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New Frontiers in Pathology

New Frontiers in Pathology. Case #7 August 3, 2012 Michael H. Roh, MD, PhD University of Michigan Health System. Disclosure Information. The speaker has no relationship representing a possible conflict of interest with respect to the content of this presentation. History.

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New Frontiers in Pathology

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  1. New Frontiers in Pathology Case #7 August 3, 2012 Michael H. Roh, MD, PhD University of Michigan Health System

  2. Disclosure Information The speaker has no relationship representing a possible conflict of interest with respect to the content of this presentation.

  3. History • The patient is a 77 year old man with a recent history of melanoma on his left earlobe. • A CT scan of the chest revealed the presence of enlarged mediastinal lymph nodes concerning for metastatic disease. • The slides represent smears prepared from an endobronchial ultrasound-guided fine-needle aspiration of a station 4R lymph node.

  4. Napsin-A

  5. Napsin-A TTF-1

  6. Napsin-A TTF-1 S100

  7. Diagnosis 4R Lymph Node, Fine-Needle Aspiration: • Positive for adenocarcinoma, consistent with pulmonary origin.

  8. EGFR L858R Control

  9. The Big Picture • The increased emphasis on personalized medicine has led to an increasing number of ancillary molecular tests with prognostic and therapeutic implications. • In patients with advanced cancer, a small biopsy such as an FNA may be the only opportunity to obtain tissue. • Pathologists must meet this challenge by successfully and reliably triaging FNA material in an optimal fashion.

  10. Select Examples of Molecular Applications to Cytology Specimens • FNAs of non-small cell lung carcinoma (NSCLC): • EGFR mutations • KRAS mutations • ALK rearrangements • FNAs of metastatic melanoma: • BRAF mutations

  11. Select Examples of Molecular Applications to Cytology Specimens • FNAs of non-small cell lung carcinoma (NSCLC): • EGFR mutations  geftinib & erlotinib • KRAS mutations • ALK rearrangements  crizotinib • FNAs of metastatic melanoma: • BRAF mutations  vemurafenib

  12. Cell Blocks • Cytologic cell blocks have traditionally been used for molecularassays and immunocytochemistry. • However, cell blocks may contain limited tissue leading to inconsistent results.

  13. FNAs are not all “created equal” Cellularity of lesion Composition of lesion Effective sampling of lesion Precision in targeting lesion

  14. FNAs are not all “created equal” Cellularity of lesion Composition of lesion Effective sampling of lesion Precision in targeting lesion Post-procedural handling of needle-rinse Proper embedding and sectioning of cell block

  15. FNAs are not all “created equal” Cellularity of lesion Composition of lesion Effective sampling of lesion Precision in targeting lesion Post-procedural handling of needle-rinse Proper embedding and sectioning of cell block

  16. Efficacy of Molecular Assays • Each FNA sample characterized by two parameters: overall cellularity & tumor purity. DNA purification Input: High cellularity High tumor purity

  17. Efficacy of Molecular Assays • Each FNA sample characterized by two parameters: overall cellularity & tumor purity. DNA purification PCR amplification Input: High cellularity High tumor purity

  18. Efficacy of Molecular Assays • Each FNA sample characterized by two parameters: overall cellularity & tumor purity. DNA purification PCR amplification Input: High cellularity High tumor purity

  19. Efficacy of Molecular Assays • Each FNA sample characterized by two parameters: overall cellularity & tumor purity. DNA purification PCR amplification Output Input: High cellularity High tumor purity

  20. Efficacy of Molecular Assays • An aspirate with low tumor purity could lead to “drowning out” of the mutant signal. DNA purification Input: High cellularity Low tumor purity

  21. Efficacy of Molecular Assays • An aspirate with low tumor purity could lead to “drowning out” of the mutant signal. DNA purification PCR amplification Input: High cellularity Low tumor purity

  22. Efficacy of Molecular Assays • An aspirate with low tumor purity could lead to “drowning out” of the mutant signal. DNA purification PCR amplification Input: High cellularity Low tumor purity

  23. Efficacy of Molecular Assays • An aspirate with low tumor purity could lead to “drowning out” of the mutant signal. DNA purification PCR amplification Input: High cellularity Low tumor purity Output

  24. Efficacy of Molecular Assays • An aspirate with low tumor purity could lead to “drowning out” of the mutant signal. DNA purification PCR amplification or Input: High cellularity Low tumor purity Output

  25. Direct Smears • Direct-smeared slides provide an alternative source of cellular material to perform ancillary techniques (both molecular studies and immunocytochemistry).

  26. Pooled specimen

  27. Molecular Testing Using Direct Smears

  28. Molecular Mutational Analysis of EGFR and KRAS on Direct Smears of Lung Cancer • Freshly prepared and archived, decoverslipped Diff-Quik smears. • EGFR mutations were evaluated via PCR-based fragment analysis and verified by direct sequencing. • KRAS mutations evaluated by direct sequencing (codons 12, 13, 61). Betz et al (2011) American Journal of Clinical Pathology

  29. Molecular Mutational Analysis of EGFR and KRAS on Direct Smears of Lung Cancer • Freshly prepared and archived, decoverslipped Diff-Quik smears. • EGFR mutations were evaluated via PCR-based fragment analysis and verified by direct sequencing. • KRAS mutations evaluated by direct sequencing (codons 12, 13, 61). EGFR and KRAS mutations were mutually exclusive. Betz et al (2011) American Journal of Clinical Pathology

  30. Molecular Mutational Analysis of BRAFon Direct Smears of Metastatic Melanoma Pre Diff-Quik Post

  31. Molecular Mutational Analysis of BRAFon Direct Smears of Metastatic Melanoma Hookim et al. (2012) Cancer Cytopathology Bernacki et al. (Submitted) American Journal of Clinical Pathology

  32. Concluding Remarks • Cytologic specimens are versatile with respect to specimen preparation methods. • Direct-smeared slides provide a rich source of cellular material to perform ancillary molecular techniques. • DNA extraction from freshly prepared and archived direct smears is easy, reliable, and provides satisfactory molecular test results. • As the cellularity of smears can be directly assessed at the time of FNA, the pathologist can be more confident that adequate material has been obtained for diagnosis and necessary ancillary molecular studies.

  33. Acknowledgments University of Michigan, Department of Pathology Stewart Knoepp, M.D., Ph.D. Bryan Betz, Ph.D. Xin Jing, M.D. Judy Pang, M.D. Jeremiah Placido, M.D. Kim Hookim, M.D. Lindsay Schmidt, M.D. Sara Farmen, M.D. Jason Carvalho, M.D. Joseph Willman, M.D. Jon McHugh, M.D. Tina Fields, B.S. Helmut Weigelin, M.S. University of Michigan, Department of Internal Medicine Doug Arenberg, M.D. Christopher D. Lao, M.D., M.P.H. Greg Kalemkerian, M.D. Bruce G. Redman, D.O. Anthony Courey, M.D.

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