Sources/Clones
Dako (124), Immunotech (124) and Zymed (BCL2-100).
Fixation/Preparation
Antibodies to bcl-2 are reasonably robust and work very well on paraffin-embedded tissue. Staining is not too dependent on fixation protocols and good results may be obtained with formalin-fixed, B5-fixed, methacarn-fixed and fresh-frozen tissues. Staining is enhanced by the use of antigen retrieval with either microwave or pressure-cooking pretreatment. The bcl-2 antibody may be used for labeling acetone-fixed cryostat sections or fixed-cell smears.
Background
The bcl-2 gene was identified more than a decade ago with the discovery and analysis of the t(14; 18) (q32; q21) translocation (Bakhshi et al, 1985). This translocation occurs in 70-80% of follicular lymphoma, comprising juxtaposition of the bcl-2 gene with the immunoglobulin heavy chain (IgH) gene on chromosome 14q32 (Chen-Levy et al, 1989). This results in an overexpression of the translocated bcl-2 allele induced by enhancers in the IgH region, although the translocation is not a prerequisite for bcl-2 protein expression, since this occurs in many cases without this rearrangement (Pezzella et al, 1990). The bcl-2 polypeptide is a 26 kD protein that is found on intracellular (mitochondrial and nuclear) membranes and in the cytosol (on the smooth endoplasmic reticulum), rather than on the cell surface. bcl-2 is not an oncogene and has no effect on cell replication. bcl-2 protein does, however, prevent cells from undergoing apoptosis, conferring a survival advantage on cells harboring the t(14; 18) translocation. In normal lymphoid tissue, bcl-2 antibody reacts with small B lymphocytes in the mantle zone and many cells within T-cell areas. In the thymus many cells in the medulla are stained, with weak/negative reaction in the cortex (Chetty et al, 1997).
Applications
The most relevant use of bcl-2 immunostaining lies in the distinction of reactive follicular lymphoid hyperplasia from follicular lymphoma (Pezzella & Gatter, 1995; Veloso et al, 1995 Cooper & Haffajee, 1996). Positive staining is cytoplasmic in location. Follicular lymphomas show striking bcl-2 expression in neoplastic follicles, whilst only isolated individual cells within the reactive follicle centers are positive (mostly T-cells). This difference in staining pattern is not due to down regulation or decreased bcl-2 mRNA, but largely to a posttranslational mechanism that results in decreased protein levels. Furthermore, bcl-2 protein expression is demonstrated in all grades of follicle center cell lymphomas in both small and large cells (Cooper & Haffajee, 1997). Strongly bcl-2-positive lymphoid aggregates in the bone marrow of patients previously diagnosed with nodal follicular lymphoma are indicative of lymphoma involvement (Chetty et al, 1995). However, there is no practical value in applying the bcl-2 antibody for classification of a malignant lymphoid infiltrate, since many different lymphoma types can be bcl-2 positive. Nevertheless, it has been demonstrated that non-Hodgkin's lymphoma with bcl-2 expression has a significantly higher relapse rate and a lower cause-specific survival than those without (Hill et al, 1996).
Expression of bcl-2-has been studied in many epithelial neoplasms (Pezzella et al, 1993; Lu et al, 1993). In general, better prognoses accompany bcl-2 positive neoplasms than negative ones, with some prostatic cancers being the exception to the rule (Colombel et al, 1993). A reciprocal relationship has been demonstrated between bcl-2 reactivity and p53 overexpression in 65% of colorectal neoplasia, with a bcl-2+ve/p53-ve subgroup showing a strong correlation with negative lymph node status, implying a less aggressive pathway of neoplastic transformation (Kaklamanis et al, 1996). Recently, bcl-2 protein was detected in all grades of cervical intraepithelial neoplasia, with a striking increase in the number of positive cells with increasing severity of CIN, in combination with a mild increase in staining intensity (Harmsel et al, 1996).
