3/31/2016 1 Immunohistochemistry on Limited Tissue Samples: Do’s and Don’ts Andrew M Bellizzi, M.D. Department of Pathology University of Iowa Hospitals and Clinics andrew‐[email protected]Disclosures • I fretted over this . . . • Last night I was sleepless . . . in Seattle Disclosures
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Immunohistochemistry Limited Tissue Samples: Do’s Don’tshandouts.uscap.org/2016_cm06_belli_1.pdf• Performing a second IHC on a previously IHC‐ stained slide (if negative) Dabbs
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Immunohistochemistry on Limited Tissue Samples:
Do’s and Don’tsAndrew M Bellizzi, M.D.Department of Pathology
• To make an accurate, specific diagnosis with asfew immunostains as possible
• To provide (prognostic and) predictive info• To avoid less useful immunostains• To perform clinically valid immunohistochemistry• To triage tissue for other ancillary studies
Outline
• Competing interests: other ancillary studies• Technical aspects of immunocytochemistry• Enemies of the state: non‐specific immunos• Next‐generation immunohistochemistry• Favorite markers/panels
– Carcinoma of unknown origin– Lung adenocarcinoma vs. squamous cell carcinoma– Mesothelioma– Solid pancreatic tumors– Sarcoma– Lymphoma
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63‐year‐old woman with liver, lung, and adrenal masses; presumed lung primary based on FNA of liver lesion 4‐months prior; EGFR/ALK/ROS1wild‐type; progression on platinum‐based chemotherapy; biopsy for cancer mutation profiling
TTF‐1
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Taher,Thanks for raising this issue in the setting of our increasingly engaging in this era of personalized medicine.This was my case, as staff pathologist.I ordered the TTF‐1 because I believed it was necessary to the diagnosis. In fact, in this case, it was negative. I had already ordered it BEFORE your call to the hot seat. So, there was no communication breakdown.I was aware of the prior diagnosis. I read your note while I was evaluating the glass slides. I was aware that the primary driver of this biopsy was to perform NGS.There was actually much more tumor present in the immunostained slide than on the initial H&E, fortunately. I was pessimistic of the prospects for NGS success based on the initial H&E.I was acutely sensitive of the purpose of this biopsy, which is why I limited myself to ONE critical immunostain. Taking one 5 micron section off the tissue block (for IHC) DOES NOT compromise the ability to perform molecular, especially if the sections for molecular are taken simultaneously.
The anatomic pathologist is first and foremost responsible that a correct diagnosis is rendered. So, in this case I have to reasonably reassure myself that this is, in fact, metastatic lung adenocarcinoma.In this instance, both purposes should be served. Your note on the consult sheet WAS CRITICAL. If you hadn't made this comment, I would have performed a more extensive immunopanel.As always, communication is key.I would be happy to discuss this issue at greater length or in person.Best,Andrew
Tension Between Diagnosis and Predictive Testing is Increasing
• Use of IHC in pre‐op lung cytologic specimens
• >600% increase in IHC utilization
Ocque R, Tochigi N, Ohori P, Dacic S. Am J Clin Pathol 2011;136:81‐7.
Study Period Diagnosis of AdCAIHC Frequency
Diagnosis of SCCIHC Frequency
2000‐2004 14% (22/156) 11% (5/46)
2005‐2010 86% (134/156) 89% (41/46)
Ancillary Tests in FNA/Small BiopsyTumor Type Standard Applications Extended Applications
Breast Cancer ER, PR, HER2
Esophagus/Gastric AdCA HER2
Oropharygeal SCC and Head and Neck SCC of Occult Origin
p16 or HPV ISH
Lung Adenocarcinoma EGFR, ALKRET, MET, LKB1, PTEN
Colon Cancer KRAS, NRAS, BRAF, MSI PIK3CA, PTEN, EGFR CN
Mesothelioma
Thyroid Aspirate BRAF, KRAS
Pancreatic Cyst Aspirate Cyst fluid analysis KRAS, DNA content
Melanoma BRAF KIT
Sarcoma FISH or RT/PCR (for specific diagnosis)
Hematolymphoid Flow cytometry, IgH/TCR gene rearrangements, FISH (for diagnosis or prognosis)
p16 FISH
ROS1, PD‐L1NGS Multigene Panel
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Aisner DL, Sams SB. Diagn Cytopathol 2012;40:511‐24.
