1. LYMPH NODE FNAB & ANCILLARY TESTING Drs. E. Filter, D. Morrison & M. Weir

2. INTRODUCTION Lymphadenopathy in setting of thyroid nodule or post thyroidectomy Lymphadenopathy in setting of thyroid nodule or post thyroidectomy - requires imaging, work-up - sometimes diagnosis - FNAB LN diagnosis & staging important LN diagnosis & staging important - changes management Ancillary testing - useful with FNAB Ancillary testing - useful with FNAB

3. OBJECTIVES For Lymph Node FNABs: 1. State roles of ancillary testing for - thyroglobulin, flow cytometry 2. List reasons for non-correlation between FNAB & thyroglobulin

4. CASE 1 LPP 51 year old female LPP 51 year old female Hx pap thyroid ca 1995, surgery, RAI Hx pap thyroid ca 1995, surgery, RAI Serum Tg undetectable, Stim Tg <1, WBS - N Serum Tg undetectable, Stim Tg <1, WBS - N Suspicious left neck level 3 lymph node Suspicious left neck level 3 lymph node FNAB & Tg testing on sample FNAB & Tg testing on sample

6. Nuclear atypia Comparison to benign thyroid epithelium Pap society atlas

7. CASE 1 Jan 2014 Jan 2014 - FNAB: positive for papillary carcinoma - Tg on FNAB sample: abnormal QUESTIONS: why do the Tg at all? why not do just Tg (omit FNAB) why not do just Tg (omit FNAB)

8. CASE 1 QUESTIONS: 1. Why do the Tg at all? - literature to support reflex testing - cost conscious, limited resources 2. Why not do just Tg alone (omit FNAB)? - it is only for DTC (foll, pap ca) - + FNAB has high PPV

9. LITERATURE Thyroglobulin Wash Testing in the Surveillance of Patients with Thyroid Carcinoma: Proposal for a Reflex Test Thyroglobulin Wash Testing in the Surveillance of Patients with Thyroid Carcinoma: Proposal for a Reflex Test Anastasiya Pigal, Rossitza Draganova- Tacheva, Charalambos C. Solomides, Marluce Bibbo.

10. Bibbo paper Retrospective study Retrospective study Determine if Tg sample contributed to management of cases with + FNAB Determine if Tg sample contributed to management of cases with + FNAB 30 susp/pos LN or thyroid bed FNABs 30 susp/pos LN or thyroid bed FNABs Hx of or subsequent histologically proven differentiated thyroid ca (DTC – foll or pap ca) Hx of or subsequent histologically proven differentiated thyroid ca (DTC – foll or pap ca) Lit review (DTC only) Lit review (DTC only)

11. Bibbo paper Susp/Pos FNABTg > 1 ng/mLTg < 1 ng/mL Tg negative DTC on F/U 30 Bibbo 23 LN, 7 bed 22 (73%) 8 (27%) 7 577 Lit review 548 LN, 29 bed 557 (97%) 20 (3%) 14 All treated regardless of Tg outcomes

12. Bibbo paper Their data & literature review: Their data & literature review: - recommend reflex Tg testing of FNAB ONLY for neg/indeterminate cytology ONLY for neg/indeterminate cytology - + Tg in setting of susp/pos FNAB - no change management

13. PRACTICE REFLECTION Now: Now: - in Plasmalyt: divide for cyto/Tg - all samples sent are tested Should we change our practice? Should we change our practice? - medium to store Tg? - hold Tg until FNAB reported? - who initiates Tg testing?

14. CASE 2 VB VB R thyroid nodules R thyroid nodules - lower 2.6 cm nodule, microcalcifications - 2.2 cm R cervical LN > 4 cm R cervical LN > 4 cm Lung nodules Lung nodules FNABs x3 (2 nodules, LN) FNABs x3 (2 nodules, LN)

15. Case 2 Thyroid Nodules Thyroid Nodules: ● Location: Right Mid Lobe ● Approximate Size: 1.6 x 2.2 x 2.4 cm ● Location: Right Lower Lobe ● Approximate Size: 1.7 x 2.6 cm x ?(went below clavicle) ● Echogenicity: Both Hypoechoic ● Taller than Wide (transverse) Ratio: Yes ● Vascularity: Yes ● Margins: Indistinct

