Warning! You won't be able to use the quotation basket until you enable cookies in your Web browser.
Warning! Your Web browser is no longer supported. Please upgrade to a modern browser.

Protein Expression Education Series - Lung

ALK

General

ALK (Anaplastic Lymphoma Kinase) is a transmembrane molecule that is only normally expressed in some neural tissues. It has characteristic expression in a significant proportion of Anaplastic Large Cell Lymphoma (ALCL) cases. It has also been expressed in cases of pleomorphic liposarcoma, inflammatory myofibroblastic tumor and a small subset of diffuse large B-cell lymphomas.

A subset (1-5%) of non-small cell lung carcinomas (usually adenocarcinomas) have an ELM4-ALK mutation, which is often sensitive to the tyrosine kinase inhibitor (TKI) crizotinib (Pfizer). Newer TKIs, including ceritinib (Novartis) and alectinib (Hoffmann-La Roche), have also been found to be effective.

Historically, ALK translocations have been identified by FISH analysis. IHC is also an accepted method with an FDA approved test (IHC CDx Assay). PCR is being studied as an alternative. ALK antibody clones 5A4 (Novocastra, Leica Biosystems, Buffalo Grove, Illinois), ALK1 (Dako, Santa Clara, California), 1A4 (Origene, Rockville, Maryland) and D5F3 (Cell signaling Technology, Danvers, Massachusetts) have been successfully used to identify ALK mutated lung tumors, with the 5A4 & D5F3 having equivalent sensitivity. The ALK1 clone is not as sensitive and the 1A4 clone lacks specificity compared to other antibodies. Please consult the current medical literature for FDA approved tests for ALK translocation identification in non-small cell lung carcinomas.

Normal Expression

  • Neural Tissue

Abnormal Expression

  • Anaplastic Large Cell Lymphoma
  • Pleomorphic Liposarcoma
  • Subset of Diffuse Large B-Cell Lymphoma
  • Inflammatory Myofibroblastic Tumors
  • Merkel Cell Carcinoma (KIF5B-ALKrearrangement)

Interpretation

ALK may stain in a cytoplasmic and/or nuclear pattern. In ALCL the combined pattern of cytoplasmic and nuclear staining is associated with the t(2;5).

In lung ALK staining/expression is cytoplasmic. Like other markers (e.g. Napsin A), staining may be present in macrophages. Necrotic tumor, extracellular mucin, and cells of neural origin may also stain.

References

Thunnissen E, Allen TC, Adam J, Aisner DL, Beasley MB, Borczuk AC, et al. Immunohistochemistry of Pulmonary Biomarkers: A Perspective From Members of the Pulmonary Pathology Society. Arch Pathol Lab Med. 2018;142: 408–419. doi:10.5858/arpa.2017-0106-SA

Chan, J. K. C., Ip, Y. T., &Cheuk, W. (2013). The Utility of Immunohistochemistry for Providing Genetic Information on Tumors. International Journal of Surgical Pathology, 21(5), 455–475. doi:10.1177/1066896913502529

Chan, J. K. C. (2013). Newly Available Antibodies With Practical Applications in Surgical Pathology. International Journal of Surgical Pathology, 21(6), 553–572. doi:10.1177/1066896913507601

Paik, J. H., Choe, G., Kim, H., Choe, J.-Y., Lee, H. J., Lee, C.-T., et al. (2011). Screening of Anaplastic Lymphoma Kinase Rearrangement by Immunohistochemistry in Non-small Cell Lung Cancer: Correlation with Fluorescence In Situ Hybridization. Journal of Thoracic Oncology : Official Publication of the International Association for the Study of Lung Cancer, 6(3), 466–472. doi:10.1097/JTO.0b013e31820b82e8

Yi, E. S., Boland, J. M., Maleszewski, J. J., Roden, A. C., Oliveira, A. M., Aubry, M.-C., et al. (2011). Correlation of IHC and FISH for ALK Gene Rearrangement in Non-small Cell Lung Carcinoma: IHC Score Algorithm for FISH. Journal of Thoracic Oncology : Official Publication of the International Association for the Study of Lung Cancer, 6(3), 459–465. doi:10.1097/JTO.0b013e318209edb9

Mino-Kenudson, M., Chirieac, L. R., Law, K., Hornick, J. L., Lindeman, N., Mark, E. J., et al. (2010). A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 16(5), 1561–1571. doi:10.1158/1078-0432.CCR-09-2845

Reichard, K. K., McKenna, R. W., & Kroft, S. H. (2007). ALK-positive diffuse large B-cell lymphoma: report of four cases and review of the literature. Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc, 20(3), 310–319. doi:10.1038/modpathol.3800742

Medeiros, L. J., &Elenitoba-Johnson, K. S. J. (2007). Anaplastic Large Cell Lymphoma. American Journal of Clinical Pathology, 127(5), 707–722.

Takeuchi K, Choi YL, Togashi Y, et al. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res. 2009;15(9):3143–3149. doi:10.1158/ 1078-0432.CCR-08-3248.

CK5

General

CK5 is a high molecular weight cytokeratin that stains stratified squamous epithelium (e.g. skin, tongue mucosa, etc.), basal cells of the prostate gland, myoepithelial cells in breast tissue, and mesothelial cells/mesothelioma. Therefore CK5 is a versatile marker, which can be used in a variety of diagnostic applications.

CK5 is useful in the diagnosis of squamous cell carcinoma, urothelial carcinoma, mesothelioma, thymic tumors, and skin appendage tumors. It is also helpful in identifying the myoepithelial layer in breast tissue, and the basal layer in prostate tissue. The combined expression of p63 and CK5 in poorly differentiated tumors is highly suggestive of squamous origin (Kaufmann, O., et al).

Lung

CK5 will stain approximately 70-80% of squamous cell carcinomas with a cytoplasmic staining pattern. p63 is considered slightly more sensitive, but may give varying positivity in cases of lung adenocarcinoma that can be confusing. Therefore, CK5 and p63 are often used as part of a panel to diagnose squamous cell carcinoma. Since p63 is a nuclear marker, these two stains can be performed as a double stain on a single slide with one or two different chromogens.

