VOLUME

23

NUMBER

16

JUNE

2005

JOURNAL OF CLINICAL ONCOLOGY

O R I G I N A L

R E P O R T

Gastric Mucosa-Associated Lymphoid Tissue

Lymphoma Detected by Clonotypic Polymerase Chain
Reaction Despite Continuous Pathologic Remission
Induced by Involved-Field Radiotherapy
Ariela Noy, Joachim Yahalom, Leah Zaretsky, Ian Brett, and Andrew D. Zelenetz
From the Lymphoma Disease Management Team and Laboratory of Molecular
Hemato-Oncology, Memorial SloanKettering Cancer Center, New York, NY.
Submitted October 6, 2004; accepted
March 3, 2005.
Authors disclosures of potential conflicts of interest are found at the end of
this article.
Address reprint requests to Ariela Noy,
MD, Memorial Sloan-Kettering Cancer
Center, 1275 York Ave, New York, NY
10021; e-mail: noya@mskcc.org.
2005 by American Society of Clinical
Oncology
0732-183X/05/2316-3768/$20.00
DOI: 10.1200/JCO.2005.10.018

Purpose
Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is indolent and often associated with Helicobacter pylori bacterial infection. H pyloriindependent MALT develops
either in the absence of the bacteria or persists after bacterial eradication. We have
previously demonstrated long-term pathologic remission after involved-field radiotherapy
therapy (IFRT). We determined molecular remission status by clonotypic polymerase chain
reaction (PCR).
Patients and Methods
Twenty-four consecutive patients with stage I to IIE gastric MALT lymphoma who obtained
a pathologic remission after IFRT alone were evaluated. All had at least two follow-up
endoscopic gastroduodenal biopsies at Memorial Sloan-Kettering Cancer Center. IFRT
median dose was 30 Gy (range, 28.5 to 43.5 Gy). Post-treatment biopsies were subjected to
semi-nested clonotypic PCR.
Results
All patients obtained a complete response based on routine immunohistochemical pathologic analysis of random post-treatment gastric biopsies. Median follow-up from completion
of IFRT was 63 months (range, 19 to 117 months). Event-free survival was 96%; 23 of 34
patients remained in clinical and pathologic complete remission. Baseline DNA extraction
yielded 17 clone-specific primer pairs. At the first follow-up test, 14 of 17 pairs were PCR
positive. Eight remained persistently positive; and one was persistently negative. Others
were intermittently positive.
Conclusion
Despite sustained biopsy-proven remissions for as long as 117 months after radiation, the
vast majority of patients remain positive by clonotypic PCR. This suggests that the malignant
clone is present but missing either an internal or external signal essential to the cancer
phenotype. One possibility is that radiation eradicates the polyclonal H pylorispecific T cells
eliminating critical local factors necessary for proliferation of the monoclonal B cells.
J Clin Oncol 23:3768-3772. 2005 by American Society of Clinical Oncology

INTRODUCTION

Gastric marginal zone lymphoma of mucosaassociated lymphoid tissue (MALT) is an indolent lymphoma often associated with
Helicobacter pylori bacterial infection. Antigen
stimulation leads to a polyclonal T-cell re-

sponse promoting a B-cell monoclonal proliferation.1,2 The idiotype of the B cell is not
specific for H pylori. Eradication of the bacteria can lead to pathologic lymphoma regression in many cases (reviewed in Zucca and
Cavalli3). However, the long-term efficacy
of antibiotic therapy has not been fully

