What is the difference between IHC and ICC?In the 1930s, the principles of immunohistochemistry (IHC) were already known, but it wasn’t until 1942 that the first IHC research results were officially published. This is regarded as a milestone in immunofluorescence technology. Albert Coons from Harvard Medical School published an article in the Journal of Immunology, using FITC-labeled antibodies to identify pneumococcal antigens in infected tissues. Since then, methods for tissue fixation, detection labels, and microscopy have continuously improved, making immunohistochemistry an essential tool in diagnosis and research.In pathology, using specific tumor markers, doctors diagnose whether tumors are benign or malignant through IHC, determine the stage and grade of tumors, and identify cell types and sources of metastasis to locate the primary tumor. Similarly, IHC can also be used in drug development to assess drug efficacy by detecting the upregulation or downregulation of disease targets.Immunohistochemistry refers to the detection of antigens (such as proteins) in tissue sections based on the principle of specific binding between antibodies and antigens. The root ‘immuno’ comes from the antibodies used in the process, while ‘histo’ means tissue. A similar technique is immunocytochemistry (ICC). These two terms are often used interchangeably, but there are some differences.For IHC, tissues are taken from patients or animals and are frozen or embedded in paraffin. These tissues are sliced into approximately 4μm thick sections, processed after sealing. Through this method, researchers can observe the localization of cellular components while maintaining the original structure of the surrounding tissues.For ICC, most extracellular matrix and other matrix components are removed, leaving only whole cells for staining. The source of ICC can be cell suspensions from patients or animals (such as blood smears, swabs, etc.) or cell lines cultured in the laboratory.Aside from biological sources, IHC and ICC also differ in the extent of sample processing. ICC requires permeabilization, either through the fixation process or as a separate permeabilization step, so that antibodies can bind to intracellular targets. IHC may not require a separate permeabilization step, depending on the thickness of the sections and the fixation method. IHC sections embedded in paraffin must be further processed before antibody staining. Once the samples are prepared, the staining operations for IHC and ICC are almost identical. Of course, in terms of staining, optimizing based on the antibodies used is still essential.Tumor Cell Immunohistochemical Drug Resistance Prognostic MarkersMalignant tumor immunohistochemical drug resistance prognostic markers, a complete set of 4 items: P-gp, GSTπ, TOPOⅡ, Ki-67.Breast cancer immunohistochemical drug resistance prognostic markers, a complete set of 7 items: P-gp, GSTπ, TOPOⅡ, Ki-67, ER, PR, C-erbB-2.1, P-glycoprotein (P-gp)–Drug pump effect–Cell membrane/cytoplasm–The higher the positive rate, the stronger the resistance to the following drugs: Doxorubicin, Epirubicin, Mitoxantrone, Vincristine, Vinblastine, Taxotere.2, Glutathione S-transferase (GST π)–Detoxification effect–Cytoplasm–The higher the positive rate, the stronger the resistance to the following drugs: Doxorubicin, Cisplatin, Nitrogen mustard, Cyclophosphamide, Tumor necrosis factor.3, Topoisomerase II (TOPOⅡ)–Target effect–Nucleus–The higher the positive rate, the more effective against the following drugs: Anthracycline antibiotics and Vinca alkaloids, such as VP-16, Teniposide, Mitoxantrone, Doxorubicin, VM26. Those with high positive rates are particularly effective against VP-16.4, Estrogen Receptor (ER)–Sex hormone effect–Nucleus–The higher the positive rate, the more effective the tumor is to endocrine therapy, and the better the prognosis.5, Progesterone Receptor (PR)–Sex hormone effect–Nucleus–The higher the positive rate, the more effective the tumor is to endocrine therapy, and the better the prognosis.6, C-erbB-2–Oncogene product–Cytoplasm–The higher the positive rate, the higher the malignancy of the tumor. Those who are ER and PR positive but also C-erbB-2 positive do not respond well to Tamoxifen treatment.7, Ki-67–Cell proliferation marker–Nucleus–The higher the positive rate, the faster the tumor proliferates, the higher the malignancy.Ki-67 is a marker of cell proliferation, expressed in all phases of the cell cycle G1, S, G2, and M, absent in G0 phase, closely related to tumor differentiation, infiltration, metastasis, and prognosis.8, PCNA (Proliferating Cell Nuclear Antigen).9, CEA Most adenocarcinomas express CEA.10, Rb (Retinoblastoma) is a tumor suppressor gene that regulates the cell cycle.11, P53 In immunohistochemistry, it is usually mutated; the higher the positive rate, the worse the prognosis. The wild-type has a very short half-life.12, Nm23 is