Domenico Delia

Acute lymphoblastic leukaemia associated antigen. IV Expression on non-leukaemic ‘lymphoid’ cells

Abstract The cellular specificity of the common ALL (cALL) membrane (gp100) antigen and the p28, 33 (Ia-like) antigen has been further explored. Non-leukaemic cells with a variable and usually weak expression of the cALL antigen can be demonstrated in foetal haemopoietic tissue, in bone marrow of normal children, in the marrow of children with a variety of non-leukaemic haematologic and non-haematologic disorders. In normal children the cALL antigen positive cells appear to be restricted to the bone marrow. These cells are regularly detected in the marrow of many leukaemic (ALL, AML) and non-leukaemic children with post-chemotherapy associated lymphocytosis, in marrow transplant recipients, and in neonatal ‘leukaemoid’ reactions. When the number of cALL + cells is monitored in children following cessation of maintenance chemotherapy for ALL, some prognostic correlation can be observed; in general, however, the numbers of ‘lymphoid’ cells bearing these leukaemia associated antigens have no clear predictive value with respect to relapse. Analysis by simultaneous markers coupled with flow microfluorimetry and sorting indicates that the majority of cALL antigen + cells (> 90%) are positive for the p28, 33 (Ia-like) antigen but do not express markers of mature lymphocytes (T antigen, E sheep, E mouse rosettes, surface immunoglobulin), binding sites for complement and IgG or a myeloid membrane antigen. More than 90% of pre-B cells (i.e. cyt IgM + , SmIg − ) are p28, 33 positive but only a small proportion (5–15%) are cALL antigen positive. These and additional data support the view that the cALL and p28, 33 (Ia) antigens are normal ‘early’ differentiation antigens of the lymphoid lineage.

Expression of the OKT monoclonal antibody defined antigenic determinants in malignancy

Abstract Monoclonal antibodies OKT1,3,4,6,8,9,10,11 and 11A have been screened by immunofluorescence (FACS and microscopy) and 125 I-binding assays for their cellular specificity in human leukaemia in relation to other cell markers (e.g. cALL gp100 antigen, HLA-DR, TdT, E rosettes). In a series of 69 T cell malignancies a distinctive correlation was observed between phenotype and clinical diagnosis. In every instance, T-ALL cells have an immature or early thymic phenotype and T non-Hodgkin lymphoma have a predominant cortical thymic phenotype indicating that these two malignancies originate from T cell precursors. In contrast, adult T-Sezary leukaemia, T-CLL and T-PLL have mature T cell subset phenotypes, the majority being T3 + T4 + T8 − /TdT − “helper/inducer” cell types and are presumed to originate in mature immunocompetent T cells. OKT11 and OKT11A give concordant reactions with E (sheep) rosettes and appear to react with the same receptor structure. The composite phenotype of several T-ALL cell lines can be modulated in vitro by phorbol ester (TPA). HPB-ALL, for example, loses TdT and OKT4 binding, decreases OKT9, maintains OKT8 and increases OKT1, HLA-ABC and OKT11 binding and E rosettes. OKT9 reacts preferentially in T leukaemias with T-ALL but is not T specific. It binds to many types of leukaemias and to malignant cell lines e.g. teratocarcinoma, melanoma, colon carcinoma and neuroblastoma. Normal foetal thymus, foetal liver and lectin activated T cells are also positive. Cell separation experiments indicate that OKT9 determinant is clearly associated with cell proliferation status although not cycle phase specific. When leukaemic cells (such as HL-60, HPB-ALL) are induced to mature in vitro by retinoic acid or TPA, respectively, the OKT9 determinant is lost. Biochemical studies indicate that OKT9 reacts with a gp 90K dalton dimer which appears to be the receptor for transferrin. This monoclonal antibody defined structure is probably the same as that described by Onary with monoclonal B3/25 and by Bramwell and Harris. The latter believed the structure to be a specific marker of malignancy. Some other OKT monoclonals are not T lineage specific. OKT10 binds to leukaemias and normal marrow cells of the myeloid and B cell lineages. OKT6 binds to neuroblastoma cells as does a similar monoclonal, NA134/HTA-1.

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.

Lineage relationship of chronic lymphocytic leukemia and hairy cell leukemia: Studies with TPA

The tumor promoting agent TPA (phorbol ester; 1.6 X 10(-8)M) was used to induce the differentiation in vitro of B-chronic lymphocytic leukemia (B-CLL) cells from 14 untreated patients. The uninduced phenotype was SIg+, Mrbc+, RFT-1+, RFA-4-, FMC7-. After 72 h incubation with TPA, B-CLL cells became RFA-4+, FMC7+ and lost the capability of Mrbc rosetting. Large proportions of the induced cells also showed morphological and ultrastructural changes, such as undulating membranes and bleblike protusions and became strongly positive for tartrate resistant acid phosphatase (TRAP+) and also contained cytoplasmic immunoglobulins. These features are very similar to the features of hairy cell leukemia (HCL). These observations confirm previous clinical findings that B-CLL and HCL are related disorders of the B lineage. The development of hairy features in induced B-CLL and in HCL seems to be a malignancy-associated feature because the Mrbc+ normal B cells (B-CLL-equivalent cells) isolated from tonsil also develop TRAP positivity but no membrane aberrations.