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MALT Lymphoma Detected by Clonotypic PCR

established, and in approximately half of pathologic complete

remissions, the malignant clone can be detected in endoscopic
gastroduodenal (EGD) biopsies by polymerase chain reaction
(PCR) targeted to the immunoglobulin heavy-chain gene.4
In contrast, H pyloriindependent MALT develops either in the absence of the bacteria or persists after bacterial
eradication. We have previously demonstrated long-term clinical and pathologic remission after involved-field radiotherapy
(IFRT).5 Seventeen patients were treated with a median total
radiation dose of 30 Gy (range, 28.5 to 43.5 Gy) delivered in
1.5-Gy fractions within 4 weeks to the stomach and adjacent
lymph nodes. All patients obtained a biopsy-confirmed complete response. At a median follow-up time of 27 months
(range, 11 to 68 months) from completion of radiotherapy,
event-free survival rate was 100%.
These results were updated recently6 to include 51 patients, five of whom had persistent (n 3) or relapsed (n 2)
disease after chemotherapy. A biopsy-proven complete response was obtained in 49 (96%) of 51 patients. Three patients
relapsed (at 14, 27, and 29 months); one patient who relapsed
was successfully treated with salvage therapy with additional
IFRT. Three patients died of other malignancies (melanoma,
bladder, and lung) that were all outside the radiation field and
with no evidence of recurrent MALT lymphoma. At a median
follow-up of 4 years, the freedom from treatment failure rate
was 89% 5%, and the cause-specific survival rate was 100%.
The current study was undertaken to determine whether these
patients achieve molecular remission after radiotherapy.
PATIENTS AND METHODS
Patient Population
The radiation oncology database files of Memorial SloanKettering Cancer Center (MSKCC) were reviewed to identify all
patients (N 51) with stage I to IIE gastric MALT lymphoma receiving radiation therapy from 1992 to 2001. Patients (n 24) were
included in the current study if they had not received prior chemotherapy or radiation therapy and if they had at least two follow-up
EGD biopsies at MSKCC. All diagnoses were reviewed by MSKCCdedicated hematopathologists. When sufficient material was available, immunoperoxidase studies were performed, in addition to
hematoxylin and eosin sections, using a biotin-avidin peroxidase
complex method according to the manufacturers instructions (Ventana Medical Systems, Inc, Tucson, AZ). The monoclonal antibodies
used were CD20, CD5, CD3, CD10, and Bcl-2.
Staging included total body computed tomography with oral
and intravenous contrast and bilateral bone marrow biopsies.
Patients enrolled after 2000 often had positron emission tomography scans performed as well.
The frequency of EGD biopsy was dictated by the treating
physician, but typically, it was performed 2 to 3 months after IFRT,
between 6 and 12 months during the first 2 years, and annually
thereafter. Almost all posttreatment biopsies included samples of
any suspicious-appearing sites and random sampling of the cardia,
greater and lesser curvature, and antrum of the stomach. The
study was reviewed by the institutional review board and determined to be exempt research as per 45 CFR 46.101.b.4

