Future directions of Liquid Biopsy in monitoring of melanoma patients

Size: px

Start display at page:

Download "Future directions of Liquid Biopsy in monitoring of melanoma patients"

Buck Armstrong
5 years ago
Views:

1 Future directions of Liquid Biopsy in monitoring of melanoma patients Verena Haselmann, MD PhD Institute for Clinical Chemistry University of Mannheim, Germany

2 Circulating tumor DNA (ctdna)

3 ctdna - advantages High compliance Minimal invasive, few risks High availability Archival tissue, degraded, different mutation profile Patient/Oncologist Physician Specialist e.g. Surgeon Accessible No selection bias Assess primary tumor and metastases with one sample, tumor heterogeneity not a problem Monitoring possible Pathology Clinical Laboratory Real-time

Future development: MRD 1st line: Tumor Tissue (FFPE)

on a regular basis Analyis of biomarkers and liquid

4 Future development: MRD 1st line: Tumor Tissue (FFPE) Targeted resequencing (NGS) only performed once Identification of an individual tumor mutational profile 2nd line: Plasma Follow up for MRD-Diagnostic - performed on a regular basis Analyis of biomarkers and liquid profiling of individual tumor mutations Reduce lead time compared to imaging?

Future development: Companion Diagnostics 1st line: Plasma Targeted reanalysis of ctdna Identification of pharmacogentic relevant tumor mutations in real-time Follow up of the identified mutations

5 Future development: Companion Diagnostics 1st line: Plasma Targeted reanalysis of ctdna Identification of pharmacogentic relevant tumor mutations in real-time Follow up of the identified mutations for monitoring of treatment response Gene Mutation BRAF BRAF V600E (T1799A) BRAF V600K (GT1798_1799AA) EGFR EGFR L858R (T2573G) EGFR Del 19 (including E746_S752>V ( D) EGFR L747_A750>P ( D) EGFR L747_T751del ( D) EGFR L747_P753>S ( D) EGFR E746_A750del* ( D, D) KRAS G12S (G34A) KRAS KRAS G12R (G34C) KRAS G12C (G34T) KRAS G12D (G35A) KRAS G12A (G35C) KRAS G12V (G35T) KRAS G13D (G38A) KRAS G61H (A183C/T) NRAS NRAS Q61K (C181A) NRAS Q61R (A182G) NRAS Q61L (A182T) NRAS Q61H (A183T) PIK3CA PIK3CA E542K (G1624A) PIK3CA E545K (G1633A) PIK3CA E545G (A1634G) PIK3CA Q546K (C1636A) PIK3CA M1043I (G3129T) PIK3CA H1047Y (C3139T) PIK3CA H1047R (A3140G) PIK3CA H1047L (A3140T)

ctdna technical challenges Netherlands; 2 Denmark; 1 Italy; 3 Switzerland; 1

$Netherlands Austria Switzerland Czech Republic ctdna fraction very low E.g.$ less than 0.02 % Risk of contamination E.g.

6 ctdna technical challenges Netherlands; 2 Denmark; 1 Italy; 3 Switzerland; 1 Austria; 4 Belgium; 5 Highly fragmented Mean size bp Czech Republic; 2 Germany France; 4 Germany; 12 Greece; 3 Greece France Belgium Italy Denmark Netherlands Austria Switzerland Czech Republic ctdna fraction very low E.g. less than 0.02 % Risk of contamination E.g. with wildtype DNA from leukocytes National Cancer Institute Schwarzenbach et al 2011, Nat Rev Cancer Thiery et al 2013, Nat Medicine

ctdna detection methods Detection Capability (mutant DNA/ total DNA) 100% 10% Sanger Sequencing Pyrosequencing 1% Real-time PCR Next Generation Sequencing 0.

