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Originally published as JCO Early Release 10.1200/JCO.2006.06.7165 on September 11 2006 © 2006 American Society of Clinical Oncology. Detection of Subclinical Systemic Disease in Primary CNS Lymphoma by Polymerase Chain Reaction of the Rearranged Immunoglobulin Heavy-Chain Genes
From the Departments of Hematology, Oncology, and Transfusion Medicine, and General Pathology and Reference Center for Hematopathology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin; and the Department of Radiation Therapy and Medical Oncology, Pius Hospital Oldenburg, Oldenburg, Germany Address reprint requests to Kristoph Jahnke, MD, Oregon Health and Science University, Department of Neurology, Blood-Brain Barrier and Neuro-Oncology Program, 3181 SW Sam Jackson Park Rd, L603, Portland, OR 97239-3098; e-mail: kristoph.jahnke{at}charite.de
PURPOSE: To search for subclinical systemic disease in bone marrow and peripheral blood in patients with primary CNS lymphoma (PCNSL) to elucidate whether extracerebral relapse may represent a sequel of initial occult systemic disease rather than true extracerebral spread. PATIENTS AND METHODS: Bone marrow and peripheral-blood specimens of 24 PCNSL patients were examined using polymerase chain reaction (PCR) for analysis of clonally rearranged immunoglobulin heavy-chain (IgH) genes. RESULTS: Identical dominant PCR products were found in bone marrow aspirates, blood samples, and tumor biopsy specimens of two patients, indicating that the same tumor cell population is present in the CNS and in extracerebral sites. Follow-up IgH PCR performed in one of these patients in complete remission 24 months after diagnosis yielded a persistent monoclonal product in the blood. An oligoclonal IgH rearrangement pattern was found in the tumor specimen of two other patients, whereas bone marrow and blood samples demonstrated the same dominant PCR products. Follow-up PCR showed a persistent monoclonal amplificate in blood in one of these patients 27 months after diagnosis. CONCLUSION: It could be demonstrated for the first time that subclinical systemic disease can be present in PCNSL patients at initial diagnosis. Our findings may have an impact on the understanding of PCNSL pathogenesis and the extent of staging and treatment.
Primary CNS lymphoma (PCNSL) is defined as a non-Hodgkin's lymphoma (NHL) confined to the CNS at the time of diagnosis.1 Relapse of PCNSL almost invariably occurs within the CNS, with a systemic relapse rate of only approximately 7%.1,2 Extracerebral relapse of PCNSL may derive from occult systemic disease at primary diagnosis rather than from true extracerebral dissemination. This is supported by detection of occult systemic disease in up to 13% of patients with thorough conventional staging procedures in some studies.3,4 The extent of systemic staging to distinguish PCNSL from secondary CNS spread of systemic lymphoma is a controversial issue. Polymerase chain reaction (PCR) for analysis of clonally rearranged immunoglobulin heavy-chain (IgH) genes may be used to demonstrate lymphoma manifestations below the detection limit of routine staging procedures in extracerebral NHL (so-called minimal residual disease5-7) but has never been evaluated in PCNSL for the detection of subclinical systemic disease. In this study, we examined bone marrow and peripheral blood in newly diagnosed PCNSL using a new advanced IgH PCR with primers for all three framework regions (FRs) to prove our hypothesis that some patients have concomitant systemic lymphoma not detectable by routine staging measures.
Patients Approval for this study was obtained from the institutional ethics committee. We examined specimens (both bone marrow and blood in 20 patients, blood or bone marrow in two patients each, and tumor in seven patients) from 24 consecutive HIV-negative patients (10 men and 14 women; median age, 62 years; range, 43 to 83 years) with newly diagnosed PCNSL presenting at five German institutions. Data on corticosteroid pretreatment were available in 20 patients, 10 of whom received corticosteroids. All tumors were classified according to WHO as diffuse large B-cell lymphomas.8 Original pathology reports were reviewed in all patients. Ocular and CSF involvement was initially excluded by slit-lamp examination and CSF examination in all patients. IgH PCR analysis of CSF and vitreal aspirates was not performed. Systemic manifestations were excluded in all patients. Staging procedures included blood count with differential, four-color flow cytometry of peripheral blood, liver enzymes, bilirubin, creatinine, lactate dehydrogenase, bone marrow examination, and computed tomography scans of the neck, chest, and abdomen. Positron emission tomography scans at baseline were performed in one patient only. High-dose methotrexate was administered to all patients, and adjuvant whole-brain irradiation (WBI) was performed in four patients.
