Predictive Biomarker Testing in Cytology and Small Biopsy Specimens. Natasha Rekhtman, MD, PhD Memorial Sloan Kettering Cancer Center New York, NY

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1 Predictive Biomarker Testing in Cytology and Small Biopsy Specimens Natasha Rekhtman, MD, PhD Memorial Sloan Kettering Cancer Center New York, NY

2 Outline Small thoracic specimen acquisition: What s new? Specifics of biomarker testing on cytology State of sufficiency of small samples for molecular testing: Clinical experience Practical solutions for doing more with less

3 Outline Small thoracic specimen acquisition: What s new? Bronchoscopic Percutaneous (transthoracic) Specifics of biomarker testing on cytology State of sufficiency of small samples for molecular testing: Clinical experience Practical solutions for doing more with less

4 Bronchoscopic samples: Conventional New EBUS core needle (Cook Medical): Yield TBD Forceps biopsy (endo & trans bronchial) Conventional (Wang) trans bronchial needle aspiration Endobronchial ultrasound (EBUS) trans bronchial needle aspiration + brush/wash/lavage Until recently: only FNAs treferences/esc WM EN pdf

5 Bronchoscopic samples: Other recent developments Electromagnetic navigational bronchoscopy Cryobiopsy real time GPS like navigation system, which guides the locatable sensor to the lesion allows sampling of peripheral lesions tissue adheres to cryoprobe after freezing/thawing cycles biopsy diameters >1 cm reported Weiser TS et al Ann Thorac Surg Poletti V et al Respirology. 2014

6 Bronchoscopy Guidelines: increased tissue acquisition for biomarker testing is widely advocated Respiration 2014;88: Standard recommendations include: EBUS TBNA: at least 3 passes per positive LN station

7 Transthoracic (percutaneous) samples Dominant Bx type (85% of small Acquisition of multiple samples and sample size limited by concerns for complications: Pneumothorax: ~30% of procedures; <10% require aspiration/chest tube Pulmonary hemorrhage: 5 15% of procedure Hemothorax: <2% Techniques vary widely by operator and institution: Sample type Core biopsy vs FNA vs both Number of passes Instruments Single needle vs coaxial (stationary guide needle + sampling needle) Needle gauge

8 Interventional Radiology guidelines for thoracic specimen acquisition:

9 Interventional Radiology guidelines for thoracic specimen acquisition:

10 Interventional Radiology guidelines for thoracic specimen acquisition: void in guidelines (or best practice recommendation ) regarding the need for greater tissue acquisition (when clinically feasible)

11 CT guided core MSKCC 2012 (representative case) 2015 (representative case)

12 Specifics of biomarker testing on cytology Air dried, Diff Quik (Wright Giemsa) stained smears Needle rinse EtOH fixed, Papanicolaou (Pap) stained smears Liquid based (ThinPrep TM, Hologic; SurePath TM, BD Diagnostics) variety of collection media (CytoLyt, SurePath, Formalin, RPMI, etc) Cell block (paraffin embedded)

13 Specifics of biomarker testing on cytology Air dried, Diff Quik (Wright Giemsa) stained smears Extracted DNA based FISH Needle rinse EtOH fixed, Papanicolaou (Pap) stained smears Liquid based (ThinPrep TM, Hologic; SurePath TM, BD Diagnostics) variety of collection media (CytoLyt, SurePath, Formalin, RPMI, etc) Cell block (paraffin embedded)

14 Specifics of biomarker testing on cytology Air dried, Diff Quik (Wright Giemsa) stained smears Extracted DNA based FISH Needle rinse EtOH fixed, Papanicolaou (Pap) stained smears Liquid based (ThinPrep TM, Hologic; SurePath TM, BD Diagnostics) variety of collection media (CytoLyt, SurePath, Formalin, RPMI, etc) Cell block (paraffin embedded)

15 Molecular testing on cytology: smears vs cell blocks Smears: Pros: No formalin fixation (no DNA cross linking) better DNA quality FISH: no nuclear truncation true number of FISH signals/nucleus 1 Cons: Destroys original slide (medico legal issues CLIA slide retention policy, though exemption in CAP 14) IHC suboptimal on smears cell block still usually needed Some commercial labs only accept paraffin embedded material Cell blocks: Pros: Operational simplicity: and same ALK workflow testing, as with surgical cell specimens blocks being (serial recuts) No destruction of diagnostic slides Cons: next slide Arch Pathol Lab Med : Expert Consensus Opinion. Cytologic samples are suitable for EGFR preferred over smear preparations. 1. Savic and Bubendorf Acta Cytol. 2012

16 The state of cell block variation and satisfaction in the era of molecular diagnostics and personalized medicine. Crapanzano JP et al Saqi A. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY Cytojournal Mar 20;11:7 Survey of 90 pathologists and cytotechnologists (37 academic, 40 community/private, 6 commercial lab, 7 other) Over 10 methods utilized in practice: Plasma thrombin (33%) HistoGel (27%) Cellient (8%) Other (including Zinc formalin known to interfere with molecular assays) 44% of responders reported being dissatisfied with their cell block method low/inconsistent cellularity reported as #1 cause other reported causes process perceived as time consuming, labor intensive, subject to interoperator variability, etc