7 ctdna detection methods Detection Capability (mutant DNA/ total DNA) 100% 10% Sanger Sequencing Pyrosequencing 1% Real-time PCR Next Generation Sequencing 0.1% COLD-PCR, ASA, ARMS, % BEAMing, ddpcr, TAmSeq, SafeSeq,... Li et al. 2006, Nat Methods Li et al. 2008, Nat Medicine Forshew et al Sci Trans Med

8 ctdna detection methods Detection Capability (mutant DNA/ total DNA) 100% 10% Sanger Sequencing Pyrosequencing 1% Real-time PCR Next Generation Sequencing 0.1% COLD-PCR, ASA, ARMS, % BEAMing, ddpcr, TAmSeq, SafeSeq,... Li et al. 2006, Nat Methods Li et al. 2008, Nat Medicine Forshew et al Sci Trans Med

9 Study design pharmacogenetic target Hodis et al., Cell. 2012

10 Study design pharmacogenetic target Hodis et al., Cell. 2012

11 Study design detection method sysmex.co.jp

12 Study design validation of detection method

13 Study design currently available study data Yankovitz et al., 2007, Journal of Molecular Diagnostics: 26 stage IV melanoma patients, concordance 59%, ASA, blood-samples stored on ice before plasmaseparation, thawed plasma was centrifuged and the pellet (gdna from leukocytes) was used for analysis Aung et al., 2009, Journal of Molecular Diagnostics: 221 stage IV melanoma patients, specificity 94%, sensitivity 42%, ARMS, no information regarding blood-draw, partly used serum Board, 2009, British Journal of Cancer: 96 stage IV melanoma patients, concordance 56%, serum, ARMS Pinzani et al., 2010, Clinica Chimica Acta: 55 melanoma patients, different stages, concordance 80%, selfdeveloped real-time-based approach, assay sensitivity 0.03% Fusi et al, 2011, European Journal of Cancer: 21 stage IV melanoma patients, concordance 91%, selfdeveloped assay, no information regarding pre-analytical sample handling Ascierto et al, 2013, Clinical Oncology: 76 stage IV melanoma patients, sensitivity 79%, specificity 100%, BEAMing, no information regarding pre-analytical sample handling, assay conducted by company Lipson et al., 2014, Journal for ImmunoTherapy of Cancer: 12 stage IV melanoma patients, BEAMing, ctdna versus imaging Tsao et al., 2015, Scientific Reports: 6 stage IV melanoma patients, ddpcr, ctdna versus LDH Sanmamed et al., 2015, Clinical Chemistry: 20 melanoma patients under BRAFi, concordance 84%, ddpcr, only one centrifugation step, too high g-forces A. Santiago-Walker et al., 2015, Clinical Cancer Research: 732 melanoma patients, different stages, e.g. stage IV, concordance 76%, BEAMing, no information regarding pre-analytics, assay conducted by company Gonzalez-Cao et al., 2015, Melanoma Research: 92 stage III and IV melanoma patients, sensitivity 57%, specificity 100%, serum and plasma, self-developed assay, no information regarding storage duration until plasma separation Gray et al, 2015, Oncotarget: 48 stage IV melanoma patients, concordance 65%, ddpcr, plasma separation within 24h, different plasma volumes Schreuer et al, 2016, Journal of Translational Medicine: 35 stage IV melanoma patients, sensitivity 75%, no wild-type patients included, ASA, 1ml plasma used, 1 centrifugation step

