G. Delsol

Expression of the ALK Tyrosine Kinase Gene in Neuroblastoma

ALK (anaplastic lymphoma kinase) is a tyrosine kinase receptor, expressed as part of the chimeric NPM-ALK protein, in anaplastic large cell lymphomas (ALCLs) exhibiting the t(2;5)(p23;q35) translocation. As a result of this translocation, the NPM (nucleophosmin) gene is fused to the portion of the ALK gene encoding its intracytoplasmic segment. In normal mouse tissues, mRNA encoding the Alk receptor has been found only in neural cells, suggesting involvement of this receptor in the development of the nervous system. The purpose of the present study was to examine the presence of ALK transcripts and protein in normal human tissues and a variety of cell lines and human tumors. Emphasis was placed on neuroblastomas because other tyrosine kinase receptors are expressed in human neuroblastomas. Fifty-six cell lines, including 29 lines of neural origin, and lymphoid and nonlymphoid tissue specimens, including 24 neuroblastomas, were investigated for ALK expression, using reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. The results confirmed that mRNA encoding ALK protein was not detectable in any normal or neoplastic hematopoietic tissue tested, except for t(2;5)-positive ALCL. The salient finding was that 13 of the 29 cell lines of neural origin and 22 of 24 neuroblastomas were found to express ALK transcripts and ALK protein. However, no correlation was evident between any known prognostic factors and the level of ALK expression.

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 Hodgkins 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 Hodgkins 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 Hodgkins disease from other subtypes. The lymphocytic and histocytic cells of nodular lymphocyte predominant Hodgkins 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-Steinberg cells and Hodgkins 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 Hodgkins cells in Bouins- or formalin-fixed tissues. Pleomorphic T-cell lymphomas, which could mimic Hodgkins 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 Hodgkins cells in Hodgkins 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 Hodgkins disease.

Anaplastic large‐cell lymphomas of B‐cell phenotype are anaplastic lymphoma kinase (ALK) negative and belong to the spectrum of diffuse large B‐cell lymphomas

There is controversy in the literature as to whether anaplastic large‐cell lymphoma of B‐cell phenotype is related to the t(2;5)‐positive T‐ or ‘null’ cell lymphoma of the same morphology. We report a study of 24 lymphomas with morphological features of anaplastic large‐cell lymphoma which expressed one or more B‐cell markers and lacked T‐lineage markers. Clinical features were more in keeping with large B‐cell lymphoma than with classical t(2;5)‐positive anaplastic large‐cell lymphoma, and immunostaining for anaplastic lymphoma kinase (ALK) protein provided no evidence for the (2;5) translocation (or one of its variants). The staining patterns for CD20 and CD79 were typical of diffuse large B‐cell lymphoma, CD30 expression was variable, and most cases (15/22) lacked epithelial membrane antigen (EMA). These findings support the view that ‘B‐cell anaplastic large‐cell lymphoma’ is unrelated to t(2;5)‐positive (ALK‐positive) lymphoma, and that it represents a morphological pattern occasionally encountered among diffuse large B‐cell lymphomas. By the same reasoning, most tumours diagnosed as ‘ALK‐negative anaplastic large‐cell lymphoma of T‐cell or null phenotype’ probably belong to the spectrum of peripheral T‐cell lymphomas.

Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas

The anaplastic lymphoma kinase (ALK), tyrosine kinase oncogene is implicated in a wide variety of cancers. In this study we used conditional onco-ALK (NPM-ALK and TPM3-ALK) mouse MEF cell lines (ALK+ fibroblasts) and transgenic models (ALK+ B-lymphoma) to investigate the involvement and regulation of angiogenesis in ALK tumor development. First, we observed that ALK expression leads to downregulation of miR-16 and increased Vascular Endothelial Growth Factor (VEGF) levels. Second, we found that modification of miR-16 levels in TPM3-ALK MEF cells greatly affected VEGF levels. Third, we demonstrated that miR-16 directly interacts with VEGF mRNA at the 3′-untranslated region and that the regulation of VEGF by miR-16 occurs at the translational level. Fourth, we showed that expression of both the ALK oncogene and hypoxia-induced factor 1α (HIF1α) is a prerequisite for miR-16 downregulation. Fifth, in vivo, miR-16 gain resulted in reduced angiogenesis and tumor growth. Finally, we highlighted an inverse correlation between the levels of miR-16 and VEGF in human NPM-ALK+ Anaplastic Large Cell Lymphomas (ALCL). Altogether, our results demonstrate, for the first time, the involvement of angiogenesis in ALK+ ALCL and strongly suggest an important role for hypoxia-miR-16 in regulating VEGF translation.