17 Improving Cell Blocks: MSKCC experience Standard HistoGel method was used in practice, and results were highly variable particularly for scant FNAs Modified method: HistoGel + 95% EtOH + other (manuscript in preparation) Ex vivo split samples: Standard method Modified method Clinical experience: Consecutive FNAs Standard method (n=100) Modified method (n=100) 82% 97% Cellular cell blocks *sufficient for at least some molecular studies (data shown only for positive FNAs) P Mean >8 fold increase in cellularity with modified method Rudomina, Rekhtman, Lin ACS 2013

18 Testing for IHC based biomarkers on cytology Standard IHC validated is on FFPE tissue Alcohol fixation may affect antigenicity: MIB1 antibodies either incompatible or inhibited by CytoLyt (Konno, Rekhtman. USCAP 2015) PTEN (clone 138G6; Cell Signaling) appears to be inhibited by CytoLyt (Pandya, Moreira. USCAP 2015) Studies show equivalence in alcohol fixed cytologic samples: 1 ALK (D5F3) EGFR L858R EGFR Ex19 All IHC biomarkers must be separately validated on non formalin fixed cytologic preparations. 1. Moreira, Hasanovic. Acta Cytol 2012

19 State of sufficiency of small thoracic samples for molecular studies: Clinical experience Rekhtman et al J Thorac Oncol Many studies report on success rates of molecular testing in small specimens received in molecular laboratories, but this does not take into account upfront drop off due to insufficiency as decided at the time of diagnostic evaluation Key question is sufficiency rates in consecutive samples

20 Clin Cancer Res 2013 Re biopsy protocol at the time of acquired resistance to erlotinib ( ) N=162 samples (155 pts) Resections (24; 15%) Core (87; 54%) FNA (35; 22%) Effusion (16; 9%) USS DNA extraction Fragment analysis for Ex 19 Sequenom (8 gene multiplex) Sufficiency: 95% Insufficient: Resection: 2/24 (8%) Core 4/87 (5%) FNA: 1/35 (3%) MET FISH Sufficient: 46%

21 Lung Cancer Mutation Consortium (LCMC) Prospective multi institutional (14 site) study; 1007 patients 10 biomarker analysis of advanced stage lung adenocas Dates: Goal: Establish frequency, treat based on genotype, analyze survival (feasibility of testing) Methods Multiplex genotyping (Sequenom, SNaPshot, Sanger) for mutations in 8 genes (EGFR, KRAS, BRAF, MEK1, ERBB2, PIK3CA, AKT1, NRAS) Sizing electrophoresis for EGFR deletions and HER2 (ERBB2) insertions FISH for ALK rearrangements and MET amplification

22 Patients with stage IV or recurrent adenoca Unstained tissue not available (28%) Sufficiency rates 91% Sholl et al J Thor Oncol Epub 2015: Failure rate by specimen type (based on 470 specimens from a single site): Resection (268): 5% Biopsy (136): 26% Cytology (66): 35% 82% 76% 67%

23 Clinical experience: Sufficiency in consecutive samples Reference Site N Small sample type Yu et al Clin Cancer Res 2013 Arcila et al Clin Cancer Res 2011 Kris et al JAMA 2014 Sequist et al Ann Oncol 2011 Cardarella et al J Thor Oncol 2012 Kim et al Cancer Discovery 2011 Coley et al Ca Cytopathol 2015 Schneider et al AJCP 2015 Feretti et al Lung Ca 2013 MSKCC 162 (155 pts) MSKCC 153 (121 pts) LCMC (14 sites) 87 CNB 35 FNA 16 effusion 95 CNB 14 FNA 34 effusion Assays Sufficiency for molecular Sequenom, sizing, FISH CNB: 95% FNA: 97% Sequenom, sizing, FISH CNB: 89% FNA: 79% Effusion: 71% 1007? type Molecular (multiplex), sizing (2), FISH (2) BWH/DF % small (? type) MDACC (BATTLE) Columbia U Sufficiency for 2+ platforms 46% 31% 82 91% overall 67% 255 CNB Sequencing (3), FISH (2), IHC (6) 83% CNB 18 FNA UPMC CNB 120 FNA Grenoble, France Bottom line: MGH Overall, 589insufficiency 63% small rate SNaPshot, of small ALK FISHthoracic specimens 94% for 89% multi assay testing is (? type) substantial, and increase in the number of tested Sanger (5),ALK FISH 81% overall 74% biomarkers by separate assays is not sustainable. ARMS, ALK FISH CNB: 95% FNA: 94% Sequencing (2), ALK FISH CNB: 67% FNA: 57% CNB: 67% FNA: 46% 91 CNB Pyrosequencing (2), ALK FISH 72% ( ) 92% ( )*