14 Study design currently available study data Yankovitz et al., 2007, Journal of Molecular Diagnostics: 26 stage IV melanoma patients, concordance 59%, ASA, blood-samples stored on ice before plasmaseparation, thawed plasma was centrifuged and the pellet (gdna from leukocytes) was used for analysis Aung et al., 2009, Journal of Molecular Diagnostics: 221 stage IV melanoma patients, specificity 94%, sensitivity 42%, ARMS, no information regarding blood-draw, partly used serum Board, 2009, British Journal of Cancer: 96 stage IV melanoma patients, concordance 56%, serum, ARMS Pinzani et al., 2010, Clinica Chimica Acta: 55 melanoma patients, different stages, concordance 80%, selfdeveloped real-time-based approach, assay sensitivity 0.03% Fusi et al, 2011, European Journal of Cancer: 21 stage IV melanoma patients, concordance 91%, selfdeveloped assay, no information regarding pre-analytical sample handling Ascierto et al, 2013, Clinical Oncology: 76 stage IV melanoma patients, sensitivity 79%, specificity 100%, BEAMing, no information regarding pre-analytical sample handling, assay conducted by company Lipson et al., 2014, Journal for ImmunoTherapy of Cancer: 12 stage IV melanoma patients, BEAMing, ctdna versus imaging Tsao et al., 2015, Scientific Reports: 6 stage IV melanoma patients, ddpcr, ctdna versus LDH Sanmamed et al., 2015, Clinical Chemistry: 20 melanoma patients under BRAFi, concordance 84%, ddpcr, only one centrifugation step, too high g-forces A. Santiago-Walker et al., 2015, Clinical Cancer Research: 732 melanoma patients, different stages, e.g. stage IV, concordance 76%, BEAMing, no information regarding pre-analytics, assay conducted by company Gonzalez-Cao et al., 2015, Melanoma Research: 92 stage III and IV melanoma patients, sensitivity 57%, specificity 100%, serum and plasma, self-developed assay, no information regarding storage duration until plasma separation Gray et al, 2015, Oncotarget: 48 stage IV melanoma patients, concordance 65%, ddpcr, plasma separation within 24h, different plasma volumes Schreuer et al, 2016, Journal of Translational Medicine: 35 stage IV melanoma patients, sensitivity 75%, no wild-type patients included, ASA, 1ml plasma used, 1 centrifugation step

15 Study design currently available study data Yankovitz et al., 2007, Journal of Molecular Diagnostics: 26 stage IV melanoma patients, concordance 59%, ASA, blood-samples stored on ice before plasmaseparation, thawed plasma was centrifuged and the pellet (gdna from leukocytes) was used for analysis Aung et al., 2009, Journal of Molecular Diagnostics: 221 stage IV melanoma patients, specificity 94%, sensitivity 42%, ARMS, no information regarding blood-draw, partly used serum Board, 2009, British Journal of Cancer: 96 stage IV melanoma patients, concordance 56%, serum, ARMS Pinzani et al., 2010, Clinica Chimica Acta: 55 melanoma patients, different stages, concordance 80%, selfdeveloped real-time-based approach, assay sensitivity 0.03% Fusi et al, 2011, European Journal of Cancer: 21 stage IV melanoma patients, concordance 91%, selfdeveloped assay, no information regarding pre-analytical sample handling Ascierto et al, 2013, Clinical Oncology: 76 stage IV melanoma patients, sensitivity 79%, specificity 100%, BEAMing, no information regarding pre-analytical sample handling, assay conducted by company Lipson et al., 2014, Journal for ImmunoTherapy of Cancer: 12 stage IV melanoma patients, BEAMing, ctdna versus imaging Tsao et al., 2015, Scientific Reports: 6 stage IV melanoma patients, ddpcr, ctdna versus LDH Sanmamed et al., 2015, Clinical Chemistry: 20 melanoma patients under BRAFi, concordance 84%, ddpcr, only one centrifugation step, too high g-forces A. Santiago-Walker et al., 2015, Clinical Cancer Research: 732 melanoma patients, different stages, e.g. stage IV, concordance 76%, BEAMing, no information regarding pre-analytics, assay conducted by company Gonzalez-Cao et al., 2015, Melanoma Research: 92 stage III and IV melanoma patients, sensitivity 57%, specificity 100%, serum and plasma, self-developed assay, no information regarding storage duration until plasma separation Gray et al, 2015, Oncotarget: 48 stage IV melanoma patients, concordance 65%, ddpcr, plasma separation within 24h, different plasma volumes Schreuer et al, 2016, Journal of Translational Medicine: 35 stage IV melanoma patients, sensitivity 75%, no wild-type patients included, ASA, 1ml plasma used, 1 centrifugation step