Prostate

CK5 is an excellent marker for the basal layer, and stains in a similar pattern to 34betaE12. Again, CK5 can be combined with p63, which also stains the basal layer of prostate glands.

Bladder

Urothelial carcinoma will typically express p63 diffusely with variable reports of CK5 or CK5/6 expression in urothelial carcinoma.

Breast

CK5 will stain the myoepithelial layer of breast tissue, but other markers such as smooth muscle myosin and calponin are typically used more commonly. Basal-like breast carcinomas usually express CK5, especially with the monoclonal CK5 antibody compared to CK5/6 (Bhargava, R, et al). Metaplastic/sarcomatoid breast carcinomas have also been reported to express CK5.

Skin

CK5 will stain the stratified squamous epithelium. If there are cells in the squamous epithelium, which may be Paget cells or melanocytes, CK5 can be used along with CK7 or Melan A/HMB-45 to differentiate between odd appearing keratinocytes, Paget's disease, or melanocytes.

CK5 vs. CK5/6

There is still occasionally discussion as to which is a better marker. Some feel CK5/6 is a better marker since it has more than one cytokeratin in its cocktail. However, there is a very strong consensus among pathologists who have used both that CK5 performs significantly better than CK5/6, and is therefore, the preferred antibody.

Common expression patterns in carcinoma (Chu, PG, et al).

Tumor Expression % Differentiation
Skin: BCC & SCC 100% Squamous
Squamous Salivary Gland Tumors 100% Myoepithelial
Mesothelioma 76% Mesothelial
Urothelial Ca. 62% Transitional
Endometrial Ca. 50% Squamous
Ovarian Ca. (Serous) 75% Serous Epithelium

Note: other references note a much higher + expression pattern in mesotheliomas This may be dependent upon the mix of epithelial and sarcomatous variants.

CK5 is a high molecular weight cytokeratin expression by stratified epithelium (epidermis) and myoepithelial/basal layer cells.

p63/CK5 (double stain) with p63 nuclear expression (DAB) and CK5 cytoplasmic expression (red)

Lung squamous cell carcinoma with p63 nuclear expression (DAB) and cytoplasmic CK5 expression (red).

CK5/6 expression in a lung squamous cell carcinoma. CK5/6 tends to have more varied expression compared to the monoclonal CK5 antibody.

References

Fichtenbaum EJ, Marsh WL, Zynger DL. CK5, CK5/6, and double-stains CK7/CK5 and p53/CK5 discriminate in situ vs invasive urothelial cancer in the prostate. Am J ClinPathol. 2012;138: 190–197. doi:10.1309/AJCP5ZC4GQVNWTYR

Bhargava, R., Beriwal, S., Mcmanus, K., & Dabbs, D. J. (2008). CK5 is more sensitive than CK5/6 in identifying the “basal-like” phenotype of breast carcinoma. American Journal of Clinical Pathology, 130(5), 724–730. doi:10.1309/AJCP3KFF1LTYWQIY

Mukhopadhyay, S., &Katzenstein, A.-L. A. (2011). Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. The American Journal of Surgical Pathology, 35(1), 15–25. doi:10.1097/PAS.0b013e3182036d05

Kaufmann, O., Fietze, E., Mengs, J., &Dietel, M. (2001). Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. American Journal of Clinical Pathology, 116(6), 823–830. doi:10.1309/21TW-2NDG-JRK4-PFJX

Chu, P. G., & Weiss, L. M. (2002). Expression of cytokeratin 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases. Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc, 15(1), 6–10. doi:10.1038/modpathol.3880483

Sutton LM, Han JS, Molberg KH, Sarode VR, Cao D, Rakheja D, et al. Intratumoral expression level of epidermal growth factor receptor and cytokeratin 5/6 is significantly associated with nodal and distant metastases in patients with basal-like triple-negative breast carcinoma. Am J ClinPathol. 2010;134: 782–787. doi:10.1309/AJCPRMD3ARUO5WPN

Hadi, AIMM Annual Meeting, "The Thirty Most Important Antibodies", presentation, 2011.

Mod Path 15:6-10, 2002.

Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol. 2008;129: 705–733. doi:10.1007/s00418-008-0435-6

CK7

CK7 is an intermediate filament with a selective expression pattern in different carcinomas, which when combined with CK20 is useful in the work up of carcinomas of unknown primary origin.

CK7 is part of a group of cytokeratins often referred to as low-molecular weight keratins (LMWK). In general these cytokeratins (CK7, CK20 CK8, CK18, and CK19) are expressed in simple epithelium (as opposed to high molecular weight cytokeratins expressed in stratified epithelium). The distribution of expression of these LMWK is dependent upon the organ system. This differential expression can be helpful when narrowing a differential diagnosis for a carcinoma of unknown primary site (CUPS). Most commonly CK7 is combined with CK20 in a panel, which dependent upon the differential expression pattern, more specific markers can be added to narrow the diagnostic possibilities (see CK7/CK20 tables below).

Common expression patterns in carcinoma (Dennis, JL, et al).

Tumor Expression %
Breast >80%
Colon <10%
Lung >90%
Ovary >80%
Pancreas >80%
Stomach 35-70%
Prostate ~10%

Cytokeratin expression in various tumors (Moll, RT, et al. )

Tumor CK8/CK18 CK19 CK7 CK20 CK5
Hepatocellular Ca. + +/- +/- +/- =
Colorectal ACA + + +/- + =
Stomach ACA + + +/- +/- =
Pancreas Ductal ACA + + + +/- +/-
Lung ACA + + + = =
Breast Inv. Ductal + + + = +/-
Endometrium ACA + + + = +/-
Ovary ACA + + + = =
RCC, Clear Cell Type + +/- = = =
RCC, Papillary Type + + + = =
RCC, Chromophobe + +/- + = =
Mesothelioma + + +/- = +
Lung, Small Cell Ca. + +/- = = =
Merkel Cell Ca. + + = + =
Urothelial Carcinoma + + + +/- +/-
Squamous Cell Ca. +/- +/- = = +

Key: "+/-“, focal staining in some cases. “=“, negative, “+”, positive.

