Organ-specific inhibition of metastatic colon carcinoma by CXCR3 antagonism.

Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Recent research has indicated that CXCR3/chemokines interactions that orchestrate haematopoetic cell movement are implicated in the metastatic process of malignant tumours, including that of CRC cells to lymph nodes. To date, however, the contribution of CXCR3 to liver and lung metastasis in CRC has not been addressed. To determine whether CXCR3 receptors regulate malignancy-related properties of CRC cells, we have used CXCR3-expressing CRC cell lines of human (HT29 cells) and murine (C26 cells) origins that enable the development of liver and lung metastases when injected into immunodeficient and immunocompetent mice, respectively, and assessed the effect of CXCR3 blockade using AMG487, a small molecular weight antagonist. In vitro, activation of CXCR3 on human and mouse CRC cells by its cognate ligands induced migratory and growth responses, both activities being abrogated by AMG487. In vivo, systemic CXCR3 antagonism by preventive or curative treatments with AMG487 markedly inhibited the implantation and the growth of human and mouse CRC cells within lung without affecting that in the liver. In addition, we measured increased levels of CXCR3 and ligands expression within lung nodules compared with liver tumours. Altogether, our findings indicate that activation of CXCR3 receptors by its cognate ligands facilitates the implantation and the progression of CRC cells within lung tissues and that inhibition of this axis decreases pulmonary metastasis of CRC in two murine tumour models.

Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.

Establishment of a novel anaplastic large-cell lymphoma-cell line (COST) from a 'small-cell variant' of ALCL.

Anaplastic large-cell lymphoma (ALCL) is a distinct biological and cytogenetic entity with a broad spectrum of morphological features (common type, small-cell variant and lymphohistiocytic variant). Few cell lines of ALCL are available and they all originate from primary tumors demonstrating the common type morphology (ie large-sized lymphoma cells). We established a new ALCL cell line (COST) from the peripheral blood of a patient with a small-cell variant of ALCL, at diagnosis. Cells growing in vitro and in SCID mice consisted of two populations, that is, small- and large-sized cells as seen in the patient's tumor. Both large and small malignant cells were positive for CD43/MT1 T-cell associated antigen, perforin, granzyme B and TIA-1, but negative for CD2, CD3, CD5, CD7, CD4 and CD8 antigens. Standard cytogenetic studies as well as multiplex FISH confirmed the presence of the canonical t(2;5)(p23;q35) translocation, but also revealed additional numerical and structural abnormalities. The COST cell line is the first ALCL small-cell variant cell line, and thus provides a potentially useful tool for further functional and molecular studies that should improve our understanding of the small-cell variant of ALCL, which is more frequently complicated by a leukemic phase.

Monoclonal antibodies in the diagnosis of Hodgkin's disease. The search for a rational panel.

A novel, comprehensive panel of monoclonal antibodies was tested in a large series of routinely processed lymph node biopsy specimens from patients with Hodgkin's disease (69 cases), with the object of developing either definitive or adjunctive diagnostic criteria. B- and T-cell lymphomas and reactive states that could mimic Hodgkin's disease were also assessed with the same monoclonal antibody panel. In addition to the popularly used anti-Leu-M1 (CD15), the panel included the recently produced Ber-H2 (CD30) antibody, which detects a formalin-resistant epitope of the Ki-1 antigen. The other monoclonal antibodies were directed against epithelial membrane antigen (Dako-EMA) and leukocyte common antigen (Dako-LC) (CD45), as well as B-cell (LN-1 and LN-2) and T-cell (MT1) associated antigens. The results showed clear phenotypic separation of nodular lymphocyte predominant subtype of Hodgkin's disease from other subtypes. The lymphocytic and histiocytic cells of nodular lymphocyte predominant Hodgkin's disease were reactive for LN-1 (all cases) and anti-EMA (most cases) but negative for anti-Leu-M1 and Ber-H2. Within the other subtypes--i.e. nodular sclerosis and mixed cellularity--nearly all Reed-Sternberg cells and Hodgkin's cells were positive for both anti-Leu-M1 and Ber-H2. Ber-H2 monoclonal antibody was observed to react more frequently with Reed-Sternberg cells and Hodgkin's cells in Bouin's- or formalin-fixed tissues. Pleomorphic T-cell lymphomas, which could mimic Hodgkin's disease on morphology, created the same problem on phenotypic analysis. However, MT1 identified a significant proportion of T-cell lymphomas with Reed-Sternberg-like cells, having proven negative for Reed-Sternberg cells and Hodgkin's cells in Hodgkin's disease. Thus, a combination of anti-Leu-M1, Ber-H2, anti-EMA, LN-1, and MT1 monoclonal antibodies appears at present to be the most useful panel for the diagnosis and the differential diagnosis of Hodgkin's disease.