Bcl-2 expression has been demonstrated in 79% (15 of 19) of synovial sarcoma cases (Hirakawa et al, 1996), but was negative in 20 leiomyosarcomas, four malignant peripheral nerve sheath tumors and four fibrosarcomas. However, in another study, bcl-2 protein was expressed in seven rhabdomyosarcomas and 5/7 leiomyosarcomas, four epithelioid leiomyomas and 6/14 leiomyomas (Soini & P«kk 1996).
References
•Bakhshi A, Jensen JP, Goldman P, et al 1985. Cloning the chromosomal breakpoint of t(14; 18) human lymphomas: clustering around J on chromosome 14 near a transcriptional unit on chromosome 18. Cell H 41: 899-906.
•Chen-Levy Z, Nourse J, Cleary ML 1989. The bcl-2 candidate protooncogene is a 24 kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14; 18) translocation. Molecular and Cell Biology 9: 701-710.
•Chetty R, Echezarreta G, Comley M, Gatter K 1995. Immunohistochemistry in apparently normal bone marrow trephine specimens from patients with nodal follicular lymphoma. Journal of Clinical Pathology 48: 1035-1038.
•Chetty R, Dada MA, Gatter KC 1997. bcl-2: Longevity personified. Advances in Anatomic Pathology 4:134-138.
•Colombel M, Symmans F, Gill S et al 1993. Detection of the apoptosis-suppressing oncoprotein bcl-2 in hormone refractory human prostate cancer. American Journal of Pathology 143:390-400.
•Cooper K, Haffajee Z 1996. bcl-2 immunohistochemistry distinguishes follicular lymphoma from follicular hyperplasia in formalin-fixed tissue with microwave antigen retrieval. Journal of Cellular Pathology 1: 52-56.
•Cooper K, Haffajee Z 1997. bcl-2 and p53 protein expression in follicular lymphoma. Journal of Pathology 182: 307-310.
•Harmsel BT, Smedts F, Kruijpers J, Jeunink M, Trimbos B, Ramaekers F 1996. bcl-2 immunoreactivity increases with severity of CIN: a study of normal cervical epithelia, CIN, and cervical carcinoma. Journal of Pathology 179:26-30.
•Hill ME, MacLennan KA, Cunningham DC et al 1996. Prognostic significance of BCL-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin's lymphoma: a British National Lymphoma investigation study. Blood 88: 1046-1051.
•Hirakawa N, Naka T, Yamamoto I, Fukuda T, Tsuneyoshi M 1996. Overexpression of bcl-2 protein in synovial sarcomas. Human Pathology 27: 1060-1065.
•Kaklamanis L, Savage A, Mortensen N et al 1996. Early expression of bcl-2 protein in the adenoma-carcinoma sequence of colorectal neoplasia. Journal of Pathology 179:10-14.
•Lu Q-L, Elia G, Lucas S, Thomas JA 1993. bcl-2 proto-oncogene expression in Epstein-Barr virus-associated nasopharyngeal carcinoma. International Journal of Cancer 53: 29-35. virus-associated nasopharyngeal carcinoma. International Journal of Cancer 53: 29-35.
•Pezzella F, Gatter K 1995. What is the value of bcl-2 protein detection for the histopathologist? Histopathology 26: 89-93.
•Pezzella F, Tse AGD, Cordell JL, Pulford KAF, Gatter KC, Mason DY 1990. Expression of the bcl-2 oncogene protein is not specific for the 14; 18 chromosomal translocation. American Journal of Pathology 137: 225-232.
•Pezzella F, Turley H, Kuzu I et al 1993. bcl-2 protein expression in non-small cell lung carcinoma. Immunohistochemical evidence for abnormal expression and correlation with survival. New England Journal of Medicine 329: 690-694.
•Soini Y, P«kk P 1996. bcl-2 is preferentially expressed in tumours of muscle origin but is not related to p53 expression. Histopathology 28: 141-145.
•Veloso JD, Rezuke WN, Cartun RW, Abernathy EC, Pastuszak WT 1995. Immunohistochemical distinction of follicular lymphoma from follicular hyperplasia in formalin-fixed tissues using monoclonal antibodies MT2 and bcl-2. Applied Immunohistochemistry 3: 153-159.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Dako (124), Immunotech (124) and Zymed (BCL2-100).