16. Case 2 Lymph Node Biopsy Lymph Node: ● Location: Right Cervical Level 4. ● Approximate Size: 2.8 x 3.4 x >4cm ● Echogenicity: Hypoechoic ● Appearance: Round with no fatty hilum ● Vascularity: Yes ● Margins: Regular

18. + CALCITONIN Pap society atlas

19. CASE 2 FNAB FNAB - medullary carcinoma (thyroid, LN) Tg on FNAB sample not elevated Tg on FNAB sample not elevated Discrepancy between +FNAB & -Tg Discrepancy between +FNAB & -Tg - not DTC, so Tg expected to be neg Note: Tg alone would be false neg

20. CASE 2 Reasons for negative Tg when +FNAB? Reasons for negative Tg when +FNAB?

21. CASE 2 Reasons for negative Tg when +FNAB Reasons for negative Tg when +FNAB - not papillary or follicular carcinoma - sampling (false neg) - passes different material - low cellularity/unsatisfactory - test issue (false neg) - collection tube type - circulating antithyr Ab Roseneide et al Thyroid 2014 Thyroglobulin in the Washout Fluid of Lymph Node Biopsy

22. Summary 1 (Cases 1&2) Role of Tg testing: Role of Tg testing: - not for susp/pos LN outcomes - use with FNAB, not alone Reasons for non-correlation LN FNAB & Tg: Reasons for non-correlation LN FNAB & Tg: - not DTC - sampling, test issue

23. CASE 3 OH OH Left thyroid nodule Left thyroid nodule Left level 4 lymph node Left level 4 lymph node FNABs x2 FNABs x2

26. THYROID FNAB Few groups, focal atypia crowding pseudoinclusions grooves

27. CASE 3 Thyroid FNAB Thyroid FNAB - Indeterminate for pap ca (AUS) LN FNAB LN FNAB - Indeterminate - low cellularity, few epithelial groups Tg on FNAB elevated Tg on FNAB elevated

28. CASE 3 Role of Tg Role of Tg - best use: indeterminate/neg FNAB - has high sensitivity in this setting - will impact management

29. CASE 3 Reasons for positive Tg when neg/ind FNAB? Reasons for positive Tg when neg/ind FNAB?

30. CASE 3 Reasons for positive Tg when neg/ind FNAB Reasons for positive Tg when neg/ind FNAB - sampling - go thru thyroid for central LN - cystic LN, low cellularity/unsat - thyroid bed residual thyroid tissue - test issues - saline collection – matrix effect - high serum Tg - thyroid palpation effect (Tg released) Roseneide 2014

31. CASE 4 HK HK Hx papillary carcinoma 2006 Hx papillary carcinoma 2006 Thyroid nodule in bed query LN Thyroid nodule in bed query LN FNAB FNAB

34. CASE 4 ?LN FNAB ?LN FNAB - low cell, negative (lymphocytes) - may not be representative Tg on FNAB not elevated Tg on FNAB not elevated

35. CASE 4 Is a neg Tg reassuring when FNAB neg? Is a neg Tg reassuring when FNAB neg? - maybe - beware sampling: - FNAB low cellularity or unsat - Tg sample may not be representative

36. Tg TESTING ISSUES Lack consensus, international standards Lack consensus, international standards for performance & interpretation Measure Tg FNAB same method as sTg Measure Tg FNAB same method as sTg Tg free serum in Tg assay kit preferable to washing in saline Tg free serum in Tg assay kit preferable to washing in saline Use 1 mL fluid, plain serum tubes Use 1 mL fluid, plain serum tubes Cut off for measurement - controversial Cut off for measurement - controversial Roseneide 2014 Thyroid

37. PRACTICE REFLECTION Now: Now: - in Plasmalyt, not serum tube - variable volumes Should we change our practice? Should we change our practice? - Tg free serum? - serum tubes - 1 mL

38. Summary 2 (Cases 3&4) Role of Tg testing: Role of Tg testing: - use for neg/indeterminate FNABs - will impact management - beware of neg Tg when unsat/lo cell FNAB Reasons for non-correlation LN FNAB - Tg: Reasons for non-correlation LN FNAB - Tg: - sampling, test issues

