LC3A-Positive Light Microscopy Detected Patterns of Autophagy and Prognosis in Operable Breast Carcinomas

Autophagy is a self-degradation mechanism by which cells recycle their own cytoplasmic constituents and dispose of excess or defective organelles after starvation and oxygen deprivation. An antibody to the microtubule-associated protein 1 light chain 3 (LC3A), recognizing both the soluble (LC3A-I) and the membrane-bound form (LC3A-II) of the protein, was used to detect autophagic activity in 102 breast carcinomas. Three distinct patterns were recognized: (1) diffuse cytoplasmic, (2) cytoplasmic/juxta-nuclear, and (3) “stone-like” pattern – dense, rounded, amorphous structures, 5 μm on average, typically enclosed within cytoplasmic vacuoles. The diffuse cytoplasmic pattern showed a direct association with estrogen and progesterone receptor expression. The juxta-nuclear pattern indicated a similar association with hormone receptors, an inverse association with tumor size, and a favorable prognosis. By contrast, an increased number of stone-like structures, probably representing an excessive autophagic response, was related to high-grade tumors and a less favorable outcome. Interestingly, 60 additional epithelial tumors of nonbreast origin disclosed identical autophagic patterns, and so did MDA231 breast cancer xenografts and HCT116 colon tumor spheroids (also analyzed by electron microscopy). Moreover, MCF-7 human breast cancer cell lines confirmed induction of LC3A by anoxia and Thapsigargin. It is concluded that autophagy can be readily recognized in breast carcinomas by light microscopy, after immunohistochemical staining with LC3A, but the significance of the various patterns expressed would need further evaluation.Cancer cells survive the adverse conditions of the extracellular milieu (i.e., hypoxia, nutrient deprivation, and reduced growth factors) through angiogenesis and anaerobic glycolysis.¹ Yet rapidly proliferating malignant neoplasms, having high metabolic demands, are not sufficiently supplied by these processes.^2,3,4,5,6 An alternative metabolic pathway for providing energy, when both oxygen and glucose are depleted, is autophagy—a self-degradation mechanism by which cells recycle their own cytoplasmic constituents and dispose of excess or defective organelles resulting in protein and ATP synthesis.^7,8,9 However, autophagy when prolonged can also cause cell death, but how the outcome is determined and the relationship of autophagy to clinical outcome is, at present, poorly understood.Angiogenesis and anaerobic glycolysis (i.e., the shifting from oxidative phosphorylation to anaerobic metabolism) have both been studied extensively in relation to malignant disease, including breast cancer.^{10,11,12,13,14,15} The activation of antiapoptotic pathways, as a complementary cell surviving mechanism, has also been considered in a number of studies.^16,17 By contrast, the phenomenon of autophagy only recently received attention for its biological significance in human malignancies. It is now known, for example, that under suboptimal microenvironmental conditions, cytoplasmic constituents are first entrapped by a membrane sac, called the isolation membrane, which subsequently closes to form double membrane structures, called autophagosomes or autophagic vacuoles. These, and specifically their outer membrane, are fused with lysosomes producing autolysosomes.^8,18 Lysosomal hydrolases degrade the cytoplasmic contents of the autophagosome, together with its inner membrane,^8,18 and the resulting macromolecules are recycled. Failure to accomplish this self-degradation process, probably because of overload of cytoplasmic components, enzyme exhaustion, or lysosomal enzyme defects, leads to accumulation of debris and massive destruction of tumor cells.^18,19,20,21Increased new blood vessel formation is associated with rapidly advancing tumors.^10,12,13 Equally, high levels of hypoxia are indicative of tumor aggressiveness.^11,22,23,24 Besides, an activated antiapoptotic pathway contributes to tumor growth and progression.²⁵ Tumor hypoxia and accelerated angiogenesis have both been used as markers of poor prognosis for a variety of tumors, including breast cancer.^{10,11,12,13,22,26} Nonetheless, the role of autophagy remains, by and large, obscure in this respect. Is it a favorable or an unfavorable prognostic indicator in human breast malignancies?This study was designed to investigate this question in a series of 102 operable breast carcinomas. However, rather than using electron or fluorescence microscopy, this study uses light microscopy after immunohistochemical staining with an antibody recognizing the autophagy-related protein Atg8 (microtubule associated protein 1 light chain 3 MAP1LC3A or LC3A), an essential component of the autophagic machinery.^27,28 LC3A-II is derived from a proLC3 30-KDa protein after cleavage by autophagin Atg4 to produce the active cytosolic form LC3A-I (18 kDa). After activation by Atg7, LC3A-I is transferred to Atg3 and subsequently converted into the membrane-bound form LC3A-II.^8,28 The latter localizes on the isolation membranes and the complete spherical autophagosomal and autolysosomal membranes, forming a suitable marker of autophagic activity.     

