Immunohistochemistry is an important complimentary tool for diagnosis of cancer"


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  • When morphology and routine staining cannot provide all the diagnostic answers, pathologists turn to advanced staining techniques like Special Stains, Immunohistochemistry (IHC)  and In Situ Hybridization (ISH).
  • Immunohistochemistry has become the “Gold Standard” for many diagnostic situations. However, the vast majority of cases do not require any additional stains other than the H & E. or special Stains.
    In the words of Dr.Rodney Miller : “….a Pathologist must be committed to remaining current with ever-expanding IHC literature, since awareness of the specificities of the antibodies and expected patterns of reactivity in tissue is critical to employ this technique effectively and avoid diagnostic or interpretive errors.”.
  • In most cases YES. But we need all three. Special stains and IHC both compliment H&E in making the correct diagnosis. In the following few slides I will show cause why and how special stain and IHC compliment H&E and then I will move on to Immunohistochemistry and panels in diagnosis of cancer.
  • Special stains remain an important tool for many Pathologists providing a powerful complement to H&E.
    Micro-organisms are easily and clearly identified using Special Stains than with IHC.
  • Special stain gives a better contrast and morphology of the spirochetes than IHC.
  • Adenocarcinoma stained with special Stain and MUC-1
  • When Pathologists come across a case of poorly differentiated carcinoma or undifferentiated carcinoma of unknown origin, an additional test like special stains or IHC or both is necessary for a correct diagnosis of the disease.
    IHC is also very useful when it is important to know the sub-type of the cancer for selecting the drug of choice or to monitor treatment based on the progress of the disease.
    1) Sometimes it is difficult to identify if the tumor is from epithelial or from connective tissues or from plasma cells or lymphatic or from mixed cell types. IHC is very useful in these cases to identify if it is a carcinoma or sarcoma or lymphoma or mixed like carcinosarcoma or adeno-squamous carcinoma etc
    2) IHC is a reliable tool in deciding if the tumor originated as a primary or it is metastatic from another organ.
    3)In Treatments: BRCA1,BRCA2 for Hereditary BrCa, Her2 for Herceptin treatment, BCR for CML(Gleevac)
    4) IHC play an increasingly important role in the clinical management of breast cancer(ER,Ki67),VEGF in Melanoma, P63 in Head and neck cancer.
    5) without IHC it is difficult to diagnose correctly any Hematolymphoid proliferations.
    6)However PSAP will stain carcinoid tumors of unknown origin so should not conclude that it is from prostate. If you include an nuero endocrine group antibody you will get the correct Dx
    7) Intraoperative SLN evaluation by imprint cytology with immunohistochemistry achieves a more accurate diagnosis of metastasis than imprint cytology alone
  • These classifications provide doctors with valuable information about how the tumor acts and what kind of treatments may work best.
    In general, surgical and radiation treatments are similar for these different types of breast cancer. But drug treatments -- such as chemotherapy, endocrine therapies, and other medications -- are usually different. These treatments are targeted to the specific type of cancer.
  • Markers for Tissue of origin
  • The immunohistochemical reactions can be used in different situations within research or pathological anatomy laboratories. The most important are: 1) histogenetic diagnosis of morphologically non-differentiated neoplasias ; 2) subtyping of neoplasias (such as lymphomas, for example); 3) characterization of primary site of malignant neoplasias; 4) research for prognostic factors and therapeutic indications of some diseases; 5) discrimination of benign versus the malignant nature of certain cell proliferations ; identification of structures, organisms and materials secreted by cells.
  • An Algorithm can be designed broadly or based on information available to the Pathologist about the patient he or she can design a simple algorithm to R/O some doubts and confirm some Dx.
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    Example of most common
    -Applying this panel and the algorithm correctly classified 88% of the original known tumors.(352 known Ad ca were used)However,HCC expressed none of these markers. If you do TTF-1 first then remember that TTF-1 will show cytoplasmic reactivity in a granualr pattern in most of Prostate Ad Ca. True staining should be Nuclear. Some tumors had similar profiles that were difficult to separate: pancreas and stomach (but we can say with some degree of confident that if CK7 is negative ca of pancreas unlikely, CA 125 pos and mesothelin pos then more likely pancreas. ER,mesothelin and CA125 together indicate ovary.Cocktails: Mammaglobin + ER is good cocktail for br ca.