a metastasis suppressor gene, and its positive expression is negatively correlated with tumor metastasis. It has been widely used in the detection of various malignancies such as breast cancer, non-small cell lung cancer, gastric cancer, colorectal cancer, liver cancer, and laryngeal cancer. Almost all studies have shown that patients with high expression of nm23 protein have a relatively low rate of lymph node metastasis and a longer survival period.13, E-Cadherin is a transmembrane glycoprotein that mediates intercellular adhesion, and its loss of function leads to the destruction of connections between cells, mainly used in studies of tumor invasion and metastasis.14, PS2 is an estrogen-regulated protein, its expression is related to ER expression, and it can serve as one of the indicators for endocrine therapy and prognosis judgment.15, CK18 is a low molecular weight keratin, mainly marking various single-layer epithelium including glandular epithelium, while stratified squamous epithelium is often negative, mainly used for adenocarcinoma diagnosis.16, CK19 is distributed in single-layer epithelium and mesothelium, commonly used for adenocarcinoma diagnosis, hepatocytes do not express it, while bile ducts show positive reaction.17, Hep Par 1 is a hepatocyte antigen, positive in normal hepatocytes and well-differentiated hepatocellular carcinoma, while poorly differentiated hepatocellular carcinoma is often weakly positive or negative.18, CK20 is used for diagnosing gastrointestinal adenocarcinoma, ovarian mucinous tumors, and Merkel cell carcinoma of the skin. Squamous carcinoma, breast cancer, lung cancer, endometrial and non-mucinous ovarian tumors are often negative.19, CK7 is commonly positive in ovarian, lung, and breast epithelium, while negative in colon, prostate, and gastrointestinal epithelium.20, Villin is a brush border protein, typically expressed only in cells with a brush border, such as gastrointestinal epithelial cells, pancreatic and bile duct epithelial cells, and epithelial cells of the renal parenchyma (especially the proximal tubules).Villin has a high expression rate in gastrointestinal cancer, pancreatic cancer, gallbladder cancer, and bile duct cancer tissues. If a tumor with a significant glandular structure shows no villin expression, it is highly unlikely to originate from the gastrointestinal tract, pancreas, gallbladder, or bile duct. Breast cancer often becomes a disease to rule out in cases of unknown primary metastatic cancer in female patients. If significant villin immunohistochemical positive staining is observed in metastatic cancer tissues, it is extremely unlikely that this tumor is of breast origin.Other tumors that usually show negative villin immunohistochemical staining include: serous ovarian cancer, transitional cell carcinoma of the urethra, and prostate cancer. Mesothelioma is also often negative for villin expression; therefore, in some cases, villin can also be used as an antibody to differentiate between mesothelioma and adenocarcinoma.However, there are some non-gastrointestinal tumors that can express villin, such as endometrioid adenocarcinoma, mucinous ovarian cancer, renal cell carcinoma, and a small portion of lung cancer. Some experts have also reported villin expression in certain cases of cervical endometrial adenocarcinoma.Diagnosis of liver cancer: Villin immunohistochemical staining can show the structure of bile ducts, so it may also be very useful in expressing tubular structures in certain liver cancers. Polyclonal CEA is the first reagent used for this purpose, and CD10 (CALLA) is also very useful in expressing this structure in liver cancer. The expression of polyclonal CEA, villin, and CD10 (CALLA) in liver cancer cases does not conflict with each other; therefore, if liver cancer is suspected, it is recommended to use these three antibodies together to assist in the diagnosis of difficult cases.Villin is also very helpful in the study of neuroendocrine tumors. It is well known that carcinoids and pancreatic islet cell tumors have similar morphological characteristics, and it is almost impossible to distinguish between these two tumors morphologically. Villin is particularly useful in this case because literature reports that 85% of gastrointestinal carcinoid cases express villin, while no positive expression has been reported in islet cell tumors. The expression of villin in carcinoids is usually membrane-positive.Additionally, there is some evidence that villin expression in small cell carcinoma of the stomach and lower gastrointestinal tract is higher than in small cell carcinoma at other sites, such as lung, esophagus, bladder, or prostate. Literature reports that about 40% of lung carcinoid cases are villin positive, and there is also villin expression in some other neuroendocrine tumors, such as medullary thyroid carcinoma and a few Merkel cell carcinomas.21, MRP1 is a