Microsatellite instability in gastric MALT lymphomas and other associated neoplasms
Dipartimento di Scienze Cliniche e Biologiche e Servizio di Anatomia Patologica, Universila dell'Insubria e Ospedale di Circolo, Varese; 'Istituto Oncologico delta Svizzera Italiana, IOS1, Divisione di Oncologia Medica, Ospedale San Giovanni
Centro inlerdisciplinare di Oncologia, Ospedale di Circolo
Summary Background: Microsatellite instability (MSI), caused by a reduced efficacy of the DNA mismatch repair (MMR) machinery, represents a type of genomic instability frequently detected in HNPCC spectrum cancers and in a subset of sporadic carcinomas. The involvement of MSI in the pathogenesis of gastric lymphoma of mucosa-associated lymphoid tissue (MALT) has never been conclusively investigated. In this study, we tested the presence of MSI in tumor samples of patients harboring both MALT lymphomas and other types of malignancies. Materials and methods: We examined 10 microsatellite loci (D3S11, D3S1261, D3S1265, D6S262, D6S193, BAT-26, BAT25, D17S250, APC, D2S123) out of a total of 34 primary tumors from 14 patients with MALT lymphomas and one or more additional neoplasms. The patients' MSI results were also tested for an association with a positive family history of cancer. Results: MSI, defined by the presence of microsatellite alterations in more than 40% of the examined loci, was scored negative in all tumors studied, and pedigree analysis failed to identify any condition of familial cancer among the patients examined. Conclusions: The present study suggests that defects in DNA mismatch repair do not contribute significantly to the molecular pathogenesis of MALT lymphomas and associated neoplasms.
B-cell lymphomas of mucosa-associated lymphoid tissue
(MALT) comprise a distinct clinico-pathological entity
among the extra-nodal non-Hodgkin's lymphomas .
Gastric lymphoma is the most common type of MALT
lymphoma and there is strong evidence that
Helicobacter pylori infection is critical in the pathogenesis of
this tumor . However, it is not clear whether H. pylori
alone causes the evolution from chronic gastritis to
MALT lymphoma, or if other factors interfering with
pathogenetic mechanisms of these tumors are required.
The genetic background underlying the pathogenesis of
MALT lymphoma is still poorly understood.
Some authors have identified trisomy 3 as the most
frequent numerical chromosome aberration in
lowgrade MALT lymphomas [3-6]. In a recent report Ott
et al. , studying a large series of low- and high-grade
MALT lymphomas with a sensitive bicolor fluorescence
in situ hybridization (FISH), identified aneusomies of
chromosomes 3, 7, 12 and 18 only in high-grade cases.
In addition, reciprocal translocation such as
t(ll;18)(q21;q21)  and genomic rearrangement of bcl-6 
have been reported to be the most common aberrations
in low-grade MALT lymphomas.
In 1993, the discovery of microsatellite instability
(MSI) in colorectal cancer and its link to hereditary
nonpolyposis colorectal cancer (HNPCC) highlighted a
new oncogenic mechanism . MSI, defined as the
presence of tumor-associated alterations in the germline
size of microsatellite repeats, is the result of a reduced
efficacy of the DNA mismatch repair (MMR)
machinery. MSI has also been found in a wide variety of
human sporadic cancers [11-13] and may be the most
likely cause of multiple primary tumors in non-HNPCC
Although MSI is rarely involved in lymphomas [15,16],
Peng et al. in 1996  reported a surprisingly high
frequency of MSI in both low- (50%) and high- (56%)
grade MALT lymphomas and suggested that MMR
defects may cause the accumulation of mutations in
cancer-related genes such as TP53 . Du et al. 
proposed that MSI may also explain the increased
incidence of additional tumors (such as carcinomas or
other types of lymphomas) observed in patients with
MALT lymphomas  and represent additional
evidence that MALT tumors are a distinct
clinico-pathological and genetic entity .
In sharp contrast to these findings, other studies [21,
22] failed to identify MSI as a frequent aberration in
gastric MALT lymphomas.
The aim of this work was to define the frequency of
Lymph node (75)
Adrenal gland (62)
Breast DCa (43)
Pancreas DCa (48)
Breast DCa (64)
Bladder PTCa (62)
Larynx Ca (50)
Rectum Ca (59)
Stomach Ca (70)
Stomach Ca (62)
Bilateral kidney PCa (62)
Patient MALT lymphoma site MALT lymphoma grade Additional lymphomas
Abbreviations: LG - low-grade MALT lymphoma; HG - high-grade MALT lymphoma; DLBCL - diffuse large B-cell lymphoma; DCa - ductal
carcinoma; PTCa - papillary transitional carcinoma; Ca - carcinoma; PCa - papillary carcinoma; CCa - clear cell carcinoma.