Gailey MP, et al. Cancer Cytopathol 2015. 123;30‐9.Knoepp SM, Roh MH. Cancer Cytopathol 2013. 121:120‐8.
Snow AM, et al. BMC Clin Pathol 2014. 14;30.
What’s Old is New
Hunt JL, et al. Diagn Cytopathol 1998;18:377‐80.
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Lessons Learned
• Do consider every biopsy of a tumor to be a potential molecular diagnostics specimen
• Do consider cutting unstained sections up‐front for molecular testing, if ordering IHC
• Do communicate with your clinical colleagues about priorities for the specimen
• Do not give up hope if you’ve exhausted a specimen: you may be able to recover DNA from routinely processed material
FNA of a Mural‐Based Gastric MassCell block
KIT on subsequent resectionDOG1
KIT KIT and DOG1 Intensity by FNA Method
• Cell blocks of 52 GIST, 24 LMS, 10 other
• No significant difference in number of cells in EUS vs CT cellblocks
• EUS‐FNA: FNA collected into CytoLyt (methanol‐based fixative)
• CT‐FNA: FNA collected into RPMI
• plasma and thrombin added to produce cell block, fixed in 10% formalin, processed for paraffin embedding
Hwang DG, et al. Am J Clin Pathol 2011;135:448‐53.
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The staining of cytologic preparations with a battery of antibodies to the various cell components or products is very costly and rarely rewarding . . . The results of ICC vary according to the batch of antibodies and the technical skills of the laboratories, the interpretationof the results is not always easy, and the problem with borderline positive stains is often perplexing.
Koss LG. The future of cytology. The Wachtel lecture for 1988. Acta Cytol 1990;34:1‐9.
We This Might Be So . . .
• Surgical Pathologists order IHC on FFPE tissue
• Cytopathologists may request IHC on:– Direct Smears
• Air‐dried– Unfixed– Post‐fixed
• Alcohol‐fixed– Unstained– Stained– Destained
– Cytospins– Monolayer Preparations– Cell Blocks
Cell Block
• Mandelbaum FS. Diagnosis of malignant tumors by paraffin sections of centrifuged exudates. J Lab ClinMed. 1917; 2:580.
Cell Blocks: Advantages and Disadvantages
• Advantages– Processed similarly to surgical pathology material– Facilitates multiple sections– Easy to store
• Disadvantages– Not amenable to on‐site adequacy assessment– May be pauci/acellular
• 10.6% of 246 lung/thoracic FNA’s (Rafael OC, et al 2014)• 57% of 76 consecutive EBUS FNA’s (Knoepp, Roh 2013)
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Performance in Other Cytology Specimen Types Potential Sources of Pre‐Analytic Variation
• Delay in fixation• Type, concentration, pH of fixative• Fixation time• Tissue Processing• Paraffin impregnation (paraffin melting point)• Block/slide storage
Engel KB, Moore HM. Arch Pathol Lab Med 2011;135:537‐43.
Potential Sources of Analytic Variation
• Antigen retrieval– Yes or no– Enzyme or heat‐induced
• Optimization: determination of provisional assay conditions, which is most often involves staining a single case or small number of cases at varying assay conditions
• Validation: testing and appropriate tissue set to determine analytic sensitivity and specificity, to reasonably assure that the test performs as expected
Goldsmith J. CAP Today Q&A. July 13, 2013.Fitzgibbons PL, et al. Arch Pathol Lab Med 2014;138:1432‐43.
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Goldstein NS, et al. Appl Immunohistochem Mol Morphol 2007;15:124‐33.