    MC5 + will increase the specificity upto 98%
    Explanation: GCDFP 15 +, CDX2 (-), at this stage if we do MC5 and if that is positive then we can say with 98% certainity that the primary tumor is breast.
    Here is another scenario:
    GCDFP15(-), CDX2(-),ER + BUT Mesothelin (-) = Breast.
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    Dx difficult when tumor involves the lower uterine segmnt or upper endocx.It becomes more difficult in the mucinous subtype in which stroma is absent, (one of the feature that suggests an EM origin).
    This differentiation is very important due to its clinical significance,that is their differences in management of the disease and prognosis. While the treatment of EM ca starts with surgical staging and intraoperative assessment of the grade and extent of tumor in the uterus, primary ECA is treated by an initial radical hysterectomy and pelvic lymph adenectomy with or without adjuvant radiation.
    All four ab as a panel gives 100% differential Dx. However any two markers MUC1 and p16, MUC1 with ER/PR also gives best results.
    You can include Vim also EMMA(70%) and ECA(7%)
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    Panel of Abs is very useful when you have a dilemma to treat or not decision to make in a not so sure diagnosis.
    Is it BPH (Benign Prostatic Hyperplasia? Or PIN Prostate Intra epithelial Neoplsm - a precursor to Prostate cancer?
  • <number>
    In most cases of a poorly differentiated carcinoma within the area of prostate/urinary bladder, morphological features on the H & E alone is sufficient. However if overlapping morphologic features of both neoplsms are present, making determination of site of origin is very difficult and may necessitate IHC confirmation.
  • For example: BRCA1/2 predisposes to Breast cancer.Over expression of HER2/neu in 30% of Breast cancer patients, amplification of RAS in colorectal cases and over expression and suppression of PTEN vs AKT in cancer modulations. Presence of HPV in Head and neck Sq.Cell Carcinoma and Genital warts and cervical cancer.
    CML-Chronic Myelogenous Leukemia results in Philadelphia Chromosome( Drug: Imatinib
    CRC:ColoRectal Cancer
  • PanCK(AE1/AE3) can detect most of carcinoma. CD45 will be positive for 95% of Lymphoma, mMelanoma will be 95 to 100% positive with HMB45. There are a few exemptions to the above. However, PanCK is positive for Gilial Tumors and Schwanoma and mesenchymal tumors. And..Negative for Hepato Cellular Carcinoma. PanCK is also positive for mixed tumors like Sarcomatoidcarcinoma. In Renal Cell carcinoma, Endometrial adenoCarcinoma, Ovarian carcinoma and Thyroid carcinoma, Vimentin is co-expressed with Cytokeratins.
  • Expression of CK7 and CK20 can differentiate most of Carcinoma from each other.
  • Sq CC Vs AdCa
  • Primary and Addl Markers
  • However each group can be further differentiated by using specific Antibodies for each disease. (Please refer to hand out for details).
    Algorithmic immunohistochemical analysis of undifferentiated carcinomas. CA indicates carcinoma; adenoCA, adenocarcinoma; SmCC,
    small cell carcinoma; SCC, squamous cell carcinoma; RCC, renal cell carcinoma; HCC, hepatocellular carcinoma; ¶, seminoma is keratin negative,
    OCT3/4 positive; *NE markers, neuroendocrine markers, including synaptophysin, chromogranin, and CD56; , undifferentiated anaplastic thyroid
    carcinoma is often negative for thyroid transcription factor 1 (TTF-1) and thyroglobulin; and , characteristic canalicular pattern.
  • Here is another algorithm. As you can see most of the antibodies used are common ones routinely used in cancer diagnosis. Diagnostic algorithm to characterize metastatic adenocarcinoma from unknown primary. (CK: cytokeratin; TTF1: thyroid
    transcription factor 1; ER: estrogen receptor; CA125: cancer antigen 125; tireo: tireoglobulin; VIM: vimentin; PSA: prostate specific antigen)
  • With the help of IHC as a tool in diagnosis 90% of cancer can be correctly guessed.
  • KEY: + (few -) = Large majority of cases positive, a few negative cases may be seen
    - (foc +) = Largely negative, but scattered positive tumor cells may be seen
    +/- = Tumor may be either positive or negative
    + (rare-) = Tumor positive, but rare negative cases may be encountered
  • Liver Lesion panel
  • J. Ye, J. J. Findeis-Hosey, Q. Yang et al., “Combination of napsin A and TTF-1 immunohistochemistry helps in differentiating primary lung adenocarcinoma from metastatic carcinoma in the lung,” Applied Immunohistochemistry and Molecular Morphology, vol. 19, no. 4, pp. 313–317, 2011.