CK7 expression in skin highlighting duct epithelium of eccrine/adnexal structures. CK7 staining pattern is the inverse of CK5 expression.

CK7 expression in eccrine/adnexal structures from a skin section. CK7 with stain in an “inverse” pattern with epithelium compared to CK5

CK7 expression in lung adenocarcinoma

References

Hadi, AIMM Annual Meeting, "The Thirty Most Important Antibodies", presentation, 2011.

Dennis, J. L., Hvidsten, T. R., Wit, E. C., Komorowski, J., Bell, A. K., Downie, I., et al. (2005). Markers of adenocarcinoma characteristic of the site of origin: development of a diagnostic algorithm. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 11(10), 3766–3772. doi:10.1158/1078-0432.CCR-04-2236

Moll, R., Divo, M., &Langbein, L. (2008). The human keratins: biology and pathology. Histochemistry and Cell Biology, 129(6), 705–733. doi:10.1007/s00418-008-0435-6

Moll R. Cytokeratins as markers of differentiation. Expression profiles in epithelia and epithelial tumors. Veroffentlichungenaus der Pathologie. 1992.

CK20

CK20 is an intermediate filament with a selective expression pattern in different carcinomas, which when combined with CK7 is useful in the work up of carcinomas of unknown primary origin. CK20 has also been used in combination with other markers to evaluate potentially neoplastic urothelium based on proliferation and epithelial maturation characteristics. In other settings, loss of normal expression (e.g. CK20/CDX-2 expression loss in colon adenocarcinoma) has been associated with prognostic and molecular characteristics (e.g. BRAF mutation).

Common expression patterns in carcinoma (Dennis, JL, et al).

Tumor Positive (%)
Breast <10%
Colon >80%
Lung <10%
Ovary <10%
Pancreas 35-50%
Stomach 30-50%
Prostate <10%

Moll, RT, et al. Cytokeratin expression in various tumors.

Tumor CK8/CK18 CK19 CK7 CK20 CK5
Hepatocellular Ca. + +/- +/- +/- =
Colorectal ACA + + +/- + =
Stomach ACA + + +/- +/- =
Pancreas Ductal ACA + + + +/- +/-
Lung ACA + + + = =
Breast Inv. Ductal + + + = +/-
Endometrium ACA + + + = +/-
Ovary ACA + + + = =
RCC, Clear Cell Type + +/- = = =
RCC, Papillary Type + + + = =
RCC, Chromophobe + +/- + = =
Mesothelioma + + +/- = +
Lung, Small Cell Ca. + +/- = = =
Merkel Cell Ca. + + = + =
Urothelial Carcinoma + + + +/- +/-
Squamous Cell Ca. +/- +/- = = +

Key: "+/-“, focal staining in some cases. “=“, negative, “+”, positive.

CK20 expression (or lack thereof) in lung adenocarcinoma (CK7+/CK20-).

CK20 expression in colon adenocarcinoma.

CK20 expression in underlying colon adenocarcinoma, and normal expression in overlying benign mucosa (increased expression as it becomes more superficial).

References

Hadi, AIMM Annual Meeting, "The Thirty Most Important Antibodies", presentation, 2011.

Dennis, J. L., Hvidsten, T. R., Wit, E. C., Komorowski, J., Bell, A. K., Downie, I., et al. (2005). Markers of adenocarcinoma characteristic of the site of origin: development of a diagnostic algorithm. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 11(10), 3766–3772. doi:10.1158/1078-0432.CCR-04-2236

Moll, R., Divo, M., &Langbein, L. (2008). The human keratins: biology and pathology. Histochemistry and Cell Biology, 129(6), 705–733. doi:10.1007/s00418-008-0435-6

Wee A. Diagnostic utility of immunohistochemistry in hepatocellular carcinoma, its variants and their mimics. ApplImmunohistochemMolMorphol. 2006;14: 266–272.

Yin H, He Q, Li T, Leong AS-Y. Cytokeratin 20 and Ki-67 to distinguish carcinoma in situ from flat non-neoplastic urothelium. ApplImmunohistochemMolMorphol. 2006;14: 260–265.

Kim J-H, Rhee Y-Y, Bae JM, Cho N-Y, Kang GH. Loss of CDX2/CK20 expression is associated with poorly differentiated carcinoma, the CpG island methylator phenotype, and adverse prognosis in microsatellite-unstable colorectal cancer. Am J SurgPathol. 2013;37: 1532–1541. doi:10.1097/PAS.0b013e31829ab1c1

CK7+ / CK20+

Tumor CK7 CK20
TCC (Urothelial) + +
Ovarian Ca. (mucinous) + +
Pancreatic Ca. (subset) + +
Cholangiocarcinoma (subset) + +
Gastric Adenoca. (subset) + +
Sm. Int. (non-Ampullary) Ca. + + (60-70%)

Follow-up Markers for CK7+/CK20+

IHC Stain Comment
Uroplakin Supportive of urothelial carcinoma
Thrombomodulin Expression may be seen in urothelial carcinoma or adenocarcinoma of the bladder
p63 Expression supports urothelial carcinoma
CK5/6 Expression supports urothelial carcinoma
CEA May be supportive of pancreatic ductal carcinoma
CA19-9 May be supportive of pancreatic ductal carcinoma
CDX-2 +/- expression seen in pancreatic ductal carcinoma, adenocarcinoma of the bladder, and ovarian mutinous carcinoma
GATA-3 Expression supports urothelial carcinoma

References

Arch Pathol Lab Med. Vol 134, February 2010.

Hadi, AIMM Annual Meeting, “Carcinomas of Unknown Primary”, presentation, 2011.