PCR Amplification of Rearranged

Immunoglobulin H Genes
DNA extraction for PCR amplification of immunoglobulin
(Ig) H genes was performed on diagnostic pretreatment specimens. Three PCR quality curls totaling 300 M each were cut from
EGD-derived biopsies. DNA extraction was performed using a
QiaAmp DNA mini kit (Qiagen, Valencia, CA) with overnight
octane paraffin dissolution. The rearranged VH gene from B-cell
tumors was amplified, as previously described, using a set of
primers based on Fr2a and Fr3a consensus sequences with a downstream semi-nested pair of JH primers (LJH and VLJH), as previously described, with minor modifications.7-9
Because of the small nature of the biopsy samples, quantification of input DNA was not performed so as to preserve the
sample. PCR reactions were carried out in 50-L volumes containing 1 and 5 L DNA template, 0.5 mol/L each of the 5 and 3
primers, 200 mol/L each deoxynucleotide triphosphate, 10
mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2,
and 0.625 U Taq DNA polymerase (Roche Diagnostics, Penzberg,
Germany). The cycling conditions using a Perkin Elmer 9700
thermal cycler (Perkin Elmer, Wellesley, MA) were as follows: an
initial denaturation step at 95C for 7 minutes, followed by three
cycles of denaturation (45 seconds at 93C), annealing (45 seconds
at 50C), and elongation (110 seconds at 72C). After the last cycle,
a final elongation step for 7 minutes at 72C was performed. The
second round of amplification with the semi-nested 3 was carried
out under the same conditions without the initial 7-minute denaturation at 95C using 1 L of a 1:1,000 dilution of the first-round
product. PCR fragments (10 L) were separated on 4% agarose
gels (2% Nu-Sieve and 2% Metaphor; Camprex Bioscience, Rockland, ME) in 90 mmol/L Tris-borate and 2 mmol/L EDTA (Trisborate-EDTA buffer) and visualized with ethidium bromide staining.
All experiments were run with a negative and a positive control.
The former consisted of all the PCR reagents in the absence of a DNA
template. The latter used alternative primer pairs for a pml gene
fragment (PML 2733 5-gaggttctcttaagccaccg-3 and PML 2734 5aagcgtcaacactaggcagg-3). All oligos were purchased from Sigma
Genosys (the Woodlands, TX) and amplified under the same
conditions as the Ig VH familyspecific leader primers, resulting in
a 220-bp fragment. The positive control verified the integrity of
the DNA template. To prevent contamination, simultaneous amplification of a positive control template was not performed. PCR
set ups were carried out in a dedicated hood into which amplified
PCR products were never introduced.
Clone-Specific Primer Design
Nested primer pairs unique to the malignant clone for the
detection of minimal residual disease (MRD) were created based
on the sequence of the third complementarily determining region
(CDR-III) region of the malignant clone as previously described.10
The initial PCR product reflecting the monoclonal Ig rearrangement was gel purified and directly sequenced using a Taq DNA
polymerase based sequencing strategy (Dye Terminator Cycle
Sequencing Reaction Kit; Perkin Elmer) on an automated sequencer (ABI Prism, Foster City, CA). Bidirectional sequencing
was performed for verification. The compatibility and expected
performance characteristics of these primers with the appropriate
downstream VLJH primer were tested using the software package
Oligo 8.0 (Molecular Biology Insights, Cascade, CO). Thermodynamic properties, including annealing temperatures, were assessed before implementation, increasing the likelihood that PCR
using these primers would be successful.
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Noy et al

Clonotypic PCR
Conditions for clonotypic PCR were identical to those used
for isolating the initial V-D-J sequence with the exception of the
annealing temperatures, which were modified for each primer pair
based on the PCR predicted by Oligo 6.0. Primer pairs were first
tested on 1 and/or 5 L of the diagnostic sample from which the
initial V-D-J was amplified. In the first round of amplification, the
outer 5 CDR-III clone-specific primer was used. In the second round
of amplification, 1 L of a 103 dilution of the first-round PCR
product served as a template with the nested CDR-III clone-specific
primer. PCR products were run on a 4% Metaphor gel (FMC BioProducts, Rockland, ME) with a 10-bp ladder (Invitrogen, Carlsbad,
CA). The predicted sizes of both products served to verify the specificity of the reaction products. Negative controls included a no DNA
template and a polyclonal template reaction. The latter also served to
test the specificity of the primers. Positive control reactions used the
pml primers described earlier. In the event that primer pairs failed to
demonstrate a size-appropriate band in the baseline material or the
polyclonal control demonstrated a band of the same size, additional
primer pairs were designed. If more than 2 primer sets failed, the
patient was considered unassessable by this method.
Endoscopic Biopsy and Post-treatment PCR
Post-treatment EGD biopsies were evaluated for MRD, with
each biopsy site tested separately in duplicate. Although the exact
location and number of biopsies were dictated by the endoscopist,
at most time points, four separate biopsies were taken, typically
from the fundus, antrum, and greater and lesser curvature.
Clonotypic PCR was performed on each biopsy site separately.
No genomic templates were amplified simultaneously. Clonotypic
PCR was performed on 1 and 5 L of extracted DNA. Controls for
contamination, DNA integrity, and primer performance were used.
Experiments were performed in multiple replicates. Two replicates
with the same result were reported as positive or negative, as appropriate. If disparate results were obtained on the same sample, a third
replicate was performed. The majority result was reported.
A fractional PCR score was derived to reflect the fraction of all
biopsy sites at a given time point. For example, a 3/4 score denotes
that three of four biopsy sites were positive; a numerical score of
0/4 denotes that none of four sites was positive. In addition, a
composite PCR score was derived to reflect the overall time point.
If any biopsy was positive, the composite PCR was positive.

