Jan-Willem Vaandrager

Visualization of mono-allelic chromosomal aberrations 3' and 5' of the cyclin D1 gene in mantle cell lymphoma using DNA fiber fluorescence in situ hybridization.

In mantle cell lymphoma (MCL), in addition to the characteristic t(11;14)(q13;q32), rearrangements on the 3′ side of the cyclin D1 gene have been described. In a series of 32 MCL we found three cases with 3′-rearrangements by Southern blot analysis with a probe representing the 3′ untranslated region of the cyclin D1 gene. All three were characterized by the absence of the 4.5 kb transcript, and instead, two cases showed overexpression of the 1.7 kb and a third case (MCLp14) an aberrant 2.5 kb transcript. At the genomic level, fine mapping experiments showed in the 3′ untranslated region a 2 kb deletion in MCLp14 and a breakpoint in the other two cases. Fiber FISH analysis showed that in all three cases the 3′ aberration co-exists with a regular t(11;14) breakpoint 5′ of cyclin D1 on a single allele. Fiber FISH analysis of 16 additional MCL revealed two other such cases with breakpoints 5′ and 3′ of cyclin D1. These mono-allelic aberrations affecting the cyclin D1 gene suggest that the t(11;14) as a first step switches on transcription, and then sequences in the untranslated region of cyclin D1 are affected to stabilize the message.

In situ analysis of the variable heavy chain gene of an IgM/IgG-expressing follicular lymphoma - Evidence for interfollicular trafficking of tumor cells

It is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain the capacity of somatic hypermutation and are not being selected on basis of the quality of their mIgV regions. To extend these findings, we investigated the follicular organization and class switching in a FL that consisted of both IgM- and IgG-expressing tumor cells with a high somatic mutation load and significant intraclonal V(H) gene diversity. V(H)-C(mu) and V(H)-Cgamma gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells, isolated from frozen tissue sections by laser microdissection. We identified many different subclones and obtained limited evidence of subclone dominance in individual follicles. Remarkably, several subclones were found scattered over different follicles. All samples contained IgM- and IgG-expressing tumor cells with, in general, non-identical mutation patterns, which is not in support of ongoing class switching. Accordingly, no switch circle recombination products were found. The findings indicate that the neoplastic follicles lack the organization and functions typical of reactive germinal centers.

Chromosomal markers in lymphoma diagnosis

Many Band T-cell lymphomas and leukaemias are characterized by specific genetic abnormalities. These abnormalities include chromosomal abnormalities visible by banding analysis like numerical abnormalities, gross deletions, amplifications and chromosomal translocations. In addition, an increasing number of submicroscopical abnormalities such as small interstitial deletions, amplifications and mutations are identified in various types of lymphomas by molecular methods. An overview is given in Table 1. At present a wide array of (molecular) methods is available for the detection of genetic abnormalities; these methods include the detection of DNA alterations such as chromosome banding analysis; metaphase-, interphaseand DNA fibre(fluorescent) in situ hybridization (FISH); pulsed-field gel electrophoresis; Southern blot analysis; PCR, SSCP and DNA sequencing. Methods are also available for the detection of quantitative and qualitative alterations of RNA expression such as Northern blot analysis, RT-PCR and RNA in situ hybridization, and finally, for the detection of (altered) protein expression like Western blot analysis and immunohistochemistry. More than one test can often be used to detect a particular DNA or RNA alteration. It is often difficult for the pathologist to make the best choice. Many pathologists will tend to prefer PCR because it represents a relatively straightforward, simple and rapid method. However, for many targets, other methods may be much more appropriate. New FISH techniques such as interphase FISH and DNA fibre FISH have recently been developed which are superior to the established molecular methods such as Southern blotting. Some characteristics of individual tests are listed in Table 2.