CK7+ / CK20-

Tumor CK7 CK20
Breast Ca. (>95%) + - (0-16% are +)
Lung Adenoca. (90%) + -
Ovarian Ca. (Serous) (>90%) + -
Pancreatic Ca. (Subset) + -
Mesothelioma + -
Endometrial Adenoca. + -
Lung Small Cell Ca. (60-80%) + (dot-like) -
Cholangiocarcinoma + -
Thyroid Carcinoma + -
Gastric Carcinoma (subset) + -

Follow-up Markers for CK7+/CK20-

IHC Stain Comment
TTF-1 Expression seen in lung adenocarcinoma, small cell carcinoma, and thyroid carcinoma (medullary, papillary and follicular)
MOC31 Expression commonly seen in lung adenoma., but not mesothelioma.
CK5/6 Expression commonly seen in mesothelioma, but NOT lung adenoca.
Chromogranin Expression supports neuroendocrine origin or differentiation (small cell carcinoma).  Medullary thyroid ca. is also positive.
Synaptophysin Expression Supports neuroendocrine origin or differentiation (small cell carcinoma).  Medullary thyroid ca. is also positive.
ER / PR Expression may be seen in breast carcinoma, ovarian serous carcinoma, and endometrial adenocaricnoma.
GCDFP15 Expression is fairly specific for breast carcinoma.
Mammaglobin Expression is fairly specific for breast carcinoma.
WT-1 Expression is often seen in mesothelioma and ovarian serous carcinomas.
CEA Expression may be seen in cholangiocarcinoma, breast carcinoma, and lung adenocarcinoma.  Expression is NOT usually seen in ovarian serous carcinoma, endometrial adenocarcinoma, or mesothelioma.
Thyroglobulin Specific marker for follicular and papillary thyroid carcinoma.  Medullary ca. does not express Thyroglobulin.
Calretinin Expression usually found in mesothelioma.
Calcitonin Expression is typical for medullary (thyroid) ca.

References

Arch Pathol Lab Med. Vol 134, February 2010.

Hadi, AIMM Annual Meeting, “Carcinomas of Unknown Primary”, presentation, 2011.

CK7- / CK20+

Tumor CK7 CK20
Colon Adenocarcinoma (75-95%) - + (0-15% are -)
Merkel Cell Carcinoma - + (dot-like)

Follow-up Markers for CK7- / CK20+

IHC Stain Comment
CDX-2 Expression supports colon origin
TTF-1 Expression is not typically seen in Merkel cell ca.

CK20 negativity in colon adenocarcinoma may be due to incomplete fixation, or in some tumors CK20 and CDX-2 loss of expression may be associated with BRAF mutations.

References

Hadi, AIMM Annual Meeting, “Carcinomas of Unknown Primary”, presentation, 2011.

Arch Pathol Lab Med. Vol 134, February 2010.

CK7- / CK20-

Tumor CK7 CK20
Hepatocellular Ca. - (9-17% are +) -
Renal Cell Ca. - -
Prostate Ca. - - (0-23% are +)
Neuroendocrine Ca. - -
Squamous Cell Ca. -(+/-) -
Adrenal Cortical Ca. - -
Gastric Adenoca. (subset) - -

Follow-up Markers for CK7-/CK20-

IHC Stain Comment
PSA Expression supports prostatic adenocarcinoma
PSAP Expression supports prostatic adenocarcinoma
CEA CEA is NOT commonly expressed in prostate adenocarcinoma, renal cell carcinoma, or adrenal cortical carcinoma.
CK5/6 CK5/6 is NOT typically expressed in prostate adenocarcinoma, which is in contrast to squamous cell carcinoma that usually shows expression.
Vimentin Expression is commonly seen in renal cell carcinoma and adrenal cortical carcinoma.
RCC Ma. Commonly expressed in renal cell carcinoma.
EMA Expression commonly seen in renal cell carcinoma but not adrenal cortical carcinoma.
HepPar1 Expression supports hepatocyte origin.
CD10 RCC shows cytoplasmic expression, and a canalicular expression pattern is often seen in HCC.
pCEA Canalicular patter is common in HCC
MOC-31 HCC is negative for MOC-31.  Cholangiocarcinoma (and many other carcinomas) are positive.
CK19 Expression is NOT seen in HCC.
p63 Expression is seen in squamous cell carcinoma.
Inhibin Expression supports adrenal cortical origin
Melan-A Expression often seen in adrenal cortical carcinoma.

References

Arch Pathol Lab Med. Vol 134, February 2010.

Hadi, AIMM Annual Meeting, “Carcinomas of Unknown Primary”, presentation, 2011.

Napsin A

Napsin A is an aspartic proteinase, and is normally found in lung pneumocytes and alveolar macrophages, pancreatic acini and ducts, and renal tubules. Neoplastic tissue which stains with Napsin A includes: lung adenocarcinoma, renal cell carcinoma (clear cell and papillary), thyroid (papillary) carcinoma, and ovarian clear cell carcinoma.

Napsin A is primary used in combination with TTF-1 in identifying lung adenocarcinomas (TTF-1 appears to be a little more sensitive in poorly differentiated tumors). While approximately 97% of Napsin A expressing lung adenocarcinomas express TTF-1, up to 13% of TTF-1 negative cases express Napsin A. The more recent description of Napkin A expression in 100% of ovarian clear cell carcinomas by Kandalaft, et al. further emphasizes the importance of using an appropriate panel of IHC stains for a given differential diagnosis to obtain optimal sensitivity and specificity.

In the setting of ovarian tumors (Kandalaft, et al.), Napsin A was 100% sensitive for ovarian clear cell carcinoma. Endometrioid carcinomas of the ovary had focal Napsin A expression in 10% of cases, but no cases of high grade serous carcinoma or serous borderline tumor demonstrated expression.

Napsin A and TTF-1 expression in poorly differentiated non-small cell carcinomas (Mukjopadhyay, S, et al).

Tumor Napsin A TTF-1
Adenocarcinoma 11/19 (58%) 16/20 (80%)
Squamous Cell Carcinoma 0/15 (0%) 0/15 (0%)
Large Cell Carcinoma 0/4 (0%) 2/4 (50%)

Napsin A and TTF-1 expression in various tumor types (Bishop, JA, et al).

