Genetic Changes in MALT lymphoma
Cytogenetic analysis has proved useful by demonstrating similar alterations in MALT lymphomas from different anatomic sites. The common translocations that characterize MALT lymphomas include t(11:18)(q21:q21), t(1;14)(p22;q32) and t(14;18)(q32;q21) [12, 13, 14] . Cloning of the breakpoints of the t(11;18)(q21;q21) showed the fusion of the apoptosis inhibitor-2 (API2) gene and the MALT lymphoma-associated translocation (MALT1) gene at the 11q21 and 18q21 breakpoints, respectively [15, 16, 17] . The immunoglobulin heavy chain gene (IGH) enhancer deregulates the BCL10 gene in the t(1;14)(p22;q32) and the MALT1 gene in the t(14;18)(q32;q21) [18, 19, 20, 21] . Recent investigations have shown that these disparate translocations affect a common signalling mechanism involving BCL10 and MALT1, and thus unify all three under a common pathogenesis, resulting in the activation of the NF- k B pathway [22, 23, 24, 25] . Very recently Streubel and colleagues have described a forth translocation, t(3;14)(p14.1;q32), involving IGH and forkhead box protein P1 (FOXP1) at 3p14.1 in MALT lymphoma [26]. Preliminary data suggest that this translocation upregulates the transcription factor FOXP1 and that the oncogenic activity may not be linked to the NF- k B pathway. Taken together, the four chromosome translocations in MALT lymphoma either result in transcriptional deregulation (BCL10, MALT1, FOXP1) or in production of a chimeric protein (API2-MALT1).

Numerical aberrations (aneuploidy), most commonly trisomy 3 and/or trisomy 18, frequently occur in MALT lymphoma but may also be detected in other B-cell lymphomas, albeit at much lower frequency [27, 28, 29] .

Table 1. Genetics of MALT lymphoma

1989	Levine et al. [30]	t(11;18)(q21;q21) in gastric and lacrimal gland lymphoma
1990	Wotherspoon et al. [31]	t(1;14)(p22;q32) found in a pulmonary MALT lymphoma
1997	Ott et al. [32], Auer et al. [14]	t(11;18)(q21;q21) is associated with MALT lymphoma
1999	Dierlamm et al. [15]	t(11;18)(q21;q21) generates the API2-MALT1 fusion
1999	Willis et al. [18]	Cloning of t(1;14)(p22;q32) breakpoint and identification of BCL10
2003	Streubel et al. [20]Sanchez-Izquierdo et al. [21]	t(14;18)(q32;q21) is associated with MALT lymphoma and involves MALT1
2004	Streubel et al. [26]	t(3;14)(p14.1;q32) is associated with MALT lymphoma and involves FOXP1

Detection of genetic changes in MALT lymphoma
The t(11:18)(q21:q21) can be detected either by the identification of the API2-MALT1 fusion transcript by RT-PCR (reverse transcriptase-polymerase chain reaction) or by fluorescence in situ hybridisation (FISH). Importantly, both methods do not require fresh tissue but work well on routinely processed, formalin-fixed and paraffin embedded material, as outlined briefly below.

RT-PCR for the API2-MALT1 fusion transcript (according to Inagaki et al. [33]). In a first step total RNA is extracted from paraffin sections by protein K digestion. Next RNA is subjected to first-round multiplex one-tube RT-PCR, then to second-round nested multiplex PCRs. The final products are run on polyacrylamide gels. By this means variant fusion products resulting from all known breakpoints each in the API2 and MALT1 gene can be detected.

FISH for the detection of the four MALT lymphoma-associated translocations
To overcome the scoring problems on thin sections, FISH is usually performed on single nuclei isolated from thick paraffin slices or from needle core biopsies taken from paraffin blocks [34]. Two principal types of directly labeled FISH probes can be used to detect chromosome translocations: (1) split-apart or break-apart probes labeled in different colors that bind to regions on either side of a DNA breakpoint; and (2) co-localizing or dual-fusion probes that label each gene in a single color providing fusion signals in a translocation positive cell. One advantage of the split-apart probe design is that variant translocations are also detectable. For the detection of aneuploidy, in the context with MALT lymphomas most commonly trisomy 3 and 18, centromere-specific probes for chromosomes 3 and 18 are used.

Comparison of the two detection methods for the t(11;18)(q21;q21), the RT-PCR and FISH, yielded 100% concordance in a large series of cases, indicating that the RT-PCR is probably the ideal tool for screening purposes [26].

The Significance of Malt Lymphoma-associated Genetic Aberrations

Genetic aberrations and pathogenesis.
The overall distribution of MALT lymphoma associated genetic aberrations is shown in Figure 1:



Figure 1. Graphical demonstration of frequencies of genetic aberrations detected by FISH or RT-PCR in 255 MALT lymphomas^* [26, 35] .

^*Please note that the numbers given for trisomies 3/18 refer to cases that carry this aberration only, that is, are negative for any of the four translocations. It has to be emphasized, that the numbers given are influenced by the site distribution of the cases. For instance, series comprising a higher proportion of pulmonary and/or gastric MALT lymphomas may provide a higher proportion of t(11;18)+ cases.

