1. Best Pathology Practice Tissue specimens should be managed not only for diagnosis but also to maximize the amount of tissue available for molecular studies To guide therapy for patients with advanced lung adenocarcinoma, each institution should develop a multidisciplinary team that coordinates the optimal approach to obtaining and processing biopsy/cytology specimens to provide expeditious diagnostic and molecular results International association for the study of lung cancer/American thoracic society/European respiratory society international multidisciplinary classification of lung adenocarcinoma. Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K, Austin JH, Asamura H, Rusch VW, Hirsch FR, Scagliotti G, Mitsudomi T, Huber RM, Ishikawa Y, Jett J, Sanchez-Cespedes M, Sculier JP, Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton P, Henderson D, Johnson B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R, Saijo N, Thunnissen E, Tsao M, Yankelewitz D. J Thorac Oncol. 2011 Feb;6(2):244-85.

2. Best Pathology Practice When paired cytology and biopsy specimens exist, they should be reviewed together to achieve the most specific and nondiscordant diagnoses Cell blocks should be prepared from all cytology samples including pleural fluids International association for the study of lung cancer/American thoracic society/European respiratory society international multidisciplinary classification of lung adenocarcinoma. Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K, Austin JH, Asamura H, Rusch VW, Hirsch FR, Scagliotti G, Mitsudomi T, Huber RM, Ishikawa Y, Jett J, Sanchez-Cespedes M, Sculier JP, Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton P, Henderson D, Johnson B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R, Saijo N, Thunnissen E, Tsao M, Yankelewitz D. J Thorac Oncol. 2011 Feb;6(2):244-85.

3. Criteria for Morphologic Diagnosis of ADC and SQC on H&E Stained Slides Morphologic features of glandular (adenomatous) differentiation: – Gland formation – Mucin production Morphologic features of squamous differentiation: – Intercellular bridges – Single cell keratinization – Keratin production (keratin pearl formation)

4. Large Cell Carcinoma This diagnosis should not be made on small samples (biopsies or cytology) The diagnosis should be NSCLC NOS

5. What Is The Rate Of NSCLC NOS? Study of California Cancer Registry: 175, 298 lung cancer patients diagnosed from 1989 to 2006 On histology: 22.1% (may be higher) On cytology: 37% The rate increases with decrease in sample size The rate has risen over time Ou, S and Zell, J (2009) J Thorac Oncol 4(10): 1202.

6. Reproducibility of Pathologic Diagnosis in NSCLC Digitalized H&E from 94 surgically resected specimens were reviewed by 24 Pathologists (12 lung expert Pathologists, 12 community Pathologists) Inter-rater agreement on differentiating SQC vs. non-SQC Kappa 0.55 for all pathologists Kappa 0.64 among lung expert pathologists Kappa 0.41 among non-expert community pathologists Grilley-Olson, J et al (2009) ASCO Meeting Abstracts 27(15S): 8008.

7. IHC as a Diagnostic Tool in NSCLC Review of Literature No marker is 100% sensitive or specific Most frequently cited stains are: – For ADCTTF-1, mucin histochemical stains – For SQCp63, CK5/6, HMWK 34bE12 (MA903) Travis, W et al (2010) J Thorac Oncol 5(4): 4.

8. IHC as a Diagnostic Tool in NSCLC Mucin Histochemical Stains in Lung ADC PASMucicarmineAlcian Blue

9. IHC as a Diagnostic Tool in NSCLC TTF-1 in Lung ADC Nuclear staining Sensitivity54%* Specificity97%* Positive PV88%* Negative PV83%* Loo, P et al (2010) J Thorac Oncol 5(4): 442.