Further phenotypic evidence that nodular, lymphocyte-predominant Hodgkin's disease is a large B-cell lymphoma in evolution.

Five cases of nodular, lymphocyte predominant Hodgkin's disease (nLP HD), in which an association with (n = 3) and transformation to (n = 2) large cell lymphoma (LCL) were found, were studied with monoclonal antibodies against B-, T-, and Reed-Sternberg (R-S) cell-associated antigens and epithelial membrane antigen (EMA) on paraffin sections. Both lymphocytic (L) and histiocytic (H) cells of nLP HD and lymphoma cells of LCL expressed multiple B-cell-associated antigens (detected by LN-1/CDw75, L26, MB2, DBB.42, DBA.44, DND.53, DNA.7 antibodies) but did not react with antibodies against T-cell-associated (MT1, UCHL1/CD45RO) (one exception for CD45RO in LCL) and R-S cell-associated (Leu-M1/CD15, Ber-H2/CD30) antigens. EMA was expressed by L and H cells in all cases and conserved in LCL cells, emphasizing the frequent expression of EMA by the diagnostic cells of nLP HD. An antibody (BNH9) against blood group-related antigens (H and Y oligosaccharide antigens) that does not normally react with lymphoid cells was found to be reactive with few L and H cells in two of five and most LCL cells in four of five cases. The finding might be indicative of abnormal activation of lymphoid cells. The data reinforce current implications that nLP HD is a B-cell malignancy in evolution and that it is not truly representative of Hodgkin's disease in terms of biological and clinical behavior.

A new monoclonal anti-CD3epsilon antibody reactive on paraffin sections.

We generated a new monoclonal antibody (MAb), F7.2.38, by immunizing mice with CD3varepsilongammadelta/CD3omega complexes purified from human T-cells by OKT3 MAb-Sepharose affinity chromatography. Immunoprecipitation experiments and Western blotting analysis showed that MAb F7.2.38 recognized the CD3varepsilon chain in CD3varepsilon cDNA-transfected FOX B-cells and in various T-cell lines. Using flow cytometry on permeabilized or intact cells, the epitope was found to be located in the cytoplasmic tail of the CD3varepsilon chain. Immunohistochemical staining on paraffin-embedded sections showed that the reactivity of MAb F7.2.38 was comparable to that of the commercially available anti-CD3varepsilon polyclonal antibody. Of the 52 well-characterized T-cell lymphomas, 41 were positive for F7. 2.38 (79%), whereas all 37 B-cell lymphomas and 69 non-lymphoid tumors were unreactive. This new anti-CD3varepsilon antibody would be particularly useful for phenotyping T-cell lymphomas on routinely processed paraffin-embedded tissue sections.

Hairy cell leukemia. Diagnosis of bone marrow involvement in paraffin-embedded sections with monoclonal antibody DBA.44.