multi-drug resistance-associated protein 1, affecting chemotherapy sensitivity and is related to prognosis.22, MDR multi-drug resistance gene23, TS is thymidylate synthase, an important target for 5-FU; if it is highly expressed, positive reaction ++ or above indicates that tumor cells are resistant to 5-FU.24, Syn is a synaptophysin neural tissue marker.25, S-100 is a neural tissue marker, present in neural tissues, pituitary, carotid body, adrenal medulla, salivary glands, and some mesenchymal tissues, commonly used for diagnosing neurofibromas, malignant melanomas, liposarcomas, and chondrosarcomas.26, NSE is mainly used for diagnosing neuroendocrine tumors.27, Chr is chromogranin, highly concentrated in the adrenal medulla, used to differentiate between adrenal medulla and cortex, for diagnosing neuroendocrine tumors.28, CKH is high molecular weight keratin, mainly marking squamous cell tumors.29, CKL is low molecular weight keratin, mainly marking single-layer epithelium and glandular epithelium.30, EMA is epithelial membrane antigen, a glycoprotein widely distributed in various epithelium and their tumors.31, Vim is vimentin, a mesenchymal tissue marker.32, P504 is a formyl-CoA racemase, with a sensitivity of 97% and specificity of 100% for diagnosing prostate cancer.33, AMACR has the advantage of being cancer-specific, found only in cancerous tissues. Rubin states that AMACR can also be used as a diagnostic marker for other cancers. After examining various cancer cells, it was found that colorectal cancer, ovarian cancer, breast cancer, bladder cancer, lung cancer, lymphoma, and melanoma all overexpress AMACR, with the highest expression in colorectal and prostate cancers.34, CD117 is gastrointestinal stromal tumor.35, CD10 is a common acute lymphoblastic leukemia antigen, mainly expressed in immature lymphocytes, with application value in diagnosing hematological diseases such as Burkitt lymphoma and chronic myeloid leukemia. In recent years, it has been found that this antigen is expressed in certain tumors outside the hematopoietic system, such as endometrial stromal sarcoma and malignant melanoma. The antibody has certain reference value in diagnosing and differentiating renal cell carcinoma.36, CD15 is a cell adhesion molecule, which has good marking effect on Reed-Sternberg cells in Hodgkin lymphoma (HD) and is considered an important marker for HD. In addition to the differential diagnosis of HD, studies on the expression of CD15 in gastric cancer, colorectal cancer, thyroid cancer, breast cancer, etc., have found that CD15 expression significantly increases with the decrease in cancer cell differentiation, and the increase in lymph node metastasis and clinical staging. It is believed that CD15 expression is a good indicator for judging tumor progression, predicting lymph node metastasis, and prognosis.Immunoelectron microscopy observations show that CD15 antigen is mainly distributed in the boundary membrane, endoplasmic reticulum, Golgi apparatus, and near the nuclear membrane of colorectal cancer cells. CD15 may influence and participate in the formation and metastasis of tumors by affecting the binding conformation of the substrate.37, SMA is smooth muscle actin, marking smooth muscle.38, CD56 is a neural cell adhesion molecule, mainly distributed in most neuroectoderm-derived cells, often used for diagnosing astrocytomas, neuroblastomas, and neuroendocrine tumors. It is also an important marker for NK cell tumors and marks small cell lung cancer.39, Des is desmin, widely distributed in smooth muscle, cardiac muscle, skeletal muscle cells, and myoepithelial cells, highly expressed in well-differentiated tumors and lowly expressed in poorly differentiated tumors.40, MSA is muscle-specific actin, widely distributed in almost all muscle-type cells.41, CD68 is present in macrophages in bone marrow and various neural tissues, used for diagnosing granulocytic leukemia, various monocytic tumors, including malignant fibrous histiocytoma (preferred).42., CD34 is expressed in early lymphoid hematopoietic stem cells, progenitor cells, endothelial cells, embryonic fibroblasts, and certain neural tissue cells, mostly used to mark vascular endothelial cells, with a diagnosis of vascular tumors, GIST 80-90%.43, CD31 also marks vascular endothelium.44, CD44 is a widely distributed transmembrane glycoprotein molecule, divided into CD44s and CD44v. CD44s mainly acts as a receptor for hyaluronic acid, affecting tumor growth and metastasis after binding to hyaluronic acid. CD44v is mainly expressed in metastatic tumor cells.Li Daoming et al. used immunohistochemical LSAB method to detect the expression of CD44v in 42 cases of esophageal squamous carcinoma, finding that the positive expression rate in the lymph node metastasis group was 76.19% (16/21), while the non-metastatic group had a positive rate of 42.86% (9/12), showing a significant difference