Tumor Napsin A (% positive) TTF-1 (% positive)
Lung Tumors
Adenocarcinoma 79/95 (83%) 69/95 (73%)
     -  Well-differentiated 42/47 (89%) 38/47 (81%)
     -  Moderately-differentiated 27/32 (84%) 24/32 (75%)
     -  Poorly-differentiated 11/16 (69%) 7/16 (44%)
Squamous Cell Carcinoma 0/46 (0%) 0/48 (0%)
Large Cell Carcinoma 3/9 (33%) 4/9 (44%)
Small Cell Carcinoma 0/3 (0%) 1/3 (33%)
Atypical Carcinoid Tumor 0/1 (0%) 1/1 (100%)
Typical Carcinoid Tumor 0/2 (0%) 1/2 (50%)
Nonpulmonary Adenocarcinomas
Colon Adenocarcinoma 0/5 (0%) 0/5 (0%)
Pancreas Adenocarcinoma 0/31 (0%) 0/31 (0%)
Breast Adenocarcinoma 0/17 (0%) 0/17 (0%)
Mesothelioma (all types) 0/38 (0%) 0/38 (0%)
Renal Cell Carcinomas
Clear Cell 14/41 (34%) 0/41 (0%)
Papillary 34/43 (79%) 0/43 (0%)
Chromophobe 1/34 (3%) 0/34 (0%)
Thyroid Lesions
Papillary Carcinoma 2/38 (5%) 37/38 (97%)
Follicular Carcinoma 0/15 (0%) 15/15 (100%)
Follicular Adenoma 0/28 (0%) 28/28 (100%)

Napsin A – Lung Adenocarcinoma

Napsin A expression in benign pneumocytes and histiocytes, and absence of staining in the squamous cell carcinoma (right side of image).

Napsin A (red) and TTF-1 (DAB) double stain in a lung adenocarcinoma. Note the granular staining characteristics of Napsin A.

Napsin A expression in benign pneumocytes and histiocytes, and absence of staining in the squamous cell carcinoma (right side of image). Don’t mistake scattered intra-tumoral staining histiocytes as focal expression in the tumor.

Napsin A (red) and TTF-1 (DAB) double stain in a lung adenocarcinoma.

Napsin A expression in lung adenocarcinoma. Note the granular characteristics of the cytoplasmic staining.

References

Eisen, RN, et al. Appl Immunohistochem Mol Morphol. Vol. 19, No. 6, December 2011.

Bishop, J. A., Sharma, R., & Illei, P. B. (2010). Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Human Pathology, 41(1), 20–25. doi:10.1016/j.humpath.2009.06.014

Mukhopadhyay, S., & Katzenstein, A.-L. A. (2011). Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. The American Journal of Surgical Pathology, 35(1), 15–25. doi:10.1097/PAS.0b013e3182036d05

Köbel M, Duggan MA. Napsin a: another milestone in the subclassification of ovarian carcinoma. Am J Clin Pathol. 2014;142(6):735–737. doi:10.1309/AJCPAVGZKA1A1HVC.

Kandalaft PL, Gown AM, Isacson C. The lung-restricted marker napsin a is highly expressed in clear cell carcinomas of the ovary. Am J Clin Pathol. 2014;142(6):830–836. doi:10.1309/AJCP8WO2EOIAHSOF.

Fadare O, Desouki MM, Gwin K, Hanley KZ, Jarboe EA, Liang SX, et al. Frequent Expression of Napsin A in Clear Cell Carcinoma of the Endometrium: Potential Diagnostic Utility. Am J Surg Pathol. 2013. doi:10.1097/PAS.0000000000000085

p63

p63 is a nuclear transcription marker used in the identification of tumors subtypes and tissue structures such as: myoepithelial cell layer (breast), basal cell layer (prostate), salivary gland tumors, squamous cell carcinoma (most sensitive marker), skin appendage tumors, and urothelial carcinoma. As a general rule, transcription markers like p63 show a strong diffuse nuclear expression diagnostically.

Diagnostic Applications

Cell Differentiation Comments
Myoepithelial Cells Will stain cells in tumors of myoepithelial origin such as salivary gland tumors.
Urothelial Cells 70-100% of urothelial carcinomas will express p63
Squamous Cells Squamous Cell Carcinomas from most locations will mark with p63 resulting in a high sensitivity.
Stromal Invasion p63 can be very helpful to define stromal invasion, as it normally marks myoepithelial cells in breast tissue and basal cells in prostate tissue, which are lost in invasive carcinomas.

p63 expression in selected tumors, may have prognostic implications for Breast, Lung, and Bladder carcinomas.

GU Pathology

p63 is expressed in a high percentage of urothelial carcinomas, but is negative in renal cell carcinomas. A small percentage of collecting duct carcinomas (14%) have been reported to express p63. p63 may be helpful in differentiation of renal urothelial carcinoma and poorly differentiated renal cell carcinoma (RCC).

Arch Pathol Lab Med-Vol.135, January, 2011

Tumor p63 Expression (%)
Urothelial Carcinoma 70-100%
Renal Cell Carcinoma 0%
Sarcomatoid Urothelial Carcinoma 50%
Collecting Duct Carcinoma 14%

Placental Sit Trophoblastic Tumor

p63 is expressed in intermediate trophoblasts associated with placental site nodules, but is negative in placental site trophoblastic tumors (epithelioid trophoblastic tumors also express p63) [Mittal, et. al.].

Undifferentiated Carcinomas

CK5/6 co-expression with p63 is a sensitive (77%) and specific (96%) marker for squamous cell carcinoma. p63 appears to be very specific for squamous and urothelial origin. Only rare other non-squamous cell carcinomas show >50% expression of p63. [Kaufman, O., et. al.]

Lung Non-Small Cell Carcinoma

p63 is very sensitive (80-100%) in identifying squamous differentiation. It is often used along with CK5, TTF-1 and Napsin A to help subclassify non-small cell carcinomas (squamous cell carcinoma is classically CK5+ and p63+). As a nuclear transcription marker, it is important to see consistent diffuse expression (usually strong) to consider “positive.” It is not uncommon to find variable dim to moderate expression in a subset of the tumor cells in lung adenocarcinomas.