24 Next generation sequencing (NGS) on thoracic biopsies and cytology NGS = breakthrough technology, which consolidates multi assay testing into a single platform (detects multiple mutations, deletions and copy number alterations in a single assay). MSK IMPACT TM (Integrated Mutation Profiling of Actionable Cancer Targets): 410 gene Illumina HiSeq capture based platform in clinical use at MSKCC since May Early MSKCC experience with NGS on lung carcinoma samples (courtesy of Maria Arcila, MD): Lung cancer samples (n=244) % Failure rates (for specimens received in molecular lab) Mean DNA yield (Qubit) Resection: 30% 3% 2.8 ug (3 5 slides) Small Bx: 49% 7% 0.8 ug (15 20 slides) Cytology: 21% 12% 0.6 ug (15 20 slides)

25 NGS on thoracic biopsies and cytology: Data in consecutive samples Hagemann et al Cancer 2015 Scarpa et al PLoS 2014 Site Platform N Cell block vs smears vs other Wash U Illumina 381 consecutive cases 100 CNB 33 FNA U Verona, Italy Ion Torrent 38 consecutive trans thoracic FNAs DNA input? 150ng 21 smears 17 fresh (FineFix) 10ng SUFFICIENCY RATE CNB: 60% sufficient FNA: 33% sufficient 5% insufficient

26 NGS on thoracic biopsies and cytology: Data in consecutive samples Hagemann et al Cancer 2015 Scarpa et al PLoS 2014 Site Platform N Cell block vs smears vs other Wash U Illumina 381 consecutive cases 100 CNB 33 FNA U Verona, Italy Ion Torrent 38 consecutive trans thoracic FNAs DNA input? 150ng 21 smears 17 fresh (FineFix) 10ng SUFFICIENCY RATE CNB: 60% sufficient FNA: 33% sufficient 95% sufficient

27 Practical solutions for doing more with less : optimizing specimen processing

28 acquisi on Sections for diagnosis (H&E, IHC, etc) Sections for molecular and FISH

29 acquisi on optimizing on site assessment Sections for diagnosis (H&E, IHC, etc) Sections for molecular and FISH

30 On site assessment for small specimens FNAs: on site assessment of FNAs smears is a well established practice, which decreases non diagnostic rate by up to 22% 1. Biopsies: Recently, touch preparations (aka imprints) have gained popularity for CT guided cores. 1. Santambrogio L et al Chest 1997.

31 Depletion of tumor cells from core biopsies by vigorous touch preps Touch Prep Residual core MSKCC clinical experience: complete depletion in 9/342 (3%) of lung cores. 1 Other recent series: complete depletion in 9/140 (8.2%) of cores Tong LC, Rudomina D, Rekhtman N, Lin O. Cancer Cytopathol Moghadamfalahi M et al Cytojournal. 2014

32 Touch Preps: Ex vivo study Increase of cell (and DNA) content with progressively more vigorous Touch Preps TP cell # as % of core p= (n=14 each) Up to 1.3 g of DNA Bottom line: critical to avoid excessively forceful touch preps + important to procure additional cores/fnas whenever clinically feasible Rekhtman, Kazi et al Archives of Pathology Epub 2014

33 acquisi on loss of cellularity for touch preps Recuts for diagnosis (H&E, IHC, other) Recuts for molecular and FISH

34 Separate embedding of multiple cores Diagnostic core Molecular core

35 acquisi on loss of cellularity for touch preps separate embedding of multiple cores # of upfront H&E sections 1 # of times block is faced (preparing USS upfront or paired with IHC) 1 diagnostic IHC to most efficient panels 2 1. Travis WD, Rekhtman N. Semin Respir Crit Care Med Rekhtman N et al Mod Pathol 2011

36 acquisi on loss of cellularity for touch preps separate embedding of multiple cores # of upfront H&E sections 1 # of times block is trimmed (preparing USS upfront or paired with IHC) 1 diagnostic IHC to most efficient panels 2 1. Travis WD, Rekhtman N. Semin Respir Crit Care Med Rekhtman N et al Mod Pathol 2011

37 +FNA acquisi on loss of cellularity for touch preps separate embedding of multiple cores # of upfront H&E sections # of times block is faced (preparing USS upfront or paired with IHC) diagnostic IHC to most efficient panels sensi vity, DNA input pla orms

38 Conclusions Biomarker testing on small thoracic specimens presents great challenges, but also great opportunities to evolve established processes, setting the paradigm for other organ systems Cytology is an underused and undervalued resource for molecular testing, but wider adoption requires optimization of cell blocks and an update in regulatory policies on smear retention Increasing sufficiency of small samples for biomarker testing requires multidisciplinary effort from specimen acquisition to handling in pathology laboratories to evolution of molecular technology to higher sensitivity/low DNA input platforms

39 Thanks to Thoracic pathology & cytopathology William D. Travis Andre L. Moreira Oscar Lin Molecular pathology Maria E. Arcila Snjezana Dogan Marc Ladanyi Pulmonology & Interventional Radiology Mohit Chawla Jeremy Durack

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