The new monoclonal antibody DBA.44 recognizes an unknown fixation-resistant B-cell differentiation antigen expressed by mantle zone lymphocytes, reactive immunoblasts, monocytoid B cells, and a small proportion of high- and low-grade lymphomas. Among node-based lymphomas, the strongest membrane staining was observed in centroblastic, immunoblastic, and monocytoid B-cell lymphomas. In studying bone marrow biopsy specimens from 166 patients with hairy cell leukemia, strong positive staining of surface membrane 'hairy' features of leukemic cells was observed in routinely fixed and decalcified bone marrow biopsy specimens of nearly all cases. The antibody distinguished hairy cell leukemia from the more common B-cell chronic lymphocytic leukemia and bone marrow infiltrates of typical lymph node-based lymphomas by immunomorphologic criteria. DBA.44 was valuable to (1) confirm the diagnosis of hairy cell leukemia, (2) estimate the bone marrow density of hairy cell leukemia before and after treatment, and (3) make the diagnosis of hairy cell leukemia in ambiguous cases, which are all properties that indicate its usefulness in the practice of diagnostic hematopathology.

Production and characterisation of an antimelanoma monoclonal antibody KBA.62 using a new melanoma cell line reactive on paraffin wax embedded sections.

AIMS--To generate new monoclonal antibodies directed against melanoma associated antigens using a new melanoma cell line, KAL. METHODS--The melanoma cell line was established in culture from a lymph node metastasis of malignant melanoma. Normal Balb/c mice were immunised with KAL cells. Splenocytes were used for fusion experiments using standard techniques. Hybridoma supernatants were tested for antibody binding activity using an indirect immunoperoxidase method on frozen sections from KAL tumour cells xenografted onto nude mice and human tonsils. KBA.62 was selected because of its reactivity with melanocytic proliferations on both frozen and paraffin wax sections. RESULTS--On immunoblotting, KBA.62 reacted with three bands of 140, 135 and 128 kD and two weak bands of 88 and 73 kD. In normal human tissues basal melanocytes in the epidermis did not react with this antibody and only occasional labelling of endothelial cells was noted. Of the human tumours, KBA.62 reacted strongly and uniformly with the majority of benign (21/21) and malignant (75/86) melanocytic proliferations. Staining was localised predominantly to the cell membrane with little or no cytoplasmic reactivity. Negative staining was observed in the majority of human non-melanocytic neoplasms, the exceptions being some carcinomas (11/89), particularly the well differentiated squamous cell type. This, however, was not thought to present a diagnostic problem. CONCLUSIONS--KBA.62 appears to be potentially useful in ascertaining the immunomorphological diagnosis of malignant melanoma in routinely processed paraffin wax sections.

Telomerase activity in Hodgkin's disease.

Telomerase activity has been demonstrated in various types of cancers including lymphoid neoplasms. The tumour specificity of telomerase activity has been a subject of debate since the description of detectable enzymatic activity in non neoplastic tissues. We and others have demonstrated that such activity was present in non Hodgkin's lymphomas as well as in reactive hyperplastic lymph nodes and tonsils. However, we at the same time were surprised by the absence of telomerase activity in most cases of Hodgkin's disease. We have extended our previous study by adding additional cases and confirm that in the majority of cases, Hodgkin and Reed-Sternberg cells lack telomerase activity (only 2 cases positive out of 20). These results may indicate that the tumour cells of Hodgkin's disease evolve different pathways for maintaining the length of their telomeres during the process of becoming immortal.

A new monoclonal anti-CD7 antibody reactive on paraffin sections.

CBC.37 monoclonal antibody (mAb) was generated using balb/c mice immunized with CEM T cell line. It was selected because of its strong reactivity on T lymphocytes on paraffin tissue sections. The anti-CD7 specificity of CBC.37 mAb was assessed by immunohistochemistry, cross-blocking, and cross-immunoprecipitation experiments using CBC.37 and the anti-CD7 mAb DK24. CBC.37 mAb immunoprecipitated a 40-kDa protein. Cross-blocking and cross-immunoprecipitation experiments demonstrated that the two antibodies recognized the same molecule. Immunostaining of a large number of reactive lymph nodes and B and T cell lymphomas confirms that CBC.37 mAb was directed against T cells. As expected, on reactive lymph nodes the staining pattern was comparable to that of CD3. Among the 110 T cell lymphomas examined, all T lymphoblastic lymphomas were positive (15+/15; 100%). As a result of the frequent loss of CD7 antigen, only 25+/95 (26%) of peripheral T cell neoplasms were found to be positive for CBC.37. A marked reduction in the number of CBC.37-positive T cells was observed in 7 of the 60 cases of benign inflammatory dermatoses studied (approximately 12%). CBC.37 was unreactive with all healthy and neoplastic non-lymphoid samples examined. Because the lack of CD7 expression in T cell lymphomas is of diagnostic value, CBC.37 mAb in association with other anti-T cell antibodies working on paraffin sections could be of particular value in asserting the diagnosis of T cell lymphomas in routine histopathology.