Small Cell Carcinoma

Small cell carcinomas express p63 in approximately 77% of cases (n=14). [Au, NH, et. al.] This could be a potential pitfall if one is considering a poorly differentiated squamous cell carcinoma and not a poorly differentiated neuroendocrine carcinoma.

Other

Thymomas express p63 along with a subset of lymphomas.

 

 

Variable subset nuclear expression of p63 in a lung adenocarcinoma, which is characteristic in a subset of cases. SCC expression should be diffuse and consistent.

p63 highlighting the basal layer of benign prostate gland epithelium.

Co-expression of CK5 (red) and p63 (DAB) in a lung squamous cell carcinoma. Note the diffuse consistent expression of p63.

PIN-4 expression in benign prostate. HMWCK and p63 highlighting basal layer without significant expression of AMACR (red) in the epithelial cells.

p63 highlighting nuclear expression in the myoepithelial cell layer surrounding LCIS. Best used in combination with another cytoplasmic myoepithelial marker (e.g. smooth muscle myosin, calponin, etc.).

References

Mukhopadhyay S, Katzenstein A-LA. Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. Am J Surg Pathol. 2011;35: 15–25. doi:10.1097/PAS.0b013e3182036d05

Di Como CJ, Urist MJ, Babayan I, Drobnjak M, Hedvat CV, Teruya-Feldstein J, et al. p63 expression profiles in human normal and tumor tissues. Clin Cancer Res. 2002;8: 494–501.

Gualco G, Weiss LM, Bacchi CE. Expression of p63 in anaplastic large cell lymphoma but not in classical Hodgkin's lymphoma. Hum Pathol. 2008;39: 1505–1510. doi:10.1016/j.humpath.2008.02.016

Au NHC, Gown AM, Cheang M, Huntsman D, Yorida E, Elliott WM, et al. P63 expression in lung carcinoma: a tissue microarray study of 408 cases. Appl Immunohistochem Mol Morphol. 2004;12: 240–247.

Kargi A, Gurel D, Tuna B. The diagnostic value of TTF-1, CK 5/6, and p63 immunostaining in classification of lung carcinomas. Appl Immunohistochem Mol Morphol. 2007;15: 415–420. doi:10.1097/PAI.0b013e31802fab75

Liu H. Application of immunohistochemistry in breast pathology: a review and update. Arch Pathol Lab Med. 2014;138: 1629–1642. doi:10.5858/arpa.2014-0094-RA

Hadi, AIMM Annual Meeting, "The Thirty Most Important Antibodies", presentation, 2011.

Truong, LD, et. al. "Immunohistochemical Diagnosis of Renal Neoplasms." Archives of Pathlogy and Laboratory Medicine. 2011;135:92-109.

Mittal, K, et. al. "Aplplication of Immunohistochemistry to Gynecologic Pathology." Archives of Pathology and Laboratory Medicine. Vol. 132, March 2008.

Kaufmann, O., et. al. "Value of p63 and CK 5/6 as IHC Markers for the Differential Dx. of Poorly Differentiated and Undifferentiated Carcinomas." American Journal of Clinical Pathology. 2001;116:823-830

Au NHC, Gown AM, Cheang M, et al. P63 expression in lung carcinoma: a tissue microarray study of 408 cases. Appl Immunohistochem Mol Morphol. 2004;12(3):240–247.

p40

p40 is a member of the p53 family, and is an isoform of p63. It is also known as delta-Np63. p63 can have different isoforms, and p40 reacts with truncated dominant isoforms of the p63 gene. (Brown, et. al.; Bishop, et. al.; and Ross, et. al.) p40 performs in a similar fashion to p63, but with so-called better specificity. (Bishop)

p40 appears very sensitive for lung squamous cell carcinomas (>90%) and urothelial carcinomas (>88%) from multiple studies. (Gaily, et. al. and Bishop, et. al.) Rarely (<5%) breast, biliary tract/pancreas, endometrium, and prostate showed reactivity, and evaluation of multiple cases (40-100 each) of renal cell carcinoma, thyroid, hempatocellular carcinoma, colon adenocarcinoma, stomach adenocarcinoma, and ovarian epithelium failed to show any reactivity (negative). (Brown, et. al.)

General Comments

Ross, et. al. favors the performance of p40 over p63 because only 3% of lung adenocarcinomas showed any positivity with p40 compared to 31% with p63. To an inexperienced pathologist using p63 in lung cancer subcategorization, partial positivity with p63 may lead to comments like “focal squamous differentiation” or “cannot exclude a component of squamous cell carcinoma”. These comments should be avoided, if possible. Often oncologists will not give the patient bevacizumab, which is an important therapy option, if there is any evidence of squamous differentiation due to the risk of pulmonary hemorrhage.

It is important to remember a basic tenant (general rule) in interpreting nuclear transcription markers like p40 and p63; expression should be consistent and diffuse in cells of interest to be considered significant.

References

Brown AF, Sirohi D, Fukuoka J, Cagle PT, Policarpio-Nicolas M, et al. (2013) Tissue-preserving antibody cocktails to differentiate primary squamous cell carcinoma, adenocarcinoma, and small cell carcinoma of lung. Arch Pathol Lab Med 137: 1274–1281. doi:10.5858/arpa.2012-0635-OA.

Bishop JA, Teruya-Feldstein J, Westra WH, Pelosi G, Travis WD, et al. (2012) p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol 25: 405–415. doi:10.1038/modpathol.2011.173.

Rossi G, Pelosi G, Barbareschi M, Graziano P, Cavazza A, et al. (2013) Subtyping Non-Small Cell Lung Cancer: Relevant Issues and Operative Recommendations for the Best Pathology Practice. International Journal of Surgical Pathology 21: 326–336. doi:10.1177/1066896913489346.

Gailey MP, Bellizzi AM (2013) Immunohistochemistry for the novel markers glypican 3, PAX8, and p40 (ΔNp63) in squamous cell and urothelial carcinoma. Am J Clin Pathol 140: 872–880. doi:10.1309/AJCP4NSKW5TLGTDS.