High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease

Hodgkins disease is commonly associated with EBV latent infection. The incidence of EBV reactivation (active infection or EBV infection with replicative cycle) was evaluated in a series of 30 patients with untreated Hodgkins disease (except for one case with chronic lymphocytic leukemia) by quantitation of EBV DNA and titration of anti‐ZEBRA antibodies in serum samples. DNA was detected in serum (>2.5 × 102 genomes/ml) in 15 of 30 patients and was more frequent in Hodgkins disease with EBV‐positive Reed‐Sternberg cells (10/12) than in EBV‐negative cases (5/18), (P < 0.01). Of interest was the demonstration that viremia correlated well with increased titers of anti‐ZEBRA IgG and/or standard serological profiles of EBV reactivation (12/15), (P < 0.05). However the lack of EBV replicative cycle in Reed‐Sternberg cells (negative for ZEBRA antigen and early antigen BHLF1) suggests that the viral replication occurs in a nonneoplastic cell compartment rather than in tumor cells. The measurement of EBV DNA loads and the titration of anti‐ZEBRA antibodies shed new lights on the link between activation of EBV replication and Hodgkins disease: these serological markers together with the determination of the EBV status of the tumor suggest that replication of the viral genome occurs with a decreased efficiency of the immune system, thus allowing progression of the tumor. J. Med. Virol. 57:383–389, 1999.© 1999 Wiley‐Liss, Inc.

Leukaemic presentation of small cell variant anaplastic large cell lymphoma: report of four cases

We report four cases of a rare subtype of CD30‐positive anaplastic large cell lymphoma (ALCL) with a predominant small cell component (small cell variant of ALCL) presenting with a leukaemic feature. Lymph node biopsy showed malignant cells of varying size with a predominant population of small to medium‐sized malignant cells associated with large anaplastic cells strongly positive for CD30 and epithelial membrane antigen (EMA). Both large and small cells were reactive with antibody ALK1, which recognizes the chimaeric NPM‐ALK protein associated with the t(2;5)(p23;q35). All patients presented with hyperleucocytosis with atypical small lymphocytes. Bone marrow involvement was detected on both aspirate and bone marrow trephine where scattered malignant cells were only demonstrated by immunostaining for CD30 and ALK protein. Atypical cells in peripheral blood, lymph node and skin biopsies showed a T or null cell phenotype. Cytogenetic analysis of blood, bone marrow and/or lymph node revealed the t(2;5)(p23;q35) characteristic of ALCL. The patients responded to chemotherapy but showed early relapse without abnormal cells in peripheral blood.

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

Five cases of nodular, lymphocyte predominant Hodgkins 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-l/CDw75, L26, MB2, DBB.42, DBA.44, DND.53, DNA.7 antibodies) but did not react with antibodies against T-cell-associated (MTl, UCHLl/ CD45RO) (one exception for CD45RO in LCL) and R-S cell-associated (Leu-Ml/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 Hodgkins disease in terms of biological and clinical behavior.

