H[ )5$0(B SHIFT 'HO , **$ 7 * Somatic <H V +10 FA T 1 UV FKU H[ MISSENSE SNP 9* AC *HUPOLQH nn +10 FA T 1 UV FKU H[ MISSENSE SNP 9* AC *HUPOLQH nn +10 NOTCH1 FKU H[ MISSENSE SNP /5 AC Somatic <H V +1B0 TP53 FKU H[ )5$0(B SHIFT 'HO $* A Somatic <H V +1B0 TP53 UV FKU H[ MISSENSE SNP 5 / CA Somatic <H V +10 TP53 UV FKU H[ MISSENSE SNP *6 CT Somatic <H V +10 FA T 1 UV FKU H[ MISSENSE SNP 6 / * A *HUPOLQH nn +10 FA T 1 UV FKU H[ MISSENSE SNP 6 / * A *HUPOLQH nn +10 PIK3CA FKU H[ MISSENSE SNP 09 A * *HUPOLQH nn +10 PIK3CA FKU H[ MISSENSE SNP 09 A * *HUPOLQH nn Panel A CNAs Sample HN472-M4 Region TT S T Loss/Gain Loss Loss Loss Chromosomal location

/DZU HQFH et al. /DZU HQFH et al. No Sample HN539-M2 Region T Loss/Gain Loss Chromosomal location

No Sample VU-preSCC-M3 Region S T S T Loss/Gain Loss *DLQ Loss *DLQ *DLQ Chromosomal location :KROHDUP :KROHDUP :KROHDUP :KROHDUP :KROHFKU RPRVRPH

/DZU HQFH et al. /DZU HQFH et al. /DZU HQFH et al. Leemans et al. Leemans et al.

Gene d bSNP ID Chr . Exon Function class T ype A A Ref. allele Alt. allele V A F Read depth Germline/ somatic &RQÀUPHG Sanger +1B0 MLL2 FKU H[ MISSENSE SNP /) C * *HUPOLQH nn +10D NSD1 chr5 H[ MISSENSE SNP 6) CT Somatic No +10D NOTCH1 FKU H[ MISSENSE SNP 5& * A Somatic <H V +10D TP53 UV FKU H[ MISSENSE SNP 5& * A Somatic <H V (continued) (continued)

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Gene dbSNP ID Chr . Exon Function class T ype A A Ref. allele Alt. allele V A F Read depth Germline/ somatic &RQÀUPHG Sanger 98 SUH6&&0 NOTCH1 FKU H[ )5$0(B SHIFT 'HO &$* C Somatic <H V 98 SUH6&&0 TP53 FKU H[ NONSENSE SNP : CT Somatic <H V 98 SUH6&&0 TP53 FKU H[ MISSENSE SNP / 4 AT Somatic <H V ' CASP8 UV chr2 H[ NONSENSE SNP 5 CT na <H V ' PIK3CA UV FKU H[ MISSENSE SNP (. * A na <H V ' FBXW7 UV FKU H[ MISSENSE SNP 5* * C na <H V ' TP53 FKU H[ NONSENSE SNP < * T na <H V ' FA T 1 FKU H[ )5$0(B SHIFT 'HO AACA T A na <H V ' TP53 UV FKU H[ MISSENSE SNP <& TC na <H V ' TP53 UV FKU H[ MISSENSE SNP 5 / CA na <H V ' PIK3CA UV FKU H[ MISSENSE SNP (. * A na <H V ' CDKN2A UV FKU H[ NONSENSE SNP 5 * A na <H V ' MLL2 UV FKU H[ MISSENSE SNP 9, CT na <H V ' TP53 UV FKU H[ MISSENSE SNP 54 CT na <H V ' TP53 UV FKU H[ MISSENSE SNP &6 C * na <H V ' TP53 UV FKU H[ MISSENSE SNP 70 * A na <H V QDQRWDVVHVVDEOH QQQRWQHFHVVDU\ (continued)