16 Study design currently available study data Yankovitz et al., 2007, Journal of Molecular Diagnostics: 26 stage IV melanoma patients, concordance 59%, ASA, blood-samples stored on ice before plasmaseparation, thawed plasma was centrifuged and the pellet (gdna from leukocytes) was used for analysis Aung et al., 2009, Journal of Molecular Diagnostics: 221 stage IV melanoma patients, specificity 94%, sensitivity 42%, ARMS, no information regarding blood-draw, partly used serum Board, 2009, British Journal of Cancer: 96 stage IV melanoma patients, concordance 56%, serum, ARMS Pinzani et al., 2010, Clinica Chimica Acta: 55 melanoma patients, different stages, concordance 80%, selfdeveloped real-time-based approach, assay sensitivity 0.03% Fusi et al, 2011, European Journal of Cancer: 21 stage IV melanoma patients, concordance 91%, selfdeveloped assay, no information regarding pre-analytical sample handling Ascierto et al, 2013, Clinical Oncology: 76 stage IV melanoma patients, sensitivity 79%, specificity 100%, BEAMing, no information regarding pre-analytical sample handling, assay conducted by company Lipson et al., 2014, Journal for ImmunoTherapy of Cancer: 12 stage IV melanoma patients, BEAMing, ctdna versus imaging Tsao et al., 2015, Scientific Reports: 6 stage IV melanoma patients, ddpcr, ctdna versus LDH Sanmamed et al., 2015, Clinical Chemistry: 20 melanoma patients under BRAFi, concordance 84%, ddpcr, only one centrifugation step, too high g-forces A. Santiago-Walker et al., 2015, Clinical Cancer Research: 732 melanoma patients, different stages, e.g. stage IV, concordance 76%, BEAMing, no information regarding pre-analytics, assay conducted by company Gonzalez-Cao et al., 2015, Melanoma Research: 92 stage III and IV melanoma patients, sensitivity 57%, specificity 100%, serum and plasma, self-developed assay, no information regarding storage duration until plasma separation Gray et al, 2015, Oncotarget: 48 stage IV melanoma patients, concordance 65%, ddpcr, plasma separation within 24h, different plasma volumes Schreuer et al, 2016, Journal of Translational Medicine: 35 stage IV melanoma patients, sensitivity 75%, no wild-type patients included, ASA, 1ml plasma used, 1 centrifugation step

17 Study design pre-analytics K3 EDTA-blood Plasma separation within 6h (2. centrifugation steps, 1st at 1600g, 2nd at 3000g) Storage plasma at -80 C 2ml EDTA-plasma for each analysis Isolation cfdna with Qiagen Circulating Nucleic Acid Kit Storage isolated cfdna at -20 C Quantification isolated ctdna with LINE1 real-time PCR Mutational analysis with BEAMing (including negative, positive and non-template control)

18 Study design cohorts Study 1: 1216 prospectively collected EDTA blood-samples from 441 melanoma patients Cohort Mannheim: stage III and IV melanoma patients, mutational data from archival tumor specimens being available Cross-section cohort: all stages BRAFi cohort: patients undergoing active or imminent therapy with BRAFi and MEKi Study 2: comprised 71 retrospectively analyzed EDTA blood samples from metastatic melanoma patients

19 1. Concordance study Cohort Mannheim Cohort Essen Number of Patients n=136 (%) n=71 (%) sex m 80 (58.8) 46 (64.8) f 56 (41.2) 25 (35.2) Age Median 65 63,3 range Stage IV 72 (52.9) 71 (100) IIIc 13 (9.6) 0 (0) IIIb 29 (21.3) 0 (0) IIIa 22 (16.2) 0 (0) Tissue-based analysis BRAF V600E mutation 62 (45.6) 27 (38.0) other BRAF mutation 4 (2.9) 9 (12.7) NRAS mutation 11 (8.1) 12 (16.9) ckit mutation 4 (2.9) n.a. no mutation 55 (40.4) 23 (32.4) Plasma-based number of samples analysis analysed mean number of tests per patient BRAF V600E mutation 40 (29.4) 25 (35.2) no mutation 96 (70.6) 46 (64.8)