[Detection of residual disease in follicular lymphomas using the PCR technique: importance of clono-specific probes]. / Détection de la maladie résiduelle dans les lymphomes folliculaires par la technique de PCR: intérêt des sondes clono-spécifiques.

Follicular lymphoma constitutes 30-40% of non-Hodgkin's lymphomas. Most patients have widespread disease at diagnosis. The clinical course is generally indolent, and it is not usually curable with available treatment. The source of relapse in patients who achieve complete clinical remission is residual neoplastic cells that are present below the limits of detection using standard techniques. With the development of PCR technology, the presence of these residual malignant cells [Minimal Residual Disease (MRD)] has been demonstrated clearly. Recently, an association of high-dose chemotherapy with autologous bone marrow or peripheral blood progenitor cell autograft appeared promising in the treatment of these lymphomas. In the search of clonal markers for the detection of MRD in follicular lymphomas, two strategies can be used. In the cases associated with the t(14;18) (q32;q21) chromosomal translocation, the bcl-2/JH junctional regions are amplified by PCR in approximately equal to 50% of cases and then sequenced in order to synthesize an anti-junction oligonucleotide probe specific for each patient's malignant clone (clonospecific probe). In the cases negative for this translocation, an alternative strategy consists in the amplification of immunoglobulin high chain (IgH) gene rearrangement (approximately equal to 75% of cases). The present review highlights the value of molecular markers such as bcl-2/JH and VH/JH rearrangements to follow the neoplastic clone and to detect MRD in patients with follicular lymphomas.

[Immunochemical diagnosis of hepatic localizations in malignant lymphoid hematologic diseases. Study of 80 cases]. / Diagnostic immunohistochimique des localisations hépatiques des hémopathies lymphoïdes malignes. Etude de 80 cas.

The diagnostic value of immunohistochemistry using monoclonal antibodies was assessed in 100 liver biopsy specimens. The majority of these cases were hepatic localizations of lymphoid malignancies. Ten normal and reactive inflammatory liver biopsies were used as controls. Some monoclonal antibodies directed against leukocyte antigens revealed unexpected reactivities with normal liver structures: biliary tract (anti-CD10, anti-B MB2) and hepatocytes (anti-B LN1). In 12/17 cases of hepatic involvement by large cell malignancy, immunohistochemistry allowed the diagnosis of non Hodgkin's lymphoma (NHL); the remaining 5 cases were metastatic undifferentiated carcinoma. It was difficult to differentiate small cell liver NHL from reactive inflammatory infiltration. New anti-B (MB1, MB2, 4KB5, LN1 and LN2) and anti-T (MT1 and UCHL1) monoclonal antibodies suitable for use on paraffin sections were of value to phenotype NHL when only fixed material was available. But, information was too limited to distinguish malignant from reactive infiltrates. Immunohistochemistry on frozen sections was often necessary to diagnose inflammatory infiltrates and to phenotype NHL. Most NHL were of B cell origin (11/13 cases) and showed monotypic surface immunoglobulins as well as B cell-associated antigens (CD22+). The expression of the T CD5 antigen by B-cell NHL may have some diagnostic value. When monotypic surface immunoglobulins could not be demonstrated (due to background staining) the expression of this antigen by B lymphocytes was considered to be highly indicative of their neoplastic nature. Hairy cell leukemia exhibited a pathognomonic phenotype on frozen sections (CD11c+, CD22+, CD25+). T NHL were rare (2 cases) and difficult to diagnose due to the lack of clonal markers. The diagnosis of Hodgkin's disease in liver (15/20 cases) was facilitated by using paraffin sections of both monoclonal antibodies anti-CD15 (Leu M1) and anti-CD30 (Ber-H2) which detect fixation-resistant antigens expressed by Sternberg cells.