IgH PCR Three different FR primer sets (FR1, FR2, and FR3) were applied separately to all samples (50 ng of DNA) in conjunction with a joining segment of the IgH chain gene (JH) consensus primer (JH22). All specimens were amplified at least twice in completely independent PCR runs, and PCR products were analyzed by a high-resolution separation technique allowing the discrimination of amplificates differing by only one base pair (Applied Biosystems, Weiterstadt, Germany). With each PCR, positive controls (DNA from a B-cell line and from a tonsil) and negative controls (water instead of DNA; one negative control after every three DNA samples) were run to exclude false-positive results (ie, contamination) and to prove the reliability of each PCR assay. For quality control, a PCR approach based on five amplificates of different sizes (100 to 600 base pairs) obtained from single-copy genes was performed. If the 300–base pair amplificate was not detectable in the control PCR, the DNA was not regarded suitable for further analysis.9
All 51 samples contained amplifiable DNA. Polyclonal and/or oligoclonal IgH PCR products without dominant amplificates were found in the blood and bone marrow specimens of 17 patients. In three patients, however, dominant amplification products were found that differed in size in repeated IgH PCR runs of the same sample as well as between bone marrow and blood (pseudoclonality). In another two patients (patients 1 and 3; Table 1, Figs. 1-3), IgH PCR yielded dominant PCR products in bone marrow and blood samples that were identical to those found in the corresponding tumor specimens, indicating the presence of the same clonal B-cell population. Finally, an oligoclonal IgH rearrangement pattern was found in the tumor specimen of two other patients (patients 2 and 4; Table 1, Figs. 1-3), whereas bone marrow and blood samples demonstrated the same dominant PCR products. This is most likely a result of lack of tumor cells in the remaining investigated tissue samples.
The reproducible dominant PCR products were sequenced, and the presence of the same monoclonal B-cell population was confirmed. All bone marrow aspirates and blood samples were reviewed, and no cytologic or immunophenotypic indication for lymphoma was detectable. One patient (patient 3) with monoclonal PCR product in blood and bone marrow and nine patients without PCR-detectable systemic involvement experienced relapse after a median follow-up time of 25 months. All relapses were intracerebral. Follow-up PCR was performed in blood and bone marrow of patient 1 and in blood of patient 4 at last follow-up in complete remission (CR) 24 and 27 months, respectively, after diagnosis and treatment with high-dose methotrexate and yielded a persistent monoclonal amplificate in the blood of both of the patients and a polyclonal product in the bone marrow of patient 1. In both patients, no lymphoma cells were detected in blood and/or bone marrow by morphologic and immunocytologic examination. Patient 2, who was in persisting CR after 24 months, refused follow-up PCR. Patient 3, who experienced relapse after 12 months and responded to WBI, died of pulmonary embolism in CR before follow-up PCR could be performed. Follow-up PCR performed in the blood of four patients without initial extracerebral monoclonal PCR products after 12 to 27 months in persisting CR yielded polyclonal amplificates in all instances.
