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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 microm 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.

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

The cellular response to hypoxia includes the hypoxia-inducible factor-1 (HIF-1)-induced transcription of genes involved in diverse processes such as glycolysis and angiogenesis. Induction of the HIF-regulated genes, as a consequence of the microenvironment or genetic changes, is known to have an important role in the growth of experimental tumors. Hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) are known to dimerize with the aryl hydrocarbon receptor nuclear translocator in mediating this response. Because regulation of the alpha chain protein level is a primary determinant of HIF activity, our aim was to investigate the distribution of HIF-1alpha and HIF-2alpha by immunohistochemistry in normal and pathological tissues using monoclonal antibodies (mAb). We raised a new mAb to detect HIF-1alpha, designated 122, and used our previously validated mAb 190b to HIF-2alpha. In the majority of solid tumors examined, including bladder, brain, breast, colon, ovarian, pancreatic, prostate, and renal carcinomas, nuclear expression of HIF-1alpha and -2alpha was observed in varying subsets of the tumor cells. HIF-2alpha was also strongly expressed by subsets of tumor-associated macrophages, sometimes in the absence of any tumor cell expression. Less frequently staining was observed in other stromal cells within the tumors and in normal tissue adjacent to tumor margins. In contrast, in normal tissue neither molecule was detectable except within subsets of bone marrow macrophages, where HIF-2alpha was strongly expressed.

Fatal Plasmodium falciparum malaria is accompanied by systemic endothelial activation. To study endothelial activation directly during malaria and sepsis in vivo, the expression of cell adhesion molecules on dermal microvascular endothelium was examined in skin biopsies and correlated with plasma levels of soluble (circulating) ICAM-1, E-selectin, and VCAM-1 and the cytokine tumor necrosis factor (TNF)-alpha. Skin biopsies were obtained from 61 cases of severe malaria, 42 cases of uncomplicated malaria, 10 cases of severe systemic sepsis, and 17 uninfected controls. Systemic endothelial activation, represented by the up-regulation of inducible cell adhesion molecules (CAMs) on endothelium and increased levels of soluble CAMs (sCAMs), were seen in both severe and uncomplicated malaria and sepsis when compared with uninfected controls. Plasma levels of sICAM-1, sVCAM-1, and sE-selectin correlated positively with the severity of malaria whereas TNF-alpha was raised nonspecifically in malaria and sepsis. Immunohistochemical evidence of endothelial activation in skin biopsies did not correlate with sCAM levels or disease severity. This indicates a background of systemic endothelial activation, which occurs in both mild and severe malaria and sepsis. The levels of sCAMs in malaria are thus not an accurate reflection of endothelial cell expression of CAMs in a particular vascular bed, and other factors must influence their levels during disease.

Neoplastic growth is usually dependent on blood supply, and it is commonly accepted that this is provided by the formation of new vessels. However, tumors may be able to grow without neovascularization if they find a suitable vascular bed available. We have investigated the pattern of vascularization in a series of 500 primary stage I non-small-cell lung carcinomas. Immunostaining of endothelial cells has highlighted four distinct patterns of vascularization. Three patterns (which we called basal, papillary, and diffuse) have in common the destruction of normal lung and the production of newly formed vessels and stroma. The fourth pattern, which we called alveolar or putative nonangiogenic, was observed in 16% (80/500) of the cases and is characterized by lack of parenchymal destruction and absence of both tumor associated stroma and new vessels. The only vessels present were the ones in the alveolar septa, and their presence highlighted, through the whole tumor, the lung alveoli filled up by the neoplastic cells. This observation suggests that, if an appropriate vascular bed is available, a tumor can exploit it and grows without inducing neo-angiogenesis. This could have implications for strategies aimed at inhibiting tumor growth by vascular targeting or inhibition of angiogenesis.

The sequestration of parasitized erythrocytes in the microvasculature of vital organs is central to the pathogenesis of severe Plasmodium falciparum malaria. This process is mediated by specific interactions between parasite adherence ligands and host receptors on vascular endothelium such as intercellular adhesion molecule-1 (ICAM-1) and CD36. Using immunohistochemistry we have examined the distribution of putative sequestration receptors in different organs from fatal cases of P. falciparum malaria and noninfected controls. Receptor expression and parasite sequestration in the brain were quantified and correlated. Fatal malaria was associated with widespread induction of endothelial activation markers, with significantly higher levels of ICAM-1 and E-selectin expression on vessels in the brain. In contrast, cerebral endothelial CD36 and thrombospondin staining were sparse, with no evidence for increased expression in malaria. There was highly significant co-localization of sequestration with the expression of ICAM-1, CD36, and E-selectin in cerebral vessels but no cellular inflammatory response. These results suggest that these receptors have a role in sequestration in vivo and indicate that systemic endothelial activation is a feature of fatal malaria.