[T lymphocyte populations of the intestinal mucosa in celiac disease in children. Immunohistochemical study]. / Populations lymphocytaires T de la muqueuse intestinale au cours de la maladie coeliaque chez l'enfant. Etude immunohistochimique.

In order to study the distribution of lymphocyte subpopulations in a pathologic intestinal mucosa, the authors, instead of using the classic method by counting the number of lymphocytes, present an original method permitting the exploitation of quantified data from labelled surface cells by texture analyser coupled with a computerized system. We investigated 25 children presenting with chronic diarrhea and villous atrophy and 5 control subjects. Fifteen of the 25 children had celiac disease (10 active with total villous atrophy and 5, celiac disease in remission with healing mucosa), 5 cow's milk protein intolerance with total or partial villous atrophy and 5, chronic diarrhea with partial villous atrophy. Immunohistochemical study with monoclonal antibodies was carried out on frozen sections using a three-step immunoperoxidase technique. Compared with the 5 controls, patients with food intolerance (celiac disease and cow's milk protein intolerance) showed a significant increase of T suppressor lymphocytes (p less than 0.01 and p less than 0.05) in the epithelium, whereas there were more T helper lymphocytes in the lamina propria (p less than 0.05 and p less than 0.01). Non-treated celiac disease was distinguished from treated celiac disease by a marked increase in intra-epithelial T cytotoxic-suppressors. These results suggest that T cytotoxic-suppressors may be the mediators of the lesions observed in celiac disease.

[Value of immunoperoxidase with monoclonal antibodies after dismounting of previously stained sections]. / Intérêt de l'immunopéroxydase avec anticorps monoclonaux après démontage de coupes déjà colorées.

Immunoperoxidase using monoclonal antibodies may be applied to paraffin sections previously stained. The precise nature of cells found by routine staining may thus be investigated. It also enables, with small biopsies, immunohistochemical identification of a tissue zone lost by serial sectioning.

[Techniques for detection of telomerase activity in tissue samples. Diagnostic and prognosis importance]. / Techniques de détection de l'activité télomérase dans des échantillons tissulaires. Intérêt diagnostique et pronostique.

Telomerase activation has been shown to be an almost universal property of malignant tumors evoking its role in the immortalisation process. We used the recently described sensitive and rapid detection assay called telomeric repeat amplification protocol (TRAP) to detect telomerase activity in neoplastic and non neoplastic tissues. Human telomerase is a ribonucleoprotein which functions as a telomere terminal transferase by adding multiple repeats of the TTAGGG hexamer at the 3'-OH ends of either telomeres or oligonucleotide specifically designed for the TRAP assay. Whenever present, telomerase activity is revealed on acrylamide gel by a six nucleotide ladder of extension products. Detection of telomerase activity may be evaluated for differentiating neoplastic from non neoplastic tissues. In addition, quantitation of telomerase activity may serve as prognostic marker for malignant tumors.

Production of monoclonal antibodies using spleen cells from nude mice bearing human tumors.