Recommendation
• Tissue should be fixed in 10% neutral‐pH, phosphate‐buffered formalin for a minimum of 8 hours. Non‐formalin‐based fixatives and or alternative fixation methodologies are strongly discouraged in regard to IHC, in large part because performance data are limited and extrapolation from formalin‐fixed data is unreliable.
Recommendation
• Antigen retrieval (AR) is presumed to “restore” the antigenicity after the formalin fixation. The parameters of an AR protocol must be balanced to match the unique length and type of tissue fixation of the individual laboratory and the characteristics of the individual antibody.
Antigen Retrieval
• Air‐dried smears theoretically should require (less or) no AR
• Alcohol‐fixed smears (coagulation, rather than cross‐linking, fixation) theoretically should require (less or) no AR
• Over‐AR produces high‐background staining and strong edge staining
• Under‐AR produces false negative IHC
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Fitzgibbons PL, et al. Arch Pathol Lab Med 2014;138:1432‐43.
Recommendations
• Laboratories must validate all IHC tests before placing them into clinical service
• For initial analytic validation of nonpredictivefactor assays, laboratories should test a minimum of 10 positive and 10 negative tissues
Recommendations
• “If IHC is regularly done on cytologic specimens that are not processed in the same manner as the tissues used for assay validation (eg, alcohol‐fixed cell blocks, air‐dried smears, formalin‐postfixed specimens), laboratories should test a sufficient number of such cases to ensure that assays consistently achieve expected results . . . The strength of evidence was inadequate to address the criteria and number of samples needed for validation with cytology specimens. If an assay has not been fully validated on cytologic specimens, laboratories may include a disclaimer in their report that results should be interpreted with caution.”
Controls
• “Unless the laboratory has a large bank of similarly prepared cytology material for positive and negative IC controls (non‐formalin‐fixed cytology specimen by the exact same preparation method as the current sample being tested), then criteria for having “proper controls” is not met and any interpretation of IC results should be suspect.”
Fowler LJ, Lachar WA. Arch Pathol Lab Med 2008;132:372‐83.
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Controls
• “In our experience, unstained direct smears in high quantities can be prepared using centrifuged cellular material for effusion specimens and these can be used for positive control ICC reactions.”
Knoepp SM, Roh MH. Cancer Cytopathol 2013. 121:120‐8.
Multiplexing
• Double‐staining, triple‐staining, etc.• Performing a second IHC on a previously IHC‐stained slide (if negative)
Dabbs DJ, Wang X. Diagn Cytopathol 1998;18:166‐9.
Lessons Learned
• Do perform ICC on cell blocks, if possible• Do examine cell block technique if “ICC performing suboptimally
• Do consider including cytology specimens in clinical IHC validations
• Do include similarly processed positive controls on ICC runs
• Do not perform ICC on other cytology specimen types unless the procedure has been specifically optimized for them (esp. antibody dilution, antigen retrieval)
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Myths
• CA125 is specific for Müllerian origin• CA19‐9 is specific for pancreatic origin• CK19 is specific for pancreatobiliary origin• MOC‐31 is an adenocarcinoma marker• RCC is specific for renal origin • Vimentin is specific for sarcoma
CA125 Expression Tumor Site (adenocarcinoma unless otherwise noted)
% Positive(at ≥10% cells staining with ≥ moderate intensity)
McCluggage WG, et al. Int J Gynecol Pathol. 2002 Jan;21(1):11‐5. McCluggage WG, Jenkins D. Int J Gynecol Pathol. 2003 Jul;22(3):231‐5.
Vimentin p16
ER, PRUterine cervical mass
Results of immunopanel favor endometrial origin
Lessons Learned
• Do not extrapolate the results of differential‐specific markers beyond the differential (e.g., CK19, MOC‐31)
• Do not order CA125, CA19‐9, RCC (No No Never)• Vimentin??? If you must . . . sparingly . . . But please don’t tell me that you did . . .
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Next‐Generation Immunohistochemistry
• Mine developmental biology and molecular genetic literature to find:– Lineage‐restricted transcription factors– Markers identified by gene expression profiling – Protein correlates of molecular genetic events
• Bottom line: our markers keep getting better
Gene Expression Profiling Comparing Urothelial, Kidney, and Prostate Cancer
Higgins JPT, et al. Am J Surg Pathol. 2007 May;31(5):673‐80.