The ages of tumor onset are indicated in brackets.
MSI in tumor samples of patients harboring both
MALT lymphomas and other types of malignancies.
According to the literature, these patients with different
multiple malignancies might have an acquired and/or
inherited risk for defects in their DNA mismatch repair
systems . The patients' MSI results were also tested
for an association with a positive family history.
Materials and methods
Case selection and histological investigations
A total of 34 primary tumors from 14 patients with MALT lymphomas
and one or more additional malignancies from which material for MSI
analysis was available were selected from a consecutive series of
MALT lymphomas observed in our institutions betweeen 1986 and
1998 (Table 1). The clinical data of some of the patients were analysed
in a previous paper . All patients were from a geographic area that
is characterized by a high incidence of MALT lymphomas (southern
Switzerland and northern Italy).
Microdissection and DNA extraction
For each patient. 10 um-thick sections were obtained from paraffin
blocks of formalin-fixed tumor tissues and from at least two blocks of
histologically normal tissue including gastric mucosa, bone-marrow,
lymph nodes and spleen.
We performed a careful dissection of all of the pathology
specimens under microscopic guidance to ensure the presence of at least
70% of malignant cells in the selected area.
Genomic DNA was obtained by xylene deparaffinization followed by
proteinase 1C digestion and purification by MicroSpin S-200 Columns
(Pharmacia Biotech, Italy).
Microsatellite markers and MSI analysis
We analyzed MSI status by examining two mononucleotide and eight
dinucleotide microsatellite markers: five of these (D3SI1, D3S1261,
D3S1265, D6S262) were selected for the high rates of instability in
MALT lymphomas reported by Peng et al.  and for chromosome
location (D6S193; ). In addition, in accord with the recent
guidelines for MSI analysis , we included in our study the panel of
markers (BAT-26, BAT-25, D17S250, APC, D2SI23) recommended by
the National Cancer Institute Workshop of Bethesda 1997  for their
high sensitivity and specificity in the detection of MSI tumors.
The PCR mixture (50 ul) included 100-200 ng of genomic DNA,
10 mM Tris. 50 mM KC1, 1-2 mM MgCl2, 200 uM dNTPs and 50
pmol of each primer, 2U Taq Polymerase (Perkin-EImer, Italy).
Samples were denatured at 94 "C for 5 minutes followed by 35
cycles of denaturation (94 =C, 30'), annealing (55 C, 30'), extension
(72 C, 30') and a final extension of 5 minutes at 72 C in a
PerkinEImer Gene Amp Thermal 2400. PCR products were electrophoresed
on 6% denaturing polyacrilamide sequencing gels for two hours at
1600 V, 40 mA, 50 "C. Polyacrilamide gels, fixed on glass plates with
methacryloxypropyltrimethoxysane, were silver-stained using the
DNA Silver Staining System (Promega, Italy). Silver-stained gels were
reproduced on EDF films (Kodak, USA).
A three-generation family history of cancer was analyzed during the
genetic counselling of all 14 patients.
Fourteen patients with MALT lymphomas and one or
more additional malignancies (Table 1) were tested for
repeat instability by microsatellite analysis.
A total of 34 tumors (21 MALT lymphomas, 11
carcinomas, 2 diffuse large B-cell lymphomas) were studied
at 10 microsatellite loci including two mononucleotide
repeats and eight dinucleotide repeats.
The results of microsatellite analysis are summarized
in Table 2 and representative cases are illustrated in
Figures 1 and 2. Microsatellite alterations at three loci
on chromosome 3 were observed in both gastric MALT
lymphoma and lung DLBCL of patient 5 and a
novellength allele for a single locus was detected in the gastric
MALT lymphomas of patients 4, 9 and 10. In one
carcinoma (a papillary transitional carcinoma of the
urinary bladder) size alterations at loci D2S123 and
D3S1265 were observed (case 10, Table 2). All
microsatellite alterations showed only a single new band
resulting from slight expansion or contraction of the
original allele and were classified as type II according to
Thibodeau et al. .
None of the MALT or associated tumors exhibited
alterations at more than 40% of the ten loci tested and,
as a consequence, MSI was scored negative in all tumors
studied according to the Bethesda guidelines . In
contrast, according to the criteria for MSI adopted by
reported in other families but no typical association
with well known familial cancer syndromes was