Moleculaire diagnostiek van melanocytaire laesies

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1 Moleculaire diagnostiek van melanocytaire laesies Léon van Kempen Universitair Medisch Centrum Groningen Afd. Pathologie Laboratorium voor Moleculaire Pathologie

2 Week Disclosure belangen spreker (Potentiële) belangenverstrengeling Voor bijeenkomst mogelijk relevante relaties met bedrijven Sponsoring of onderzoeksgeld Honorarium of andere (financiële) vergoeding Aandeelhouder Andere relatie, namelijk nanostring, Abbott Geen / Zie hieronder Bedrijfsnamen nanostring, Janssen pharmaceutica, BAYER april 2019, Veenendaal

3 Genomische classificatie van het cutane melanoom (2015) Cell. 2015; 161(7):

4 WHO 2018 moleculaire classificatie van melanocytaire tumoren Low CSD High CSD Low to no CSD Pathway I II III IV V VI VII VIII IX Benign neoplasm Naevus? IMP? IMP Spitz naevus? Acral naevus? Melanosis CN Blue naevus? Naevus Intermediate Low grade dysplasia High grade dysplasia BIN DPN? IAMP? IAMP Atypical Spitz tumor IAMP / dysplasia Atypical melanosis / dysplasia / IAMPUS Nodule in CN Atypical CBN MELTUMP LM (MIS) MIS STUMP / MELTUMP Acral MIS Mucosal MIS MIS in CN Atypical CBN? BIM DPM PEM? Malignant neoplasm SMM (VGP) Melanoma in LMM (VGP) Desmoplastic BIN DPN PEM Malignant Spitz tumor / Spitz Acral (VGP) Mucosal lentiginous (VGP) Melanoma in CN Melanoma in BN Uveal Common mutations V600E or or RAS MAP2K1 or non V600E or NF1 NF1 amp ERBB2 amp MAP2K1 amp MAP3K1 amp EGFR amp MET amp HRAS rearr ALK rearr. ROS1 rearr. RET rearr. NTRK1 rearr. NTRK3 rearr. or MET rearr HRAS ALK rearr. NTRK1 rearr. or NF1 KRAS or rearr. V600 (small lesions) or rearr. GNAQ GNA11 or CYSLTR2 GNAQ GNA11 CYSLTR2 or PLCB4 Other alterations TP53 PTEN BAP1 CTNNB1 or APC PRKAR1 A or PRKCA TP53 PTEN RAC1 NFKBIE prom PIK3CA PTPN11 TERT amp CCND1 amp GAB2 amp NF1 SF3B1 CCND1 amp CDK4 amp MDM2 amp BAP1 EIF1AX SF3B1 BAP1 EIF1AX SF3B1 Bold : mutations identified in malignant lesions Red: Gain of function mutation Blue: Loss of function mutation Green: Change of function mutation

5 WHO 2018 moleculaire classificatie van melanocytaire tumoren Low CSD High CSD Low to no CSD Pathway I II III IV V VI VII VIII IX Benign neoplasm Naevus? IMP? IMP Spitz naevus? Acral naevus? Melanosis CN Blue naevus? Naevus Intermediate Low grade dysplasia High grade dysplasia BIN DPN? IAMP? IAMP Atypical Spitz tumor IAMP / dysplasia Atypical melanosis / dysplasia / IAMPUS Nodule in CN Atypical CBN MELTUMP LM (MIS) MIS STUMP / MELTUMP Acral MIS Mucosal MIS MIS in CN Atypical CBN? BIM DPM PEM? Malignant neoplasm SMM (VGP) Melanoma in LMM (VGP) Desmoplastic BIN DPN PEM Malignant Spitz tumor / Spitz Acral (VGP) Mucosal lentiginous (VGP) Melanoma in CN Melanoma in BN Uveal Common mutations V600E or or RAS MAP2K1 or non V600E or NF1 NF1 amp ERBB2 amp MAP2K1 amp MAP3K1 amp EGFR amp MET amp HRAS rearr ALK rearr. ROS1 rearr. RET rearr. NTRK1 rearr. NTRK3 rearr. or MET rearr HRAS ALK rearr. NTRK1 rearr. or NF1 KRAS or rearr. V600 (small lesions) or rearr. GNAQ GNA11 or CYSLTR2 GNAQ GNA11 CYSLTR2 or PLCB4 Other alterations TP53 PTEN BAP1 CTNNB1 or APC PRKAR1 A or PRKCA TP53 PTEN RAC1 NFKBIE prom PIK3CA PTPN11 TERT amp CCND1 amp GAB2 amp NF1 SF3B1 CCND1 amp CDK4 amp MDM2 amp BAP1 EIF1AX SF3B1 BAP1 EIF1AX SF3B1 Bold : Mutations identified in malignant lesions Red: Gain of function mutation Blue: Loss of function mutation Green: Change of function mutation