20 1. Concordance study Cohort Mannheim Cohort Essen Number of Patients n=136 (%) n=71 (%) Plasma-based analysis Positive for BRAFV600E 34 (25.0) 24 (33.8) Negative for BRAFV600E 92 (67.6) 42 (59.2) False-negative* 5 (3.7) 4 (5.6) False positive# 5 (3.7) 1 (1.4) Statistical analysis Positive agreement 34/39 (87.2) 24/28 (85.7) Negative agreement 92/97 (94.8) 42/43 (97.7) Overall agreement 126/136 (92.6) 66/71 (92.3) kappa 0,82 0,85 SE of kappa 0,055 0,064 95% CI from to from to Number of observed agreements 126 (92.65%) 66 (92.96%) Number of aggreemnets expected by chance 80.4 (59.09%) 37.7 (53.12%) *wildtype instead of BRAFV600E mutant #BRAFV600E mutant instead of wildtype results considered concordant according to clinical situation at the time of the blood draw for Cohort Mannheim

21 1. Concordance study Patients of interest Number of patients n=24 (%) Beaming negative with known tissue BRAF mutation* post resection of melanoma at time of blood draw 14 (58.3) CR/PR under treatment at time of blood draw 6 (25.0) Beaming positive with known tissue BRAF wildtype status at time of blood draw* Secondary BRAF mutant neoplasia 2 (8.3) Secondary BRAF mutant melanoma False negative histologic examination 1 (4.2) 1 (4.2) *results adjusted according to clinical situation at time of blood draw

22 2. Cross-section study Cohorte crosssection analysis Number of Patients n=440 (%) sex m 239 (54.3) f 201 (45.7) Age Median 66 range Stage IV 65 (14.8) IIIc 22 (5.0) IIIb 51 (11.6) IIIa 44 (10.0) Iic 8 (1.8) Iib 33 (7.5) Iia 44 (10.0) Ib 99 (22.5) Ia 68 (15.5) other skin tumor 6 (1.4) Tissue-based analysis BRAF V600E mutation 83 (18.9) other BRAF mutation 4 (0.9) NRAS mutation 11 (2.5) ckit mutation 4 (0.9) no mutation 80 (18.2) n.a. 258 (58.6) Plasma-based analysis number of samples analysed 1211 mean number of tests per patient 2,8 BRAF V600E mutation 57 (13.0) no mutation 383 (87.0) Melanoma subtype cutaneous n.a. mucosal n.a. occult n.a. other n.a. n.a. n.a.

23 2. Cross-section study Cohort cross-section analysis Number of Patients n=440 (%) Plasma-based analysis Positive for BRAFV600E 39 (8.9) Negative for BRAFV600E 377 (85.7) False-negative* 6 (1.4) False positive# 18 (4.1) Statistical analysis Positive agreement 39/45 (86.7) Negative agreement 377/395 (95.4) Overall agreement 416/440 (94.5) kappa 0,734 SE of kappa 0,051 *wildtype instead of BRAFV600E mutant #BRAFV600E mutant instead of wildtype 95% CI from to Number of observed agreements 416 (94.55%) Number of aggreemnets expected by chance (79.47%) *results adjusted according to clinical situation at time of blood draw

24 2. Cross-section study Patients of interest Number of patients n=34 (%) Beaming negative with known BRAF mutation* post resection of melanoma at time of blood draw CR/PR under treatment at time of blood draw 20 (58.8) 6 (17.6) Beaming positive with known BRAF wildtype status at time of blood draw* Secondary BRAF mutant neoplasia 2 (5.9) Secondary BRAF mutant melanoma 5 (14.7) False negative histologic examination 1 (2.9)

25 3. BRAFi study BRAF before treatment 15 weeks of treatment Chapman et al 2011, NEJM Hirth et al. 2012, Nat Drug Discov Wagle et al., JCO 2011

26 3. BRAFi study BRAF before treatment 15 weeks of treatment relapse 23 weeks of therapy Chapman et al 2011, NEJM Hirth et al. 2012, Nat Drug Discov Wagle et al., JCO 2011