between the two groups.The cancer cells around the cancer nest, interstitially infiltrating cancer cells, mitotic cancer cells, cancer cells in emboli, and cancer cells infiltrating the vascular wall all show strong positive expression. Zhang Chengwu et al. detected the expression of CD44v6 in 20 cases of normal gastric mucosal epithelium, 43 cases of atypical hyperplasia, and 85 cases of gastric cancer, finding no expression in normal gastric mucosa, while positive rates in atypical hyperplasia and gastric cancer tissues were 30.2% and 74.1%, respectively, with expression intensity closely related to the depth of gastric cancer infiltration, lymph node metastasis, tumor growth pattern, vascular and lymphatic invasion, and distant metastasis. The above results indicate that high expression of CD44v constitutes the aggressiveness and ease of metastasis of tumor cells.45, NESTIN is extremely abundant in neural stem cells.46, Ost is osteopontin, secreted by osteoblasts.47, AAT is anti-trypsin from fibroblast-derived tumors.48, ACT is anti-chymotrypsin.49, GFAP is glial fibrillary acidic protein, a neural tissue marker, commonly used for diagnosing astrocytomas.50, Tg is thyroglobulin, positive in thyroid cancer.51, CT is calcitonin, positive in medullary thyroid carcinoma.52, PH is parathyroid hormone, positive in parathyroid tumors.53, N-myc enhanced expression in small cell lung cancer and neuroblastoma lacks response to chemotherapy and progresses rapidly;54, bcl-2 has a drug resistance mechanism due to anti-apoptotic effects; those with high expression are resistant to most anticancer drugs/radiation therapy.55, TGA72 is a tumor-associated antigen 72, expressed in various malignant epithelial tumors, especially breast cancer, ovarian cancer, and colon cancer. Normal epithelial cells, sarcomas, and lymphohematopoietic system tumors are usually TGA72 negative. The TGA72 antibody has been used extensively in breast cancer research, with its high expression usually associated with larger tumor size, poor differentiation of lymph node metastatic tumor cells, and high proliferative activity.56, GA733 is a tumor-associated antigen encoding epithelial glycoprotein 40, an epithelial cell adhesion molecule (EP-CAM), playing an important role in the growth and differentiation of epithelial cells. Various tumors can express GA733, especially breast cancer, colon cancer, and lung cancer.Kubuschok et al. used GA733 to detect occult micrometastases in surgically removed lymph nodes of non-small cell lung cancer, finding that the detection of occult foci is an independent prognostic factor for overall survival. The expression of GA733 in colorectal cancer is related to tumor prognosis, with cell membrane and cytoplasm expression being worse than that on the basement membrane side.57, TTF-1 is thyroid transcription factor-1, expressed in thyroid gland epithelial cells and lung epithelial cells. In lung tumor studies, it has been found that most small cell lung cancers, primary and metastatic lung adenocarcinomas, a small portion of undifferentiated large cell lung cancers, and most atypical neuroendocrine tumors show positive immunohistochemical results for TTF-1, while lung squamous carcinoma and most typical carcinoids are TTF-1 negative. In papillary thyroid carcinoma, TTF-1 is also positive, while TTF is negative in other tissues. Therefore, TTF-1 can be used to differentiate between lung adenocarcinoma and squamous carcinoma and assist in differentiating it from metastatic adenocarcinoma of the lung.TTF-1 expression in thyroid and its tumorsTTF-1 is mainly expressed in thyroid follicular cells and chief cells of the parathyroid gland. TTF-1 is the basis for the differentiation of the thyroid and the secretion of thyroglobulin, promoting the transport of thyroid peroxidase, iodine/sodium, and is related to the activity of serum TSH, with active TSH-R enhancing TTF-1 expression.The expression of TTF-1 in normal and benign thyroid tissues is high, but it is low in papillary and follicular thyroid carcinomas, and not expressed in undifferentiated carcinomas. The expression intensity of TTF-1 in malignant thyroid lesions increases with age, and the longer the tumor exists, the higher the recurrence rate.TTF-1 expression in lung cancer75% of non-small cell lung cancers (NSCLCs) show positive expression, with adenocarcinoma (ACs) significantly higher than squamous cell carcinoma (SCC), over 90% of primary small cell lung cancers (SCLC) express positive, and TTF-1 positive expression intensity in non-small cell lung cancer (NSCLCs) is negatively correlated with patient prognosis, serving as an independent prognostic indicator.Typical carcinoids of the lung (TCS) are all negative, indicating that small cell lung cancer and non-small cell lung cancer may have a theory of a common origin that is different from TCS.
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