Relationship between classic Hodgkin lymphoma and overlapping large cell lymphoma investigated by comparative expressed sequence hybridization expression profiling.

There is a diagnostic grey zone between classic Hodgkin lymphoma (cHL) and some non-Hodgkin lymphoma (NHL), including primary mediastinal B cell lymphoma, diffuse large B cell lymphoma, and anaplastic large cell lymphoma. They all have some morphological and/or phenotypic features in common. To investigate this, we undertook an expression profiling study of these lymphomas using comparative expressed sequence hybridization. This technique detects chromosomal regions that are differentially expressed between a test and a reference tissue in a manner similar to comparative genomic hybridization, and is particularly suitable when the number of informative biopsies is limited. Using this approach, we identified a unique expression profile for all lymphoma types investigated. Unsupervised hierarchical cluster analysis of the acquired data showed that cHL separates from all investigated NHLs, including ALCL-like HL. Moreover, anaplastic lymphoma kinase (ALK)-negative ALCL clustered in a separate branch together with ALCL-like HL. Thus, analysing the neoplastic cells concurrently with their microenvironment, ALK-negative ALCL and ALCL-like HL seem to be related to each other, while cHL constitutes a separate lymphoma entity.

Real-time reverse transcription-PCR and fluorescence in-situ hybridization are complementary to understand the mechanisms involved in HER-2/neu overexpression in human breast carcinomas.

AIMS: To evaluate the HER-2/neu status at the mRNA and DNA level of breast carcinomas and to compare it with HER-2/neu receptor overexpression by immunohistochemistry (IHC). METHODS AND RESULTS: In 32 invasive breast carcinomas, frozen tissue was available for real-time detection of HER-2/neu mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR). Corresponding paraffin sections were examined by IHC and fluorescence in-situ hybridization (FISH). Thereby, different IHC and FISH procedures were compared. Using microwave epitope retrieval, all 32 cases scored 3+ on IHC, whereas only 28 out of 32 cases scored IHC 3+ using water bath epitope retrieval. All of these 28 cases showed increased levels of HER-2/neu mRNA. Dual-colour FISH analysis showed corresponding gene amplification in all 28 cases, with two cases showing a peculiar amplification pattern. In the remaining four cases, scoring IHC 2+ using water bath epitope retrieval, mRNA levels were not elevated. Three cases did not have gene amplification and one case showed low-level HER-2/neu gene amplification. All four carcinomas showed chromosome 17 polysomy. CONCLUSIONS: Real-time RT-PCR is accurate in selecting breast carcinoma cases scoring 3+ by IHC with high-level gene amplification. Results obtained by dual-colour FISH suggest that mechanisms leading to HER-2/neu receptor overexpression may be different between carcinomas scoring 2+ and 3+ on IHC, with polysomy 17 found in the former and gene amplification in the latter.

A phase II trial with rosiglitazone in liposarcoma patients.

Agents of the thiazolidinedione drug family can terminally differentiate human liposarcoma cells in vitro by activating genes responsible for lipocyte differentiation. One study has shown clinical activity of troglitazone treatment in liposarcoma patients. We sought to find further evidence for this result. In all, 12 patients with a liposarcoma received rosiglitazone 4 mg b.d. They were followed clinically and with repeated biopsies for histological and biological studies. At the molecular level the mRNA translation of three genes that are induced by this treatment (peroxisome proliferator-activated receptor gamma (PPARgamma), adipsin and fatty acid binding protein) was determined. Nine patients were eligible for evaluation. One patient had to stop treatment due to hepatotoxicity. The mean time to progression was 6 months (2 - 16 months), with one patient still on treatment. We did not see any significant change in histologic appearance of the liposarcomas by the treatment. The level of gene expression changed significantly in two patients, but this did not result in a clinical response. Based on this study, rosiglitazone is not effective as an antitumoral drug in the treatment of liposarcomas. Increased PPARgamma activity does not correlate with the clinical evolution.

The NPM-ALK and the ATIC-ALK fusion genes can be detected in non-neoplastic cells.

Anaplastic large cell lymphoma (ALCL) is frequently associated with the t(2;5)(p23;q35) translocation. It creates a NPM-ALK fusion gene, fusing the anaplastic lymphoma kinase (ALK) gene (2p23) and the nucleophosmin (NPM) gene (5q35). Other rearrangements involving the ALK gene have recently been shown to be associated with ALCL, among which the ATIC-ALK rearrangement resulting from the inv(2)(p23q35) translocation is probably the most recurrent. The aims of the present study were to investigate the presence of NPM-ALK and ATIC-ALK fusion genes in ALCL, using a real-time 5' exonuclease-based reverse-transcription polymerase chain reaction (RT-PCR). This sensitive technique was also applied to investigate whether both fusion genes might be detected in Hodgkin's disease cases and in reactive lymphoid tissue. Results of the RT-PCR were compared to ALK immunostaining, cytogenetics, and fluorescence in situ hybridization (FISH) results. RT-PCR detected the NPM-ALK and ATIC-ALK fusions at high levels in 8 and 3 of a total of 13 ALK-positive ALCL cases. One ALK-positive ALCL case was negative for both fusion genes analyzed but revealed a new ALK-related translocation t(2;17)(p23;q25) by cytogenetic and FISH analysis. In addition, of the eight ALK-positive ALCL cases that were strongly positive for the NPM-ALK fusion, three cases also showed the presence of the ATIC-ALK fusion, although at much lower levels. Similarly, out of the three strongly positive ATIC-ALK cases, one case was positive for the NPM-ALK fusion, at low levels. Finally, the NPM-ALK and the ATIC-ALK fusions were detected, at equally low levels, respectively in 13 and 5 ALK-negative ALCL cases, in 11 and 5 Hodgkin's disease cases and in 20 and 1 non-neoplastic lymphoid tissues. The distinction between the high- and low-level detection was confirmed by relative quantitative RT-PCR for a representative number of cases. Of interest is the fact that the high-level detection coincided with the presence of ALK gene rearrangement detected by cytogenetics and FISH and may reflect a central role of the transcript in the oncogenic mechanism of ALK-positive ALCL. Low-level detection is not supported by cytogenetics and FISH, presumably due to the presence of the transcripts in only a small minority of normal cells not detectable by these techniques. Our findings demonstrate that NPM-ALK and ATIC-ALK fusion transcripts may be detected in conditions other than ALK-positive ALCL including reactive lymphoid tissues, although at low levels, suggesting the presence of the transcripts in normal (bystander) cells. Moreover, they suggest that the ALK gene rearrangement by itself might be insufficient to induce tumor formation. They further question the validity of quantitative real-time RT-PCR for monitoring minimal residual disease in ALCL. Finally, the newly identified translocation t(2;17)(p23;q25) can be added to the list of ALK gene rearrangements occurring in ALK-positive ALCL.