ROS-1

ROS-1 rearrangements with at least 12 different partner proteins have been identified in a small subset of lung non-small cell carcinomas (1–2%), which shows susceptibility to tyrosine kinase inhibitors (TKIs) similar to ALK rearranged tumors. ROS-1 is considered a oncogene found on chromosome 6. The exact mechanism of activation of this gene protein product with the various gene rearrangement partners is not understood. The protein function is similar to that of the ALK family, which is why this mutation was studied for possible response to ALK inhibitors (crizotinib).

Recently, ROS-1 mutated tumors have been approved for TKI therapy with identification of a rearrangement by FISH analysis. Like ALK, ROS-1 FISH utilizes a break apart probe to identify the presence of a gene rearrangement. Other successful modalities for identification of ROS-1 rearrangements include 'next generation' sequencing (NGS) and immunohistochemistry.

Immunohistochemistry (IHC) has been studied as an alternative to FISH as a screening modality. Based on multiple studies, the sensitivity of IHC appears to be near 100% with the specificity of at least 92%. These studies were performed using the D4D6 rabbit monoclonal antibody clone (Cell Signaling Technology, Danvers, Massachusetts).

Stain Interpretation

Unlike ALK, there is no known normal tissue counterpart which can be used as a control. Therefore, known ROS-1 positive tumors or cell lines (HCC78 cell line with the SLC34A2-ROS1 rearrangement) are generally used. ROS-1 expression is cytoplasmic with described expression ranging from finely granular to globular cytoplasmic staining and membranous staining. No consensuses has been established as to the minimal threshold of positivity.

Possible interpretation pitfalls include weak staining of type II pneumocytes and alveolar macrophages along with osteoclast-type giant cells in bone biopsies. Like any immunostain, contextual evaluation is critical.

References

Thunnissen E, Allen TC, Adam J, Aisner DL, Beasley MB, Borczuk AC, et al. Immunohistochemistry of Pulmonary Biomarkers: A Perspective From Members of the Pulmonary Pathology Society. Arch Pathol Lab Med. 2018;142: 408–419. doi:10.5858/arpa.2017-0106-SA

Shaw AT, Ou S-HI, Bang Y-J, Camidge DR, Solomon BJ, Salgia R, et al. Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J Med. 2014;371: 1963–1971. doi:10.1056/NEJMoa1406766

Bubendorf L, Büttner R, Al-Dayel F, Dietel M, Elmberger G, Kerr K, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch. 2016;469: 489–503. doi:10.1007/s00428-016-2000-3

Boyle TA, Masago K, Ellison KE, Yatabe Y, Hirsch FR (2015) ROS1 immunohistochemistry among major genotypes of non- small-cell lung cancer. Clin Lung Cancer 16(2):106–111. doi:10.1016/j.cllc.2014.10.003

CaoB,WeiP,LiuZ,BiR,LuY,ZhangL,ZhangJ,YangY,Shen C, Du X, Zhou X (2016) Detection of lung adenocarcinoma with ROS1 rearrangement by IHC, FISH, and RT-PCR and analysis of its clinicopathologic features. Onco Targets Ther 9:131–138. doi:10.2147/OTT.S94997

Sholl LM, Sun H, Butaney M, Zhang C, Lee C, Janne PA, Rodig SJ (2013) ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas. Am J Surg Pathol 37(9):1441–1449. doi:10.1097/PAS.0b013e3182960fa7

Yoshida A, Tsuta K, Wakai S, Arai Y, Asamura H, Shibata T, Furuta, K, Kohno T, Kushima R (2014) Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung can- cers. Mod Pathol 27(5):711–720. doi:10.1038/modpathol.2013.192

Rogers TM, Russell PA, Wright G, Wainer Z, Pang JM, Henricksen LA, Singh S, Stanislaw S, Grille J, Roberts E, Solomon B, Fox SB (2015) Comparison of methods in the detection of ALK and ROS1 rearrangements in lung cancer. J Thorac Oncol 10(4):611–618. doi:10.1097/JTO.0000000000000465

Rimkunas VM, Crosby KE, Li D, Hu Y, Kelly ME, Gu TL, Mack JS, Silver MR, Zhou X, Haack H (2012) Analysis of receptor tyro- sine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion. Clin Cancer Res 18(16): 4449–4457. doi:10.1158/1078-0432.CCR-11-3351

Mescam-Mancini L, Lantuejoul S, Moro-Sibilot D, Rouquette I, Souquet PJ, Audigier-Valette C, Sabourin JC, Decroisette C, Sakhri L, Brambilla E, McLeer-Florin A (2014) On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas. Lung Cancer 83(2):168–173. doi:10.1016/j. lungcan.2013.11.019

Shan L, Lian F, Guo L, Qiu T, Ling Y, Ying J, Lin D (2015) Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR. PLoS One 10(3): e0120422. doi:10.1371/journal.pone.0120422

TTF-1

TTF-1 is a nuclear transcription factor that is expressed in thyroid and respiratory epithelium. It is a useful marker for lung adenocarcinomas and thyroid malignancies. In the setting of neuroendocrine carcinomas, TTF-1 expression is not specific as to the site of origin, except that Merkel cell carcinomas of the skin do not usually express TTF-1 (some have reported focal expression). The expression pattern of TTF-1, like other transcription markers, is generally strong and diffuse. Lower levels of positivity should bring caution to the interpretation.

TTF-1 use has become more important to sub-classify lung tumors on small biopsy specimens. It is generally considered the most sensitive and specific individual marker for lung adenocarcinomas, but is often used in combination with Napsin A to maximize sensitivity and specificity for the determination of primary lung adenocarcinomas. Current recommendations are to test non-small cell lung carcinomas (non-squamous cell carcinoma) for ALK, ROS-1 and EGFR mutations, as targeted therapies are available (this list is ever expanding and should be verified with current medical literature).

Expression in Lung Tumor Subtypes

Lung Tumor Subtype Expression (%)
Squamous Cell Carcinoma 0-5%
Adenocarcinoma 80%
BAC (non-mucinous) 90%
BAC (mucinous) <10%
Carcinoid Tumor 0-35%
Atypical Carcinoid >85%
Small Cell Carcinoma >95%
Large Cell Undifferentiated 50-70%

Napsin A and TTF-1 expression in poorly differentiated non-small cell carcinomas (Mukjopadhyay, S, et al).