10. IHC as a Diagnostic Tool in NSCLC TTF-1 in Lung ADC Cytoplasmic staining should be interpreted as NEGATIVE

11. False Positive Immunostaining —TTF-1 Type II Pneumocytes 200x

12. TumoursNapsin A (% positive) TTF-1 (% positive) Adenocarcinoma79/95 (83%)69/95 (73%) Well differentiated42/47 (89%)38/47 (81%) Moderately differentiated27/32 (84%)24/32 (75%) Poorly differentiated11/16 (69%)7/16 (44%) Squamous cell carcinoma0/46 (0%)0/48 (0%) Large cell carcinoma3/9 (33%)4/9 (44%) “Non-small cell carcinoma,” Not otherwise specified 5/12 (41.7%)3/12 (25%) Renal cell carcinomas Clear cell14/41 (34%)0/41 (0%) Papillary34/43 (79%)0/43 (0%) Chromophobe1/34 (3%)0/34 (0%) Thyroid lesions Papillary carcinoma2/38 (5%)37/38 (97%) Follicular carcinoma0/15 (0%)15/15 (100%) Follicular adenoma0/28 (0%)28/28 (100%) Summary—Napsin A and TTF-1 IHC Staining (I) Bishop, J et al (2010) Hum Pathol 41(1): 20. Sensitivity seems ≥ TTF-1 Often positive in renal neoplasms Rarely positive in thyroid neoplasms Complementary to TTF-1? Not ready for prime time

13. IHC as a Diagnostic Tool in NSCLC p63 in Lung SQC Nuclear staining Sensitivity92%* Specificity74%* Positive PV82%* Negative PV88%* Loo, P et al (2010) J Thorac Oncol 5(4): 442.

14. IHC as a Diagnostic Tool in NSCLC CK5/6 in Lung SQC Cytoplasmic staining Sensitivity 84%* Specificity 79%* Positive PV84%* Negative PV79%* Loo, P et al (2010) J Thorac Oncol 5(4): 442.

15. Modern Pathology (2012) 25, 405–415

16. IHC as a Diagnostic Tool in NSCLC—Review of Literature Predictive scoring cutoffs TTF-1 Moderate > 50% or strong > 10% CK5/6 and p63 Moderate or strong > 10% AB/PAS Any droplet Proportion 1 : 1-10% 2 : 10-50% 3 : > 50% Proportion 1 : 1-10% 2 : 10-50% 3 : > 50% S100 A7CK 5/6 Proportion 01230123 Intensity 0 0 11 22 33 HMWCKp63 Proportion 01230123 Intensity 0 0 11 22 33 Predictor of adenocarcinoma: TTF1 Proportion 0123Not predictive Intensity 0 1Predictive 2 3No data Loo, P et al (2010) J Thorac Oncol 5(4): 442.

17. Diagnostic Algorithm for Classification of NSCLC in Small Biopsy Samples Malignant bronchial biopsy (NSCLC) Malignant bronchial biopsy (NSCLC) H&E stain IHC and AB/PAS 75% subtyped (87% accuracy-SQC; 80% accuracy-ADC) 75% subtyped (87% accuracy-SQC; 80% accuracy-ADC) 18% have subtype predicted (86% accuracy) 18% have subtype predicted (86% accuracy) 7% not typed “ Null ” IHC on biopsy 7% not typed “ Null ” IHC on biopsy “ Null ” Case Resection histology “ Null ” Case Resection histology 14% Squamous cell (1% total) 14% Squamous cell (1% total) 43% Adenocarcinoma (3% total) 43% Adenocarcinoma (3% total) 43% Large cell (3% total) 43% Large cell (3% total) Loo, P et al (2010) J Thorac Oncol 5(4): 442.

18. IHC as a Diagnostic Tool in NSCLC Review of Literature Large cell carcinoma of the lung: an endangered species? Pardo J, Martinez-Peñuela AM, Sola JJ, Panizo A, Gúrpide A, Martinez-Peñuela JM, Lozano MD. Appl Immunohistochem Mol Morphol. 2009 Oct;17(5):383.