Breast cancer GCDFP‐15Mammaglobin
Historically, diagnostic armamentarium geared toward cytoplasmic or membranous differentiation markers; reduced sensitivity in poorly differentiated tumors
GATA‐3: highly expressed, regardless of differentiation
Primacy of lineage‐restricted transcription factors
Hematolymphoid Markers Validated at U of Iowa in Last 2‐Years
Name Diagnostic Application
Tryptase Mast cells
CD163 Monocyte‐macrophage lineage marker (more specific than CD68)
c‐MYC Burkitt lymphoma and c‐MYC activated DLBCL
SOX11 Mantle cell lymphoma (w/ emphasis on identifying CycD1‐ cases)
Lessons Learned• Do perform a limited panel of IHC to assign tumor type/site of origin based on clinical and morphologic differential (“big 3” + appropriate “next‐gen” markers)
• Do not equate p63+ with SCC; do consider p40
• Do consider using “next‐gen” markers to make specific sarcoma and lymphoma diagnoses
Thank you!!!
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U of Iowa Development QueueName Diagnostic ApplicationAndrogen receptor Sebaceous CA, salivary duct CA, prostate CA; Pitfall: low specificity
NECs expressed a median of 8 TFs (range 0‐18) out of 38 examined
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76‐year‐old man with past tobacco use and asbestos exposure p/w pleural effusion; at thoracotomy diaphragm encased by nodular fibrous tissue; multiple plaque like areas throughout pleural cavity Pan‐keratin
Calretinin, WT‐1, CK5/6, MOC‐31, Ber‐EP4, STAT6
Mesothelioma‐Marker Expression in Sarcomatoid Mesothelioma
Chirieac LR, et al. Am J Cancer Res. 2011;1(1):14‐24. All of these were prospectively dx’d as pancreatic neuroendocrine tumor . . . only one of them is
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Cellular Epithelioid Neoplasms of the Pancreas
• Solid neoplasms composed predominantly of neoplastic elements with little stroma– Pancreatic neuroendocrine tumor (PNET)– Solid‐pseudopapillary neoplasm (SPN)– Acinar cell carcinoma (ACC)– Pancreatoblastoma (PB)
• On cytology: dyshesive, monomorphic– SPN: 3 of 6 misdiagnosed as PNET (Bardales et al ‘04)– ACC: 2 of 4 misdiagnosed as PNET (Stelow et al ‘06)– ACC: 14/29 misdiagnosed, 5 as PNET, 5 as ductal AdCA
(Sigel et al ‘13)
Immunophenotype of Cellular Epithelioid Neoplasms of the Pancreas
Core biopsy from a 40 cm retroperitoneal tumor demonstrates undifferentiated neoplasm composed of sheets of epithelioid cells.After performing 17 immunostains a diagnosis of “malignant neoplasm” indeterminate for sarcoma, carcinoma, or lymphoma was rendered.
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MDM2, CDK4
Most consistent with dedifferentiated liposarcoma Resection of similar case
Abrupt transition
Well‐differentiated component
Dedifferentiated component
Keratin and/or EMA‐positivity do not assure a diagnosis of carcinoma
Potential Pitfall #1
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Leiomyosarcoma Smooth muscle actin, desmin+ Keratin AE1/AE3+: keratin, EMA expression in 30‐40% LMS
33‐year‐old woman with 1‐year h/o R hip pain; large SQ mass
EMA+, AE1/AE3+, GATA‐3 focal + p63, CK5/6, ER, PR, TTF‐1, WT‐1, CD31, S‐100, HMB45 all ‐Conclusion: Favor metastatic carcinoma, ? breast or urothelial
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INI1: absent expression epithelioid sarcoma, proximal‐type63‐year‐old man with increasing hip pain x 1 month; proximal femur lesion with soft tissue extension
Undifferentiated Malignant Neoplasm with Osteoclast‐like Giant Cells