RESULTS

From 1992 to 2001, 51 patients with stage I to IIE MALT

lymphoma were treated with radiation therapy at MSKCC.
The median total radiation dose was 30 Gy (range, 22.5 to
43.5 Gy) delivered in 1.5-Gy fractions within 4 weeks to the
stomach and adjacent lymph nodes. Eight patients were excluded because of persistent (n 3) or relapsed (n 2) disease
after chemotherapy. Not all patients had subsequent endoscopies performed at MSKCC. Twenty-four consecutive patients
with stage I to IIE gastric MALT without prior chemotherapy
or radiation and who had at least two follow-up EGD biopsies
at MSKCC were identified and studied.
Treatment was well tolerated, with no significant shortor long-term side effects. All patients obtained a biopsy3770

confirmed histologic complete response. One patient (unique

patient identification number [UPIN] 4) received 22.5 Gy, had
a pathologic partial response followed by an additional 15 Gy,
and remains without evidence of disease. All but one patient
remained asymptomatic and without evidence of disease at
the last follow-up (median time from completion of radiotherapy, 63 months; range, 19 to 117 months). Diseasespecific event-free survival rate was 96%, with all but one
patient remaining in clinical and pathologic complete remission. One patient (UPIN 14) may have developed pulmonary MALT after 108 months of follow-up.
DNA was extracted from the baseline diagnostic EGDdirected biopsies from the 24 eligible patients. PCR of the
heavy-chain Ig gene rearrangement and sequencing yielded
17 clone-specific primer pairs. In two patients, the baseline
DNA extracted was of poor quality and could not be amplified by the control pml sequences. In five patients, a monoclonal sequence could not be obtained from the PCR
amplimer despite repeated attempts. Thus, 17 patients were
assessable by clonotypic PCR.
Endoscopy was typically performed 2 months after
radiation to confirm pathologic remission and then every 6
to 12 months thereafter at the discretion of the physician.
Most patients continued to undergo endoscopy annually
throughout the period of follow-up. The median time from
diagnosis to the last EGD was 39 months (range, 9 to 104
months). Biopsy sites were dictated by the endoscopist,
although typically, four separate biopsies were taken from
the fundus, antrum, and greater and lesser curvature.
Figure 1 demonstrates the PCR results by time point.
The fraction of biopsy sites testing positive by clonotypic
PCR is given in Figure 1, and a graphical representation of
the composite score for the time point is also shown. The
first endoscopy was typically shortly after the radiation, and
the second endoscopy was typically the first to be evaluated
by PCR. At the first time point tested, 14 of 17 patients were
positive by clonotypic PCR. Three patients (UPIN 16, 19,
and 24) had only one time point evaluated, and all were
positive. The most common pattern was seen in eight patients, for whom the clonotypic PCR remained persistently
positive with a median follow-up of 39 months (range, 11 to
74 months). Two patients (UPIN 7 and 20) were intermittently positive with 52 and 104 months of follow-up. UPIN
23 was initially negative but became positive at two subsequent biopsies. One patient (UPIN 14) was positive at the
first two follow-up biopsies but converted to negative at the
subsequent two biopsies (the last biopsy was approximately
4.5 years after radiation). Finally, only one patient was
persistently negative at all time points up to 39 months; for
this patient, the clonotypic PCR primers were effective on
the baseline specimen (data not shown).
At any given time point, multiple sites were biopsied in
most patients. In the 67 time points evaluated, a mean of 3.4
and a median of four sites were biopsied. Three or more
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MALT Lymphoma Detected by Clonotypic PCR