27 3. BRAFi study BRAFi cohort Number of Patients n=35 (%) sex Men 18 (51 4) Women 17 (48 6) Age Median 59 4 range Stage IIIC (unresectable) 4 (11 4) Stage IV 31 (88 6) M1a 1 (2 9) M1b 4 (11 4) M1c 26 (74 3) LDH 20 (57 1) Brain metastases 18 (51 4) BRAF status V600E 35 (100) BEAMing positive 35 (100) Plasma-based analysis number of samples analysed 134 mean number of tests per patient 3 83 Mean cfdna level (GE/ml) 95,540 Mean ctdna level (GE/ml) 2,789 Mean ctdna fraction (%) 1 27 BRAFi/MEKi treatment V+C 7 (20 0) D+T 10 (28 6) V mono 15 (42 9) D mono 3 (8 6) Median Duration of Treatment (months) 17 7 Median Time to Resistance (months) 9 1 Median Overall Survival (95% CI) (months) 23 0 ( ) Line of Treatment First-line 32 (91 4) Second-line 2 (5 7) Third-line 1 (2 9) Best overall response Complete response 4 (11 4) Partial response 11 (31 4) Stable disease 9 (25 8) Progressive disease 11 (31 4)

Tumor S-100 volume µg/l mm LDH S-100 U/l µg/l LDH U/l Tumor volume mm Study 3: BRAFi Mutant fragments per ml plasma Case 1: Patient #52 Patient #52 500 1 BRAFi therapy 3 400 300 2 200 100 80 60 40 20

(brain) 100 0 120-100 0 100 200 300 400 Time since BRAFi in days Mutant Mutant fragments fragments per ml per plasma ml plasma Mutant fragments per ml plasma Mutant fragments per ml plasma Patient

20 300 100 100 0.05 300 200 160 0 0 0.15 0.00 0-100 -100 00 100 200 300 400 400 200 100 Time since BRAFi in days 140 0.10 1 2 3 3 0 Time since BRAFi in days 200 180 160 140 100 0 0.

28 Tumor S-100 volume µg/l mm LDH S-100 U/l µg/l LDH U/l Tumor volume mm Study 3: BRAFi Mutant fragments per ml plasma Case 1: Patient #52 Patient # BRAFi therapy Time since BRAFi in days Mutant fragments per ml plasma Patient # BRAFi therapy Mutant fragments 400 per ml plasma Tumor volume mm 300 (complete) 200 Tumor volume mm (brain) Time since BRAFi in days Mutant Mutant fragments fragments per ml per plasma ml plasma Mutant fragments per ml plasma Mutant fragments per ml plasma Patient #52 Patient # BRAFi therapy Mutant fragments Patient # per ml plasma Patient # LDH Time since BRAFi in days Time since BRAFi in days Time since BRAFi in days Mutant fragments per Mutant ml plasma fragments per ml plasma Tumor volume mm (complete) S-100 Tumor Mutant volume fragments mm (brain) Mutant per ml plasma fragments per LDHml plasma S-100

29 3. BRAFi study Plasma-based testing versus imaging techniques: Mean lead time reduction: 110 days (in cases a PD was detected earlier: 170 days)

Acknowledgement Institut für Klinische Chemie, UMM Prof. Dr.

Sysmex, Hamburg Prof. Dr. Harmut Juhl Dr. Frank Diehl Dr.

Jones Klinik für Dermatologie, UMM DFKZ, Heidelberg Prof. Dr.

30 Acknowledgement Institut für Klinische Chemie, UMM Prof. Dr. Michael Neumaier Ingrid Brechtel Angelika Duda Maximillian Kittel Sysmex, Hamburg Prof. Dr. Harmut Juhl Dr. Frank Diehl Dr. Barbara Behrens Dr. Frederick S. Jones Klinik für Dermatologie, UMM DFKZ, Heidelberg Prof. Dr. Jochen Utikal Dr. Christoffer Gebhardt Dr. Tim Holland-Letz Klinik für Dermatologie, UK-Essen Prof. Dr. Dirk Schadendorf Dr. Antje Sucker

Disclosure. Summary. Circulating DNA and NGS technology 3/27/2017. Disclosure of Relevant Financial Relationships. JS Reis-Filho, MD, PhD, FRCPath

Disclosure. Summary. Circulating DNA and NGS technology 3/27/2017. Disclosure of Relevant Financial Relationships. JS Reis-Filho, MD, PhD, FRCPath Circulating DNA and NGS technology JS Reis-Filho, MD, PhD, FRCPath Director of Experimental Pathology, Department of Pathology Affiliate Member, Human Oncology and Pathogenesis Program Disclosure of Relevant