Comparative Antigenic Phenotypes of Normal and Leukemic Hemopoietic Precursor Cells Analysed with a “Library” of Monoclonal Antibodies

A dominant paradigm of cancer research is that alterations in the cell surface are of paramount importance to tumour cell behavior (Wallach 1978; Marchesi 1976). It is widely held that this is in part reflected in the regular expression of neo-antigens resulting from gene derepression [or “retrogressive differentiation” (Coggin 1978)], mutation (Baldwin 1974) or altered processing [e.g. glycosylation (Hakomori 1975)]. The search for novel antigens or other cell surface features of human tumour cells has an obligatory control demand which is frequently ignored or inadequately dealt with, i.e. that the appropriate cellular controls be analysed in parallel. Since most epithelial carcinomas and acute leukemias probably arise from tissue stem cells and, moreover, frequently have a maturation arrest imposed upon them, it should be selfevident that (a) many or most of the consistent phenotypic features of leukemic cells (and tumour cells in general) will be a reflection of their immature cell origins and (b) the significance of potentially unique biochemical or molecular features of tumour cells cannot be interpreted until we have access to normally infrequent tissue precursor cells.

Immunohistochemical Reactivity of Anti-Myeloid/Stem Cell Workshop Monoclonal Antibodies in Thymus, Lymph Node, Lung, Liver, and Normal Skin

Monoclonal antibodies (mAbs) against human monocytoid cells are “relatively specific” (1,3) and spurious cross-reactivity with other cell types can be detected “in vitro” in experimental models (3). Thus, instead of a single mAb it is customary to identify a specific cell type by the use of a panel of mAbs. Among the system of mononuclear phagocytes (MPS) (2,4), however, some cell types show characteristic histological distribution in the various organs and the specificity of a mAb for each single cell type is relatively simple to assess by immunohistochemical methods. In order to test the reactivity of the anti-myeloid stem cell mAbs of the Workshop, various tissues, including most of the cell types belonging in the mononuclear phagocytic system, were studied.* In addition GR positivity was easily detected in the tissues studied.

Integrins and Homing Receptors in Cutaneous Lymphomas

The aim of this study was to evaluate the expression on neoplastic cell membranes of some receptors involved in intercellular and cell-matrix adhesion. We used monoclonal antibodies (mAbs) directed against VLA-3, VLA-4, LFA-1, ICAM-1, Leu-8 and CD44 antigens, with an alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemical method, in 40 cases of cutaneous B-cell lymphomas (CBCL) and 60 cases of cutaneous T-cell lymphomas (CTCL), which had been previously characterized employing a large panel of mAbs. Our results showed, in CBCL, consistent expression of CD44, CDlla, ICAM-1 and VLA-4 molecules. LEU-8 and VLA-3 receptors were detected, respectively, in 50% and in 25% of the cases examined. No significant difference was noted between the pattern of adhesion molecules of our CBCL and of 70 cases of nodal follicular lymphomas. In CTCL, CD44 reactivity was demonstrated in all cases; VLA-4, CDlla and ICAM-1 were negative in a case of γδ T-cell lymphoma and in a case of highly malignant CD8+ lymphoma. Furthermore, anti-VLA-4 mAb did not label a case with an NK-like phenotype, nor a case of Ki-1+ lymphoma. Interestingly anti-VLA-3 mAb, normally weakly expressed on T-cell membranes, was strongly positive on blastic cells with an activated phenotype, particularly in Ki-1+ lymphomas, including lymphomatoid papulosis. Reed-Sternberg cells in 5 cases of Hodgkin disease examined were VLA-2 negative. VLA-3 receptor seems to be involved not only in cell-matrix and cell-cell adhesion, but also in lymphocyte activation.

Immunohistochemical Reactivity of Anti-B Cell Monoclonal Antibodies in Thymus, Lymph Node, and Normal Skin

Post-medullary differentiation of B cell lineage largely depends on activation of secondary follicles in different lymphoid organs like spleen, tonsils, and lymph nodes. In all these organs the lymphatic follicles show the same basic structure and cytology. The phenotype of follicular lymphocytes is mainly related to different stages of B cell activation, and at present no organ-specific patterns of immunoreactivity have been reported (1). In the present study the immunohistochemical analysis of follicular reactivity of Workshop mAbs was restricted to the lymph node, whereas thymus and normal skin have been included to screen cross-reactivities against T lymphocytes and extra-medullary structures.

Light and Electron-Microscopic Immunocytochemical Study of Cytoplasmic Immunoglobulins in Non-Hodgkin’s Lymphomas

Sixteen selected cases of non-Hodgkin’s lymphomas (NHL), representing different stages of B-cell maturation, were studied by an avidin-biotin complex technique modified for electron microscopy. A large number of neoplastic cells reflecting the early stages of maturation toward plasma cells, such as chronic lymphocytic leukemia (2 cases), centrocytic lymphoma (2 cases), and centroblastic lymphoma (1 case), showed labeling of immunoglobulins on the membranes of the perinuclear and rough endoplasmic reticulum cisternae with minimal immunoglobulin accumulation within the cisternae. Only a few centrocytes of centroblastic-centrocytic follicular and diffuse NHL (3 cases) showed weak labeling of intracytoplasmic membranes. The cells with evident plasmablastic differentiation of lymphoplasmacytoid NHL (3 cases) and of 2 cases of immunoblastic NHL showed immunoglobulins on the membranes and within the cisternae of the rough endoplasmic reticulum. However, the centrocytes of one of the cases of lymphoplasmacytoid NHL, which revealed features of a follicular center cell lymphoma with plasmacytic differentiation, showed immunostaining of intracytoplasmic membranes without immunoglobulin accumulation. One additional case of immunoblastic NHL showed labeling of the intracytoplasmic membranes and of the periphery of Russell bodies, whereas diffuse intracisternal immunoglobulin accumulation was not observed. Burkitt’s lymphoma (1 case) showed immunoglobulins within the vesicles of the Golgi complex of numerous cells, whereas one case of Burkitt-like lymphoma was characterized by accumulation of immunoglobulins in the rough endoplasmic reticulum cisternae of a few cells with plasmablastic differentiation. Labeling of intracytoplasmic membranes was observed in both cases.