Fig 1. Clonotypic polymerase chain reaction (PCR) in gastric mucosa-associated lymphoid tissue lymphoma after involved-field
radiotherapy (IFRT). At each time point, up to
four biopsies were assessed independently;
results are shown numerically below the
composite PCR result. Solid circles (F): at
least one biopsy positive by clonotypic PCR;
unfilled squares (): no biopsy positive. For
UPIN 4, solid vertical bar represents a second course of IFRT.

biopsies were obtained 82% of the time at any given time

point. No particular pattern was discernible for a given
biopsy site. Even in patients who had a persistently positive
composite score, a particular site could be alternatively
positive and negative (data not shown).
DISCUSSION

Despite sustained clinical- and biopsy-proven pathologic

remissions lasting as long as 117 months after radiation, the
vast majority of patients (94%) has evidence of clonal MRD
by clonotypic PCR. Indeed, in our series, only one patient

was persistently negative at all time points tested, and three

additional patients were negative at the last time point
tested after 2 to 6 years of persistent positivity. The majority
of patients with serial assessable biopsies remained persistently positive, whereas three patients were positive at last
follow-up but had intermittently negative clonotypic PCR.
In most cases, multiple biopsies were performed at
each time point; three or more biopsies were obtained at
82% of the time points. This decreases the chance that a
false-negative composite score was obtained for any given
time point. However, the fact that a given site (antrum, for
example) did not remain persistently positive even in the
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Noy et al

overall persistently positive patient could be a sampling error caused by the minute nature of the EGD-directed biopsies and the relative scarcity of residual lymphoma cells.
Previous studies have suggested that molecular evidence of clonal disease persists in roughly 50% of patients
after antibiotic eradication of H pylori and subsequent histologic remission.4,11-13 More recently, Alpen et al14 reported
resolution of monoclonal B-cell proliferation in a variety of
gastric lymphomas after combined chemoradiotherapy. These
studies varied in technique from the study reported here in
that consensus primers directed at detecting Ig gene rearrangements were used to evaluate follow-up time points rather than
clone-specific primers. In one study,11 monoclonal amplimers
were sequenced to verify a persistent clone identical to the
original clonal disease, whereas in another study,14 sequencing
demonstrated that clones did not persist. The clonotypic PCR
assay used in the current study improves sensitivity approximately 100-fold to at least 1 in 106 cells10; thus, we would
anticipate that this technique would reveal a higher percentage
of patients with molecular MRD after antibiotic therapy alone.
Therefore, the apparent superiority of antibiotics to eradiate
MRD compared with the current results with radiation therapy is likely a consequence of the assays for MRD.
Similar to the data presented after H pylori eradication
and histologic regression of gastric MALT lymphoma, our
patients did extremely well despite persistent evidence of
molecular residual disease. Indeed, during a median of 63
months of follow-up, only one patient (UPIN 14) may have
had a relapse. This patient developed pulmonary disease
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3772

that was felt clinically to be consistent with recurrently MALT

lymphoma, although a biopsy was not performed. The last
EGD biopsy was 4 years prior, with that biopsy and the previous EGD biopsy showing no molecular evidence of disease.
The clinical and biologic relevance of monoclonal populations in the absence of disease has been described in
other situations as well. For example, the t(14:18)(q32;q21)
has been demonstrated in circulating mononuclear cells after
radiation for localized follicular lymphoma in patients thought
to be cured of their disease.15 Various tumor-associated translocations have also been noted in healthy volunteers.16,17
In the case of gastric MALT, the persistence of a malignant clone in such a high proportion of patients without clinical relapse is surprising. This suggests that the malignant clone
is present but missing either an internal or external signal
essential to the cancer phenotype. One possibility is that radiation eradicates the polyclonal H pylorispecific T cells, thus
eliminating paracrine cytokine-mediated B proliferation. Activation of the API2 and MLT genes in MALT lymphoma
results in resistance to apoptosis.18,19 This may explain a longlived B-cell population, but in the absence of a proliferative
signal, they remain quiescent. In either case, long-term
follow-up of these patients seems warranted.

Authors Disclosures of Potential

Conflicts of Interest
The authors indicated no potential conflicts of interest.

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