19. IHC as a Diagnostic Tool in NSCLC Review of Literature 101 LCC TMA, including 82 « classic » LCC 31 monoclonal antibodies Reclassification of 82 « classic » LCC – 33% ADC TTF1(+) CK7(+) CK19(+) p63(-) – 37% SQC 34bE12(+) p63(+) TM(+) CD44v6(+) – 20% AS* Hybrid AC-SQC immunophenotype Panel of 7 useful antibodies – TTF-1, CK7, CK19, p63, 34bE12, TM, CD44v6 – < 10% of residual LCC – > 90% reclassified ADC, SQC or ASC Pardo, J et al (2009) Appl Immunohistochem Mol Morphol 17(5): 383. ADC: Adenocarcinoma; SQC: Squamous cell carcinoma; LCC: Large cell carcinoma; ASC: Adenosquamous carcinoma; NOS: Not Otherwise Specified; IHC: Immunohistochemistry

20. Reporting Small Lung Samples The term NSCLC-NOS should be used as little as possible, and be applied only when a more specific diagnosis is not possible by morphology and/or special stains The term large cell carcinoma should not be used for diagnosis in small biopsy or cytology specimens and should be restricted to resection specimens where the tumor is thoroughly sampled to exclude a differentiated component International association for the study of lung cancer/American thoracic society/European respiratory society international multidisciplinary classification of lung adenocarcinoma. Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K, Austin JH, Asamura H, Rusch VW, Hirsch FR, Scagliotti G, Mitsudomi T, Huber RM, Ishikawa Y, Jett J, Sanchez-Cespedes M, Sculier JP, Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton P, Henderson D, Johnson B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R, Saijo N, Thunnissen E, Tsao M, Yankelewitz D. J Thorac Oncol. 2011 Feb;6(2):244-85.

21. SCLC NSCLC, ?LCNEC Algorithm for adenocarcinoma diagnosis in small biopsies and/or cytology POSITIVE BIOPSY (FOB, TBBx, Core, SLBx) STEP 1 NE morphology, large cells, NE IHC+ NE morphology, small cells, no nucleoli, NE IHC+, TTF-1 +/-, CK+ Keratinization, pearls and/or intercellular bridges Classic Morphology: SQCC No clear ADC or SQCC morphology: NSCLC-NOS Apply ancillary panel of One SQCC and one ADC marker +/OR Mucin STEP 2 Classic morphology: ADC POSITIVE CYTOLOGY (effusion, aspirate, washings, brushings) Histology: Lepidic, papillary, and/or acinar architecture(s) Cytology: 3-D arrangements, delicate foamy/vacuolated (translucent) cytoplasm, Fine nuclear chromatin and often prominent nucleoli Nuclei are often eccentrically situated

22. Algorithm for adenocarcinoma diagnosis in small biopsies and/or cytology NSCLC, favor SQCC Apply ancillary panel of One SQCC and one ADC marker +/OR Mucin STEP 2 SQCC marker +ve ADC marker -ve/or Mucin -ve IHC -ve and Mucin -ve ADC, marker or Mucin +ve; as well as SQCC marker +ve in different cells NSCLC, NOS, possible adenosquamous ca NSCLC NOSNSCLC, favor ADC ADC marker and/or Mucin +ve; SQCC marker -ve (or weak in same cells)

23. Algorithm for adenocarcinoma diagnosis in small biopsies and/or cytology NSCLC, NOS, possible adenosquamous ca NSCLC NOS Molecular analysis: e.g. EGFR mutation † NSCLC, favor ADC If tumor tissue inadequate for molecular testing, discuss need for further sampling – back to STEP 1 STEP 3 Classic morphology: ADC

24. Chr. 2p23-22 Perner, S et al. (2008) Neoplasia 10(3): 298.Mano, H (2008) Cancer Sci 99(12): 2349. Oncogenic Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S, Watanabe H, Kurashina K, Hatanaka H, Bando M, Ohno S, Ishikawa Y, Aburatani H, Niki T, Sohara Y, Sugiyama Y, Mano H. Nature. 2007 Aug 2;448(7153):561.