  • Hodgekin’s:NLPHL with LCA+, CD20+, EMA+/-,
    EBER –
    CHL with a LCA -, CD30+, CD15+,
  • Breast
    ER, + or -
    PR, + or -
    GCDFP ;gross cystic fluid protein, 75 to 95% +
    CK7, CK20 + or – from Colon or Lung
    E-cadherin + DCIS and - ILC
    Lung: Ber-EP4 + reactive mesothelial cells from cancer cells.
  • I thank NYU for giving me an opportunity to work on Immunohistochemistry from its infant stages (1984 …)
    I thank DAKO for hiring me to develop new antibodies which were flooding the market after completion of human genome mapping.
    I also thank Quest Dx for asking me to join their Clinical Trial team for Bio-Marker assay development for leading Bio Pharmas like Pfizer, Scherring-Plough, Merck, Astra-zenka, Biogen-Idec, Roche etc etc
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  • Immunohistochemistry is an important complimentary tool for diagnosis of cancer"

    1. 1. “ IHC is an Important Complimentary Tool for Diagnosis of Cancer” Lawrence T. Richards M.S.,H.T(ASCP), QIHC(ASCP) Consultant, Bio Marker Assay Development, Santa Barbara, California. For teaching purpose only
    2. 2. “ The Diagnostic Power of any Immunohistochemical Procedure is no Greater than the wisdom of the Pathologist interpreting it.” Dr.Allen M.Gown
    3. 3. IHC as a compliment to H&E • Is H&E alone not enough? • Is Special Stain not enough? • Is IHC alone enough?
    4. 4. Special Stain Antibody for Yeast Budding yeast GMS Stain and H&E (Blastomycosis). AFB HP Alcian YellowToluidine Blue
    5. 5. Special stain vs IHC H.pylori Alcian Yellow-Toluidine Blue H.Pylori antibody
    6. 6. Usefulness of IHC tests. • Poorly differentiated tumors and mixed carcinomas • Undifferentiated tumors of unknown origin • Treatment based on sub-type of cancer: Personalized Medicine eg., Breast Ca • Monitoring progress of cancer (Predictive and Prognosis) • In Malignant Lymphoma, • In identifying Carcinoid (Neuroendocrine) tumors • In Cytologic specimens
    7. 7. IHC helps in deciding Treatments ER/PR About 75% of all breast cancers are “ER positive.” They grow in response to the hormone estrogen. About 65% of these are also “PR positive.” They grow in response to another hormone, progesterone. If your breast cancer’s cells have a significant number of receptors for either estrogen or progesterone, your cancer is considered hormone-receptor positive and likely to respond to endocrine therapies Breast cancer tumors that are ER/PR-positive are 60% likely to respond to endocrine therapy. Tumors that are ER/PR negative are only 5% to 10% likely to respond to endocrine therapy Her2 In about 20% to 25% of breast cancers, the cancer cells make too much of a protein known as HER2/neu. These breast cancers tend to be much more aggressive and fast-growing. For women with HER2-positive breast cancers, the drug Herceptin has been shown to dramatically reduce the risk of recurrence Triple Negative Some breast cancers -- estimates range between 10% and 17% -- are known as “triple negative” because they lack estrogen and progesterone receptors and do not overexpress the HER2 protein. The majority of breast cancers associated with the breast cancer gene known as BRCA1 are triple negative. These cancers generally respond well to adjuvant chemotherapy
    8. 8. Histiogenic Dx of Neoplasm • ID of proliferation of cells Epithelium NeuroEndocrine Melanocyte A) Expression of cytokeratin AE1/AE3 in lung carcinosarcoma ; B) chromogranin expression in gastric neuroendocrine carcinoma ; C) HMB 45 immunostainning in melanoma .