Monoclonal antibodies were generated with spleen cells from nude mice bearing human tumors. Three grafted tumors were selected because of their difference in metastatic ability in nude mice. Two were non-metastasizing carcinomas and one a highly metastasizing adenocarcinoma of the lung (Bur-tumor). Two normal nude mice were used as controls. Culture supernatants were screened by immunoperoxidase using frozen sections from both the immunizing human tumor and normal human tonsil to detect unexpected monoclonal antibodies. Two kinds of monoclonal antibodies were obtained. The first were directed against miscellaneous membrane and/or cytoplasmic antigens expressed by normal cells (e.g. normal tonsillar epithelium). Most of these antibodies corresponded to auto-antibodies and their frequency did not appear to be influenced by the fact that the mice were with or without grafted human tumors. The second type of antibodies were directed against tumor-associated antigen and generated only by fusing splenocytes from nude mice bearing the metastasizing lung adenocarcinoma. On frozen sections Bur-1-anti-tumor antibody stained all but one lung carcinoma. Occasional carcinomas originating from other organs such as pancreas and breast were also labelled. On the other hand, more than half of the cases of so-called "malignant histiocytosis" (Ki-1 lymphoma) seemed to express this epithelial antigen. Some normal cells in the lung, pancreas (acini), and kidney (distal tubules) also bound Bur-1 antibody. These results suggested that Bur-1 antibody could be related to some antibodies already described, directed against epithelial membrane antigens. When Bur-tumor was analysed by immunoblotting with Bur-1 antibody a positive reaction was obtained with material migrating in the kD-45kD molecular-weight region.

[Cells of the phenotype HNK-1 + (CD 57 +) in reactive adenopathies, lymphomas and Hodgkin's disease]. / Les cellules de phénotype HNK-1 + (CD 57 +) dans les adénopathies réactionnelles, lymphomateuses et Hodgkiniennes.

The number and distribution of Leu-7 + cells, a subset of Natural Killer cells clustered as CD 57 + cells, were studied with an immunoperoxidase technique on reactive lymph nodes (n = 13), malignant lymphomas (n = 60) and Hodgkin's disease (n = 22). Results of paraffin-section immunocytochemistry were compared with those obtained of frozen sections of the same tissues. CD 57 + cells are small lymphocytes mainly located in the germinal centers of reactive lymph nodes and in their malignant counterpart, i.e. the follicular lymphomas. The paragranuloma of Hodgkin's disease, type I nodular, contained high numbers of CD 57+ cells. Nine cases of CD 57+ lymphomas are reported, which were of high grade of malignancy. Their histological subtypes were anaplastic (3 cases) immunoblastic (2 cases), pleomorphic medium and large cell (3 cases), unclassifiable (1 case). Their diverse T-cell (4 cases) or B-cell (2 cases) origin and the various expression of epithelial membrane antigen (EMA) or Kil/Ber-H2 (CD 30) suggest an aberrant CD 57+ phenotype of a subset of large cell lymphomas.

[Immunoperoxidase study of normal and pathologic lymphoid tissue. Value of monoclonal antibodies]. / Etude en immunopéroxydase du tissu lymphoïde normal et pathologique. Intérêt des anticorps monoclonaux.

Immunoperoxidase study can be performed either on fixed and paraffin embedded biopsy specimens or on frozen sections. Advantages and limits of these two methods, as well as the results obtained on normal and pathologic lymphoid tissue are presented. Immunoperoxidase on paraffin sections (PAP technic) is a simple method which allows a good morphologic analysis. However, most of the fixatives destroy proteic antigens particularly those linked to the cell membrane. Thus surface immunoglobulins (S.Ig) cannot be detected. In contrast cytoplasmic immunoglobulins remain antigenic enough to be demonstrated in routine paraffin embedded sections. In lymphomas synthesizing monotypic immunoglobulins, the percentage of labelled cells varies from 5 to 80%. Beside the background staining, which can be attenuated by trypsinisation, absorption of extracellular substances is often responsible for a false positive staining. Pathologists are mainly confronted with the passive uptake of extracellular immunoglobulins (IgG K and IgG L), as well as other serum proteins (lysozyme etc...). Immunoperoxidase on frozen sections allows the use of monoclonal antibodies. A large number of surface and cytoplasmic antigens can be detected. First, the localization of B and T lymphocytes, NK cells, interdigitating cells and dendritic reticulum cells within the normal lymph node is described. In the second part, the interest of monoclonal antibodies in differential diagnosis between lymphoma and pseudo-lymphoma, and in phenotyping of lymphomas is discussed. Now, it is possible to perform an in situ immunologic characterization of most lymphomas. B cell lymphomas have sIg associated with other antigens (Pan B+, HLA-DR+). Cells of chronic lymphoid leukaemia and centrocytic (cleaved-cell) lymphomas frequently express T65 (T 101+ or Leu 1+) antigen which is usually found on normal or neoplastic T lymphocytes. Monoclonal antibodies provide new evidence of the germinal centre origin of follicular lymphomas. Thus, monoclonal antibody directed against dentritic reticulum cells (CRD) revealed the same network of DRC in follicular lymphomas as in reactive germinal centres. This finding could account for the nodular pattern of these lymphomas, neoplastic cells being in some way, enclosed within the DRC network. On the other hand, neoplastic follicles are surrounded by a large amount of t lymphocytes. Some T lymphocytes are also found within the follicles where they are associated with NK cells. Lastly, as reactive benign follicles, neoplastic follicles are labelled by the anti-Calla antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