Fixation/Preparation
Antibodies to bcl-2 are reasonably robust and work very well on paraffin-embedded tissue. Staining is not too dependent on fixation protocols and good results may be obtained with formalin-fixed, B5-fixed, methacarn-fixed and fresh-frozen tissues. Staining is enhanced by the use of antigen retrieval with either microwave or pressure-cooking pretreatment. The bcl-2 antibody may be used for labeling acetone-fixed cryostat sections or fixed-cell smears.
Background
The bcl-2 gene was identified more than a decade ago with the discovery and analysis of the t(14; 18) (q32; q21) translocation (Bakhshi et al, 1985). This translocation occurs in 70-80% of follicular lymphoma, comprising juxtaposition of the bcl-2 gene with the immunoglobulin heavy chain (IgH) gene on chromosome 14q32 (Chen-Levy et al, 1989). This results in an overexpression of the translocated bcl-2 allele induced by enhancers in the IgH region, although the translocation is not a prerequisite for bcl-2 protein expression, since this occurs in many cases without this rearrangement (Pezzella et al, 1990). The bcl-2 polypeptide is a 26 kD protein that is found on intracellular (mitochondrial and nuclear) membranes and in the cytosol (on the smooth endoplasmic reticulum), rather than on the cell surface. bcl-2 is not an oncogene and has no effect on cell replication. bcl-2 protein does, however, prevent cells from undergoing apoptosis, conferring a survival advantage on cells harboring the t(14; 18) translocation. In normal lymphoid tissue, bcl-2 antibody reacts with small B lymphocytes in the mantle zone and many cells within T-cell areas. In the thymus many cells in the medulla are stained, with weak/negative reaction in the cortex (Chetty et al, 1997).
Applications
The most relevant use of bcl-2 immunostaining lies in the distinction of reactive follicular lymphoid hyperplasia from follicular lymphoma (Pezzella & Gatter, 1995; Veloso et al, 1995 Cooper & Haffajee, 1996). Positive staining is cytoplasmic in location. Follicular lymphomas show striking bcl-2 expression in neoplastic follicles, whilst only isolated individual cells within the reactive follicle centers are positive (mostly T-cells). This difference in staining pattern is not due to down regulation or decreased bcl-2 mRNA, but largely to a posttranslational mechanism that results in decreased protein levels. Furthermore, bcl-2 protein expression is demonstrated in all grades of follicle center cell lymphomas in both small and large cells (Cooper & Haffajee, 1997). Strongly bcl-2-positive lymphoid aggregates in the bone marrow of patients previously diagnosed with nodal follicular lymphoma are indicative of lymphoma involvement (Chetty et al, 1995). However, there is no practical value in applying the bcl-2 antibody for classification of a malignant lymphoid infiltrate, since many different lymphoma types can be bcl-2 positive. Nevertheless, it has been demonstrated that non-Hodgkin's lymphoma with bcl-2 expression has a significantly higher relapse rate and a lower cause-specific survival than those without (Hill et al, 1996).
Expression of bcl-2-has been studied in many epithelial neoplasms (Pezzella et al, 1993; Lu et al, 1993). In general, better prognoses accompany bcl-2 positive neoplasms than negative ones, with some prostatic cancers being the exception to the rule (Colombel et al, 1993). A reciprocal relationship has been demonstrated between bcl-2 reactivity and p53 overexpression in 65% of colorectal neoplasia, with a bcl-2+ve/p53-ve subgroup showing a strong correlation with negative lymph node status, implying a less aggressive pathway of neoplastic transformation (Kaklamanis et al, 1996). Recently, bcl-2 protein was detected in all grades of cervical intraepithelial neoplasia, with a striking increase in the number of positive cells with increasing severity of CIN, in combination with a mild increase in staining intensity (Harmsel et al, 1996).