6 Schijnbare willekeur aan mutaties Kanker : Multifactorieel proces van genetische veranderingen Melanomen : MAPK geactiveerde tumoren Sasha Trubetskoy

7 Schijnbare willekeur aan mutaties High CSD Low to no CSD Pathway II III IV Benign neoplasm? IMP? IMP Spitz naevus? IAMP? IAMP Atypical Spitz tumor Intermediate LM (MIS) MIS STUMP / MELTUMP Malignant neoplasm LMM (VGP) Desmoplastic Malignant Spitz tumor / Spitz Common mutations non V600E or NF1 NF1 amp ERBB2 amp MAP2K1 amp MAP3K1 amp EGFR amp MET amp HRAS rearr ALK rearr. ROS1 rearr. RET rearr. NTRK1 rearr. NTRK3 rearr. or MET rearr Other alterations TP53 PTEN RAC1 NFKBIE prom PIK3CA PTPN11 Liu et al Patel et al. 2017

8 nanostring Counting molecules: Fusion probe design J Mol Diagn. 2018; 20(1):63-77

9 nanostring Counting molecules: Imbalance probe design 4 probes on 5 4 probes on 3 WT gene transcript Kinase domain Ratio probe count 3 /5 = 1 Fusion gene transcript Fusion partner RNA Kinase domain Ratio probe count 3 /5 >>1

10 nanostring analyse voor mrna detectie RNA moleculen tellen

11 nanostring UMCG fusion transcript panel 2.0 ALK ROS1 RET NTRK1 NTRK2 NTRK3. EML4_13:ALK_20 CD74_6:ROS1_32 CCDC6_1:RET_12 MRRIP_23:NTTRK1_11 TRIM24:NTRK2 ETV6 :NTRK3 AGK_2:_8 EML4_18:ALK_20 EZR_10:ROS1_34 KIF5B_15:RET_11 LMNA_10:NTRK1_11 MYO5A:NTRK3 KIAA1549_16:_9 EML4_2:ALK_20 GOPC_4:ROS1_36 KIF5B_15:RET_12 LMNA_2:NTRK1_11 MYH9:NTRK3 TRIM24_9:_2 EML4_20:ALK_20 GOPC_7:ROS1_35 KIF5B_16:RET_12 TPM3_8:NTRK1_9 ELM4:NTRK3 EML4_6:ALK_19 GOPC_8:ROS1_35 KIF5B_22:RET_12 TPM3_8:NTRK1_11 BTBD1:NTRK3 EML4_6a:ALK_20 LRIG3_16:ROS1_35 KIF5B_23:RET_12 SQSTM1_6:NTRK1_9 EML4_6b:ALK_20 SDC4_2:ROS1_32 KIF5B_24:RET_11 NFASC_20:NTRK1_9 HIP1_21:ALK_20 SDC4_4:ROS1_34 KIF5B_24:RET_8 BCAN_13:NTRK1_11 HIP1_28:ALK_20 KIF5B_17:ALK_20 KIF5B_24:ALK_20 TFG_5:ALK_20 SLC34A2_13-:ROS1_32 SLC34A2_4:ROS1_32 SLC34A2_4:ROS1_34 TPM3_8:ROS1_35 TPR_15:ALK_20 ALK 5p-1 ROS1 5p-1 RET 5p-1 NTRK1 5p-1 NTRK2 5p-1 NTRK3 5p-1 5p-1 ALK 5p-2 ROS1 5p-2 RET 5p-2 NTRK1 5p-2 NTRK2 5p-2 NTRK3 5p-2 5p-2 ALK 5p-3 ROS1 5p-3 RET 5p-3 NTRK1 5p-3 NTRK2 5p-3 NTRK3 5p-3 ALK 5p-4 ROS1 5p-4 RET 5p-4 NTRK1 5p-4 NTRK2 5p-4 NTRK3 5p-4 ALK 3p-1 ROS1 3p-1 RET 3p-1 NTRK1 3p-1 NTRK2 3p-1 NTRK3 3p-1 3p-1 ALK 3p-2 ROS1 3p-2 RET 3p-2 NTRK1 3p-2 NTRK2 3p-2 NTRK3 3p-2 3p-2 ALK 3p-3 ROS1 3p-3 RET 3p-3 NTRK1 3p-3 NTRK2 3p-3 NTRK3 3p-3 ALK 3p-4 ROS1 3p-4 RET 3p-4 NTRK1 3p-4 NTRK2 3p-4 NTRK3 3p-4