[Diagnosis of undifferentiated tumors by means of monoclonal antibodies on paraffin sections]. / Diagnostic des tumeurs indifférenciées à l'aide d'anticorps monoclonaux utilisables sur coupes en paraffine.

The diagnostic value of 3 monoclonal antibodies applied on to routinely processed surgical biopsies was assessed. These antibodies were directed against keratin polypeptide (KL1), epithelial membrane antigen (DAKO-EMA) and leucocyte common antigen (DAKO-LC). First, using a three-step immunoperoxidase procedure, we determined the phenotype of well differentiated carcinomas (21 cases), non-Hodgkin's malignant lymphomas (44 cases), malignant histiocytoses (3 cases), melanomas (5 cases), sarcomas (6 cases) and miscellaneous tumors (16 cases). Nineteen out of the 21 carcinomas reacted with KL1 and DAKO-EMA antibodies but not with DAKO-LC. Forty out of the 44 non-Hodgkin's malignant lymphomas reacted with DAKO-LC. All these tumors were negative with KL1 antibodies but three of them, as well as 3 cases of malignant histiocytosis, expressed the epithelial membrane antigen. The value of these 3 antibodies was then assessed in the differential diagnosis of 30 undifferentiated tumors. A definite diagnosis was made in 28 cases: there were 11 undifferentiated carcinomas and 11 large cell malignant lymphomas. The phenotype of 6 tumors was highly suggestive of malignant histiocytosis, the peculiarity of which is to express both leucocyte common (DAKO-LC+) and epithelial membrane antigens (DAKO-EMA+). Only two tumors did not react with these 3 antibodies. We conclude that it is now possible to determine the nature of nearly all undifferentiated tumors on paraffin-embedded biopsy specimens.

Anti-tumor activity of obinutuzumab and rituximab in a follicular lymphoma 3D model.

Follicular lymphomas (FLs) account for 35-40% of all adult lymphomas. Treatment typically involves chemotherapy combined with the anti-CD20 monoclonal antibody (MAb) rituximab (RTX). The development of the type II anti-CD20 MAb obinutuzumab (GA101) aims to further improve treatment. Here, using FL cells we show that RTX and GA101 display a similar activity on RL cells cultured in 2D. However, 2D culture cannot mimic tumor spatial organization and conventional 2D models may not reflect the effects of antibodies as they occur in vivo. Thus, we created a non-Hodgkin's lymphoma (NHL) 3D culture system, termed multicellular aggregates of lymphoma cells (MALC), and used it to compare RTX and GA101 activity. Our results show that both antibodies display greater activity towards FL cells in 3D culture compared with 2D culture. Moreover, we observed that in the 3D model GA101 was more effective than RTX both in inhibiting MALC growth through induction of (lysosomal) cell death and senescence and in inhibiting intracellular signaling pathways, such as mammalian target of rapamycin, Akt, PLCgamma (Phospholipase C gamma) and Syk. Altogether, our study demonstrates that spatial organization strongly influences the response to antibody treatment, supporting the use of 3D models for the testing of therapeutic agents in NHL.