The differential diagnosis of hairy cell leukemia with a panel of monoclonal antibodies

A panel of monoclonal antibodies reactive with normal human lymphoid cells and with hairy cells has been applied to the immunocytochemical analysis of hairy cell leukemia. Staining was performed by immunoenzymatic methods on frozen sections of bone marrow trephines and extramedullary tissues and on cell smears. Hairy cells reacted with antibodies against HLA-DR, leukocyte common antigen, B-cell antigens (antibodies To15 and B1) and with three anti-hairy cell monoclonal antibodies (S-HCL3, HC1, and HC2). Neoplastic cells in other B-cell lymphoproliferative disorders also expressed HLA-DR, leukocyte common, and B-cell antigens but were consistently negative for the antigen detected by monoclonal antibody S-HCL3. Furthermore, hairy cells differed from other neoplastic B-cells in that they were unreactive with monoclonal antibodies against C3b receptors, anti-Leu-1, Tü1, Tü33, and lacked a meshwork of dendritic reticulum cells. These findings establish a distinctive antigenic phenotype for hairy cell leukemia and indicate that it may be diagnosed reliably by immunoenzymatic labeling of tissue sections or cell smears.     

Diffuse large B-cell lymphoma

The recently published 4th edition of the WHO Classification of Tumours of Haematopoietic and lymphoid tissues has added significant new data into previous entities of large B-cell lymphoma as well as introducing some new categories and two borderline subtypes. This review discusses each of these issues in turn with an emphasis on providing a practical interpretation to assist the diagnostic pathologist in dealing with these lesions.     

Relationship of elevated tumour thymidine phosphorylase in node-positive breast carcinomas to the effects of adjuvant CMF

Background: Thymidine phosphorylase (TP) catalyses the reversiblephosphorylation of thymidine to thymine and 2-deoxyribose-1-phosphate. Highexpression of TP in cell lines potentiates the effects of the cytotoxic drugs5-fluorouracil and methotrexate, both of which are used in thecyclophosphamide, 5-fluorouracil and methotrexate (CMF) treatment regimen ofbreast cancer.Patients and methods: We therefore examined the expression of thisenzyme in 328 invasive breast carcinomas using immunohistochemistry andassessed whether the expression of this enzyme by the tumour predicts patientresponse to CMF in node-positive patients.Results: Whereas no significant difference in either relapse-freesurvival (RFS) (P = 0.2) or overall survival (OS) (P = 0.07) wasobserved between TP-negative and -positive tumours in non-treated patients,there was a significant increase in both RFS (P = 0.02) and OS (P = 0.02) inpatients treated with CMF in TP-positive compared with TP-negativetumours. A multivariate analysis of the 134 node-positive patientsdemonstrated that in ductal carcinomas, TP was an independent variable for OS.Conclusions: This pilot study suggests that patients with TP-positivetumours have a significant survival benefit when treated with CMF and supportsthe hypothesis that TP enhances tumour sensitivity to the anti-metabolites5-fluorouracil and methotrexate.     

bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non-small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role if bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-N0,1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P=0.04) and both were inversely associated with microvessel density (P<0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P<0.01). A significant co-expression of c-erbB-2 with TP was noted (P=0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P<0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P=0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c-erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c-erbB-2-related immune response remains to be further investigated. This revised version was published online in July 2006 with corrections to the Cover Date.