    9. 9. Metastatic Adenocarcinoma of unknown origin with site specific markers PSA (-) + TTF-1 (-) + GCDFP15 + (-) CDX2 / CK20 Colon Lung CDX2 CK 7 Mesothelin (-) + (-) + Mesothelin (-) + Lysozyme + (-) CA125 + (-) (-) + ER MC5+ (98%) Prostate + Breast Stomach/Pancreas + (-) Ovary (-) Breast Pancreas,(Ovary serous) Stomach / Pancreas Stomach / Pancreas Breast / Stomach / Pancreas Source: Colon
    10. 10. Endocervical Ad.Ca & Endometrial Mucinous Ad.Ca ECA • • • • MUC-1(-) ER(-) PR(-) P16(+) EMMA • • • • MUC-1 (+) ER(+) PR(+) P16(-) Khoury T et al.BMC Clin Path 2006;6:1
    11. 11. Prostate Ca or Benign ? • Prostate Cancer • • • • • • • • EpCam + ATM + AMACR + PSA + / (-) 34ßE12 (-) almost all p63 (-) almost all Prostein + NKX3.1 + • Benign Prostate • • • • • • • • EpCam (-) ATM (-) / + AMACR (-) PSA + / (-) 34ßE12 + p63 + Prostein + NKX3.1 + ATM=ataxia-telangiectasia mutated;AMACR=alpha-methylacyl-CoA racemase;Ep-Cam=epithelial transmembrane glycoprotein Hammerich KH et al.Application of Immunohistochemistry to the Genitourinary System(Prostate, Urinary Bladder, Testis, and Kidney)Archives of Pathology and Laboratory Medicine;132:432-440,2007
    12. 12. Urothelial Ca vs Prostate Ca Urothelial ca • • • • • • • • • • EMA CK7 P63 CK5/6 EpCam CD57 PSA PAP NKX3.1 Prostei n Prostate ca + + +/0 +/0 +/0 -/+ +/0 + 0 +/0 + 0 + 0 + Hammerich KH.Archives of Pathology and Laboratory Medicine;132(3):432-440 Urothelial ca Prostate ca
    13. 13. Prognastic Risk Biomarkers (or screening biomarkers) Describe risk of cancer occurrence or cancer progression because they are implicated in neoplastic progression and include: 1. Genetic predisposition (e.g., BRCA1/2) 2. Over expression of genes (e.g., BCRABLTyrosin Kinase Inhibitor in CML, HER-2/neu in Br Ca,PTEN, RAS,Colo Rectal Cancer AKTin Pancreatic cancer) 4.Environmental factors and lifestyle (e.g., HPV or HBV infection The-use-of-Biomarkers-for-Treatment-Sessa-Fasolo.pdf
    14. 14. Undifferentiated Tumors Use of IHC to differentiate broad lineage Pan CK AE1/AE3 CD45 (LCA) HMB45 or S100 VIM Carcinoma Positive Negative Negative Negative Melanoma Negative Negative Positive Sarcoma Negative Negative Negative Positive Lymphoma Negative Positive Positive Negative Negative
    15. 15. Carcinoma Use of CK7 and CK20 CK7 + CK20 + CK7 + CK20 - CK7 – CK20 + CK7 – CK20 - Urothelial Ca Pancreatic Ad Ca OvarianMucinous Ca Ad Ca of Bladder Gastric Ad Ca Cholangio Ca subset Breast Ca EndoMetrial Ad Ca EndoCervical Ad Ca Ovarian Cerous Ca Lung Ad Ca Cholangio Ca LungSmCC Mesothelioma Thyroid Ca SCC of Cervix SalivaryGland tumors Urothelial Ca subset Pancreatic and Gastric Ad Ca subset Colorectal Ad Ca Markel Cell ca Gastric Ad Ca subset Prostate Ad Ca SCC RCC HCC Mesothelioma AdrenoCortical ca NonSeminoma LungSmCC minorSubset Gastric Ad Ca subset
    16. 16. Immunohistochemistry stains in squamous cell carcinoma and adenocarcinoma of lung. H&E: hematoxylin and eosin; CK: cytokeratin; TTF-1: thyroid transcription factor 1. Squamous carcinomas are typically positive for CK5/6 and P63, and negative for CK7 and TTF-1, with the reverse profile for adenocarcinoma although this case of squamous cell carcinoma demonstrates focal weak staining for CK7. SqCC AdCa “Ancillary Testing in Lung Cancer Diagnosis” Dublinski et al.