According to our data on 255 MALT lymphomas the four translocations are present altogether in about 36% of the cases and they occur mutually exclusive. The t(11;18) is only exceptionally accompanied by a trisomy, whereas the other translocations frequently harbor additional aberrations. As outlined in Table 2, the four translocations occur at markedly variable frequencies in MALT lymphomas of different sites. Whereas the t(11;18) is most prevalent in MALT lymphomas of the lung and stomach but rarely found in cutaneous, salivary gland and ocular adnexal/orbital tumors, the opposite is true for the t(14;18) and the t(3;14), respectively. A striking association between t(14;18) and hepatic MALT lymphoma (4 of 4 cases positive), and between t(3;14) and thyroidal MALT lymphoma (3 of 6 cases positive) has been observed.

Table 2. Frequency (%) of translocations in MALT lymphomas of different sites [26, 35] .

	n	t(11;18)	t(14;18)	t(1;14)	n	t(3;14)
Stomach	71	24	1	0	20	0
Skin	51	8	14	0	20	10
Salivary gland	42	2	12	2	20	0
Ocular adnexa/orbit	37	3	24	0	20	20
Intestine	16	12	0	12	-	-
Lung	15	53	7	7	5	0
Thyroid	6	0	0	0	6	50
Liver	4	0	100	0	0	0
Total	252				91

The significance of the relationship between a certain translocation (or pattern of translocations) with a MALT lymphoma at a given site is largely unclear but may hypothetically reflect a distinct pathogenesis. The occurrence of the t(11;18)(q21;q21) in gastric MALT lymphoma has been linked to oxidative damage caused predominantly by CagA-positive strains of Helicobacter pylori [36]. A similar etiology involving a hitherto unidentified pathogen is conceiveable in t(11;18)(q21;q21)-positive pulmonary MALT lymphoma [36]. This hypothesis is supported by the observation that t(11;18)(q21;q21)-positive pulmonary MALT lymphoma is not associated with autoimmune disease [37]. Besides the very well established relation of Helicobacter pylori infection and gastric MALT lymphoma, other microbial agents have been accused to trigger MALT lymphomas at certain sites, such as Borrelia burgdorferi cutaneous disease, and more recently Chlamydia psittaci ocular adnexal MALT lymphoma and Campylobacter jejuni an extremely rare variant of intestinal MALT lymphoma known as immunoproliferative small intestinal disease (IPSID) [9, 10, 11] .

The high frequency of trisomy 3 in MALT lymphoma, particularly in tumors arising from the intestine, salivary glands, and ocular adnexa suggests that one or more genes at chromosome 3, such as FOXP1 and BCL6, are involved in the pathogenesis of these neoplasms.

Table 3. Frequency (%) of trisomies 3 and 8 in MALT lymphomas of different sites [35].

	n	Trisomy 3	Trisomy 8
Stomach	71	11	6
Skin	51	20	4
Salivary gland	42	55	19
Ocular adnexa/orbit	37	38	14
Intestine	16	75	25
Lung	15	20	7

Diagnostic significance of MALT lymphoma-associated genetic aberrations
Four of the three translocations, the t(11;18), t(14;18), and t(1;14), are known to be specific for or are at least very closely associated with MALT lymphoma. This knowledge could be helpful in certain diagnostic situations, for instance the demonstration of the t(11;18) by RT-PCR in case of a small lung biopsy with a lymphoid infiltrate suspicious but not diagnostic of MALT lymphoma. Similarly, the close association of trisomies 3 and 18 with MALT lymphoma may be helpful in doutful cases. As shown in Table 3 the two trisomies, particularly trisomy 3, very often occur in intestinal, salivary gland, and ocular adnexal tumors. In context with the appropriate histopathologic and immunophenotypic features, the detection of one of these genetic aberrations, by RT-PCR in case of the t(11;18) or FISH in case of the other aberrations, would strongly favor the diagnosis of MALT lymphoma. Therefore, in the above mentioned context, the two techniques are more powerful than immunoglobulin gene rearrangement studies by PCR because they are able to detect not only a clonal process but additionally help to classify the disease.

Clinical significance of MALT lymphoma-associated translocations
So far, the t(11;18) in gastric MALT lymphoma is the only aberration which may influence therapeutic decision making as most translocation-positive tumors do not respond to Helicobacter pylori eradication therapy, are associated with more advanced stage of disease, and usually do not transform into aggressive lymphoma [38, 39, 40, 41] . The t(1;14) has been linked to a more aggressive clinical course, however MALT lymphomas carrying the t(1;14) are exceptionally rare (only 1.6% in our series of 255 cases) and therefore the tumors are not routinely screened for this aberration. Whether the remaining two translocations, i.e. t(14;18) and t(3;14), or any of the trisomies are important with respect to clinical parameters such as stage of disease, response to treatment and disease recurrence has to await the results of ongoing studies.

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