25. ALK Break Apart FISH ~12 Mb ALKEML4 e20

26. Clinical Activity Observed in an Updated Phase I-II Trial of an Oral C- met and ALK Inhibitor Crizotinib (PF– 02341066) * Partial response patients with 100% change have non-target disease present Progressive disease Stable disease Confirmed partial response Confirmed complete response 60 40 20 0 -20 -40 -60 -80 -100 Maximum change in tumour size (%) -30% Overall Response Rate = 57% Disease Control Rate (CR+PR+SD) at 8 weeks= 87% ONLY 3-5% OF NSCLC Bang, Y et al. (2010). ASCO Meeting Abstracts 28(18_suppl): 3.

27. (EML4)-ALK Fusion Gene Tumors Occur Mainly in Adenocarcinoma First authorAdeno (total no.) Squamous (total no.) Others (total no.) Imamura (2008)3.4% (149)0% (48)0 (24) Shinamura (2008)4% (50)0% (20)0% (7) Takeuchi (2008)4.3% (253)0% (71)0% (19) Koivunen (2008)3.8% (208)0% (88)0 (100) Rodig (2009)5.6% (358)-- Martlelli (2009)4.8% (63)8.3% (48)22.2% (9) Wong (2010)5.3% (209)0% (34)8.7% (23) Salido (2010)4.3% (69)0% (30)0% (8) Jokoji (2010)3.1% (254)-- Takahashi (2010)2.4% (211)0% (75)0% (27) Paik (2011)6.3% (423)0% (163)3.7% (27) Tumors studied2247577244

28. Histological Features of Adenocarcinoma Associated with EML4-ALK Aberration Rodig (N. America) Inamura (Japan) No. Studied342253 ALK +ve20 (5.8%)11 (4.3%) Acinar4/124 (3.2%)6/34 (17.6%) Papillary0/46 (0%)5/206 (2.4%) Solid11/134 (8.2%)0/5 (0%) BAC1/22 (4.5%)0/7 (0%) Others00/1 (0%) Signet ring Tend to be mucin producing and TTF1+ None3/295 (1%) ≤ 10%2/21 (10%) >10%12/26 (46%) Rodig SJ, et al. CCR 2009;15:5216-23; Inamura K, et al. Modern Pathol 2009;22:508-15

29. ALK + (n=54) ALK – (n=100) P value Any lepidic24%84%<0.0001 Any acinar87%49%<0.0001 Any papillary67%70%0.7170 Any solid65%33%0.0002 Cribriform80%16%<0.0001 Signet ring cells65%1%<0.0001 CRIBRIFORM SIGNET RING Am J Pathol 2011;35:1226-34

30. 3+2+ 1+ 0 ALK1 antibody with ADVANCED detection system (DAKO) 2+/3+ IHC: Sensitivity 90% Specificity 97.8% J Thorac Oncol. 2011;6: 466–472

31. J Thoracic Oncol 2011;6:466-72

32. Lung Cancer 2012 (Epub)

33. A novel, highly sensitive antibody allows for the routine detection of ALK- rearranged lung adenocarcinomas by standard immunohistochemistry. Mino-Kenudson M, Chirieac LR, Law K, Hornick JL, Lindeman N, Mark EJ, Cohen DW, Johnson BE, Jänne PA, Lafrate AJ, Rodig SJ. Clin Cancer Res. 2010 Mar 1;16(5):1561. Antibody D5F3ALK1 Sensitivity100%67% Specificity99%97%

34. Conclusions Current practice of pathology in lung cancer requires more precise classification of tumor type For poorly differentiated NSCLC, a minimal set of immunohistochemistry markers (TTF1, mucin, p63 and CK5) should reduce the number of NOS to <5%. Cell block preparation should be part of the routine practice of cytology (especially for FNA and fluid) For both small biopsy and cytology, every attempt should be made to preserve some samples for molecular studies of predictive biomarkers It is likely that in the future, histopathology/immunohistochemistry may play important role in the development of cost-effective diagnostic algorithm for molecular assays

35. ACKNOWLEDGEMENTS Dr. Diana Ionescu (British Columbia Cancer Agency) Dr. Tony Magliocco (Tom Baker Cancer Centre and Calgary Laboratory Services) Dr. Christian Couture (Université Laval) Maritz Canada & Eli Lilly Canada