    17. 17. Primary and Additional Markers For teaching purpose only For teaching purpose only
    18. 18. CK7 and CK20 For teaching purpose only
    19. 19. Metastatic AdenoCarcinoma from Unknown Primary Tumors from Unknown Primary Site For teaching purpose only
    20. 20. CUP Diagnosis (Cancer of Unknown Primary) For teaching purpose only
    21. 21. Prostate Ca vs Urothelial Ca Cancer PSA/ CKPSAP hmw CK7 CK20 Prostate + Adenoca few - few + foc + foc + Urothelial Ca + few - + rare - +/- CK17 T -Mod CEAm CD57 Leu 7 6%+ - foc + + few - +/- + 60-90% foc + + few-
    22. 22. Immunoperoxidase Panel for Liver Lesions Hepatocellular Colon Biliary Pancreas Ovary /EM Sarcoma Hepar + - - - - - AFP + - - - - - CEA - + + +/- +/- - CK7 - - + + + - CK20 - + ? ? - - AE1 AE3 +/- + +/- + + - CA 19-9 - - - + - - ER + (Not for Renal Cell Carcinoma). (?) suggests could be positive 35% to 65% or negative); and +/- dependent on subtype of histology from that organ. -
    23. 23. Napsin A + TTF-1 Napsin A, an aspartic acid protease whose expression in the lung is regulated by TTF-1, has also shown promise in helping to differentiate primary lung from metastatic adenocarcinomas. While Napsin A expression may also be seen in normal kidney and in a proportion of renal tumors, positivity for both TTF-1 and napsin A is a strong indication that an adenocarcinoma originated from lung .
    24. 24. Examples of common panels of Antibodies Used Generic T-cell Vs B-Cell : CD3, CD20, CD45 Folicular Lymphoma Vs Hyperplasia: Bcl2, Bcl6, CD3, CD10,CD20 Low Grade B Lymphoma: CD3, CD5, CD10, CD20, CD23, CD43, Bcl2, Bcl6, MALT Lymphoma: CD3, Cd5, CD20, Bcl2, ISH Kappa and Lambda Hodgkin’s Lymphoma: CD3, CD15, CD20, CD30, CD45 Myeloma: CD138, ISH Kappa and Lambda Carcinoma Vs Lymphoma: CD3, CD20, CD45, PanCK Metastatic Carcinoma: CK7, CK20, TTF-1 GIST: CD117, CD34, S100, Desmin, SMA Mesothelioma: PanCK, CK5/6, Calret, TTF-1, CEA, CD15 If male add PSA/ if female add BRST2
    25. 25. Antibodies commonly used • Breast ER, PR, gross cystic fluid protein, CK7,CK20, E-cadherin • Colon and other GI tract CEA (monoclonal), CK7, CK20 • Germ cell PLAP, -fetoprotein, -HCG, AE1/3 • Hepatocellular Hepar, -fetoprotein, CK7, CK20, AE1 and AE3 (separately) • Lung TTF-1, CK7, CK20, CEA, Ber-EP4, chromogranin, synaptophysin, S100 • Lymphoma LCA (CD45RB monoclonal), CD3, CD20, CD30, ALK-1, myeloperoxidase, and light chains, Bcl-2
    26. 26. Antibodies commonly used contd. • Mesothelioma: Calretinin, AE1, AE3 • Melanoma: S100 (when spindled cells), HMB-45 • • • • • • • (when epithelioid), MART1 Neuroendocrine : Chromogranin, synaptophysin, NSE Pancreas : AE1/3, CK7, CK20, CA 19-9, CEA, chromogranin, synaptophysin, -antichymotrypsin, CD10, PR, Ber-EP4 Prostate :PSA, CK7, CK20 Renal: EMA, CD10, HMB-45, inhibin- (to exclude adrenal /cortical) Sarcoma : Vimentin, S100, CD117 (c-Kit), CD34,SMA, myogenin, CD31, CD68, desmin,CD1a, CD99 Thyroid: TTF-1, thyroglobulin, calcitonin, CEA Urothelial : CK7, CK20,Uroplakin
    27. 27. Conclusion Thank you for giving me an opportunity to share my knowledge with you all. It is my hope that that this presentation will fulfill the goal of providing the attendees a little insight into the powerful tool of IHC in diagnosis of difficult cases and help in monitoring disease progression in patients and course correction in treatment procedures.
    28. 28. Acknowledgements New York University -Department of Environmental Medicine DAKO -Research and Development Quest Diagnostics -Clinical Trials and Bio-Marker Development