12 Analyse van chromosomale veranderingen SNP analyse Maligne tumoren zijn in het algemeen aneuploid Bepaling van chromosoom kopienummer veranderingen door middel van SNP analyse. LogR ratio Probe signaal relatief aan referentie Uveaal melanoom (man) Verlies van chr 3, Partieel verlies van chr 8p Gain van chr 8q B-allel frequentie Ratio probe signaal van de 2 allelen Van Beek et al In: Melanoma - From Early Detection to Treatment

13 Analyse van chromosomale veranderingen SNP analyse Melanocytaire leasie, histologisch passend bij Spitz nevus -> Gain chr 11p (HRAS) Partial gain 7q Anne Jansen

14 Analyse van chromosomale veranderingen SNP analyse Atypische melanocytaire proliferatie Hoeveel chromosale afwijking zijn er nodig voor de moleculaire classificatie maligne? FFPE is een uitdaging ivm fixatie artefacten -> Calibratie. Anne Jansen

15 Tissue is the issue Percentage en absoluut aantal verdachte melanocytaire cellen tov normaal

16 Tissue is the issue

17 Melanoom FISH (CE-IVD) Percentage kernen met Afkapwaarde (30 kernen) Oordeel >2 RREB1 signalen per kern >29% Amplificatie Geen afwijkingen RREB1/CEP6 >1 per kern >55% Amplificatie >2 CCND1 signalen per kern >38% Amplificatie MYB/CEP6 <1 per kern >40% Deletie CCND1 amplificatie

18 TERT- Telomerase reverse transcriptase Low CSD High CSD Low to no CSD Pathway I II III IV V VI VII VIII IX Benign neoplasm Naevus? IMP? IMP Spitz naevus? Acral naevus? Melanosis CN Blue naevus? Naevus Intermediate Low grade dysplasia High grade dysplasia BIN DPN? IAMP? IAMP Atypical Spitz tumor IAMP / dysplasia Atypical melanosis / dysplasia / IAMPUS Nodule in CN Atypical CBN MELTUMP LM (MIS) MIS STUMP / MELTUMP Acral MIS Mucosal MIS MIS in CN Atypical CBN? BIM DPM PEM? Malignant neoplasm SMM (VGP) Melanoma in LMM (VGP) Desmoplastic BIN DPN PEM Malignant Spitz tumor / Spitz Acral (VGP) Mucosal lentiginous (VGP) Melanoma in CN Melanoma in BN Uveal Common mutations V600E or or RAS MAP2K1 or non V600E or NF1 NF1 amp ERBB2 amp MAP2K1 amp MAP3K1 amp EGFR amp MET amp HRAS rearr ALK rearr. ROS1 rearr. RET rearr. NTRK1 rearr. NTRK3 rearr. or MET rearr HRAS ALK rearr. NTRK1 rearr. or NF1 KRAS or rearr. V600 (small lesions) or rearr. GNAQ GNA11 or CYSLTR2 GNAQ GNA11 CYSLTR2 or PLCB4 Other alterations TP53 PTEN BAP1 CTNNB1 or APC PRKAR1 A or PRKCA TP53 PTEN RAC1 NFKBIE prom PIK3CA PTPN11 TERT amp CCND1 amp GAB2 amp NF1 SF3B1 CCND1 amp CDK4 amp MDM2 amp BAP1 EIF1AX SF3B1 BAP1 EIF1AX SF3B1

19 Oncotarget. 2017; 8(45): Digital droplet PCR (ddpcr) Flow cytometry-like analysis of PCR products (0/1) in 20k droplets

20 TERT promotor mutaties -HEX -FAMM -FAMM Cell. 2015; 161(7): : Alleen de C228T mutaties correleert met verhoogde TERT mrna expressie