Polymerase chain reaction on cerebrospinal fluid cells in the detection of leptomeningeal involvement by B‐cell lymphoma and leukaemia: a novel strategy and its implications

In the search of B‐cell lymphoma/leukaemia dissemination to cerebrospinal fluid (CSF), we used the highly sensitive semi‐nested PCR (snPCR) for the analysis of IgH gene rearrangements. This method detects a rearranged IgH gene from a single B lymphocyte which may or may not represent the neoplastic B‐cell population. We therefore performed multiple snPCR (three to five) experiments on the same CSF sample, postulating that the detection of a band of the same size and sequence in different PCR runs was highly indicative of a clonal population. 17 consecutive cases with a differential diagnosis of primary (PcnsL) (n=10) or secondary (ScnsL) (n=7) CNS lymphoma or leukaemia were investigated by the new strategy. The clonal nature of the B‐cell population was confirmed in 3/10 of suspected PcnsL, and in six other cases the PCR study was indicative of reactive lymphocytosis. One case revealed a clonal B‐cell population in the clinical context of an autoimmune disorder. Evidence of clonal B‐cell population was found in 4/7 of suspected ScnsL. In one of these cases the detected band and its sequence proved identical to that of the primary nodal lymphoma. We believe that the evaluation of B‐cell clonality in CSF requires multiple snPCR amplification on the same sample to compare the size of the products and, if necessary, the DNA sequences to ascertain the diagnosis of malignancy in equivocal cytologic and clinical findings.

Antibody BNH9 detects red blood cell-related antigens on anaplastic large cell (CD30+) lymphomas.

Two new monoclonal antibodies--BNH9 and BNF13--were generated by using a human lung adenocarcinoma cell line and standard hybridoma techniques. Both were found to react with epithelial and endothelial cells in routinely fixed and embedded tissues. Unexpected membrane labelling of some large cell lymphomas while non-reacting with normal lymphoid cells, prompted further characterisation. The antibodies were found to recognise red blood cell-related oligosaccharide antigens. The specificities were directed towards H and Y determinants. A distinctive pattern of reactivity was found for BNH9 in studying 480 cases of various lymphoid neoplasms. Strong expression of H and/or Y antigens was observed in 65/127 (51%) cases of anaplastic large cell(ALC) (CD30+) lymphomas, which are also known to co-express epithelial membrane antigen (EMA) frequently. Only a minority (less than 6%) of other non-Hodgkins lymphomas (NHL) (CD30-,EMA-; 208 cases) and Hodgkins disease (HD) (CD30+, EMA-; 126 cases) were positive. Expression of H and Y antigens was inducible on normal lymphocytes by mitogenic stimulation and by Epstein-Barr virus infection. The data suggest remarkable biological differences of ALC lymphomas within NHL and from HD.

Detection of t(14;18) carrying cells in bone marrow and peripheral blood from patients affected by non-lymphoid diseases.

AIMS/BACKGROUND: To assess the presence of bcl-2/JH rearrangements in bone marrow and peripheral blood lymphocytes from patients affected by diseases other than malignant lymphomas. The t(14;18) (q32;q21) translocation, which juxtaposes the bcl-2 oncogene on chromosome 18 and the JH segment of the immunoglobulin heavy chain (IgH) genes on chromosome 14, is found frequently in follicular lymphomas. METHODS: A sensitive semi-nested polymerase chain reaction (PCR) was used to detect t(14;18) translocation in bone marrow aspirates and peripheral blood lymphocytes from 48 patients. In 137 additional individuals peripheral blood lymphocytes only were tested. RESULTS: Cells carrying bcl-2/JH rearrangements were detected in about a quarter of the bone marrow samples and half of the peripheral blood lymphocyte samples. In seven patients, t(14;18) positive cells were found in both the bone marrow and peripheral blood lymphocyte samples. The size of the PCR products and bcl-2/JH DNA sequence analysis showed that the same t(14;18) carrying clone was present in the bone marrow and the corresponding peripheral blood lymphocyte samples in three of these seven patients. Some patients had more than one bcl-2/JH rearrangement. There was no significant correlation between age and the translocation incidence. Cells carrying the t(14;18) translocation were present in peripheral blood lymphocyte samples with a similar incidence--between 47% and 52% in all age groups from 20 to 79 years. Patients older than 80 years had a lower (37%) but not significantly different incidence. CONCLUSIONS: These findings suggest that patients affected by non-lymphoid diseases may have several t(14;18) carrying cells and some of them undergo a clonal expansion. Whether individuals with t(14;18) positive cells are at a higher risk of lymphoid malignancies remains unanswered and further epidemiological studies are required.