Presentation of endogenous antigenic peptides to cytotoxic T lymphocytes (CTLs) is mediated by the major histocompatibility complex (MHC) class I molecules. These antigenic peptides derived from the cytoplasmic protein pool are transported by the recently described MHC-encoded transporters (TAP1 and TAP2) into a pre-Golgi region where they take part in the assembly of MHC class I molecules. Using an affinity-purified polyclonal antibody (AK1.7) for TAP1, we analyzed 81 colorectal carcinomas, 32 adenomas, and the respective nonneoplastic mucosa. Loss of the transporter molecule (TAP1) was observed in 14% (11 of 81) of the carcinomas, either complete (7 of 11) or focal (4 of 11), whereas adenomas and normal mucosa were always positive. This study adds further information to the understanding of the mechanisms related to the defective presentation of the MHC class I molecules by tumor cells.

To assess the expression of p53 in premalignant lesions, we examined by immunohistochemistry benign colorectal adenomas (n = 72, measuring more than 6 mm and less than 95 mm in diameter) from patients without (group I, n = 23) or with (group II, n = 49) concurrent sporadic colorectal carcinomas. Using a panel of three monoclonal antibodies (PAb 240, PAb 421, PAb 1801) and two polyclonal antibodies (CM1, C19) immunohistological staining was demonstrated in 26% of the cases (19 of 72 adenomas, 7 of 23 from group I and 12 of 49 from group II). In the majority of the cases, p53 positive foci in the adenomas occurred in the most dysplastic areas, although focal positivity was detected in glands that were histologically normal. Expression of p53 protein was also detected in 21 of 30 (70%) colorectal carcinomas of group II. In two cases focal positive staining was observed in the polyps but not in the concurrent carcinomas. Non-neoplastic colonic mucosa and stromal lymphoid cells were negative in all cases examined. Over-expression of p53 in neoplastic tissues detected by immunocytochemistry is generally believed to correlate with the presence of mutation in the gene. This may not be an absolute rule, because in some hemopoietic malignancies, there is evidence that p53 protein may be detectable in the absence of an underlying mutation. These findings therefore represent the highest incidence in colorectal adenomas of abnormalities in the p53 protein expression, probably largely due to underlying mutations. This study also suggests that immunocytochemical demonstration of p53 protein may be a suitable method for the routine detection of subpopulations of cells which, by clonal expansion, could acquire a growth advantage within an adenoma during the neoplastic process.

It has been reported previously that the bcl-2 protooncogene protein is detectable in neoplastic cells from cases of human lymphoma in which the 14;18 chromosomal translocation is present, but not in lymphomas that lack this chromosomal rearrangement or in normal lymphoid tissue. In the present study we confirmed, by immunohistologic labeling with polyclonal and monoclonal antibodies, that bcl-2 protein is strongly expressed in many cases of follicular lymphoma and that these neoplastic follicles differ clearly from their nonmalignant counterpart (reactive germinal centres) in which bcl-2 protein is undetectable. However we also found bcl-2 protein in normal T- and B-lymphoid cells and in a variety of lymphoproliferative disorders in which the 14;18 translocation is not present. It is therefore concluded that expression of bcl-2 protein is not a specific marker for lymphomas bearing the 14;18 chromosomal translocation and that the observations of other investigators may have reflected the inadequate sensitivity of their staining procedure.

A new monoclonal antibody, KP1, against the CD68 antigen, which labels macrophages and other members of the mononuclear phagocyte lineage in routinely processed tissue sections, has been used to stain a range of lymphoid, histiocytic, and myelomonocytic proliferations. All 20 neoplasms of myeloid, myelomonocytic, and presumed macrophage derivation reacted with antibody KP1. None of the 22 cases of T cell neoplasia had positive reactions. Although 14 of 41 B lineage lymphomas and leukaemias were stained by antibody KP1, staining was usually confined to small dots of reactivity, in contrast to the strong and extensive cytoplasmic staining seen in the neoplasms of myeloid and macrophage/monocyte origin. Furthermore, positive B cell neoplasms were almost all small cell proliferations, which are unlikely to be confused with myelomonocytic malignancies. It was concluded that antibody KP1 is a valuable addition to a panel of monoclonal antibodies for phenotyping lymphomas, particularly in routinely fixed tissues. It should assist the pathologist in the recognition of extramedullary presentation of leukaemia, aid in the diagnosis of suspected cases of true histiocytic neoplasia, and allow for quantitation of macrophages infiltrating lymphomas and other solid tumors.