For the first time, we demonstrated that PCNSL patients may have occult systemic involvement not detectable by routine staging. We used a new and highly reliable IgH PCR system (BIOMED-2)9 that has also been used in recent studies.10,11 The use of primers for all three IgH FRs helps to significantly decrease the rate of false-negative results caused by somatic hypermutation in primer binding sites of the involved VH gene segments, thus increasing the overall clonality detection rate. The systemic persistence of monoclonal B cells in two patients without other evidence of lymphoma may indicate that high-dose methotrexate and WBI are insufficient to eradicate subclinical disease in patients with PCNSL. Moreover, this finding provides an interesting insight into the biology of PCNSL. One may speculate that patients with PCNSL have not been observed long enough to see the development of (systemic) relapse detectable with conventional methods. However, a long-term quiescent behavior of residual tumor cells in aggressive lymphoma has not been reported thus far. Lack of proliferation support for tumor cells in the microenvironment of the systemic compartment or, alternatively, lack of proliferative potential of residual lymphoma cells may be a possible explanation. Clinical consequences of PCR testing for subclinical systemic disease in PCNSL remain speculative. Moreover, our results are confounded by the relatively small number of patients, the lack of positron emission tomography imaging at baseline in all but one patient, and frequent corticosteroid administration, which may have hampered detection of systemic disease. A longer prospective evaluation of a larger number of patients is necessary to determine clinical relevance of search for occult systemic disease in PCNSL. The question of whether therapy aimed at control of subclinical systemic disease will result in fewer (extracerebral) relapses and/or a better survival, as has been shown in systemic NHL,5 should then be addressed.
The authors indicated no potential conflicts of interest.
published online ahead of print at www.jco.org on September 11, 2006 Presented in part at the 42nd Annual Meeting of the American Society of Clinical Oncology, Atlanta, GA, June 2-6, 2006. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Fine HA, Mayer RJ: Primary central nervous system lymphoma. Ann Intern Med 119:1093-1104, 1993 2. Blay JY, Conroy T, Chevreau C, et al: High-dose methotrexate for the treatment of primary cerebral lymphomas: Analysis of survival and late neurologic toxicity in a retrospective series. J Clin Oncol 16:864-871, 1998[Abstract] 3. O'Neill BP, Dinapoli RP, Kurtin PJ, et al: Occult systemic non-Hodgkin's lymphoma (NHL) in patients initially diagnosed as primary central nervous system lymphoma (PCNSL): How much staging is enough? J Neurooncol 25:67-71, 1995[CrossRef][Medline] 4. Ferreri AJ, Reni M, Zoldan MC, et al: Importance of complete staging in non-Hodgkin's lymphoma presenting as a cerebral mass lesion. Cancer 77:827-833, 1996[CrossRef][Medline] 5. Zwicky CS, Maddocks AB, Andersen N, et al: Eradication of polymerase chain reaction detectable immunoglobulin gene rearrangement in non-Hodgkin's lymphoma is associated with decreased relapse after autologous bone marrow transplantation. Blood 88:3314-3322, 1996 6. Kurokawa T, Kinoshita T, Ito T, et al: Detection of minimal residual disease B cell lymphoma by a PCR-mediated RNase protection assay. Leukemia 10:1222-1231, 1996[Medline] 7. Kurokawa T, Kinoshita T, Murate T, et al: Complementarity determining region-III is a useful molecular marker for the evaluation of minimal residual disease in mantle cell lymphoma. Br J Haematol 98:408-412, 1997[CrossRef][Medline] 8. Jaffe ES, Harris NL, Stein H, et al: World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, International Agency for Research on Cancer Press, 2001 9. van Dongen JJ, Langerak AW, Bruggemann M, et al: Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17:2257-2317, 2003[CrossRef][Medline] 10. Sandberg Y, Heule F, Lam K, et al: Molecular immunoglobulin/T-cell receptor clonality analysis in cutaneous lymphoproliferations: Experience with the BIOMED-2 standardized polymerase chain reaction protocol. Haematologica 88:659-670, 2003 11. Lassmann S, Gerlach UV, Technau-Ihling K, et al: Application of BIOMED-2 primers in fixed and decalcified bone marrow biopsies: Analysis of immunoglobulin H receptor rearrangements in B-cell non-Hodgkin's lymphomas. J Mol Diagn 7:582-591, 2005 12. Marafioti T, Hummel M, Anagnostopoulos I, et al: Classical Hodgkin's disease and follicular lymphoma originating from the same germinal center B cell. J Clin Oncol 17:3804-3809, 1999 Submitted March 21, 2006; accepted June 14, 2006.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
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