21 Oncotarget. 2017; 8(45): TERT promoter ddpcr TERT C228T TERT wt TERT C250T 10% tumor DNA

22 WHO 2018 moleculaire classificatie van melanocytaire tumoren Low CSD High CSD Low to no CSD Pathway I II III IV V VI VII VIII IX Benign neoplasm Naevus? IMP? IMP Spitz naevus? Acral naevus? Melanosis CN Blue naevus? Naevus Low grade dysplasia BIN DPN? IAMP? IAMP Atypical Spitz tumor IAMP / dysplasia Atypical melanosis / dysplasia / IAMPUS Nodule in CN Atypical CBN? Intermediate High grade dysplasia MELTUMP LM (MIS) MIS STUMP / MELTUMP Acral MIS Mucosal MIS MIS in CN Atypical CBN? BIM DPM PEM Malignant neoplasm SMM (VGP) Melanoma in LMM (VGP) Desmoplastic BIN DPN PEM Malignant Spitz tumor / Spitz Acral (VGP) Mucosal lentiginous (VGP) Melanoma in CN Melanoma in BN Uveal Common mutations V600E or or RAS MAP2K1 or non V600E or NF1 NF1 amp ERBB2 amp MAP2K1 amp MAP3K1 amp EGFR amp MET amp HRAS rearr ALK rearr. ROS1 rearr. RET rearr. NTRK1 rearr. NTRK3 rearr. or MET rearr HRAS ALK rearr. NTRK1 rearr. or NF1 KRAS or rearr. V600 (small lesions) or rearr. GNAQ GNA11 or CYSLTR2 GNAQ GNA11 CYSLTR2 or PLCB4 Other alterations TP53 PTEN BAP1 CTNNB1 or APC PRKAR1A or PRKCA TP53 PTEN RAC1 NFKBIE prom PIK3CA PTPN11 TERT amp CCND1 amp GAB2 amp NF1 SF3B1 CCND1 amp CDK4 amp MDM2 amp BAP1 EIF1AX SF3B1 BAP1 EIF1AX SF3B1 Bold : Mutations identified in malignant lesions Red: Gain of function mutation Blue: Loss of function mutation Green: Change of function mutation

23 Moleculaire diagnostiek van melanocytaire tumoren Mutatie analyse + ampl. NGS,, HRAS, MAP2K1, GNAQ, GNA11,, PIK3CA, AKT1, EGFR, TP53, PTEN, RAF1, MAP3K1, CDK4, MDM2, CCND1, CTNNB1, MAP3K1, TERT, NF1, BAP1 Chromosomale veranderingen SNP array Whole genome Translocaties nanostring ALK, ROS1, RET, NTRK1-3, NGS, FISH 6q23.2 q23.3, 6p24, 11q13.3 FISH (multicolor) MYB del, RREB1 amp, CCND1 amp TERT promoter mutatie analyse ddpcr TERT C228T (-124C>T) en C250T (-146C>T)

24 Test portfolio voor aanvullende (moleculaire) diagnostiek van melanocytaire tumoren Pathology labs with a high volume of melanocytic tumours Expert centers Screening IHC MelanA, HMB45, SOX10, P16, Ki-67/MIB1, V600E, ALK1, ROS1, BAP1, Beta-catenin Basic sequencing panel exon 15, RAS exon 2 and 3 IHC PRKAR1A, Pan-TRK, MET, MITF, PNL2 other melanocytespecific antibodies Wide NGS mutation panel exon 11, exon 11, 13, 17 and 18, GNAQ exon 4 and 5, GNA11 exon 4 and 5, MAP2K1, CYSLTR2 L129Q, PLCB4, SF3B1, EIF1AX, NF1, BAP1, TERT FISH techniques 4 color test,, TERT, CCND1, MDM2 Gene fusion analysis ALK, ROS1, NTRK1, NTRK3, MAP3K8,, MET, RET, PRKCA Pangenomic CNV analysis

25 EORCT Melanoma Group recommendations (in preparation) Implementation of the WHO 2018 guidelines for the molecular analysis of melanocytic tumors in daily practice. Arnaud de al Fouchardière (Léon Bérard, FR) Willeke Blokx (UMCU, NL) Léon van Kempen (UMCG, NL) Susanna Puig (Barcelona, ESP) Daniela Massi (Univ of Florence, IT)