Bcl-2 expression has been demonstrated in 79% (15 of 19) of synovial sarcoma cases (Hirakawa et al, 1996), but was negative in 20 leiomyosarcomas, four malignant peripheral nerve sheath tumors and four fibrosarcomas. However, in another study, bcl-2 protein was expressed in seven rhabdomyosarcomas and 5/7 leiomyosarcomas, four epithelioid leiomyomas and 6/14 leiomyomas (Soini & P«kk 1996).
References
•Bakhshi A, Jensen JP, Goldman P, et al 1985. Cloning the chromosomal breakpoint of t(14; 18) human lymphomas: clustering around J on chromosome 14 near a transcriptional unit on chromosome 18. Cell H 41: 899-906.
•Chen-Levy Z, Nourse J, Cleary ML 1989. The bcl-2 candidate protooncogene is a 24 kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14; 18) translocation. Molecular and Cell Biology 9: 701-710.
•Chetty R, Echezarreta G, Comley M, Gatter K 1995. Immunohistochemistry in apparently normal bone marrow trephine specimens from patients with nodal follicular lymphoma. Journal of Clinical Pathology 48: 1035-1038.
•Chetty R, Dada MA, Gatter KC 1997. bcl-2: Longevity personified. Advances in Anatomic Pathology 4:134-138.
•Colombel M, Symmans F, Gill S et al 1993. Detection of the apoptosis-suppressing oncoprotein bcl-2 in hormone refractory human prostate cancer. American Journal of Pathology 143:390-400.
•Cooper K, Haffajee Z 1996. bcl-2 immunohistochemistry distinguishes follicular lymphoma from follicular hyperplasia in formalin-fixed tissue with microwave antigen retrieval. Journal of Cellular Pathology 1: 52-56.
•Cooper K, Haffajee Z 1997. bcl-2 and p53 protein expression in follicular lymphoma. Journal of Pathology 182: 307-310.
•Harmsel BT, Smedts F, Kruijpers J, Jeunink M, Trimbos B, Ramaekers F 1996. bcl-2 immunoreactivity increases with severity of CIN: a study of normal cervical epithelia, CIN, and cervical carcinoma. Journal of Pathology 179:26-30.
•Hill ME, MacLennan KA, Cunningham DC et al 1996. Prognostic significance of BCL-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin's lymphoma: a British National Lymphoma investigation study. Blood 88: 1046-1051.
•Hirakawa N, Naka T, Yamamoto I, Fukuda T, Tsuneyoshi M 1996. Overexpression of bcl-2 protein in synovial sarcomas. Human Pathology 27: 1060-1065.
•Kaklamanis L, Savage A, Mortensen N et al 1996. Early expression of bcl-2 protein in the adenoma-carcinoma sequence of colorectal neoplasia. Journal of Pathology 179:10-14.
•Lu Q-L, Elia G, Lucas S, Thomas JA 1993. bcl-2 proto-oncogene expression in Epstein-Barr virus-associated nasopharyngeal carcinoma. International Journal of Cancer 53: 29-35. virus-associated nasopharyngeal carcinoma. International Journal of Cancer 53: 29-35.
•Pezzella F, Gatter K 1995. What is the value of bcl-2 protein detection for the histopathologist? Histopathology 26: 89-93.
•Pezzella F, Tse AGD, Cordell JL, Pulford KAF, Gatter KC, Mason DY 1990. Expression of the bcl-2 oncogene protein is not specific for the 14; 18 chromosomal translocation. American Journal of Pathology 137: 225-232.
•Pezzella F, Turley H, Kuzu I et al 1993. bcl-2 protein expression in non-small cell lung carcinoma. Immunohistochemical evidence for abnormal expression and correlation with survival. New England Journal of Medicine 329: 690-694.
•Soini Y, P«kk P 1996. bcl-2 is preferentially expressed in tumours of muscle origin but is not related to p53 expression. Histopathology 28: 141-145.
•Veloso JD, Rezuke WN, Cartun RW, Abernathy EC, Pastuszak WT 1995. Immunohistochemical distinction of follicular lymphoma from follicular hyperplasia in formalin-fixed tissues using monoclonal antibodies MT2 and bcl-2. Applied Immunohistochemistry 3: 153-159.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