39. CASE #5: CERVICAL LYMPHADENOPATHY

40. Case #5 72 year old female 72 year old female Hx non-diagnostic thyroid nodules Hx non-diagnostic thyroid nodules No clinical risk factors, stable sizes, no suspicious sonographic features on left, benign biopsy (low cellularity) on right. No clinical risk factors, stable sizes, no suspicious sonographic features on left, benign biopsy (low cellularity) on right. Now presenting with left cervical lymphadenopathy Now presenting with left cervical lymphadenopathy FNA of lymph nodes (levels 4, 5b) obtained FNA of lymph nodes (levels 4, 5b) obtained

41. Thyroid Nodules

42. Lymph Nodes

43. Smear (Pap stain)

44. Cibas, E. Cytology, 3 rd, Ed. 2009 Air dried smear (DiffQuik stain)

45. Case #5 Flow cytometry reported 2 monoclonal B-cell populations Flow cytometry reported 2 monoclonal B-cell populations The morphologic findings and flow cytometry results are consistent with a B-cell lymphoproliferative disorder The morphologic findings and flow cytometry results are consistent with a B-cell lymphoproliferative disorder A non-Hodgkin lymphoma (NHL) is favoured A non-Hodgkin lymphoma (NHL) is favoured

46. Ancillary Studies in Lymphoma Dx Immunocytochemistry panels Immunocytochemistry panels Molecular studies Molecular studies Flow cytometry Flow cytometry

47. Flow Cytometry

48. Flow Cytometer Cibas, E. Cytology, 3 rd, Ed. 2009

49. Flow Cytometry Markedly improves the diagnostic sensitivity in cases of suspected NHLs Markedly improves the diagnostic sensitivity in cases of suspected NHLs :. recommended ancillary study if clinical DDx includes lymphoma BUT: :. recommended ancillary study if clinical DDx includes lymphoma BUT: less useful in cases of Hodgkin lymphoma and plasma cell dyscrasias less useful in cases of Hodgkin lymphoma and plasma cell dyscrasias

50. Flow Cytometry Requires submission of your sample in flow medium (RPMI) Requires submission of your sample in flow medium (RPMI) Refrigerated Refrigerated Can use Plasmalyte if RPMI not immediately available Can use Plasmalyte if RPMI not immediately available Formalin and alcohol-based solutions (e.g. Cytolyt) not optimal for flow interpretation Formalin and alcohol-based solutions (e.g. Cytolyt) not optimal for flow interpretation www.lifetechnologies.com

51. Role of FNA in Lymphoma Dx DISADVANTAGES Samples may be non-diagnostic Necrosis Fibrosis Poor technique Samples may not be representative Benign and malignant lymphoid cells can co-exist in same lymph node Reactive lymphadenopathies and malignancies can mimic lymphomas Grading of lymphomas poorly reproducible in cytology DeMay, R. The Art & Science of Cytopathology, 2 nd Ed., 2012

52. Role of FNA in Lymphoma Dx ADVANTAGES Does not interfere with subsequent histologic interpretation Can help select best node for excision Ideal in certain situations: Deep or surgically inaccessible nodes Patients of high surgical risk Rapidly progressive and/or extensive disease Can document transformation to a higher-grade lymphoma (e.g. Richter syndrome) DeMay, R. The Art & Science of Cytopathology, 2 nd Ed., 2012

53. In Summary… The diagnosis of lymphoma requires a clinicopathologic approach AND use of ancillary studies The diagnosis of lymphoma requires a clinicopathologic approach AND use of ancillary studies Although there are known pitfalls, FNA cytology can be very useful for proving or excluding a lymphoma Although there are known pitfalls, FNA cytology can be very useful for proving or excluding a lymphoma Flow cytometry is key to diagnosis Flow cytometry is key to diagnosis

54. TAKE HOME MESSAGES Role of ancillary testing for LN FNABs Role of ancillary testing for LN FNABs - Tg: ind/neg FNABs - Tg: ind/neg FNABs - flow cytometry: confirm reactive/lymphoma - flow cytometry: confirm reactive/lymphoma Reasons for non-correlation FNAB & Tg: Reasons for non-correlation FNAB & Tg: - sampling - nature of lesion - test issues