Gastric Mucosa-Associated Lymphoid Tissue Lymphoma Detected by Clonotypic Polymerase Chain Reaction Despite Continuous Pathologic Remission
http://www.100md.com
《临床肿瘤学》
the Lymphoma Disease Management Team and Laboratory of Molecular Hemato-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY
ABSTRACT
PURPOSE: Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is indolent and often associated with Helicobacter pylori bacterial infection. H pylori–independent 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 pylori–specific T cells eliminating critical local factors necessary for proliferation of the monoclonal B cells.
INTRODUCTION
Gastric marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is an indolent lymphoma often associated with Helicobacter pylori bacterial infection. Antigen stimulation leads to a polyclonal T-cell response 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 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 pylori–independent 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 Sloan-Kettering 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 MSKCC-dedicated 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 manufacturer's 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 μM 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-μL volumes containing 1 and 5 μL DNA template, 0.5 μmol/L each of the 5' and 3' primers, 200 μmol/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 95°C for 7 minutes, followed by three cycles of denaturation (45 seconds at 93°C), annealing (45 seconds at 50°C), and elongation (110 seconds at 72°C). After the last cycle, a final elongation step for 7 minutes at 72°C 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 95°C using 1 μL of a 1:1,000 dilution of the first-round product. PCR fragments (10 μL) 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 (Tris-borate-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 5'-aagcgtcaacactaggcagg-3'). All oligos were purchased from Sigma Genosys (the Woodlands, TX) and amplified under the same conditions as the Ig VH family–specific 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.
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 μL 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 μL of a 10–3 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 μL 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
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 short- or long-term side effects. All patients obtained a biopsy-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). Disease-specific 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 EGD-directed 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 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 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 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 pylori–specific 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 long-lived 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.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Hussell T, Isaacson PG, Crabtree JE, et al: Helicobacter pylori-specific tumour-infiltrating T cells provide contact dependent help for the growth of malignant B cells in low-grade gastric lymphoma of mucosa-associated lymphoid tissue. J Pathol 178:122-127, 1996
Hussell T, Isaacson PG, Crabtree JE, et al: The response of cells from low-grade B-cell gastric lymphomas of mucosa-associated lymphoid tissue to Helicobacter pylori. Lancet 342:571-574, 1993
Zucca E, Cavalli F: Are antibiotics the treatment of choice for gastric lymphoma Curr Hematol Rep 3:11-16, 2004
Bertoni F, Conconi A, Capella C, et al: Molecular follow-up in gastric mucosa-associated lymphoid tissue lymphomas: Early analysis of the LY03 cooperative trial. Blood 99:2541-2544, 2002
Schechter NR, Portlock CS, Yahalom J: Treatment of mucosa-associated lymphoid tissue lymphoma of the stomach with radiation alone. J Clin Oncol 16:1916-1921, 1998
Yahalom J, Portlock C, Gonzales M, et al: H. Pylori-independent MALT lymphoma of the stomach: Excellent outcome with radiation alone. Blood 100:160a, 2002 (abstr)
Trainor KJ, Brisco MJ, Story CJ, et al: Monoclonality in B-lymphoproliferative disorders detected at the DNA level. Blood 75:2220-2222, 1990
Ramasamy I, Brisco M, Morley A: Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms. J Clin Pathol 45:770-775, 1992
Diss TC, Peng H, Wotherspoon AC, et al: Detection of monoclonality in low-grade B-cell lymphomas using the polymerase chain reaction is dependent on primer selection and lymphoma type. J Pathol 169:291-295, 1993
Noy A, Verma R, Glenn M, et al: Clonotypic polymerase chain reaction confirms minimal residual disease in CLL nodular PR: Results from a sequential treatment CLL protocol. Blood 97:1929-1936, 2001
Savio A, Franzin G, Wotherspoon AC, et al: Diagnosis and posttreatment follow-up of Helicobacter pylori-positive gastric lymphoma of mucosa-associated lymphoid tissue: Histology, polymerase chain reaction, or both Blood 87:1255-1260, 1996
Thiede C, Wundisch T, Alpen B, et al: Long-term persistence of monoclonal B cells after cure of Helicobacter pylori infection and complete histologic remission in gastric mucosa-associated lymphoid tissue B-cell lymphoma. J Clin Oncol 19:1600-1609, 2001
Montalban C, Santon A, Boixeda D, et al: Treatment of low grade gastric mucosa-associated lymphoid tissue lymphoma in stage I with Helicobacter pylori eradication: Long-term results after sequential histologic and molecular follow-up. Haematologica 86:609-617, 2001
Alpen B, Kuse R, Parwaresch R, et al: Ongoing monoclonal B-cell proliferation is not common in gastric B-cell lymphoma after combined radiochemotherapy. J Clin Oncol 22:3039-2045, 2004
Finke J, Slanina J, Lange W, et al: Persistence of circulating t(14;18)-positive cells in long-term remission after radiation therapy for localized-stage follicular lymphoma. J Clin Oncol 11:1668-1673, 1993
Dolken G, Illerhaus G, Hirt C, et al: BCL-2/JH rearrangements in circulating B cells of healthy blood donors and patients with nonmalignant diseases. J Clin Oncol 14:1333-1344, 1996
Trumper L, Pfreundschuh M, Bonin FV, et al: Detection of the t(2;5)-associated NPM/ALK fusion cDNA in peripheral blood cells of healthy individuals. Br J Haematol 103:1138-1144, 1998
Dierlamm J, Baens M, Wlodarska I, et al: The apoptosis inhibitor gene API2 and a novel 18q gene, MLT, are recurrently rearranged in the t(11;18)(q21;q21)p6 associated with mucosa-associated lymphoid tissue lymphomas. Blood 93:3601-3609, 1999
Dierlamm J, Wlodarska I, Michaux L, et al: Genetic abnormalities in marginal zone B-cell lymphoma. Hematol Oncol 18:1-13, 2000(Ariela Noy, Joachim Yahal)
ABSTRACT
PURPOSE: Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is indolent and often associated with Helicobacter pylori bacterial infection. H pylori–independent 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 pylori–specific T cells eliminating critical local factors necessary for proliferation of the monoclonal B cells.
INTRODUCTION
Gastric marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is an indolent lymphoma often associated with Helicobacter pylori bacterial infection. Antigen stimulation leads to a polyclonal T-cell response 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 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 pylori–independent 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 Sloan-Kettering 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 MSKCC-dedicated 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 manufacturer's 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 μM 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-μL volumes containing 1 and 5 μL DNA template, 0.5 μmol/L each of the 5' and 3' primers, 200 μmol/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 95°C for 7 minutes, followed by three cycles of denaturation (45 seconds at 93°C), annealing (45 seconds at 50°C), and elongation (110 seconds at 72°C). After the last cycle, a final elongation step for 7 minutes at 72°C 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 95°C using 1 μL of a 1:1,000 dilution of the first-round product. PCR fragments (10 μL) 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 (Tris-borate-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 5'-aagcgtcaacactaggcagg-3'). All oligos were purchased from Sigma Genosys (the Woodlands, TX) and amplified under the same conditions as the Ig VH family–specific 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.
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 μL 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 μL of a 10–3 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 μL 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
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 short- or long-term side effects. All patients obtained a biopsy-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). Disease-specific 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 EGD-directed 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 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 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 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 pylori–specific 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 long-lived 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.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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