The immunoreactivity of six different monoclonal antigranulocyte antibodies (Leu M1, TG1, 3C4, BY/87a, BY/37a, and 3CD1) has been evaluated in 23 cases of Hodgkin's disease (7 lymphocyte predominant, 12 nodular sclerosing, and 5 mixed cellularity); in a variety of non-Hodgkin's lymphomas and in a series of reactive and benign lesions of lymph nodes. Applying a monoclonal antibody (PD7/26) to leukocyte common antigen (T200), we have also investigated reports that the L&H variants in nodular lymphocyte predominant Hodgkin's disease are strongly immunoreactive for leukocyte common antigen in contrast to the lack of reactivity of Reed-Sternberg (RS) cells and variants thereof in other forms of Hodgkin's disease. All six monoclonal anti-granulocyte antibodies reacted against RS cells and "Hodgkin's cells" in the nodular sclerosing (NSHD) and mixed cellularity (MCHD) types, with strong cell membrane and juxtanuclear (Golgi) staining. In contrast an anti-leukocyte antibody PD7/26 was unreactive with RS cells and variants thereof in NSHD and MCHD. On the other hand, RS cells and L&H variants thereof in the nodular L&H form of Hodgkin's disease (nodular lymphocyte predominant type) showed reactivity with PD7/26 but not with the anti-granulocyte markers. Rare L&H cells in 2 cases of diffuse lymphocyte predominant type showed reactivity with some, but not all, of the anti-granulocyte antibodies. These findings provide further support for the concept that the nodular L&H type of Hodgkin's disease represents an entity distinct from other forms of this disorder. Our studies also demonstrate the usefulness of these immunoperoxidase techniques when applied to formalinfixed, paraffin-embedded tissues.

The use of monoclonal and polyclonal antibodies for the immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue has been limited by the fact that most antigens on lymphoid cells are denatured by histologic fixation, dehydration, and embedment. In this article the authors have analyzed a small panel of antibodies which represent exceptions to this rule, in that they identify denaturation-resistant determinants on leukocyte antigens in paraffin-embedded tissue. Monoclonal antibodies L26 [corrected] and 4KB5 label preferentially B cells, monoclonal antibody UCHL1 stains predominantly T cells, and monoclonal antibody MAC 387 reacts with granulocytes and some macrophages. A polyclonal antiserum raised against purified CD3 (T3) antigen, a T-cell-specific molecule, was also employed. This antibody panel was used to immunophenotype routinely processed tissue biopsy specimens from 61 non-Hodgkin's lymphomas (all of which had been previously phenotyped in cryostat sections). The lineage of the neoplastic cells was correctly identified in 32 of 34 (94%) cases of B-cell lymphoma, in 19 of 19 (100%) cases of T-cell neoplasm, and in 2 of 4 (50%) cases of histiocytic malignancy. It is concluded that this combination of antibodies is helpful in immunophenotyping non-Hodgkin's lymphomas when only paraffin-embedded tissue sections are available, although additional reagents of higher specificity are required to improve the identification of lymphomas.

A group of 63 cases of anaplastic large cell lymphomas (variants of diffuse large cell lymphomas often diagnosed as "malignant histiocytosis") was characterized on both morphologic criteria and expression of epithelial membrane antigen (EMA) and Ki-1 antigen (CD30). On the basis of the reactivity of these tumors with anti-EMA and anti-Ki-1 antibodies, four subtypes could be distinguished. In the majority of cases (n = 49), neoplastic cells coexpressed EMA and Ki-1 antigens. Nineteen of these cases were tested for IL-2R, and all were positive (Type I, EMA+, Ki-1+, IL-2R+). In the second group (n = 5), the neoplastic cells expressed EMA but not the Ki-1 antigen. These cases were not tested for the presence of IL-2R (Type II, EMA+, Ki-1-, IL-2R?). There were tumors with similar morphology expressing only Ki-1 antigen (Type III, EMA-, Ki-1+, IL-2R-) or negative for both EMA and Ki-1 antigens (Type IV, EMA-, Ki-1-, IL-2R-). EMA appeared to occur predominantly on activated cells, as has been previously shown for Ki-1 antigen. Analysis using monoclonal antibodies to T-cell, B-cell, or macrophage-associated differentiation antigens showed that these tumors were heterogeneous in terms of cell lineage. Tumors coexpressing EMA, Ki-1, and IL-2R (Type I), were most commonly of T-cell origin (n = 12); the remainder in this type expressed B-cell markers (n = 4), a mixed B/T phenotype (n = 2), or no clear phenotype (n = 9). By contrast, tumors of Types II, III, and IV were mainly from B-cell origin (n = 6) or showed a mixed B/T phenotype (n = 1). Despite the fact that a significant proportion of these cases were initially classified morphologically as "malignant histiocytosis," only 3 of the 63 cases were possibly of histiocytic origin. These results confirm that true malignant histiocytosis is rare and that most tumors with histologic features currently regarded as being consistent with this diagnosis are lymphocytic in origin and express activation antigens such as EMA, Ki-1 antigen, and IL-2R.