Tumor Napsin A TTF-1
Adenocarcinoma 11/19 (58%) 16/20 (80%)
Squamous Cell Carcinoma 0/15 (0%) 0/15 (0%)
Large Cell Carcinoma 0/4 (0%) 2/4 (50%)

Common expression patterns in carcinoma (Dennis, et al)

Tumor Expression (%)
Breast 0%
Colon 0-10%
Lung >75%
Ovary 0%
Pancreas <5%
Stomach <5%
Prostate <5%

Napsin A and TTF-1 expression in various tumor types (Bishop, JA, et al).

Tumor Napsin A (% positive) TTF-1 (% positive)
Lung Tumors
Adenocarcinoma 79/95 (83%) 69/95 (73%)
Well-differentiated 42/47 (89%) 38/47 (81%)
Moderately-differentiated 27/32 (84%) 24/32 (75%)
Poorly-differentiated 11/16 (69%) 7/16 (44%)
Squamous Cell Carcinoma 0/46 (0%) 0/48 (0%)
Large Cell Carcinoma 3/9 (33%) 4/9 (44%)
Small Cell Carcinoma 0/3 (0%) 1/3 (33%)
Atypical Carcinoid Tumor 0/1 (0%) 1/1 (100%)
Typical Carcinoid Tumor 0/2 (0%) 1/2 (50%)
Non-pulmonary Adenocarcinomas
Colon Adenocarcinoma 0/5 (0%) 0/5 (0%)
Pancreas Adenocarcinoma 0/31 (0%) 0/31 (0%)
Breast Adenocarcinoma 0/17 (0%) 0/17 (0%)
Mesothelioma (all types) 0/38 (0%) 0/38 (0%)
Renal Cell Carcinomas
Clear Cell 14/41 (34%) 0/41 (0%)
Papillary 34/43 (79%) 0/43 (0%)
Chromophobe 1/34 (3%) 0/34 (0%)
Thyroid Lesions
Papillary Carcinoma 2/38 (5%) 37/38 (97%)
Follicular Carcinoma 0/15 (0%) 15/15 (100%)
Follicular Adenoma 0/28 (0%) 28/28 (100%)

Pitfalls

Don’t forget that thyroid neoplasms can metastasize to the lung (especially follicular carcinomas), which may occur after a significant time period from the original diagnosis. Therefore, in the absence of Napsin A expression, additional staining for thyroglobulin may be helpful to r/o a primary thyroid tumor.

An additional interesting staining pattern with TTF-1 is in cells of hepatic origin. While they do not express TTF-1, TTF-1 will stain cells of hepatic origin in a granular cytoplasmic pattern similar to HepPar1.

Recently, Aulakh, KS, et al demonstrated that primary esophageal adenocaricnomas may have similar expression patterns of both Napsin A and TTF-1 compared to primary lung adenocarcinomas, and therefore immunohistochemistry for Napsin A and TTF-1 alone is not effective in separating primary origin in this situation.

Rare primary breast carcinomas may express TTF-1 (2-3% in large study sets).

Occasional cases of endometrial adenocarcinoma may express TTF-1 (~17%).

Normal Expression Pattern

Lung

  • Alveolar epithelium
  • Non-ciliated respiratory epithelium

Thyroid

  • Follicle cells
  • C-cells

Parathyroid

Pituitary (anterior)

Brain (diencephalon)

TTF-1 (nuclear)/Napsin A (cytoplasmic) – Lung Adenocarcinoma

TTF-1 – Lung Squamous Cell Carcinoma. Benign pneumocytes at the peripheral of the tumor showing TTF-1 expression.

TTF-1 – Benign Liver

TTF-1 (Brown-Nuclear)/Napsin A (Red-Cytoplasmic) – Metastatic Lung Adenocarcinoma

TTF-1 – Lung Adenocarcinoma

References

Hadi, AIMM Annual Meeting, "Carcinomas of Unknown Primary", presentation, 2011.

Bishop, J. A., Sharma, R., & Illei, P. B. (2010). Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Human Pathology, 41(1), 20–25. doi:10.1016/j.humpath.2009.06.014

Dennis, J. L., Hvidsten, T. R., Wit, E. C., Komorowski, J., Bell, A. K., Downie, I., et al. (2005). Markers of adenocarcinoma characteristic of the site of origin: development of a diagnostic algorithm. Clinical Cancer Research : an Official Journal of the American Association for Cancer Research, 11(10), 3766–3772. doi:10.1158/1078-0432.CCR-04-2236

Inamura, K., Takeuchi, K., Togashi, Y., Hatano, S., Ninomiya, H., Motoi, N., et al. (2009). EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset. Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc, 22(4), 508–515. doi:10.1038/modpathol.2009.2

Aulakh, K. S., Chisholm, C. D., Smith, D. A., & Speights, V. O. (2013). TTF-1 and napsin A do not differentiate metastatic lung adenocarcinomas from primary esophageal adenocarcinomas: proposal of a novel staining panel. Archives of Pathology & Laboratory Medicine, 137(8), 1094–1098. doi:10.5858/arpa.2012-0305-OA

Mukhopadhyay, S., & Katzenstein, A.-L. A. (2011). Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. The American Journal of Surgical Pathology, 35(1), 15–25. doi:10.1097/PAS.0b013e3182036d05

Miller, Rodney. American Academy of Oral and Maxillofacial Pathology Annual Meeting, "Immunohistochemistry in the Diagnosis of Metastatic Carcinoma of Unknown Primary Origin," presentation, 2012.

Disclaimer

This presentation was prepared by Dr. Philip Ferguson, a board certified pathologist practicing in Jonesboro, AR. Dr. Ferguson was compensated for this presentation. The presentation represents Dr. Ferguson’s views, and does not necessarily represent or reflect the views or opinions of Leica Biosystems. It is for educational purposes only and is not intended as regulatory or legal advice or as diagnostic advice for a particular patient or a particular disease or condition.”